Document G6wxY5qOMkXZkMjVebeJJkxYq
2002 OCT 18 AH 9 ^ 7
A R S .a 6 -_ l/3>
REVISED DRAFT HAZARD ASSESSMENT OF PERFLUOROOCTANOIC ACID
AND ITS SALTS
ANNEX 1 ROBUST SUMMARIES
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EPIDEMIOLOGIC DATA
Title: Identification of Fluorochemicals in Human Sera. I. American Red Cross Adult Blood Donors TEST SUBSTANCE
Identity: PFOA and 6 other fluorochemicals Remarks: This is a final report. The preliminary report was dated June 25, 2001.
METHOD
Study design: Cross-sectional
Manufacturing/Processing/Use: N/A
Hypothesis tested: To determine levels of PFOA in the serum of American Red Cross blood banks in 6 regions of the U.S.
Study period: 2000
Setting: N/A Total population: Serum pooled from 6 ARC blood banks in various geographic regions in the US: Los Angeles, CA; Minneapolis/St. Paul, MN; Charlotte, NC; Boston, MA; Portland, OR, and Hagerstown, MD. Subject selection criteria: Unknown
Total # of subjects in study: 645 donors, age 20-69 years
Comparison population: N/A
Participation rate: N/A Subject description: The only demographic factors known were age, gender and location. Health effects studied: Levels of 7 fluorochemicals in human blood serum, including PFOA. The other chemicals were: PFOS, PFOSAA, M570, M556, PFOSA, PFHS. Data collection methods: Each blood bank was requested to provide approximately 10
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samples per 10-year age intervals (20-29, 30-39, etc.) for each sex.
Details on data collection: Blood sera samples were analyzed using high-pressure liquid chromatography/electrospray tandem mass spectrometry (HPLC/ESMSMS) and evaluated versus an extracted curve from a human plasma matrix (see report for more details).
Exposure period: Unknown--PFOA serum levels used as surrogate for exposure.
Description/delineation of exposure groups/categories: 332 male donors, 313 female donors. There were 10 or more subjects for both males and females in each age category (20-29, 30-39, etc.) except the 60-69 age group where there were fewer.
Measured or estimated exposure: N/A
Exposure levels: N/A
Statistical methods: A reliability assessment was done. Geometric means, 95% confidence intervals of the geometric means, range, interquartile range, raw cumulative 90thpercentile, and frequency distributions, were calculated. Central tendency and distribution of the data by age, gender, and location were presented. Bootstrap analysis was done to calculate mean serum PFOS values for each 6 locations adjusted for 10-year age intervals, gender, and their interaction terms. Multivariable regression analyses. A reliability assessment was also done for the chemical analyses.
The geometric mean data were calculated under the assumption that for individual serum fluorochemical values < LLOQ the midpoint between zero and the LLOQ was assigned. However, PFOA had a lot of subjects with LLOQs.
Other methodological information: Seven sera samples from the preliminary report were not included in the analysis because the subjects were >70 years old.
RESULTS
Describe results: The results of the reliability analysis indicate that there was a strong correlation between the split samples (r = .9) for PFOA.
PFOA did not meet the criteria for a log-normal distribution based on the Shapiro-Wilk test. This lack of log normality may be due to the greater percentage of subjects with values less than the LLOQ (1.92).
The geometric mean of PFOA of all subjects was 4.6 ppb (95% Cl, 4.3 - 4.8). The range was 1.9 --52.3 ppb. Arithmetic means were not provided. Males had higher geometric means than females [4.9 ppb (95% Cl, 4.6 - 5.3), 4.2 ppb (95% Cl, 3.9 - 4.5), respectively]. Age was not an important predictor of adult serum fluorochemical concentrations.
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The geometric mean data were calculated under the assumption that for individual serum fluorochemical values < LLOQ, the mipoint between zero and the LLOQ was assigned. There were quite a few subjects with PFOA LLOQs (<2.1 ppb). If these values were assumed to be 10% or 90% of this range between zero and the LLOQ the respective range of geometric means became 4.0 to 4.8 ppb. These values were not substantially different than those calculated using the midpoint between zero and the <LLOQ; therefore, the midpoint between zero and the <LLOQ was used.
When stratified by geographic location, the highest geometric mean for PFOA was in the samples from Charlotte, NC (6.3 ppb, range: <LLOQ (2.1) - 29) and the lowest from Portland (3.6 ppb, range: <LLOQ (2 .1 )- 16.7). The other PFOA geometric means and ranges by location were: Los Angeles, 4.1 ppb (<LLOQ (2.1) - 34.1); Minneapolis/St. Paul, 4.5 ppb (<LLOQ (1.9) - 20); Boston, 5.4 ppb (1.5 - 13.9); Hagerstown, 4.2 ppb (<LLOQ (2.1) - 52.3). The cumulative 90% were highest for Charlotte (13.3), Minneapolis/St. Paul (9.9), Boston (9.7), and Los Angeles (9.2). Hagerstown and Portland were lower (7.6 and 6.8, respectively).
The results from a bootstrap analysis, done to calculate mean serum PFOA values for each 6 locations adjusted for 10-year age intervals, gender, and their interaction terms, resulted in similar means across the locations (4.3 to 5.3 ppb).
PFOS and PFOA were strongly correlated (r = .63). PFOA had a lower correlation with PFHS (r = .32).
PFOA (natural log), adjusted for age, gender, and their interaction, was a significant (p < .0001) predictor of PFOS in a multivariable model. Age and gender were not significant predictors in this model.
Study strengths and weaknesses: Blood donors cannot be considered representative of the general population of the US.
Research sponsors: 3M Medical Department, Corporate Occupational Medicine
Consistency of results: Geometric mean PFOA levels reported in this study are similar to those reported in other samples of elderly in Seattle and children in various locations.
CONCLUSIONS N/A
REFERENCE
Olsen, GW, Burris, JM, Lundberg, JK, Hansen, KJ, Mandel, JH, Zobel, LR. Identification of fluorochemicals in human sera. 1. American Red Cross adult blood donors. Final report. February 25, 2002.
Last updated on: 4/15/02
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EPIDEMIOLOGIC DATA
Title: Identification of Fluorochemicals in Human Sera. III. Pediatric Participants in a Group A Streptococci Clinical Trial Investigation
TEST SUBSTANCE
Identity: PFOA and 6 other fluorochemicals
Remarks: The results reported are final.
METHOD
Study design: Cross-sectional.
Manufacturing/Processing/Use: N/A
Hypothesis tested: To determine the serum concentrations of selected fluorochemicals in a sample of children to provide a more specific understanding of the distribution of these compounds in children.
Study period: Child sera samples were collected from January 1994 to March 1995. The sera samples were analyzed in Spring 1999.
Setting: N/A
Total population: Not reported
Subject selection criteria: The sera samples were provided to 3M by the University of Minnesota Department of Pediatrics. They were obtained from a large clinical trial on Group A streptococcal infections in children. The children were residents of 23 states in the US. These children presented with signs and symptoms of acute-onset pharyngitis. All of the children had positive throat cultures at the initial visit.
Total # of subjects in study: n = 599 children, age 2-12 years
Comparison population: N/A
Participation rate: N/A
Subject description: 299 male children, 300 female children from 23 states and the District of Columbia.
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Health effects studied: PFOA serum levels in blood, as well as 6 other fluorochemicals.
Data collection methods: Blood sera samples were collected using high-pressure liquid chromatography/electrospray tandem mass spectrometry (HPLC/ESMSMS). Sera were frozen at -20 degrees C prior to the request for analysis.
Exposure period: N/A
Description/delineation of exposure groups/categories: Blood sera samples were collected from children 2 - 1 2 years old.
Measured or estimated exposure: N/A
Exposure levels: N/A
Statistical methods: Arithmetic means, ranges, geometric means and 95% confidence intervals were calculated. Central tendency and di stribution of the data by age, gender, location and their respective interaction terms. A reliability assessment was also done.
The geometric mean data were calculated under the assumption that for individual serum fluorochemical values < LLOQ the midpoint between zero and the LLOQ was assigned. However, PFOA had a lot of subjects with LLOQs.
Other methodological information: N/A
RESULTS
Describe results:
The geometric mean of PFOA for all of the participants was 4.9 ppb (95% Cl, 4.7 -5 .1 ). The range was <LLOQ (1.9) to 56.1 ppb. Male children had significantly (p<01) higher geometric mean PFOA levels than females: 5.2 ppb and 4.7 ppb, respectively.
Age was significantly (p < .05) negatively associated with PFOA in both males and females when analyzed as a continuous variable in simple regression models.
The geometric mean data were calculated under the assumption that for individual serum fluorochemical values < LLOQ the mipoint between zero and the LLOQ was assigned. There were quite a few subjects with PFOA LLOQs (<1.9 ppb). If these values were assumed to be 10% or 90% of this range between zero and the LLOQ the respective range of geometric means became 4.3 to 4.9 ppb. These values were not substantially different than those calculated using the midpoint between zero and the <LLOQ; therefore, the midpoint between zero and the <LLOQ was used.
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PFOS and PFOA were highly correlated (r = .70). PFOA and PFHS were also correlated (r = .48). Adjusted for age, gender, and their interaction, PFOA remained a significant predictor of PFOS in a multivariable regression model of PFOS (p < .0001).
In bootstrap analyses, the mean of the 95% tolerance limit for PFOA was 10.1 ppb with an upper 95% confidence limit of 11.0 ppb. When analyzed by age, the youngest children (age 2 years) had the highest range (1.9 - 34.2 ppb) and cumulative 90% value (17.7 ppb) of any of the age groups. Age 4 was the next highest group (range, 2.0 - 56.1 ppb; 90% value, 10.1 ppb).
Study strengths and weaknesses: These data are cross-sectional data used to determine PFOA levels in U.S. children. Very little descriptive information about the subjects is available. To date, they are the only data available characterizing serum PFOA levels in children.
Research sponsors: 3M Medical Department, Coiporate Occupational Medicine
Consistency of results: To date, no other data have been collected on PFOS serum levels in children.
CONCLUSIONS N/A
REFERENCE Olsen, GW, Burris, JM, Lundberg, JK, Hansen, KJ, Mandel, JH, Zobel, LR. Identification of fluorochemicals in sera of children in the United States. Interim Report. June 25, 2001.
OTHER
Last updated on: 5/07/02
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EPIDEMIOLOGIC DATA
Title: Identification of Fluorochemicals in Human Sera. II. Elderly Participants of the Adult Changes in Thought Study, Seattle, Washington
TEST SUBSTANCE
Identity: PFOA and 6 other fluorochemicals
Remarks: The results reported are final.
METHOD
Study design: Cross-sectional.
Manufacturing/Processing/Use: N/A
Hypothesis tested: To determine the serum concentrations of selected fluorochemicals in a sample of elderly persons to provide a more specific understanding of the distribution of these compounds in this age group.
Study period: 9/29/2000
Setting: N/A
Total population: 238 serum samples from elderly adult donors from the Adult Changes in Thought study.
Subject selection criteria: Donors were 65-96 years old. Subjects were identified during an enrollment phase of this community-based prospective cohort study of dementia and normal aging conducted collaboratively between the U. of Washington and Group Health Cooperative (FIMO). Eligible individuals were those with no known history of neuropsychiatric disease or dementia.
Total # of subjects in study: 238
Comparison population: N/A
Participation rate: N/A
Subject description: 238 adults--118 males, 120 females. The mean age was 76 years. Female subjects had resided in the Seattle area for 53.3 years, males 50.2 years.
Health effects studied: PFOA serum levels in blood, as well as 6 other fluorochemicals.
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Data collection methods: Blood sera samples were collected using high-pressure liquid chromatography/electrospray tandem mass spectrometry (HPLC/ESMSMS).
Exposure period: N/A
Description/delineation of exposure groups/categories: 65-75 years (n= 121), 75+ to 85 years (n = 93), 85+ to 96 years (n = 24).
Measured or estimated exposure: N/A
Exposure levels: N/A
Statistical methods: Arithmetic means, ranges, geometric means and 95% confidence intervals were calculated. Central tendency and distribution of the data by age, gender, location and their respective interaction terms. A reliability assessment was also done.
Other methodological information: N/A
RESULTS
Describe results: The reliability analysis indicated moderately strong correlations for the split samples with PFOA (r = -7).
There was a weak correlation between age and years' residence in the Seattle area (r = 0.2). Analyzed independently of age, there were no significant associations between years resided in Seattle and PFOA. PFOS and PFOA were highly correlated (r = .75). PFOA was less highly correlated with PFHS (r = .36).
PFOA was not log normally distributed based on the Shapiro-Wiik test. The authors postulate that this lack of normality may be due to the greater percentage of subjects with PFOA values < LLOQ. The geometric mean of PFOA for all samples was 4.2 ppb (95% Cl, 3.9 - 4.5). Arithmetic means were not provided. The range was 1.4 - 16.7 ppb. Bootstrap analyses provided the mean of the 95% tolerance limit for PFOA was 9.7 ppb with an upper 95% confidence limit of 11.3 ppb.
Study strengths and weaknesses: These data are cross-sectional data used to determine PFOA levels in elderly. Very little descriptive information about the subjects is available. The subjects only characterize PFOA levels in the Seattle region. To date, they are the only data available characterizing serum PFOA levels in the elderly.
Research sponsors: 3M Medical Department, Corporate Occupational Medicine
Consistency of results: To date, no other data have been collected on PFOA serum levels in the elderly.
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CONCLUSIONS PFOA levels in these Seattle residents are similar to those found in adults and children in the U.S.
REFERENCE Olsen. GW, Burris, JM, Lundberg, JK, Hansen, KJ, Mandel, JH, Zobel, LR. February 25, 2002. Identification of fluorochemicals in human sera. II. Elderly participants of the Adult Changes in Thought study, Seattle, Washington. Final Report. 3M Company, Medical Department.
Last updated on: 4/24/02
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PHARMACOKINETIC STUDY
Title: The sex-related difference in perfluorooctanoate excretion in the rat
TEST SUBSTANCE
Identity: Perfluorooctanoic acid (PFOA); also referred to as [ l- l4C]Perfluorooctanoic acid (ammonium salt, APFO)
Remarks: Purity was not specified
METHOD
Method/guideline followed: Guideline not noted
Test type: Mammalian Excretion
Species/strain/cell type or line: Holtzman rats
Sex: Male and female
Age and body weight range of animals used: Not noted
Number of animals/sex/dose: Test group: 4 males, 6 females Control group: 7 females
Route of administration: Oral gavage
Vehicle: Distilled water
Doses: 2 mg of nonionic fluorine as APFO in a volume of 2 mL
Excretion routes, body fluids, and tissues monitored and/or sampled during study: Blood and urine
Statistical methods used: See methods
Method remarks: Animals were placed in individual metabolic cages and sacrificed by cardiac puncture 24 hours after gavage treatment with APFO. The blood was allowed to clot and the serum collected after centrifugation. The volume of the urine collections, including the volume of water used to rinse the metabolic cages was recorded. The ionic fluoride content of the serum and urine was determined at pH 5.0. The total fluorine content of the serum and urine was determined by the oxygen-bomb reverse extraction technique. For clearance studies of APFO and inulin the rats were anesthetized and the femoral vein was cannulated for continuous infusion of 5% mannitol in isotonic saline and the femoral artery was cannulated for drawing blood samples. The urinary bladder was also cannulated to obtain serial collections of urine. Intravenous doses of APFO and inulin were given to each animal and, following a 45-min equilibration period, blood and urine samples were collected. When collections were completed, probenecid was administered and additional clearance tests were performed to test the effects of probencid on the organic acid transport system.
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In the cumulative excretion study, the rats were prepared as described for the clearance tests except that the arterial cannulation was not needed. The rats were dosed iv with a mixture of radiolabeled APFO and unlabelcd APFO.
RESULTS
Detailed results:
24 hours after administration of the dose, female rats excreted 76 + 2.7 percent of the dose of nonionic fluorine (as APFO) in urine and had a mean serum nonionic fluorine level of 0.35 ppm.
In males, 9.2 + 3.5 percent of the dose of nonionic fluorine (as APFO) was excreted. A level of 44.0 + 1.7 ppm nonionic fluorine was in the serum.
APFO was bound to a similar extent in the plasma of male and female rats (97.5% bound).
In females, APFO was markedly reduced by probenecid (from 5.8 to 0.11 ml/min/100 g), indicating that elimination occurs by an active secretory mechanism, which is inhibited by probenecid.
The APFO/inulin clearance ratio was 14.5 for female rats. The ratio was decreased to 0.46 in females after probenecid. The APFO/inulin clearance ratio was 0.22 for males, and decreased to 0.12 after probenecid.
CONCLUSIONS
The authors concluded that the high APFO/inulin clearance ratio in females provided evidence that APFO is excreted in part by an active secretion mechanism. The decrease in the APFO/inulin clearance ratio in females from 14.5 to 0.46 after the administration of probenecid supports this conclusion. The lower APFO than inulin clearances in both sexes after administration of probenecid indicated partial tubular reabsorption of APFO in both sexes. This secretory mechanism is lacking or inactive in males and accounts for the greater toxicity of APFO in male rats.
REFERENCE
Hanhijarvi, H., R. Ophaug, and L. Singer. 1982. The sex-related difference in perfluorooctanoate excretion in the rat. Proceedings of the Society for Experimental Biology and Medicine; 171:50-55.
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PHARMACOKINETIC STUDY
Title: Elimination and Toxicity of Perfluorooctanoic Acid During Subchronic Administration in the Wistar Rat
TEST SUBSTANCE
Identity: Perfluorooctanoic acid (PFOA)
Remarks: Purity was not specified
METHOD
Method/guideline followed: No specific guideline was listed
Test type: 28-Day subchronic toxicity and elimination study
Species/strain/cell type or line: Wistar rat
Sex: Female and male
Age and body weight range of animals used: Newly-weaned
Number of animals/sex/dose: 6 animals/sex/dose
Route of administration: Oral gavage
Vehicle: 0.9% NaCl solution
Doses: 0, 3, 10, 30 mg/kg/day
Excretion routes, body fluids, and tissues monitored and/or sampled during study: During the study period, the animals were weighed once a week and 24 hour urine was collected on study days 7 and 28. The behavior and feed and water consumption were observed regularly. At the end of the study period, blood and tissue samples were collected from control and 30 mg/kg rats.
Statistical methods used: Student's t-test, Pearson product moment correlation and Fisher's exact test were the principal statistical procedures used for the evaluation of the findings.
Method remarks: PFOA treatment occurred over 28 consecutive days. The administered volume of the saline solutions was always 0.5 ml/lOOg animal weight. PFOA concentrations in blood and urine samples were analyzed by gas chromatography.
RESULTS
Detailed results from Day 7: On the seventh day of the study period, the mean urinary excretion of PFOA (mg/24 hrs/kg) was lower than the daily dose in all three groups of males. A statistically significant difference from the daily dose was found in all three groups of male rats (P<0.05 and P<0.001, t-test). In the female animals, none of the three groups excreted significantly less PFOA in the urine on day 7 than they were administered. The mean urinary PFOA concentration of the female rats in the lowest
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dose group was significantly higher (P<0.01) than that of the male animals. In the group receiving the highest daily dose of PFOA, the urinary PFOA concentration was significantly higher (P<0.05) in the male animals; however, this can be explained by the significantly lower (P<0.05) mean urinary volume of the male rats.
Detailed results from Day 28: By test day 28, the males seemed to have also reached the steady state, with the exception of the 10 mg/kg group which showed a significantly lower (P<0.05) excretion of PFOA than the administered daily dose. At each dose level, the mean PFOA concentrations in the plasma of the male rats were significantly higher than those of the female animals (P<0.001 for the two highest dose groups and P<0.01 for the lowest dose group). A detailed statistical analysis of organ weights did not reveal any differences. The weights of the liver relative to body weights of male rats showed a significant positive dose response (PO.OOl, Pearson). A possible explanation was the significant dose-related decrease in the bodyweights found in the males (PO.05, Pearson); however, the difference between the control males and the lowest-dose group was more obvious than between any of the dosed groups (PO.Ol, t-test). In the high-dose group females, one or more mild inflammatory foci were observed in four livers, which was not significantly different from control females with only one small chronic focus (Fisher's exact test).
Metabolites measured: None
CONCLUSIONS
The mean urinary excretion suggests that the average renal clearance of PFOA in the female rats is about 10 times higher than in the male animals. The sex-related difference in plasma PFOA concentrations are best explained by active tubular secretion in the female kidney. The steady state was achieved by 7 days in the female animals only. By the 28th day, both males and females excreted roughly the same amount of PFOA in 24 hours as their daily dose.
REFERENCE
Hanhijarvi, JT., M. Ylinen, A. Kojo, and V. Kosma. 1987. Elimination and toxicity of perfluorooctanoic acid during subchronic administration in the Wistar rat. Pharmacology and Toxicology; 61:66-68.
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PHARMACOKINETIC STUDY
Title: A Proposed Species Difference in the Renal Excretion of Perfluorooctanoic Acid in the Beagle Dog and Rat
TEST SUBSTANCE
Identity: Perfluorooctanoic Acid (PFOA)
Remarks: No information on supplier, lot number, or purity of substance tested was given.
METHOD
Method/guideline followed: NA
Test type: In vivo
Species/strain/cell type or line: Beagle dogs
Sex: male/female
Age and body weight range of animals used: Not stated
Number of animals/sex/dose: Group 1: 6 (3 males and 3 females) given 30 mg/kg PFOA with continuous infusion of 5% mannitol solution at 1.7 ml/min. Probenecid was then administered at 30 mg/kg intravenously. Group 2: 4 (2 males and 2 females) given 30 mg/kg PFOA only and kept in metabolic cages.
Route of administration: Intravenous
Vehicle: Not stated
Doses: 30 mg/kg - see above (under Number of animals/sex/dose)
Excretion routes, body fluids, and tissues monitored and/or sampled during study: Group 1: urine and blood samples Group 2: blood samples
Statistical methods used: Paired Student's t-test used to determine statistical significance of the reduction of PFOA clearance in each dog before and after administration of probenecid.
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Method remarks: Group 1: Dogs were anaesthetized with methoxyflurane. Catheters were fixed in both ureters after laparototomy and cystotomy. Urine was collected at 10 min intervals for 60 min. (Dosing and other methods described above.) Group 2: Dogs were kept in metabolic cages after the injection. Half-lives were determined for each animal separately. PFOA concentrations were analyzed with a GLC-method.
RESULTS
Detailed results: Probenecid effectively and statistically significantly reduced PFOA clearance in each dog (P < 0.05 for each dog). PFOA plasma half-lives (males): 473 and 541 hours PFOA plasma half-lives (females): 202 and 305 hours
Metabolites measured: None. It appears that only PFOA was measured.
CONCLUSIONS
Because of the statistical significance of the reduction of PFOA clearance by probenecid, males and females have an active secretion mechanism for PFOA. Plasma half-lives were longer in males than females.
The authors also compare the clearance rates with those in rats, noting that the glomular filtration rate of PFOA is similar in both species. However, the active tubular secretion rate of PFOA in the rat and dog are quite different.
REFERENCE
Hanhijarvi, H., M. Ylinen, T. Haaranen, and T. Nevalainen. 1988. A proposed species difference in the renal excretion of perfluorooctanoic acid in the beagle dog and rat Im_ Beynen, A.C. and H.A. Solleveld (Eds). New Developments in Biosciences: Their Implicationsfo r Laboratory Animal Science. Martinus Nijhoff Publishers. Dordrecht, Netherlands.
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PHARMACOKINETIC STUDY Title: Tissue Distribution, Metabolism, and Elimination of Perfluorooctanoic Acid in Male and Female Rats TEST SUBSTANCE Identity: [ l`l4C]Perfluorooctanoic acid (PFOA, 99% pure, specific activity 5.44 mCi/mmol) Remarks: Synthesized and purified according to methods described previously METHOD Method/guideline followed: NA Test type: In vivo Species/strain/cell type or line: Sprague-Dawley rats Sex: males and females Age and body weight range of animals used:
Age = six weeks Weight (males) = 170-195 grams Weight (females) = 130-155 grams Number of animals/sex/dose: 4 of each sex for tissue distribution experiments; 32 males and 16 females for tissue elimination experiments Route of administration: intraperitoneal Vehicle: Propylene glycol/water (1:1 v/v) Doses: 4 mg/kg Excretion routes, body fluids, and tissues monitored and/or sampled during study: urine, plasma, liver, bile, feces (e.g., tissues able to metabolize endogenous fatty acids or were target tissues for PFOA toxicity) Statistical methods used: Not stated Method remarks: none
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RESULTS
Males:
Tissue distribution - Percent dose on a per gram basis (from highest to lowest) in monitored organs was: liver, plasma, kidneys, heart, testes, fat (epididymal fat pad), gastrocnemius muscle. The liver and plasma were the primary tissues of distribution. [See Table 1 of the study for percentages by time after administration.] The high concentration in liver was examined further in males by an in situ nonrecirculating liver perfusion technique - `11% of the PFOA infused was extracted by the liver in a single pass'.
Elimination - Urine and feces were both major routes of elimination. Cumulative excretion of PFOA in urine and feces after 28 days was 36.4% and 35.1% respectively. The apparent half-life for whole-body elimination of PFOA was 15 days (360 hours).
Half-lives -
Organ Liver Plasma Kidney Heart Gastrocnemius Fat Testis Blood
Hrs 271.2 216 225.6 249.6 220.8 192 216 211.2
Days 11.3 9.0 9.4 10.4 9.2 8.0 9.0 8.8
Females:
Tissue distribution - Percent dose on a per gram basis (from highest to lowest) in monitored organs was: plasma, kidneys, liver, ovaries. The liver, plasma, and kidneys were the primary tissues of distribution. [See Table 2 of the study for percentages by time after administration.]
Elimination - Urine was the major route of elimination. Cumulative excretion of PFOA in urine after 1 day was 91 %, whereas the amount in feces was negligible. The apparent half-life for whole-body elimination of PFOA was < 1 day.
Half-lives -
Organ Liver Plasma Kidney Blood
Hrs 3.8 2.9 3.2 3.8
Metabolites measured: Defluorination of PFOA was measured to determine whether it was metabolized. Specific metabolites were not discussed.
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CONCLUSIONS
A sex difference in urinary elimination of PFOA was observed, resulting in different whole-body elimination half-lives. However, there was no apparent difference in biliary excretion - both males and females eliminated less than 1% of the PFOA dose by this route. Second, the greater persistence of PFOA in male rats was not due to greater formation of PFOA-lipid conjugates. Third, the parent compound appears to be the only tissue storage form of PFOA in both sexes as well as the only compound excreted in urine and bile; therefore, a differential rate of metabolite formation by sex was not responsible for sex differences. (The authors also discuss the unpublished results of Vanden Heuvel et al. 1992, noting the possible link between testosterone and inhibition of PFOA excretion.)
REFERENCE
Vanden Heuvel, J.P., Kuslikis, B.I., Van Rafelghem, M.L. and Peterson, R.E. 1991. Tissue distribution, metabolism, and elimination of perfluorooctanoic acid in male and female rats. J. Biochem. Toxicol. 6(2):83-92.
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PHARMACOKINETIC STUDY
Title: Renal Excretion of Perfluorooctanoic Acid in Male Rats: Inhibitory Effect of Testosterone
TEST SUBSTANCE
Identity: [l4C]-Perfluorooctanoic acid (PFOA, 99% pure, specific activity 5.44 mCi/mmol)
Remarks: Synthesized according to methods described previously
METHOD
Method/guideline followed: NA
Test type: In vivo
Species/strain/cell type or line: Sprague-Dawley rats
Sex: males and females
Age and body weight range of animals used: Age = six weeks Weight (males) = 170-195 grams Weight (females) = 130-155 grams
Number of animals/sex/dose: 4/sex/dose
Route of administration: intraperitoneally (i.p.)
Vehicle: Propylene glycol/water (1:1 v/v)
Doses: 4 mg/kg (administered as [ l 'l4C]PFOA one week after vehicle, 17beta-estradiol, or testosterone implantation)
Excretion routes, body fluids, and tissues monitored and/or sampled during study: Urine, plasma, feces, liver tissue, kidney tissue
Statistical methods used: Differences between treatment groups were determined by one-way analysis of variance (ANOVA). Duncan's t-test was performed to compare individual mean values when ANOVA showed differences. (Level of significance tested was p<0.05.)
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Method remarks: To test differences in elimination of PFOA between males and females, rats were castrated, ovariectomized, or sham operated; estrogen or testosterone was administered.
Estrogen (as 17beta-estradiol) capsules were inserted subcutaneously into castrated males (through an incision posterior to the scapulae).
Testosterone capsules were inserted subcutaneously in castrated males or ovariectomized females (through an incision made posterior to the scapulae).
Groups of sham-operated males and females, castrated males, and ovariectomized females were implanted with tubing only and served as controls for the implantation procedure.
In castrated male rats, probenecid was administered (at 65 mg/kg i.p.) 30 min prior to administration of PFOA. 7 hours after PFOA administration, rats were anaesthetized with Nembutal (50 mg/kg i.p.).
RESULTS Males:
sham/vehicle castrated/vehicle castrated/17beta-estradiol castrated/testosterone
Tissue PFOA Concentrations*
(% Dose uC-PFOA/g) **
Liver
Kidney
1.91 0.12
0.91 0.07
0.11 0.03
0.08 + 0.03
0.02 0.001
0.02 + 0.004
1.53 0.05
0.40 0.03
Plasma 1.48 + 0.29 0.021 0.006 0.006 0.002 1.07 0.05
*Significant differences in liver, kidney, and plasma concentrations of PFOA were observed among treatment groups (sham/vehicle, castrated/vehicle, castrated/17beta-estradiol, castrated/testosterone). In liver and kidneys, PFOA concentration was highest in the sham/vehicle group, and 2nd highest in the castrated/testosterone group. In plasma, PFOA was highest (and similar in concentration) in the sham/vehicle and castrated/testosterone groups. Plasma PFOA concentrations in castrated/vehicle and castrated/17beta-estradiol groups were very low. ** Source: Table 1, pg. 33
Urinary Elimination sham/vehicle castrated/vehicle
castrated/17beta-estradiol
castrated/testosterone
16% of dose in 4 days > 1 time greater than sham/vehicle > 1 time greater than sham/vehicle similar to sham/vehicle
Fecal excretion: This was less than urinary excretion. Regardless o f treatment group, the cumulative percent, o fPFOA dose eliminated in feces over 4 days was similar in sham/vehicle. castrated/vehicle. castrated/17beta-estradiol groups. Excretion was higher in the castrated/testosterone group.
Effect of administering probenecid: This had no effect on urinary PFOA elimination in sham/vehicle, hut reduced PFOA elimination in castrated groups. There were no differences in plasma concentrations o f PFOA among treatment groups after probenecid.
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Hyaline droplet formation (a testfo r PFOA binding to male-specific protein alpha2u-globulin): Histopathologic analysis did not reveal formation of hyaline droplets in the male kidney.
Females: Tissue concentrations: No significant differences in liver, kidney, and plasma concentrations of PFOA were observed among treatment groups (sham/vehicle, ovariectomized/vehicle, ovariectomized/testosterone). Urinary elimination: No significant differences; all rats eliminated about 70% of the PFOA dose in urine in 4 days. Fecal excretion: no significant differences; all excreted less than 0.5% of the dose.
Metabolites measured: None
CONCLUSIONS
These results suggest that testosterone pretreatment inhibits elimination of PFOA in the male. Also, probenecid reduced PFOA elimination in castrated male rats that had increased PFOA urinary excretion. Testosterone is a key determinant of the sex difference in PFOA elimination in rats.
Remarks: none
REFERENCE
Vanden Heuvel, J.P., Davis, J.W., Sommers, R., and Peterson, R.E. 1992. Renal excretion of perfluorooctanoic acid in male rats: Inhibitory effect of testosterone. J. Biochem. Toxicol. 7(l):31-36.
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PHARMACOKINETIC STUDY
Title: Covalent Binding of Perfluorinated Fatty Acids to Proteins in the Plasma, Liver, and Testes of Rats
TEST SUBSTANCE
Identity: [ l- ,4C]Perfluorooctanoic acid ([l-l4C]PFOA)
Remarks: 99% pure, specific activity = 51.7 mCi/mmol
METHOD
Method/guideline followed: Not specified
Test type: In vivo
Species/strain/cell type or line: Rat/Harlan Sprague-Dawley, obtained from Harlan Sprague-Dawley (Indianapolis, IN)
Sex: Male
Age and body weight range of animals used: Six-weeks, 170-195 grams
Number of animals/sex/dose: Not specified
Route of administration: Intraperitoneal (i.p.)
Vehicle: Propylene glycol/water (1:1, v/v; 1 ml/kg)
Doses: 9.4 umol/kg
Excretion routes, body fluids, and tissues monitored and/or sampled during study: Liver, plasma, testes
Statistical methods used: Differences from control were determined by one-way analysis of variance. The least significant differences test was used to compare individual means where analysis of variance indicated differences. The level of significance for all analyses was P < 0.05.
Method remarks: Test animals and test substances [1-l4C]Perfluorooctanoic acid was synthesized and purified as described by I.L. Reich et al (1987). Hemoglobin (2 x crystallized, dialyzed and lyophilized), fatty acid free bovine serum albumin, and all tissue culture media and supplements were obtained from Sigma Chemical Company (St. Louis, MO). Reagent grade solvents were obtained from Aldrich Chemical Company (Milwaukee, WI). Water purification was performed by passing distilled water through a four-bowl Milli-Q water purification system and a 0.25 Lm filter (Millipore Corp, Milford, MA). Rats were individually housed in suspended stainless-steel cages in a temperaturecontrolled room (approximately 21C). A 12-hour light and 12-hour dark photoperiod was maintained. Food (Purina Rat Chow No. 5012, Ralston Purina Co, St. Louis, MO) and water were 124
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provided ad libitum throughout the study. An acclimation period of at least 1-week was allowed before initiation of the experiment.
Covalent binding of PFOA to proteins in vivo [1 -l4C]Perfluorooctanoic acid ([1 -l4C]PFOA) was administered to rats in propylene glycol/water (1:1, v/v; 1 ml/kg) at a dose of 9.4 Lmol/kg, i.p. At designated times post-treatment, rats were anesthetized with Nembutal (50 mg/kg, i.p.); tissues were quickly removed, freeze clamped, and stored at -70C. Blood was removed in a lightly heparinized needle and plasma was separated by centrifugation. Macromolecular binding was determined on tissue homogenates, which were brought up to 10% TCA and put on ice for 10 minutes. To remove any unbound perfluorinated acid, samples were washed with 3 mL of methanol/ether (3:1) 6 times and then 3 more times using 3 mL of ethyl acetate. Washing was continued, if necessary, until no additional PFOAderived l4C could be extracted from the protein. One milliliter of 1 N NaOH was added to the protein precipitate and placed in a shaking water bath overnight at 37C to solubilize the protein precipitate. The sample was then neutralized with 1 M acetic acid and 1 mL was used for liquid scintillation counting,while another aliquot was used for protein analysis. The protein analysis was performed according to O.H. Lowry et al (1951). PFOA-derived radioactivity in all samples was quantitated using a Packard Liquid Scintillation Analyzer with quench correction performed with a Packard DPM 1-2-3 software program. Radioactivity in hydrolysates was determined in Hionic-Fluor scintillation cocktail. Binding was expressed as pmol PFOA equiv/mg protein.
Covalent binding of PFOA to hemoglobin and albumin in vitro 14C-labeled PFOA was added to tubes containing albumin or hemoglobin in 1.0 mL of phosphate buffer to give a 2, 4, 8, or 100 A.M solution. If methionine or cysteine were present, they were added in 1 mL of phosphate buffer to bring the reaction mixture up to 2 mL. All experiments were done using a final volume of buffer of 2 mL. The time of incubation was 1 hour at 37C. Protein was precipitated with 10% TCA. To remove any free unbound perfluorinated acid, samples were washed with 5 mL of acetone 5 times. Samples were vortexed for 1 minute and spun at 2500 revolutions/minute for 5 minutes after each acetone wash step. The acetone was transferred off. Protein samples were then washed with 5 mL of ether 2 additional times. Washing was continued if necessary until no additional PFOA (as determined by liquid scintillation counting) could be extracted from the protein. The protein powder was hydrolyzed with 1 mL of 1 N NaOH at 37C in a shaking water bath overnight. Binding was expressed as pmol PFOA equiv/mg of protein.
Selection of target tissues for the analysis of PFOA-binding to proteins The liver, plasma, and testis were selected as target tissues for the analysis of PFOA-binding to proteins because they are either major tissues of distribution for PFOA in rats (liver and plasma) or they are target organs for perfluorinated acid toxicity (liver and testes).
RESULTS
Detailed results: Covalent binding of PFOA to target tissues in rats The covalent binding data for PFOA for each tissue (2 hour, 1 day, and 4 days after treatment) were pooled because no time-dependent changes in the absolute and relative concentrations of covalently bound protein were observed. The tissue elimination half-life for PFOA (average tissue t|/2 = 9 days) showed that there was little difference in tissue concentrations of PFOA in the range of 2 hours to 4 days. In PFOA-treated rats, the absolute concentration of covalently bound
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PFOA was significantly higher in the plasma than in the liver. Of the tissues examined, the testes had the highest relative concentration of PFOA-derived radioactivity covalently bound to protein. Approximately 0.4% of the testes PFOA concentration was covalently bound. To determine which proteins were modified following in vivo administration of PFOA, tissues were homogenized and subjected to SDS-PAGE/autoradiography with fluorography. Due to the small amount of covalently bound l4C found within these tissues, no radioactive protein bands were detected when 50 Ag protein was separated on SDS-PAGE and the X-ray film was exposed to the gel for up to 2 months at -70C.
Covalent binding of PFOA to hemoglobin and albumin in vitro The covalent binding of PFOA to hemoglobin was diminished by the addition of cysteine with no effect of methionine in parallel incubations.
Metabolites measured: [ l- 14C]Perfluorooctanoic acid
CONCLUSIONS
Despite the metabolic inertness of the perfluorinated fatty acids, the results indicated that PFOA binds to proteins in the plasma, liver, and testes in a covalent manner. Although PFOA-derived radioactivity could be found covalently associated with protein following the organic extractions, the amount of radioactivity associated with any given protein was not enough to be seen following SDS-PAGE/autoradiography. The in vitro covalent binding of PFOA was reduced following the addition of cysteine to the incubation, but not the addition of methionine to the incubation. Thus, the ability of cysteine to inhibit PFOA covalent binding of proteins suggested that protein sulfhydryl groups may be involved; sulfhydryl groups may be the sites of covalent attachment of PFOA.
REFERENCE
Vandcn Heuvel, J.P., Kuslikis, B.I., and Peterson, R.E. 1992. Covalent binding of perfluorinated fatty acids to proteins in the plasma, liver and testes of rats. Chem.-Biol. Interact. 82:317-328.
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PHARMACOKINETIC STUDY
Title: Cholestyramine-Enhanced Fecal Elimination of Carbon-14 in Rats after Administration of Ammonium [l4C]Perfluorooctanoate or Potassium [14C]Perfluorooctanesulfonate
TEST SUBSTANCE
Identity: Ammonium [l4C]perfluorooctanoate ([l4C]-APFO)
Remarks: []4C]-APFO: specific activity = 0.51 pCI/mg, radiochemical purity >98%
METHOD
Method/guideline followed: NA
Test type: in vivo
Species/strain/cell type or line: rat, Charles River CD
Sex: male
Age and body weight range of animals used: 12 weeks, 300 - 342 grams
Number of animals/sex/dose: 5
Route of administration: intravenous
Vehicle: 0.9% NaCl
Doses: Single doses were administered as follows: animals treated with cholestyramine =13.3 mg/kg mean, animals not treated with cholestyramine =13.5 mg/kg mean (control group)
Excretion routes, body fluids, and tissues monitored and/or sampled during study: Urine, feces, plasma, red blood cells, and liver were monitored and/or sampled during the study.
Statistical methods used: Data were expressed as means standard deviations. The student's t-test was used to evaluate significance of difference between two groups at a significance level of p < 0.05.
Method remarks: Rats were housed in individual stainless-steel metabolism cages and fasted with free access to water for 24 hours prior to administration of the test substance. Carbonyl-labeled [l4C]APFO was obtained from 3M. Cholestyramine was obtained from Mead Johnson. The dosing solutions were prepared by dissolving the test substance in 0.9% NaCl (the [14C]APFO solution contained 2.1 mg/mL). Cholestyramine (dried and ground resin Z-620) was mixed, 4% by weight, with Purina Lab Chow. The radio-labeled test substance was administered as a single intravenous dose (lateral tail vein). Two mL of the dosing solution was administered to each of 10 rats. Five rats were given 4% cholestyramine in feed (ad libitum); the other rats were given normal Purina Lab Chow. In order to allow a comparison of the radiometric results on an absolute basis, the radio-labeled doses were not adjusted for individual body weights. The average doses as administered to each group were 13.3 mg/kg (cholestyramine-treated animals) and 13.5 mg/kg (control animals). Urine and feces samples were collected at intervals for individual rats 14 days after administration of the test substance. At this time, the rats were anesthetized
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with diethyl ether and exsanguinated by drawing blood from the descending aorta. Plasma and red blood cells were prepared promptly by centrifugation. The liver was collected as a whole organ and stored frozen until analysis.
RESULTS
Detailed results: After 14 days of cholestyramine treatment, the mean percentage of dose eliminated via feces (43.2 5.5) was 9.8-fold the mean percentage of dose eliminated via feces by control rat (4.4 1.0). The difference was found to be significant. After adjustment for the amount of carbon-14 excreted in the urine (67% for controls and 41% for cholestyramine-treated), the amounts of carbon-14, which remained to be excreted were 16% for chloestyramine-treated rats and 28% for control rats. The mean liver carbon14 content represented 4- and 8% of the dose for cholestyramine-treated and control rats, respectively. The mean carbon-14 concentrations of plasma and red blood cells with cholestyramine-treatment were significantly less than the mean concentration in controls. The mean data from analyses of liver, plasma, and red blood cells for carbon-14 content 14 days after [14C]APFO administration are shown in Table 1.
Table 1. Effect of Cholestyramine Treatment on Concentration of Carbon-14 in Rat Liver, Plasma, and Red Blood Cells After a Single Intravenous Dose of [l4C]APFO
Treatment Group Cholestyramine-treated
Carbon-14 Concentration (Tic ea/a tissue or mL fluid)
Liver
Plasma
Red BloodCells
12.1 2.1*
5.1 1.7*
1.8 0.7*
Controls
22.3 6.2
14.7 6.8
4.2 2.4
*Significantly different from control values (p < 0.05)
Metabolites measured: None
CONCLUSIONS The data support the possible utility of cholestyramine as a promotor of the excretion of pcrfluorooctanoate in humans.
Reviewer's Remarks: The author's conclusions appear to be supported by the data.
REFERENCE Johnson, J.D., Gibson, S.J., and Ober, R.E. 1984. Cholestyramine-enhanced fecal elimination of carbon14 in rats after administration of ammonium [14C]perfluorooctanoate or potassium [14C]perfluorooctanesulfonate. Fund. Appl. Toxicol. 4:972-976.
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PHARMACOKINETIC STUDY
Title: Metabolic Handling of Perfluorooctanoic Acid in Rats
TEST SUBSTANCE
Identity: Perfluorooctanoic Acid (PFOA)
Remarks: Perfluorooctanoic acid (a mixture of linear and branched isomers) was supplied by Minnesota Mining and Manufacturing Company, St. Paul, MN 55101. Its purity was not indicated.
METHOD
Method/guideline followed: Not specified
Test type: Fluoride determinations and ultrafiltration studies were utilized--Rat in vivo and in vitro; Human serum-m vitro
Species/strain/cell type or line: Holtzman rat; Human serum
Sex: Rat-female; Human-not specified
Age and body weight range of animals used: Rats weighed approximately 250 grams, age of rats was not specified; Human serum-age and weight of source was not specified
Number of animals/sex/dose: Fluoride determination--not specified; Ultrafiltration studies-- Rats scrum-two aliquots; Human serum-three aliquots
Route of administration: Rats were dosed via stomach intubation
Vehicle: Rats were dosed with an aqueous solution of perfluorooctanoic acid
Doses: Rats were dosed with 2 mg nonionic fluorine as perfluorooctanoic acid; additionally, four rats were fed a low fluoride (<0.5 ppm) diet; Human serum-0, 75, or 1500 pg nonionic fluorine (as perfluorooctanoic acid)
Excretion routes, body fluids, and tissues monitored and/or sampled during study: Rat-serum, urine, and feces were analyzed for fluoride determinations and ultrafiltration studies; Human-serum was analyzed for ultrafiltration studies Statistical methods used: Means were statistically compared by calculating Student's t-value. A P-value of <0.025 was chosen as indicating significance.
Method remarks:
Fluoride determinations--Rats were administered 2 mL of an aqueous solution containing 2 mg of nonionic fluorine, as perfluorooctanoic acid, by stomach intubation. The animals were placed in individual metabolic cages and fed rat chow (Purina) and tap water (lppm fluoride) ad libitum for 4.5, 8, 24, or 52.5 hours. In addition, four rats were placed in metabolic cages and fed a low fluoride (<0.5 ppm) diet and distilled water for a period of 96 hours. This resulted in a substantial decrease in ionic fluoride content of the feces and facilitated the analysis for nonionic fluorine. A few crystals of thymol were added
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to the urine containers to inhibit bacterial growth during the collection period. At the end of the experimental period, the urine and feces were collected and the anesthetized animals were sacrificed by cardiac puncture. The blood was allowed to clot and the serum was collected after centrifugation and stored frozen until analyzed. Urine, serum, and feces were also obtained from undosed animals to provide baseline data. The volumes of urine collections, which included water needed to rinse the metabolic cages, and the weight of the feces were recorded. A small known quantity of distilled water was added to the feces samples, which were then homogenized to a thick slurry. The serum and urine were analyzed for ionic fluoride at pH 5.0 with the fluoride ion-specific electrode. The ionic fluoride in the diffusate of the feces was determined at pH 5.0 with the fluoride electrode after isolation of the fluoride by diffusion from perchloric acid at 60C (the fluoride in perfluorooctanoic acid is not acid labile under these conditions). The total fluorine content of serum, urine, and feces was determined with the oxygen-bomb reverse extraction technique. Fifteen mL of redistilled water was added to the oxygen bomb (prior to combustion of the samples) to act as a fluoride trap. Up to 0.3 mL of serum or urine was pipetted onto a 0.3-gram pellet of filter paper pulp and fired without drying in the oxygen bomb. Larger volumes (up to 3 mL) of serum low in fluoride were pipetted onto the pellet and lyophilized prior to combustion. The method was slightly modified for the analysis of the feces in that 15 mL of total ionic strength activity buffer (TISAB, Orion Research, Inc.) was added to the oxygen bomb prior to firing instead of 15 mL of water. Blank and recovery samples were carried through the entire procedure.
Ultrafiltration studies--The pH of two 90-mL aliquots of human serum was adjusted to 7.4 by equilibration with a mixture of 95% air and 5% carbon dioxide. Seventy-five pg of nonionic fluorine (as perfluorooctanoic acid) was added to one aliquot and 1500 pg was added to the second. After 2 hour, 10 mL of the spiked serum was removed for determination of the initial concentration of perfluorooctanoic acid and the remaining serum containing perfluorooctanoic acid was transferred to an Amicon TCFIO ultrafiltration unit fitted with a Diaflo XM50 membrane, which retains molecules >50,000 daltons. The sample chamber was flushed with 95% air, 5% carbon dioxide, pressurized with nitrogen, and 15 mL of ultrafiltrate was collected. An ultrafiltrate was also prepared from pooled serum obtained from four rats (dosed for the fluoride determination study) 4.5 hours after receiving the 2 mg of nonionic fluorine as perfluorooctanoic acid by stomach intubation and from 75 mL of buffered (pH 7.4) isotonic saline to which 900 pg of nonionic fluorine had been added.
RESULTS
Detailed results:
Employing the oxygen bomb reverse extraction technique for determination of total fluorine, a recovery of 80 1.0 SEM% was obtained for perfluorooctanoic acid. The recovery of fluoride from perfluorooctanoic acid was increased to 94 1.5% when 15 mL of TISAB buffer, instead of water, was used as the fluoride trap inside the oxygen bomb. The quantity of nonionic fluorine was calculated by subtracting the ionic fluoride from the total fluorine determined after ashing in the oxygen bomb and dividing by either 80 or 94%. The blank for the procedure employing a 0.3-gram filter paper pulp pellet and 15 mL of redistilled water as a fluoride trap was 0.13 + 0.020 pg. The modified procedure used to determine the total fluoride content of the feces had a blank of 0.40 0.043 pg.
The results obtained in the ultrafiltration study of the binding of perfluorooctanoic acid in serum are given in Table 1. The control experiment in which perfluorooctanoic acid was added to an isotonic saline solution buffered at pH 7.4 provided evidence that the ultrafiltration membrane bound very little perfluorooctanoic acid. Virtually all of the nonionic fluorine (as perfluorooctanoic acid) added to the saline solution was ultrafilterable. The addition of perfluorooctanoic acid to human serum, in amounts
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which elevated the nonionic fluorine to levels as high as 16 ppm, resulted in at least 99% of the added nonionic fluorine being bound to serum constituents and not being ultrafilterable. Serum harvested from rats 4.5 hour after the administration of a 2-mg dose of nonionic fluorine, as perfluorooctanoic acid, had a nonionic fluorine level in excess of 13 ppm and virtually all of this was bound to components in the serum and not ultrafilterable. Prior to intubation of perfluorooctanoic acid, the ionic and nonionic fluorine levels were 0.032 and 0.07 ppm, respectively. Within 4.5 hours after the administration of the perfluorooctanoic acid, the nonionic fluorine in the serum rose to 13.6 ppm. Despite the large increase (200-fold) in the nonionic fluorine level in the serum, the ionic fluoride level remained very low (0.03 ppm). The nonionic fluorine level in the serum decreased to 11.2 and 0.35 ppm at 8 hours and 24 hours, respectively. The level observed at 24 hours was still approximately seven times the baseline level. By 96 hours, the mean level of nonionic fluoride in the serum had decreased to 0.08 ppm, a value that was not statistically different from that of the undosed animals. Throughout the entire 96 hours, the ionic fluoride level of the serum remained very low.
Table l. Fluoride Content of Serum, Serum Ultrafiltrates, and Buffered Isotonic Saline Before and After
the Addition of Perfluorooctanoic Acida
Specimen
Treatment
Specimen
Ultrafiltrate
Ionic
Total
Ionic
Total
Buffered Saline Perfluorooctanoic acid
-
12.1
-
11.1
(pH 7.4)
added
Human Serum
Aliquot 1
None
0.014
0.044
-
-
Aliquot 2
Perfluorooctanoic acid
0.015
0.88
0.017
0.040
added
Aliquot 3
Perfluorooctanoic acid
0.018
16.3
0.025
0.086
added
Rat Serum
1 Control serumb
0.032
0.11
-
-
2
Experimental serumc
0.013
13.2
0.016
0.12
aFluoride contents were reported as ppm and were the mean of two determinations.
bPooled serum from normal rats.
cPooIed serum from rats 4.5 hours after administration of a 2-mg dose of nonionic fluorine as
perfluorooctanoic acid.
The urine was analyzed for nonionic and ionic fluorine since the in vivo data showed a rapid removal of nonionic fluorine from the serum; the results are shown in Table 2. Although the urine from the undosed animals contained no detectable nonionic fluorine, within 4.5 hours after receiving the dose, an average of
749 pg or 37% of the fluorine in the administered dose was recovered in the urine. The quantity of nonionic fluorine in the urine increased to 61% of the administered dose at 8 hours and by 24 hours 76% had been excreted in the urine. Between 24 and 96 hours of the experimental period, the quantity of non ionic fluorine found in the urine increased to 89%. The rate of excretion of nonionic fluorine thus
increased from 0 pg/hour in the undosed animals to 166 pg/hour, 4.5 hours after receiving the dose of perfluorooctanoic acid. The rate of nonionic fluorine excretion rapidly decreased to 5 pg/hour over the interval from 52.5 to 96 hours. The rate of excretion of ionic fluoride in the urine for the undosed animals, based on a 24-hour urine collection, was found to be 1.73 pg F/hour. Although there was a tendency toward an increased ionic fluoride excretion in the dosed animals, ionic fluoride excretion rate was not statistically greater than that of the nondosed animals.
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Tabic 2. Urinary Excretion of Ionic and Nonionic Fluoride Following Administration of a 2-mg Dose of
Nonionic (As Perfluorooctanoic Acid) to Rats ________________________________________
Time (hr)
Ionic3(pg/hr)
Nonionic3
pg/hr
Accumulative % of
dose
Baseline
1.73 0.24b
0
0
4.5
1.73 0.22
166139.8
37 9.0
8.0
2.360.34
134133.6
61 5.8
24
2.3610.54
1913.2
7612.7
52.5
1.8310.32
5 1 1.2
81 2.7
96
C
5 1 1.3
8912.6
"Excretion rates for ionic and nonionic fluoride were calculated as fig excrcted/hr from the preceding time
point. The baseline value was based on a 24-hour urine collection.
bMean SEM
cThe ionic fluoride excretion rate for the 96-hour period is not given since these animals were fed a low
fluoride diet.
In an effort to account for the remaining nonionic fluorine, feces collected from dosed animals over 52.5and 96-hour periods were analyzed for nonionic fluorine. The percentage of the administered dose of nonionic fluorine recovered in the urine and feces of animals after 52.5 and 96 hours is shown in Table 3. After 52.5 hours, a mean of 4.5% of the administered dose of nonionic fluorine was recovered in the feces. An additional 81.5% was present in the urine, which brought the total quantity of nonionic fluorine excreted in the feces and urine to 86%. By 96 hours, however, the quantity of nonionic fluorine excreted in the feces had increased to 14.3% and the entire dose of nonionic fluorine had been excreted in the urine and feces.
Table 3. Percentage of the Administered Dose of Nonionic Fluorine Recovered in the Urine and Feces
after 52.5 and 96 hours
Time (hr)
Urine
Feces
Total
52.5
81.5 3.9
4.5 1 1.0
86.013.3
96
89.3 2.6
14.3 4.1
103.5 1 1.7
Metabolites measured: Ionic and nonionic fluorine
CONCLUSIONS
The nonionic fluorine level in the serum was increased 200-fold after administration of the dose, but returned to baseline levels by 52.5 hours. Although perfluorooctanoic acid is rapidly absorbed and bound to nonultrafilterable components in the serum, the entire dose of nonionic fluorine was recovered in the urine and feces after 96 hour. Neither the ionic fluoride level in the serum nor the rate of ionic fluoride excretion in the urine was significantly altered by the administration of perfluorooctanoic acid. Although perfluorooctanoic acid has not been identified in the urine, the available data suggest that it has been excreted intact or in, possibly, a conjugated form.
REFERENCE
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Ophaug, R.H. and L. Singer. 1980. Metabolic Handling of Perfluorooctanoic Acid in Rats. Proc Soc Exp Biol Med. 163:19-23.
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PHARMACOKINETIC STUDY
Title: Stimulation by oestradiol of the urinary excretion of perfluorooctanoic acid in the male rat
TEST SUBSTANCE
Identity: Perfluorooctanoic acid (PFOA); 98% pure.
Remarks: The test substance was dissolved in a mixture of propylene glycol-water (1:1) at a concentration of 25 mg/ml.
METHOD
Method/guideline followed: NA
Test type: in vivo
Species/strain/cell type or line: rat/Wistar
Sex: male and female
Age and body weight range of animals used: age not specified, male bw range 170-300g, female bw range 150-180g
Number of animals/sex/dose: Dosed with vehicle: 6 males and 6 females; Dosed with single intraperitoneal injection of PFOA: 6 females, 6 males, 10 castrated males, 10 castrated males treated with oestradiol, and 10 intact males treated with oestradiol
Route of administration: intraperitoneal injection
Vehicle: propylene glycol-water (1:1)
Doses: 50 mg/kg, single injection
Excretion routes, body fluids, and tissues monitored and/or sampled during study: urine, serum
Statistical methods used: one-way analysis of variance (differences between groups), Student's t-test for unpaired data (differences between means of two groups)
Method remarks: Rats were housed in metabolic cages in groups of 5. They were fed standard rat chow and tap water ad libitum. Twenty males were castrated at the age of 28 days and after 5 weeks they were used in the test. Half of the operated and 10 intact males were dosed with 500 pg/kg oestradiol valerate subcutaneously every second day for the 14 days before the test. Urine was collected in the cages during the 96 hours after a single dose of PFOA. Blood samples were collected by cardiac puncture. Serum samples were ultrafiltered to determine the protein binding of PFOA in serum. The PFOA contents of urine, serum, and ultrafiltrates were measured with gas chromatography. The free anionic and possible conjugated forms of PFOA in urine were separated and assayed for PFOA concentration.
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RESULTS
Detailed results: Castration and administration of oestradiol to the male rats had a significant stimulatory effect on the urinary excretion of PFOA. During the first 24 hours, female rats excreted 72 5% (n=6) of the dose, whereas intact males excreted only 9 4% (n=6). After the oestradiol treatment, both intact and castrated males were able to excrete PFOA in urine in similar amounts as females (61 19% and 68 14% at 24 h, respectively). The castrated males without oestradiol treatment excreted PFOA in urine faster than intact males (50 13% at 24 h), but not as fast as females or oestradiol treated males during the whole test (P < 0.01 ). At the end of the test (96 h), the concentration of PFOA in the serum of intact males was considerably higher (17-40 times) than in the serum of other groups.
Females and intact males treated with PFOA excreted slightly more urine than control animals up to 72 h (P < 0.05) after treatment. PFOA may enhance the urine volume excreted.
Ultrafiltration and analysis of the urine samples indicated that PFOA is not metabolized and is secreted in its anionic form. Over 98% of PFOA was bound to proteins in the serum of females and intact males determined in the samples taken during 12 h after dosing.
Metabolites measured: none
CONCLUSIONS
No conclusions were given.
REFERENCE
Ylinen, M., Hanhijarvi, H., Jaakonaho, J., and P. Peura. 1989. Stimulation by oestradiol of the urinary excretion perfluorooctanoic acid in the male rat. Pharmacology & Toxicology 65: 274-277.
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PHARMACOKINETIC STUDY
Title: Disposition of perfluorooctanoic acid in the rat after single and subchronic administration
TEST SUBSTANCE
Identity: Perfluorooctanoic acid (PFOA) from Aldrich-Chemie; purity was not indicated.
Remarks: The test substance was dissolved in: a) 0.9% NaCl solution for intragavage administration and b) propylene glycol-water mixture (1:1 ) for intraperitoneal administration.
METHOD
Method/guideline followed: NA
Test type: in vivo
Species/strain/cell type or line: rat/Wistar
Sex: male and female
Age and body weight range of animals used: a) newly weaned/bw not specified b) 10 weeks/bw not specified
Number of animals/sex/dose: a) 18 b) 20
Route of administration: a) intragavage b) intraperitoneal
Vehicle: a) 0.9% NaCl solution b) propylene glycol-water (T.l)
Doses: a) 3, 10, and 30 mg/kg/day, daily doses for 28 consecutive days b) 50 mg/kg, single injection, volume = 0.25 mL/lOOg
Excretion routes, body fluids, and tissues monitored and/or sampled during study: serum, brain, liver, kidney, lung, spleen, ovary, testis, and adipose tissue
Statistical methods used: The biological half-life of PFOA in the serum and other tissues was estimated from the equation (linear regression) of the linear relationship between time and concentration of PFOA in a semilogarithmic plot.
Method remarks: The animals were housed at 21C, with a dark period from 9 p.m. to 7 a.m. They were given tap water and regular rat chow ad libitum. The serum was collected by cardiac puncture. After decapitation the brain was sampled and the other tissues were sampled at necropsy. The concentrations of PFOA in the serum, tissues, and lipid extracts were determined using capillary gas chromatography.
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RESULTS
Detailed results: a) At all three dose levels in the gavage study, the levels of PFOA in the serum and other tissues analyzed were higher in males than females (p < 0.05). The distribution of PFOA was mainly to the serum, but liver, kidney, and lung tissues also had high concentrations. A significant (p < 0.05) positive correlation was observed between the dose and the concentration of PFOA found in the liver ( r = 0.996), kidney (r2= 0.933), spleen (r2 = 0.995), and lung (r2= 0.959) of females. No similar significant correlation was found in males. However, the spleen, testis, and brain concentrations of PFOA correlated positively with the concentration in the serum (r2 = 0.969, 0.971, and 0.976, respectively).
b) After intraperitoneal injection, the concentrations of PFOA in the serum and other tissues assayed were higher in males than in females during the whole test. PFOA was mainly distributed to the serum, but liver, kidney, spleen, and brain tissues also had high concentrations. Twelve hours after administration of PFOA, about 10% of the dose was found in the serum of females, compared to about 40% in males. After two weeks, about 3.5% of the dose was still left in the serum of males. In females, the PFOA levels in the serum, liver, and kidney decreased in a discontinuous fashion, indicating distinct phases. The half-lives of the concentration of PFOA in the serum were 24 h and 105 h in females and males, respectively. The half-life of PFOA in the liver of females during the first week was estimated to be 60 h, while in males, it was found to be 210 h. In males, a more linear relationship between time and PFOA concentration was observed. PFOA was not found in the assay of the lipid fraction of the liver. Similar levels of PFOA were found in the spleen as in the liver (females = 73 h and males = 170 h). In the kidney, half-lives of 145 h and 130 h were found for females and males, respectively. Metabolites measured: none
CONCLUSIONS
No conclusions were given.
REFERENCE
Ylinen, M., Kojo, A., Hanhijdrvi, H., and P. Peura. 1990. Disposition of perfluorooctanoic acid in the rat after single and subchronic administration. Bulletin of Environmental Contamination & Toxicology 44: 46-53.
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EPIDEMIOLOGIC DATA
Title: Mortality Among Employees of a Perfluorooctanoic Acid Production Plant
TEST SUBSTANCE
Identity: Perfluoroctanoic acid (PFOA)
Remarks: The cohort was exposed to PFOA during production of the chemical. Many other chemicals (e.g., benzene, asbestos) were also used or produced at the plant. The workers were exposed to these chemicals as well.
METHOD
Study design: Retrospective cohort mortality study.
Manufacturing/Processing/Use: There was no information in the study on methods used to manufacture and process PFOA. PFOA is used as a surfactant, and it is used in a large number of industrial applications and consumer products including plasticizers, lubricants, wetting agents, and emulsifiers.
Hypothesis tested: To determine whether mortality from any cause at the 3M Cottage Grove (Minnesota) manufacturing facility was associated with occupational exposure to PFOA.
Study period: The study population worked at the plant from Jan 1, 1947 to Dec 31, 1983.
Setting: Minnesota 3M plant that produced PFOA and other chemicals.
Total population: 3537 employees participated in the study (2788 males and 749 females). 1339 of this total worked in the Chemical Division. 398 (348 males and 50 females) of these employees were deceased, of which 148 males and 11 females worked in the Chemical Division.
Subject selection criteria: All workers employed for at least 6 months at the plant between Jan 1, 1947 to Dec 31, 1983.
Comparison population: In SMR analyses: U.S. general population death rates; Minnesota population death rates. Women were compared only with the U.S. population because cause- and calendar periodspecific Minnesota rates for women were not available. In proportional hazards analyses: non-Chemical division workers at the plant.
Participation rate: Six workers employed during this time were excluded because they had incomplete records, resulting in a 99.8% participation rate (3537/3543). Of this population, vital status (alive or dead) was obtained for 100% of the individuals, and death certificates were obtained for 99.5% of those who died.
Subject description: Individuals employed during the study period were identified through company personnel records. Mean age at employment was 27.3 years for males and 27.6 years for females. Mean age at death was 56.4 for males and 55.4 for females.
Health effects studied: Mortality
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Data collection methods: Vital status was obtained from the Social Security Administration for 19471982, and from the National Death Index for 1979-1989. Work histories and other employee data were obtained from company personnel records. Death certificates were obtained from the state health departments for those employees presumed to be deceased.
Exposure period: The potential exposure period was from Jan. 1, 1947 to Dec. 31, 1983.
Description/delineation of exposure groups/categories: PFOA production was restricted to the Chemical Division of the plant. Employees who worked in the Chemical Division for at least 1 month were considered exposed to PFOA. Employees who never worked in the Chemical Division or worked there for less than 1 month were considered not exposed to PFOA.
Measured or estimated exposure: Cumulative exposure to PFOA was estimated using the surrogate measure of months of Chemical Division employment.
Exposure levels: Not measured. Employees were considered either exposed or not exposed.
Statistical methods: Stratified Standardized Mortality Ratios (SMRs), adjusted for age, sex, and race, were calculated and compared to U.S. and Minnesota white death rates for men. For women, only state rates were available. The SMRs for males were stratified for 3 latency periods (10, 15, and 20 years) and 3 periods of duration of employment (5, 10, and 20 years). Cause-specific mortality rates were compared between exposed on unexposed workers using stratified SMRs.
Relative risk and 95% confidence intervals were estimated for deaths from all causes, cancer, cardiovascular diseases, and other selected causes using proportional hazard models (SAS). Age at first employment, year of first employment, and duration of employment were included as covariates in the model. The appropriateness of the proportional hazard assumptions was tested using stratified models with graphical analysis of log versus follow-up time relationships and models that tested the significance of a product term between exposure and log follow-up time.
Other methodological information: A nosologist coded the death certificates for underlying cause of death for the workers according to ICD 8.
Data collected included year of first employment, age at first employment, duration of employment, and duration of employment in the Chemical Division. Length of employment at the plant, length of employment in the Chemical Division, calendar year of first employment, and age at first employment were analyzed to detennine whether they had an effect on numbers of deaths from all causes and from specific causes.
RESULTS
Describe results: For all female employees, the SMRs for all causes and for all cancers were less than 1. The only elevated (although not significant) SMR was for lymphopoietic cancer, and was based on only 3 deaths. When exposure status was considered, SMRs for all causes of death and for all cancers were significantly lower than expected, based on the U.S. rates, for both the Chemical Division workers and the other employees of the plant.
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In male workers, the SMRs were close to 1 for most of the causes of death when compared to both the U.S. and the Minnesota death rates. When latency and duration of employment were considered, there were no elevated SMRs. When employee deaths in the Chemical Division were compared to Minnesota death rates, the SMR for prostate cancer for workers in the Chemical Division was 2.03 (95% Cl .55 4.59).
The SMR for prostate cancer for workers in the Chemical Division was 2.03 (95% Cl .55 - 4.59). This was based on 4 deaths (1.97 expected). There was also a statistically significant association with length of employment in the Chemical Division and prostate cancer mortality. Based on the results of proportional hazard models, the relative risk for a 1-year increase in employment in the Chemical Division was 1.13 (95% Cl 1.01 to 1.27). It rose to 3.3 (95% Cl 1.02 -10.6) for workers employed in the Chemical Division for 10 years when compared to the other employees in the plant. The SMR for workers not employed in the Chemical Division was less than expected for prostate cancer (.58).
Study strengths: Vital status was determined for 100% of the cohort.
Study weaknesses: There was a potential for misclassification of exposure because many of the nonChemical Division employees may have been exposed to PFOA. In addition, exposures were not measured; therefore categories of exposure were very broad (ever vs. never exposed). This exposure misclassification would bias the effect estimates toward the null. Workers were also exposed to other chemicals in the workplace. The authors also note that there are differences in the distribution of age at risk among the Chemical Division and non-Chemical Division workers and that this could confound the results of the study. There were also small numbers of deaths in many of the categories for males and especially for females in all categories. The cohort needs to be followed for many years to come in order to develop an accurate picture of the mortality experience of the employees of this plant.
Research sponsors: National Institute for Occupational Safety and Health Grant and the 3M Corporation
Consistency of results: Currently there are no other mortality studies on PFOA workers.
CONCLUSIONS Although an association between employment in the Chemical Division and prostate cancer was observed, the results must be interpreted carefully. Continued follow up of this study or other studies with direct exposure measurements might help to confirm the association.
REFERENCE
Gilliland, F.D. and Mandel, J.S. 1993. Mortality among employees of a perfluorooctanoic acid production plant. JOM. 35(9): 950-954.
OTHER This study is the second update of the mortality study. Another update is expected to cover the years through 1997. It has not yet been submitted to EPA.
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EPIDEMIOLOGIC DATA
Title: Mortality Study of Workers Employed at the 3M Cottage Grove Facility
TEST SUBSTANCE
Identity: Perfluoroctanoic acid (PFOA) and other fluorochcmicals
Remarks: This study is an update of the study published by Gilliland and Mandel, J Occup Med 1993, 950-954.
METHOD
Study design: Retrospective cohort mortality study.
Manufacturing/Processing/Use: The 3M Cottage Grove, MN plant has produced perfluorinated compounds since 1947. A primary product from this plant is ammonium perfluorooctanoate (APFO), a potent synthetic surfactant used in industrial applications. APFO rapidly dissociates in biologic media to perfluorooctanoate (PFOA).
Hypothesis tested: To determine whether occupational exposure to PFOA and other fluorochemicals is related to the mortality experience of employees of the 3M facility in Cottage Grove, Minnesota.
Study period: The study population worked at the plant for at least 1 year since Jan. 1, 1947. The cohort was followed through Dec. 31, 1997. Currently employed workers were assigned Dec 31, 1997 as their last date of employment.
Setting: 3M plant in Cottage Grove, Minnesota that produced PFOA and other chemicals.
Total population: 6678 workers were identified. Of these workers, 3992 worked at the plant for at least one year. Eighty percent of the cohort was male.
Subject selection criteria: All workers employed at the Cottage Grove plant for at least 1 year. The cohort was followed through Dec. 31, 1997. Currently employed workers were assigned Dec 31, 1997 as their last date of employment.
Comparison population: In SMR analyses, Minnesota population death rates for whites were used. Mortality reference rates from 7 regional counties were also used to rule out large variations based on regional mortality reporting differences.
Participation rate: Death certificates were obtained for 97% (n = 590) of the cohort who were deceased.
Subject description: 80% (n = 3183) of the employees in the cohort were male. The mean age at follow-up was 56.6 years, and the mean number of years worked at the plant was 12.1. The number of person-years at follow-up was 108198. There were 607 deaths identified in the cohort.
Health effects studied: Mortality
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Data collection methods: A review of employee work history records of any employee with at least 1 year employment were abstracted to record the workers' name, SSN, 3M identification number, date of birth, and dates of work history. This cohort was linked to records from the original cohort to update the employment information and verify other data. The National Death Index was searched for all of the workers. Discrepancies with the original cohort were resolved and deaths before 1979 were verified in the Social Security Death Index. A licensed nosologist coded the death certificates to ICD 8.
Exposure period: The potential exposure period was from Jan. 1, 1947 to Dec. 31, 1997.
Description/delineation of exposure groups/categories: Workers were placed into 3 exposure groups based on job history information. Those groups were: definite PFOA exposure (jobs where cell generation, drying, shipping and packaging of PFOA occurred throughout the history of the plant); probable PFOA exposure (other chemical division jobs where exposure to PFOA was possible but with lower or transient exposures); and not exposed to fluorochemicals (primarily non-chemical division jobs).
Measured or estimated exposure: estimated based on job history information.
Exposure levels: Not measured. Employees were considered exposed (492 workers), probably exposed (1685), or not exposed (1815).
Statistical methods: Standardized Mortality Ratios (SMRs) and 95% confidence intervals were derived using the PC Life Table Analysis System software developed by NIOSFI. This program computes age, gender, and race-specific SMRs using standard life table methods. The expected number of deaths are estimated by multiplying the age, gender, race, and calendar period tabulated person-years of follow up to the corresponding cause-specific mortality reference rates. Mortality rates for white Minnesotans were used as reference data.
Other methodological information:
RESULTS
Describe results: 607 deaths were identified in the cohort. 46 of these deaths were in the PFOA exposure group, 267 in the probable exposure group, and 294 in the not exposed group. The authors also stratified by a minimum of one-year exposure to PFOA in both the definitely-exposed and probably exposed groups. 182 workers (17 deaths) were definitely exposed for at least 1 year and 1673 workers (219 deaths) had probable exposure for at least one year.
When all employees were compared to the state mortality rates, SMRs were less than 1 or only slightly higherfo r all o f the causes o f death analyzed. None o f the SMRs were statistically significant at p = .05. The highest SMR reported was fo r bladder cancer (SMR = 1.31, 95% Cl = 0.42 - 3.05). Five deaths were observed (3.83 expected).
A few SMRs were elevatedfo r employees in the definite PFOA exposure group: 2 deathsfrom cancer o f the large intestine (SMR = 1.67), 1from pancreatic cancer (SMR = 1.34), and 1from prostate cancer (SMR = 1.30). In addition, employees in the definite PFOA exposure group were 2.5 times more likely to die from cerebrovascular disease (5 deaths observed, 1.94 expected; 95% Cl = 0.84 --6.03).
In the probable exposure group, 3 SMRs were notable: cancer o f the testis and other male genital organs (SMR = 2.75, 95% Cl = 0.07 - 15.3); pancreatic cancer (SMR = 1.24, 95% Cl = 0.45 - 2.70); and
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malignant melanoma o f the skin (SMR = 1.42, 95% Cl = 0.17 - 5.11). Only 1, 6, and 2 cases were observed, respectively. The SMR fo r prostate cancer in this group was 0.86 (n = 5).
There were no notable excesses in SMRs in the non-exposed group, except for cancer of the bladder and other urinary organs. Four cases were observed and only 1.89 were expected (95% Cl = 0.58 - 5.40).
The excess in prostate cancer deaths that was observed in the first study was not as strong in this updated cohort. Only 1 death was reported in the definite exposure group while 5 were observed in the probable exposure group. It is difficult to interpret these results since the exposure categories were modified since the last study. However, all of the employees in either group were assumed to have some exposure to PFOA. The new delineation of exposures further defined the chemical plant employees of the first study and placed them into 2 groups, while the film plant employees still remained in the non-exposed group. The number of years that these employees worked at the plant and/or were exposed to PFOA was not reported for prostate cancer.
The excess mortality in cerebrovascular disease noted in employees in the definite exposure group was further analyzed based on number of years of employment at the plant. Three of the 5 deaths occurred in workers who were employed in jobs with definite PFOA exposure for more than 5 years but < 10 years (SMR = 15.03, 95% Cl = 3.02 - 43.91). The other 2 occurred in employees with less than 1 year of definite exposure. The SMR was 6.9 (95% Cl = 1.39 - 20.24) for employees with greater than 5 years of definite PFOA exposure. When these deaths were further analyzed by cumulative exposure (timeweighted according to exposure category), workers with 27 years of exposure in probable PFOA exposed jobs or those with 9 years of definite PFOA exposure were 3.3 times more likely to die of cerebrovascular disease than the general population. A dose-response relationship was not observed with years of exposure.
The slight excess in bladder cancer in the cohort as a whole should be noted. PFOA exposures were reported in employee serum at the Decatur, Alabama plant, and bladder cancer mortality was 4 times higher in workers with high exposure jobs than the general population (SMR = 4.81, 95% Cl = 0.99 14.06). Three deaths were reported, and all of them occurred in the high exposure group. PFOA has been used at this plant as an elastomer in fluoropolymer production or it is produced as a by-product. It is also now being manufactured at Decatur. Manufacture has been occurring since 1999.
Study strengths/weaknesses: It is difficult to compare the results of the first and second mortality studies at the Cottage Grove plant since the exposure categories were modified. Although the authors claim that the newer exposure categories are more accurate, there is still a great chance that exposure misclassification occurred. Without measured exposures, it is difficult to judge the reliability of the exposure categories that were defined. It should be noted that in this study the chemical plant employees were sub-divided into PFOA-exposed groups and the film plant employees remained in the "nonexposed" group. Nevertheless, all of the deaths were considered to probably have had exposure to PFOA in the workplace. Seventeen death certificates were not located and therefore were not included in the study. This is important because it could greatly change the analyses for the causes of death that had a small number of cases. There are other chemicals present in the plant. In order to confirm the results regarding cerebrovascular disease (was an artifact of death certificate coding), regional mortality rates were used for the reference population. The results did not change.
Research sponsors: University of Minnesota
Consistency of results: The excess in prostate cancer deaths that was observed in the first study was not as strong in this updated cohort. Only 1 death was reported in the definite exposure group while 5 were
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observed in the probable exposure group. It is difficult to interpret these results since the exposure categories were modified since the last study. However, all of the employees in either group were assumed to have some exposure to PFOA. The new delineation of exposures further defined the chemical plant employees of the first study and placed them into 2 groups, while the film plant employees still remained in the non-exposed group. The number of years that these employees worked at the plant and/or were exposed to PFOA was not reported for prostate cancer.
CONCLUSIONS
Follow up of worker mortality at Cottage Grove needs to continue. Although there were more than 200 additional deaths included in this analysis, it is still a small number and the cohort is still relatively young. Given the results of studies on flurorchemicals in both animals and humans, further analysis is warranted. Of particular interest are bladder cancer, prostate cancer, cerebrovascular disease, cancer and disorders of the liver, and pancreatic cancer.
REFERENCE
Alexander, B.H. April 26, 2001. Mortality study of workers employed at the 3M Cottage Grove facility. Final Report. Division of Environmental and Occupational Health, School of Public Health, University of Minnesota.
OTHER
This study differs from the 1993 mortality study in that this one requires 1 year of exposure for inclusion in the cohort (as opposed to 6 months). In addition, the exposure categories were more specific. Additional cohort members were included (n = 169) that should have been included in the last study. It is not clear why these employees were not included in the 1993 study.
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EPIDEMIOLOGIC STUDY
Title: An Epidemiologic Investigation of Reproductive Hormones in Men with Occupational Exposure to Perfluorooctanoic Acid
TEST SUBSTANCE
Identity: Perfluorooctanoic Acid (PFOA)
Remarks: This paper further examines the observation initially reported by Gilliland in his doctoral thesis that total serum organic fluorine may be associated with reproductive hormone changes in PFOA production workers.
METHOD
Study design: Two cross-sectional studies, utilizing general medical surveillance and analysis of eleven hormones in serum of male employees at a PFOA production facility.
Manufacturing/Processing/Use: PFOA was produced at the plant by an electrochemical process, which involved a four-stage process: isolating and converting the chemical to a salt slurry, converting the slurry to a salt cake, drying the cake, and packaging. The greatest likelihood for exposure to PFOA occurred in the drying area. PFOA, a potent synthetic surfactant, is used in industrial applications.
Hypothesis tested: Since PFOA has been shown to result in dose-related increases in hepatic, pancreatic acinar, and Leydig cell adenomas in laboratory animals and increased serum estradiol levels, PFOA may show a similar effect in humans with regard to reproductive hormones, particularly increased estradiol or decreased testosterone serum levels.
Setting: PFOA production plant (presumed to be 3M plant in Cottage Grove, MN)
Total # of subjects in study: 1993--111 production workers, 1995--80 production workers. Sixty-eight production workers were common to both cohort years.
Comparison group/population: Employees were divided into four serum PFOA level categories, 0-<l ppm, 1-<10 ppm, 10-<30 ppm, and >30 ppm, in order to determine if an effect existed at the highest serum levels.
Participation rate: Not specified.
Subject description: Characteristics of subjects were not described.
Health effects studied: Potential changes in: serum cortisol, dehydroepiandrosterone sulfate (DHEAS), estradiol, follicle-stimulating hormone (FSH), 17a-hydroxyprogesterone (17-HP), free testosterone, total testosterone, luteinizing hormone (LH), prolactin, thyroid-stimulating hormone (TSH), and sex hormone binding globulin (SHBG)
Data collection methods: Medical surveillance consisted of a medical questionnaire, measurement of height, weight, and pulmonary function; standard biochemical and urinalysis tests; PFOA determination; and several hormone assays.
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Details on data collection: The upper limit of detection of PFOA in 1993 was 80 ppm, whereas there was no upper limit of detection in 1995. A thermospray mass spectrophotometry assay was used to determine serum PFOA levels in 1993 and 1995. Eleven hormones were assayed. Cortisol was assayed using a fluorescence polarization immunoassay. Radioimmunoassays (RIA) were used for DHEAS, estradiol, 17-HP and total testosterone. Free testosterone was determined using equilibrium dialysis. LH, FSH, and prolactin were assayed using a microparticle enzyme immunoassay. TSH was determined using a chemiluminescence immunometric assay. SHBG was assessed via a radioimmunoassay after chromatographic sample purification. Bound testosterone was calculated as total testosterone less free testosterone. The same assays were used for both 1993 and 1995 analyses.
Exposure period: Not specified.
Description/delineation of exposure groups/categories: For the stratified analyses, employees were divided into four serum PFOA level categories: 0-<l ppm, 1-<10 ppm, 10-<30 ppm, and >30 ppm in order to determine if an effect existed at the highest serum levels. Seventy-five percent of the employees with serum PFOA levels at 10 ppm or greater participated in both years.
Measured or estimated exposure: N/A
Statistical methods: Simple and stratified analyses, analysis of variance (ANOVA), Pearson correlation coefficients, and ordinary multivariable regression were used to evaluate associations between PFOA and each hormone, with adjustment for potential confounding variables. For stratified analyses, employees were divided into four serum PFOA level categories: 0-<l ppm, 1-<10 ppm, 10-<30 ppm, and >30 ppm in order to determine if an effect existed at the highest serum levels. For multivariate evaluation, PFOA, age, body mass index (BMI), alcohol use, and cigarette use were examined as both categorical and continuous variables. Regression models were fitted with PFOA entered as a continuous variable using linear, square, and square root transformations. The possible nonlinear association of estradiol, free testosterone, and bound testosterone was evaluated. Nonlinear dose-response relationships were examined by model fit and by comparing parameter estimates, using indicator and continuous variables. Stepwise selection procedures were also used. Study results were analyzed using SAS.
Other methodological information: No additional comments.
RESULTS
Describe results: The range of serum PFOA was 0 - 8 0 ppm in 1993 and 0 - 1 1 5 ppm in 1995. Serum PFOA measurements were highly correlated among the 68 employees who participated in the study during both years.
PFOA was not highly correlated with any of the hormones or with the following covariates: age, alcohol consumption, BMI, or cigarettes. Most of the employees had PFOA serum levels less than 10 ppm. In 1993, only 12 employees had serum levels > 10 ppm, and 15 in 1995. However, these levels ranged from approximately 10 ppm to over 114 ppm. There were only 4 employees in the30 ppm PFOA group in 1993 and only 5 in 1995. Therefore, it is likely that there was not enough power to detect differences in either of the highest categories. The mean age of the employees in the highest exposure category was the lowest in both 1993 and 1995 (33.3 years and 38.2 years, respectively). Although not significantly different from the other categories, BMI was slightly higher in the highest PFOA category.
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Estradiol was highly correlated with BMI (r = .41, p < .001 in 1993, and r = .30, p < .01 in 1995). In 1995, all 5 employees with PFOA levels > 30 ppm had BMIs > 28, although this effect was not observed in 1993. Estradiol levels in the 30 ppm group in both years were 10% higher than the other PFOA groups; however, the difference was not statistically significant. The authors postulate that the study may not have been sensitive enough to detect an association between PFOA and estradiol because measured serum PFOA levels were likely below the observable effect levels suggested in animal studies (55 ppm PFOA in the CD rat). Only 3 employees in this study had PFOA serum levels this high. They also suggest that the higher estradiol levels in the highest exposure category could suggest a threshold relationship between PFOA and estradiol.
Free testosterone was highly correlated with age in both 1993 and 1995. The authors did not report a negative association between PFOA serum levels and testosterone. There were no statistically significant trends noted for PFOA and either bound or free testosterone. However, 17-HP, a precursor of testosterone, was highest in the. 30 ppm PFOA group in both 1993 and 1995. In 1995, PFOA was significantly associated with 17-HP in regression models adjusted for possible confounders. However, the authors state that this association was based on the results of one employee (data were not provided in the report). There were no significant associations between PFOA and cortisol, DHEAS, FSH, LH, and SHBG.
Study strengths and weaknesses: There are several design issues that should be noted when evaluating the results of this study. First, although there were 2 study years (1993 and 1995), the populations were not independent. Sixty-eight employees participated in both years. Second, there were 31 fewer employees who participated in the study in 1995, thus reducing the power of the study. There were also very few employees in either year with serum PFOA levels greater than 10 ppm. Third, the crosssectional design of the study does not allow for analysis of temporality of an association. Since the halflife of PFOA is several years, the authors suggest that it is possible that there may be some biological accommodation to the effects of PFOA. Fourth, only one sample was taken for each hormone for each of the study years. In order to get more accurate measurements for some of the hormones, pooled blood taken in a short time period should have been used for each participant. Finally, there may have been some measurement error of some of the confounding variables.
Research sponsors: 3M Company
Consistency of results: An earlier study reported an association between total serum organic fluorine in workers and increased estradiol and decreased testosterone levels. However, it is difficult to compare the results of this study to the previous study because serum PFOA in workers was measured in this study.
CONCLUSIONS
hi two cross-sectional studies in 1993 and 1995, significant hormonal changes among the male production employees were not apparent in relation to their measured serum PFOA levels as had been previously observed in laboratory animals. However, it should be noted that PFOA serum levels in workers were much lower than those levels reported to cause effects in laboratory animals.
REFERENCE
Olsen, G. W. et al. 1998. An Epidemiologic Investigation of Reproductive Hormones in Men with Occupational Exposure to Perfluorooctanoic Acid. JOEM. 40(7):614-622.
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EPIDEMIOLOGIC STUDY
Title: An epidemiologic investigation of plasma cholecystokinin and hepatic function in perfluorooctanoic acid production workers
TEST SUBSTANCE
Identity: Perfluorooctanoic acid (PFOA)
Remarks:
METHOD
Study design: 3 cross-sectional analyses based on workers' medical surveillance data collected in 1993, 1995* and 1997
Manufacturing/Processing/Use: The substance is a synthetic surfactant that is produced via a 4-stage electrochemical process involving converting the chemical to a salt slurry and then to a salt cake.
Hypothesis tested: To determine whether a positive association exists between plasma cholecystokinin (CCK) levels and serum PFOA levels in fluorochemical production workers, and to determine whether PFOA may modulate hepatic responses to obesity and alcohol.
Study period: Medical exams were performed in 1993, 1995, and 1997
Setting: PFOA production plant, Cottage Grove, MN
Total population: Not specified
Subject selection criteria: None specified, although participation in medical surveillance at the plant was voluntary.
Total # of subjects in study: The numbers of participants in 1993, 1995 and 1997 were 111, 80, and 74, respectively. There were 68 participants in common for 1993 and 1995, 20 in common for 1993 and 1997, and 17 in common for all three years.
Comparison population: N/A
Participation rate: approx. 70%
Subject description: Workers who engaged in the production of PFOA. No other information was provided. Health effects studied: The effects of scram PFOA levels on hepatic responses to obesity and alcohol, and CCK levels.
Data collection methods: Plant workers voluntarily participated in biennial medical surveillance exams which consisted of a questionnaire, pulmonary function, height, and weight measurements, biochemical and urinalysis testing, PFOA determination, and some male reproductive hormone assays.
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Details on data collection: Hormone data were only collected in 1993 and 1995. These results are provided in another robust summary (Olsen et al., 1998).
Serum biochemical tests included: alkaline phosphatase, gamma glutamyl transferase (GGT), serum glutamyl oxaloacetic transaminase (SGOT), serum glutamyl pyruvic transaminase (SGPT), total bilirubin, direct bilirubin, cholesterol, low-density lipoproteins (LDL), high-density lipoproteins (HDL), triglycerides, blood urea nitrogen (BUN), creatinine, and glucose. Hematology tests included: hematocrit, hemoglobin, red blood cells, platelets, and white blood cells.
Plasma CCK-33 was measured by direct radioimmunoassay. Serum PFOA was determined by thermospray (1993 and 1995) and electrospray (1997) high performance liquid chromatography mass spectrometry methods.
Exposure period: Not specified.
Description/delineation of exposure groups/categories: For the stratified analyses, employees were divided into four serum PFOA level categories: 0-<l ppm, 1-<10 ppm, 10-<30 ppm, and >30 ppm in order to determine if an effect existed at the highest serum levels.
Measured or estimated exposure: N/A
Exposure levels: N/A
Statistical methods: Simple and stratified analyses, Pearson correlation coefficients, ANOVA, and ordinary multivariate regression with adjustment for potential confounding variables.
Other methodological information:
RESULTS
Describe results: The mean PFOA serum level in employees participating in the 1997 study period was 6.4 ppm (range 0.1 - 81.3 ppm). The mean CCK value was 28.5 pg/ml (range 8.8 - 86.7 pg/ml). The highest CCK values were reported in the 2 exposure categories less than 10 ppm. The means were 50% higher in these 2 categories than in the categories greater than 10 ppm (p = .06). When adjusted for potential confounders, multivariable regression models indicated a weak negative association between CCK and PFOA; however, these data were not included in the report. There were no statistically significant differences in serum chemistry values for participants with high CCK or PFOA levels compared to participants with low CCK or PFOA levels.
Serum PFOA levels were not consistently associated with any variables, such as alcohol or cigarette use, or with any hematological or chemistry parameters, and none of the values for such parameters were statistically significant at any of the PFOA levels. There was no evidence that PFOA levels modify hepatic responses to obesity and/or alcohol.
Study strengths and weaknesses: It should be noted that CCK was only measured in 1997. The authors indicate that the cross-sectional study format does not permit a direct analysis of an observed association with respect to time. Additionally, the number of participants in 1995 and 1997 was significantly lower than the number of participants in 1993.
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Research sponsors: 3M Company
Consistency of results: The results of this study are inconsistent with earlier findings that PFOA modulates hepatic responses to alcohol and obesity and that there is a positive correlation between PFOA and CCK levels.
The following explanations may indicate why this study failed to find a positive association between PFOA and CCK values:
It is possible that the hepatocarcinogenic effects of peroxisome proliferators in rodents do not act the same biochemically in humans.
The serum PFOA levels observed in workers may have been too low to detect an effect. Effects in animals were observed at higher doses than most of the serum levels found in workers.
CCK receptors may be different between rats and humans. Therefore, the monkey may be a more appropriate animal model to study the pancreatic effects of PFOA in humans.
The involvement of CCK in the initiation or promotion of pancreatic cancer is controversial. The rat may not be an appropriate model in the study of pancreatic cancer in humans, since acinar cell
malignancies induced by carcinogens in rats, are rare in humans.
CONCLUSIONS The results do not suggest that there is an association between PFOA levels and increases in plasma CCK levels, nor do they suggest the presence of hepatic toxicity at the PFOA levels observed. It is unlikely that PFOA levels observed in this study modulate hepatic responses to obesity and alcohol use.
REFERENCE
Olsen, G.W., et al. 1998. 3M Final Report: An epidemiologic investigation of plasma cholecystokinin, hepatic function and serum perfluorooctanoic acid levels in production workers. St. Paul, 3M Company. Sept 4.
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EPIDEMIOLOGIC STUDY
Title: Serum Perfluorooctanoic Acid and Hepatic Enzymes, Lipoproteins, and Cholesterol: a Study of Occupationally Exposed Men
TEST SUBSTANCE
Identity: Total serum fluorine was used as a surrogate for PFOA exposure. Serum PFOA was not measured due to the cost of analyzing the samples.
Remarks: Originally, employees were considered "unexposed" based on their job descriptions; however, when their serum levels were analyzed, these "unexposed" workers had PFOA levels higher than the general population. Therefore, total serum fluorine was used to classify workers into exposure groups.
METHOD
Study design: Cross-sectional study
Manufacturing/Processing/Use: This plant produces PFOA, as well as several other specialty chemicals
Hypothesis tested: To determine if workers exposed to PFOA experienced changes in hepatic enzymes and lipid metabolism. These effects have been observed in rodents exposed to PFOA.
Setting: PFOA production plant- 3M Chemolite plant in Cottage Grove, MN
Total # of subjects in study: 115
Subject selection criteria: Participants were recruited from all employees at the PFOA production plant who were employed during the period of 1985-1989.
Comparison group/population: All subjects potentially were exposed to PFOA. Therefore, differences in mean values of the biochemical endpoints were compared.
Participation rate: Presumed to be > 80%
Subject description: Mean age = 39.2; mean body mass index = 26.9; 76% drank less than 1 oz. alcohol per day and 17% drank 1-3 oz. alcohol/day; 74 percent smoked.
Health effects studied: Possible effects of total serum fluorine on levels of hepatic enzymes, lipoproteins, and cholesterol, including: serum glutamyl oxaloacetic transaminase (SGOT), serum glutamyl pyruvic transaminase (SGPT), gamma glutamyl transferase (GGT), cholesterol, low-density lipoproteins (LDL), and high-density lipoproteins (HDL).
Data collection methods: Participants completed a medical history questionnaire, were measured for height and weight, and donated blood samples by venipuncture.
Exposure period: Unknown.
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Description/delineation of exposure groups/categories: Workers were placed into exposure categories based on job descriptions; however, those workers presumed to have little or no PFOA exposure had total serum fluorine levels 20-50 times higher than those levels reported in the general population. Therefore, workers were classified into groups by total serum fluorine levels. Employees were placed into one of 5 groups of total serum fluorine levels: <1 ppm, 1-3 ppm, >3 - 10 ppm, >10 - 15 ppm, and > 15 ppm.
Measured or estimated exposure: Exposures were estimated. Total serum fluorine was used as a sumogate measure for PFOA exposure.
Statistical methods: Stratified analysis, ANOVA, Pearson correlation coefficients, and linear multivariate regression were used to evaluate associations between PFOA and biochemical endpoints. For stratified analyses, ANOVA procedures were used to assess differences in mean values. Age, body mass index (BMI), alcohol use, and tobacco use were included in the regression models as potential confounders. Number of cigarettes smoked per day was used in the model as a continuous variable if model fit was improved compared with the model using categorical variables.
RESULTS
Describe results: The range of the serum fluorine values was 0 to 26 ppm (mean 3.3 ppm). Approximately half of the workers fell into the > 1 - 3 ppm category, while 23 had serum levels < 1 ppm and 11 had levels > 10 ppm.
There were no significant differences between categories when analyzed using univariate analyses for cholesterol, LDL, and F1DL. PFOA was, however, associated with HDL levels in moderate drinkers. In the multivariate analysis, there was not a significant association between total semm fluorine and cholesterol or LDL after adjusting for alcohol consumption, age, BMI, and cigarette smoking. There were no statistically significant differences among the categories of total serum fluorine for SGOT, SGPT, and GGT. Increases in SGOT and SGPT occurred with increasing total semm fluorine levels in obese workers (BMI = 35 kg/m2). No workers reported hepatic disease diagnoses or signs, or symptoms consistent with hepatic disorders. No clinical cases of liver dysfunction associated with PFOA exposure were found in these workers.
Study strengths and weaknesses: PFOA was not measured directly and there is no exposure information provided on the employees (eg. length of employment/exposure). The authors state that no adverse clinical outcomes related to PFOA exposure have been observed in their employees; however, it is not clear that there has been follow-up of former employees. In addition, the standard deviations reported for the liver enzymes were very high for many of the exposure categories, indicating instability in the results. Many participants in the study were employed in production of compounds other than PFOA.
Research sponsors: A NIOSH grant and the 3M Medical Department
Consistency of results: There are no other known studies of this kind on PFOA.
CONCLUSIONS
PFOA was not associated with marked hepatic changes in humans as had been observed in rodents. However, PFOA may modulate the effect of alcohol use and obesity on hepatic lipid and xenobiotic metabolism.
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REFERENCE
Gilliland, F.D. and J.S. Mandel. 1996. Seram perfluorooctanoic acid and hepatic enzymes, lipoproteins, and cholesterol: A study of occupationally exposed men. Am. Journal of Industrial Medicine. 29: 560568.
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EPIDEMIOLOGIC DATA
Title: An Epidemiologic Analysis of Episodes of Care of 3M Decatur Chemical and Film Plant Employees, 1993-1998
TEST SUBSTANCE
Identity: POSF-based chemicals used at the Decatur plant
Remarks: Episodes of care analyses are not often used in occupational epidemiologic studies.
METHOD
Study design: Episode of care comparison
Manufacturing/Processing/Use: The 3M Decatur, Alabama plant began production in 1961. It is made up of the film plant and the chemical plant. The 3 major product groups in the chemical plant are protective chemicals, performance chemicals, and fluoroelastomers. Perfluorooctanesulfonyl fluoride (POSF) is the major sulfonate fluorochemical manufactured at Decatur and is used as the precursor to the production of a variety of perfluorinated amides, alcohols, acrylates, and other fluorochemical polymers.
Hypothesis tested: To use episodes of care methodology as a screen for morbidity outcomes associated with long-term, high exposure to POSF-based production at the 3M facility in Decatur, Alabama.
Study period: Episodes of care experience of 652 chemical employees and 659 film plant employees were analyzed for workers at the plant who were employed for at least 1 year between January 1, 1993 and December 31, 1998.
Setting: 3M plant in Decatur, Alabama.
Total population: 1311 workers were eligible for the cohort (at least 1 year of employment at the plant). The total worker population was not reported.
Subject selection criteria: All workers employed at the Decatur plant for at least 1 year between Jan. 1, 1993 and Dec. 31,1998. Episodes of care were limited to their Decatur time of employment for employees hired, terminated, or died during the study period. However, records of employees on Medicare, long-term disability or who chose HMO coverage were not in the database and would not be included in the episodes of care for that employee.
Comparison population: Chemical and fdm plant employees were analyzed separately and then compared to each other. Employee comparison groups were defined according to their potential workplace exposure to POSF fluorochemical production. Group A: all chemical plant employees and all film plant employees eligible for the cohort. Group B: all chemical plant employees who worked solely in the chemical plant and all film plant employees who worked exclusively in the film plant. Group C: all chemical plant employees with high fluorochemical exposures compared to their job counterparts in the film plant. Group D: all plant workers with high fluorochemical exposure for at least 10 years prior to the study onset compared to their job counterparts in the film plant.
Participation rate: 97% of Decatur employees were eligible for participation in the study.
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Subject description: 82% of the employees in the cohort were male (530 in the chemical plant and 558 in the film plant). The mean age was 45.1 in the chemical plant and 48.6 in the film plant. Sixty percent of the chemical plant employees had worked only in the chemical plant and a similar percentage of film plant workers had worked exclusively in the film plant. Seventy-six percent of the chemical plant workers had high exposure jobs.
Health effects studied: Morbidity. Based on animal data and epidemiologic studies on PFOA and PFOS, certain episodes of care were considered a priori. They included: liver and bladder cancer, endocrine disorders involving the thyroid gland and lipid metabolism, gastrointestinal disorders of the liver and biliary tract, and reproductive disorders.
Data collection methods: The Clinical Care Groups episode of care software developed by Ingenix, Inc. was used to provide a comprehensive grouping of all visits (inpatient and outpatient), procedures, ancillary services, and prescription drugs used in the diagnosis, treatment and management of more than 400 diseases or conditions. The software code constructs an episode of care around the index-eligible record by searching backward and forward in time for the health claims records that are related to the disease or condition on the index record. The index record consists of either procedure codes indicative of a face-to-face encounter or a pharmacy record for a delineating drug.
Exposure period: The episodes of care that were included in the study were those experienced between Jan. 1, 1993 to Dec. 31, 1998.
Description/delineation of exposure groups/categories: Workers were placed into groups according to potential workplace exposures: workers who were employed solely in the chemical or film plants, those who had high exposure jobs, and those who worked at least 10 years in jobs with high potential for fluorochemical exposure.
Measured or estimated exposure: estimated based on job history information.
Exposure levels: Not measured. Employees were placed into exposure categories based on job description.
Statistical methods: A risk ratio episode of care (RREpC) provided the estimate of risk between the observed to expected episodes of care for chemical plant employees compared to the observed to expected episodes of care among film plant employees. The expected number of episodes of care for both the film and chemical plant employees was calculated from health claims data of the 3M manufacturing population in the U.S. Because the chemical and film plant cohorts had slightly different age and gender structures, an adjusted ratio was calculated and compared to the unadjusted risk ratio. In most cases, the risk ratios were comparable. Therefore, 95% confidence intervals were only calculated for the unadjusted risk ratios.
Other methodological information: It should be noted that from an epidemiologic perspective, an episode of care could represent any and all incident cases, prevalent cases, and/or misclassified cases (both false positive and false negative). In addition, types and counts of episodes of care may differ by the software used, and it is possible that 2 different diagnoses may be assigned to the same episode. Certain services, such as lab procedures and prescriptions may not be reported for the episode. Also, the endpoint of an episode may vary among software programs. The clinical flexibility of the algorithm may differ depending on the software program.
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RESULTS
Describe results: The only increased risk of episodes for the conditions of a priori interest were for neoplasms of the male reproductive system and for the overall category of cancers and benign growths (which included cancer of the male reproductive system). There was an increased risk of episodes for the overall cancer category for all 4 comparison groups. The risk ratio was greatest in the group of employees with the highest and longest exposures to fluorochemicals (RREpC = 1.6, 95% Cl = 1.2 - 2.1). Increased risk of episodes in long-time, high-exposure employees also was reported for male reproductive cancers (RREpC = 9.7, 95% Cl = 1.1 - 458). It should be noted that the confidence interval is very wide for male reproductive cancers and the sub-category of prostate cancer. Five episodes of care were observed for reproductive cancers in chemical plant employees (1.8 expected), of which 4 were prostate cancers. One episode of prostate cancer was observed in film plant employees (3.4 expected). This finding is important because an excess in prostate cancer mortality was observed in the Cottage Grove plant mortality study. However, the update of the study did not confirm this finding.
There was an increased risk of episodes for neoplasms of the gastrointestinal tract in the high exposure group (RREpC = 1.8, 95% Cl = 1.2- 3.0) and the long-term employment, high exposure group (RREpC = 2.9, 95% Cl = 1.7 - 5.2). Most of the episodes were attributable to benign colonic polyps. Similar numbers of episodes were reported in film and chemical plant employees.
In the entire cohort, only 1 episode of care was reported for liver cancer (0.6 expected) and 1 for bladder cancer (1.5 expected). Both occurred in film plant employees. Only 2 cases of cirrhosis of the liver were observed (0.9 expected), both in the chemical plant. There was a greater risk of lower urinary tract infections in chemical plant employees, but they were mostly due to recurring episodes of care by the same employees. It is difficult to draw any conclusions about these observations, given the small number of episodes reported.
Chemical plant employees in the high exposure, long-term employment group were 2 'A times more likely to seek care for disorders of the biliary tract than their counterparts in the film plant (RREpC = 2.6, 95% Cl = 1.2- 5.5). Eighteen episodes of care were observed in chemical plant employees and 14 in film plant workers. The sub-categories that influenced this observation were episodes of cholelithiasis with acute cholecystitis and cholelithiasis with chronic or unspecified cholecystitis. Most of the observed cases occurred in chemical plant employees.
Risk ratios of episodes of care for endocrine disorders, which included sub-categories of thyroid disease, diabetes, hyperlipidemia, and other endocrine or nutritional disorders, were not elevated in the comparison groups. Conditions which were not identified a priori but which excluded the null hypothesis in the 95% confidence interval for the high exposure, long-term employment group included: disorders of the pancreas, cystitis, and lower urinary tract infections.
Study strengths and weaknesses: See "other methodological information" section for limitations of episodes of care software. The results of this study should only be used for hypothesis generation. Although the episode of care design allowed for a direct comparison of workers with similar demographics but different exposures, there are many limitations to this design. Episodes of care are reported, not disease incidence; therefore, this parameter cannot be interpreted in any other manner. The data arc difficult to interpret because a large RREpC may not necessarily indicate high risk of incidence of disease. In addition, many of the risk ratios for episodes of care had very wide confidence intervals. The analysis was limited to 6 years. Also, the utilization of health care services may reflect local medical practice patterns. Individuals may be counted more than once in the database because they can be categorized under larger or smaller disease classifications. Episodes of care may include the same
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individual several times. Not all employees were included in the database, such as those on long-term disability.
Research sponsors: 3M Company
Consistency of results: No other morbidity studies have been conducted on fluorochemicals.
CONCLUSIONS This study should only be used for hypothesis generation regarding workers employed at the Decatur plant who are employed in jobs with high exposure to POSF-based fluorochemicals.
REFERENCE Olsen, GW, Burlew, MM, Hocking, BB, Skratt, JC, Burris, JM, Mandel, JH. An epidemiologic analysis of episodes of care of 3M Decatur chemical and film plant employees, 1993-1998. Final Report. May 18,2001.
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Title: A Cross-sectional analysis of serum perfluorooctanesulfonate (PFOS) and Perfluorooctanoate (PFOA) in relation to clinical chemistry, thyroid hormone, hematology, and urinalysis results from male and female employee participants of the 2000 Antwerp and Decatur fluorochemical medical surveillance program
TEST SUBSTANCE
Identity: PFOS, PFOA
Remarks:
METHOD
Study design: cross-sectional
Manufacturing/Processing/Use: Facilities in Decatur, Alabama and Antwerp, Belgium which manufacture perfluorooctanesulfonyl fluoride products. These fluorochemicals can metabolize in the body to PFOS.
Hypothesis tested: To provide an aggregate analysis of the hematology, clinical chemistries, and hormonal parameters of volunteer employees in relation to serum PFOS and PFOA levels as measured in the medical surveillance examinations of Antwerp and Decatur employees in 2000.
Study period: March 1, 2000. End date was not reported.
Setting: Occupational. 3M plants located in Antwerp, Belgium and Decatur, Alabama.
Total population: 340 Antwerp employees and 500 Decatur employees working in the chemical plant area were eligible for inclusion in the surveillance.
Subject selection criteria: Voluntary participation in medical surveillance program in Y2000.
Total # of subjects in study: 255 Antwerp employees (206 male and 49 female) and 263 Decatur employees (215 male and 48 female).
Comparison population: N/A
Participation rate: 75% o f employees at the Antwerp plant and 50% o f the employees at the D ecatur p lant who were eligible participated. 73%> o f the participating Antwerp male employees and 75/o o f the D ecatur employees were engaged in production activities. Only 12% o f the participating Antwerp fem a le employees were engaged in production activities com pared to 63% o f the Decaturfem ale employees.
Subject description: Male Antwerp employees had lower PFOS and PFOA levels, were significantly younger than Decatur male employees, had lower BMIs, worked fewer
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years, had higher self-reported daily consumption of alcohol, had lower mean alkaline phosphatase, GGT, AST, ALT and triglyceride values and higher total bilirubin and HDL values. Comparable results were observed for Antwerp female employees vs. Decatur females.
Health effects studied: To determine if there were differences in the following parameters based on PFOS/PFOA levels: hematology (hematocrit, hemoglobin, RBCs, WBCs, platelet count), clinical chemistries (alkaline phosphatase, gamma glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, total and direct bilirubin, blood urea nitrogen, creatinine, glucose, cholesterol, low density lipoproteins, high density lipoproteins, and triglycerides), and thyroid hormones (thyroid stimulating hormone, serum thyroxine, free thyroxine, serum triiodothyronine, thyroid hormone binding ratio, and free thyroxine).
Data collection methods: Medical questionnaire, work history questionnaire, blood sera samples, measurements of height, weight, and blood pressure, urinalysis (Decatur only), and standard clinical chemistry and hematology tests, thyroid hormone measurement, and pulmonary function tests. Values usedfo r reference ranges were not provided.
Details on data collection: The site-specific work history questionnaire was administered to all participants. The data were self-reported. Questionnaire content, design, administration, etc. were not provided in this report. Data on blood collection (amount, etc.) not provided. Urinalysis was only assessed for Decatur employees via standard urine microstick analysis which tested for urine glucose, albumin, and RBCs.
TSH, free T4 and T3 were determined by immunochemiluminometric assay. T4 and THBR were determined by a cloned enzyme donor immunoassay. FT1 was calculated by multiplying T4 and THBR.
Sera samples were extracted using an ion-pairing extraction procedure. In addition to PFOA and PFOS, the extracts were also analyzed for PFHS, PFOSAA, PFOSA, and M556 (perfluorooctanesulfonamidoacetate) using high-pressure liquid chromatography electrospray tandem mass spectrometry and evaluated versus an extracted curve from a human serum matrix. All serum values for PFOS and PFOA were above the LLOQ.
Exposure period: Unknown. PFOS/PFOA serum levels indicate exposure.
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Description/delineation of exposure groups/categories: Workers were stratified by plant location as well as by serum PFOS distribution, production status (production vs. non-production workers), and gender.
Mean serum PFOS levels for all employees participating in this study at Antwerp (n = 206) and Decatur (n = 215) were 0.96 and 1.40 ppm, respectively. Levels among production employees were higher. At Antwerp, the mean PFOS level of male production employees was 1.16 ppm and 1.63 ppm at Decatur.
Mean PFOA levels for all employees were 1.03 and 1.90 ppm at Antwerp and Decatur, respectively. Levels among production employees were higher. At Antwerp, the mean PFOA level of male production employees was 1.28 ppm and 2.34 ppm at Decatur.
Measured or estimated exposure: Serum PFOS and PFOA levels were used to estimate exposure.
Statistical methods: Descriptive simple and stratified analyses, Pearson correlation coefficients, analysis of variance, and multivariable regression were used to evaluate associations between PFOS and PFOA and each hematological and clinical chemistry test and thyroid hormone assay. For stratified analyses, employees were divided into quartiles of their serum PFOS distribution. Potential confounding factors considered in the analyses included: age, BMI, alcohol consumption, cigarette use, years worked at either plant, and type ofjob.
Multivariable regression models were fitted with PFOS/PFOA analyzed as continuous variables. Natural log transformations of the dependent variables were performed, when necessary, to normalize variables and to enhance model fit. SAS was used to analyze the data.
RESULTS Describe results: Antwerp and Decatur employees were different in several ways (see "Subject description" above). Therefore, univariate analyses were initially stratified by location and then those analyses were stratified by gender and production status. They were placed into quartiles depending on production status. Therefore, the PFOS values in the quartiles are different for male production, non-production, and female employees. In addition, these analyses are not appropriate for PFOA.
Antwerp
The mean PFOS level for all employees at this plant was 0.96 ppm (range 0.04 - 6.24 ppm). When stratified by production status, the mean was 1.16 ppm for production employees and 0.42 ppm for non-production employees. The mean for female employees was 0.13 ppm.
Mean PFOA serum levels were 1.03 ppm for all male employees at the Antwerp plant. Male 160
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production employees had higher mean serum levels (1.28 ppm) than non-production employees (0.34 ppm). These values were significantly different between plants. Female employees had a much lower mean PFOA serum value at the Antwerp plant (0.07 ppm) than at the Decatur plant (1.23 ppm). Fewer of the Antwerp females worked at production jobs than the female employees at Decatur.
Male production employees were placed into the following quartiles based on PFOS levels: Q1 (mean, 0.29 ppm; range, 0.04 - 0.41 ppm), Q2 (mean, 0.58 ppm; range 0.41 - 0.78 ppm), Q3 (mean 1.18 ppm; range 0.79 - 1.66 ppm), Q4 (mean, 2.61 ppm; range, 1.67 - 6.24 ppm).
In male production employees (n = 150), the highest quartile mean serum PFOS level was 2.61 ppm (range 1.76 - 6.24 ppm) and the lowest was 0.29 ppm (range 0.04 - 0.41 ppm). Production employees in the highest quartile were significantly (p < .05) older and worked more years at Antwerp than employees in the lowest quartile. The only difference in clinical chemistries for production workers was in BUN. When compared by quartile of serum PFOS distribution, no significant (p <.05) differences among male production employees were observed for thyroid (TSI4, T4, free T4, T3, THBR, FTI) or for hematology (HCT, HGB, RBC, WBC, platelets). The same held true for non-production employees (n = 56).
For all female employees, BUN was significantly different (higher) between 1st quartile and 3rd and 4thquartile. Thyroid and hematology results were not significantly different between any of the quartiles for females.
Decatur
The mean PFOS level for all employees at this plant was 1.40 ppm (range 0 .11-10.06 ppm). When stratified by production status, the mean was 1.63 ppm for production employees and 0.73 ppm for non-production employees. The mean for female employees was 0.93 ppm. 75% of male employees worked in production jobs (n = 161) and 63% of female employees worked in production jobs.
The mean PFOA serum level for all male employees at the Decatur plant was 1.90 ppm. Male production employees had higher mean serum levels than male non-production employees (2.34 ppm and 0.59 ppm, respectively). These values were significantly different between plants. Female employees had a much higher mean PFOA serum value at the Decatur plant (1.23 ppm) than at the Antwerp plant (0.07 ppm). There were many more females at the Decatur plant who worked at production jobs.
Male production employees were placed into the following quartiles based on PFOS levels: Q1 (mean, 0.55 ppm; range, 0.11 - 0.75 ppm), Q2 (mean, 1.01 ppm; range 0.76 - 1.30 ppm), Q3 (mean 1.74 ppm; range 1.32 - 2.29 ppm), Q4 (mean, 3.22 ppm; range, 2.31 - 10.06 ppm).
When male production workers were placed into quartiles, the only significant (p < .05) difference between the quartiles was in ALT (highest quartile different from all 3 others).
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No significant differences between quartiles were observed for thyroid (TSH, T4, free T4, T3, THBR, FTI), hematology (HCT, HGB, RBC, WBC, platelets), or urinalysis (albumin, blood, sugar).
For female employees (both production and non-production employees), there were no significant differences among quartiles for demographics, thyroid, hematology, or urinalysis except for mean platelet count where the third quartile was significantly lower than the 1st quartile, but the 4th was not.
When results were analyzed by number of employees who had values above the reference range for hepatic clinical chemistry tests and liver enzyme and bilirubin tests, there was a higher percentage of male Decatur production workers in the highest PFOS quartile for ALT, GGT, and total liver panel than the other quartiles. Most notable were the results for ALT where 8% of employees in the lowest exposure group (Ql) and 28% in the highest exposure group (Q4) had values above the reference range, while the percentages for total liver panel (which includes alkaline phosphatase, AST, ALT, GGT, and total and direct bilirubin) were 18% and 35%, respectively. This trend was not evident in Decatur non-production employees (although the n was only 54), in Decatur females, or in any of the Antwerp employees. However, each sub population had a different serum PFOS quartile distribution. Therefore, they cannot be directly compared.
Analyses combining employees from both plants
When clinical chemistry results of all male employees from both plants were combined (both production and non-production) (n = 421) and placed into quartiles (n = 105 per quartile), mean values for triglycerides, alkaline phosphatase, total bilirubin, and ALT were significantly (p < .05) higher in the 4th quartile (mean PFOS level 2.69, range 1.69 - 10.06 ppm) than in the first (mean PFOS level 0.27 ppm, range 0.04 - 0.42 ppm). It should be noted that the number of Antwerp production employees were evenly distributed among the quartiles while this was not the case for Decatur employees. The highest number of Decatur employees was in the 4th quartile. In addition, overall the employees in the 1st quartile were slightly younger, had a lower BMI, and worked fewer years than employees in the other quartiles.
Thyroid results for this same group indicated that T3 was significantly higher (p < .05) and THBR was significantly lower (p < .05) in Q4 than Q l. In female employees combined for both plants (n = 97), alkaline phosphatase and GGT were significantly higher (p < .05) and total bilirubin significantly lower in Q4 than in Q l. Most of the Decatur female employees worked in production jobs while most of the Antwerp females worked in non-production jobs. Therefore, Q4 was 92% female production workers and all of them worked at the Decatur plant.
The combined plant data were analyzed for employees who had values above the reference range for alkaline phosphatase, AST, ALT, GGT, and total liver panel. For male employees for all of these measures, the levels increased from Q l to Q4. In Q l, 4% of the employees had values above the reference range for ALT and 6% for GGT, while 12% was reported for Q4 for both of these tests. For total liver panel, 14% of the employees had values above the reference range in
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Q1 as compared to 23% in Q4. The numbers of female employees with values above the reference range was very small (n = 8).
PFOS and PFQA analyses
The above data were not adjusted for potential confounders; therefore, multivariable regression analyses were conducted. There was a positive significant (p = .04) association between PFOS and cholesterol and also a positive significant (p = .05) association between PFOA and cholesterol. When both PFOS and PFOA were included in the model, neither were statistically significant at p=.05. PFOS was not significant with HDL although PFOA was negatively associated with F1DL (p = .04). Triglycerides were positively associated with PFOS (p = .01) and PFOA (p = .002). When both were left in the model, PFOA remained significant (p = .02). Total organic fluorine (TOF) was highly significant for triglycerides (p = .0009). No significant associations were observed with PFOS, PFOA, or TOF in relation to alkaline phosphatase, GGT, AST or total bilirubin. A significant (p = .02) positive association was observed for TOF and ALT. A positive significant (p = .04) association between T3 and PFOS was observed. Plant location was highly significant (p < .0001) in the model. BMI, cigarettes/day, alcohol/day were also significant. [In the univariate analyses, Antwerp employees had higher mean T3 levels than Decatur employees overall. However, for each plant (individually) T3 values increased by quartile as PFOS serum levels increased, although the differences were not statistically significant.] THBR, as well as the other thyroid hormones, were not significant in the regression analyses. Most of the thyroid hormones, including TSH, T4, THBR, and FTI, were not significantly associated with PFOS, PFOA, or TOF. However, PFOS, PFOA and TOF were positively associated (p = .04, .01, and .004, respectively) with T3.
Study strengths and weaknesses: Cross-sectional design, voluntary participation, the Decatur and Antwerp populations were significantly different in certain demographic and clinical chemistry results as well as in PFOS and PFOA serum levels, PFOA serum levels were higher than PFOS serum levels, however, employees were analyzed by PFOS levels, total organic fluorine serum levels were higher than both PFOS and PFOA levels and it is unclear how this is affecting the analyses, plant populations cannot be compared because quartiles are different for each subgroup, only one measurement at a certain point in time was collected for each test, and other perfluorinated chemicals in addition to PFOA and PFOA are present in the plants.
Research sponsors: 3M
Consistency of results: These results are somewhat consistent with those of the 1995 and 1997 cross-sectional medical surveillance data, in that all of them conclude that there were no significant abnormalities in hematological and clinical chemistry parameters of the Antwerp or Decatur workers. No decline in cholesterol levels was observed as PFOS serum levels increased. The hormone data collected in 1995 was different from that collected in 2000 and therefore cannot be compared. A longitudinal analysis of these data did not reveal any significant changes in hepatic or lipid clinical chemistry values; however, there were many limitations to the study.
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CONCLUSIONS The authors concluded that these data continue to suggest that Antwerp and Decatur fluorochemical production and non-production employees do not have significant changes in serum cholesterol, lipoproteins, or hepatic enzymes that are consistent with toxicological findings in laboratory animals. However, the study weaknesses should be considered.
REFERENCE Olsen GW, Burlew MM, Burris JM, Mandel JH. October 11, 2001. A cross-sectional analysis of serum perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) in relation to clinical chemistry, thyroid hormone, hematology and urinalysis results from male and female employee participants of the 2000 Antwerp and Decatur fluorochemical medical surveillance program. Final report. 3M Medical Department.
Last updated: 4/29/02
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EPIDEMIOLOGIC DATA
Title: A Longitudinal Analysis of Serum Perfluorooctanesulfonate (PFOS) and Perfluorooctanoate (PFOA) Levels in Relation to Lipid and Hepatic Clinical Chemistry Test Results from Male Employee Participants of the 1994/95, 1997, and 2000 Fluorochemical Medical Surveillance Program
TEST SUBSTANCE
Identity: PFOS, PFOA
Remarks:
METHOD
Study design: longitudinal
Manufacturing/Processing/Use: 3M Decatur, Alabama plant and Antwerp, Belgium plant.
Hypothesis tested: To determine whether occupational exposure to fluorochemicals over time is related to changes in clinical chemistry and lipid results in employees of 2 3M facilities.
Study period: There were 3 time periods during which medical surveillance took place at the plants-- 1994/95, 1997, and 2000.
Setting: 3M plants in Decatur, Alabama and Antwerp, Belgium.
Total population: 175 male employees participated in 2000 and at least one of the other sampling periods.
Subject selection criteria: Employees participated voluntarily.
Comparison population: n/a
Participation rate: 106/175 (61%) participated in 1994/95, 110/175 (63%) participated in 1997, and 175 participated in 2000. 24% participated in all 3 sampling periods (n = 41, Antwerp 20, Decatur 20), 37% in 1994/95 and 2000 (n = 65, Antwerp 45, Decatur 20), and 39% (n = 69, Antwerp 34, Decatur 35) in 1997 and 2000.
Subject description: Male employees volunteered to participate in biomonitoring offered at the plants. In general, Antwerp male employees were significantly younger, had lower BMIs and a higher daily consumption of alcohol reported than Decatur male employees. Antwerp male employees also had lower mean alkaline phosphatase and triglyceride values and higher total bilirubin and HDL values than the Decatur male employees.
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Health effects studied: To determine w hether w orkers' lipid and hepatic clinical chemistry results are affected by PFOS and PFOA levels.
Data collection methods: Clinical chemistries and hematology collected-- cholesterol (mg/dl), high density lipoproteins (HDL, mg/dl), triglycerides (mg/dl), alkaline phosphatase (IU/L), gamma glutamyl transferase (GGT, IU/L), aspartate aminotransferase (AST, IU/L), alanine aminotransferase (ALT, IU/L), total and direct bilirubin (mg/dl). Demographic data collected via questionnaire.
Details on data collection: Details on data collection methods including questionnaire content, design, administration, etc. and blood collection methods were not provided.
PFOS and PFOA methods o f analysis differed slightly each year. In 1994/95, the m ethod used tetrabutylammonium to ion-pair with PFOS and PFOA in the serum. The ion-pairs were then extracted with ethyl acetate and the abstraction product was then analyzed using highperformance liquid chromatograph-thermospray mass spectrometry. In 1997, the serum samples were analyzed by liquid chromatorgraphy/mass spectrometry, using selected ion monitoring in the negative-ion mode. In 2000, sera samples were extracted using an ion-pairing extraction procedure. High-performance liquid chromatography/electrospray tandem mass spectrometry was used. The samples were evaluated versus an extracted curve from a human serum matrix.
Exposure period: Unknown. PFOS, PFOA levels measured in blood serum.
Description/delineation of exposure groups/categories: The groups o f employees were stratified into subpopulations A, B, and C. "A" was comprised o f the employees who participated in all 3 years o f surveillance, "B" contained those employees who participated in 1994/95 and 2000, and "C" contained those who participated in 1997 and 2000.
Measured or estimated exposure: PFOS and PFOA levels were m easured in w orkers' blood serum. No ambient exposure data are available.
Exposure levels:
Mean PFOS levels in Males
Antwerp
Decatur
1994/95
1.87 ppm
2.62 ppm
1997
1.42 ppm
1.85 ppm
2000
1.16 ppm
1.67 ppm
1994/95 1997 2000
Mean PFOA levels in Males
Antwerp
Decatur
1.08 ppm
1.90 ppm
1.54 ppm
1.41 ppm
1.43 ppm
1.83 ppm
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Statistical methods: repeated measures incorporating the random subject effect fitted to a mixed model using SAS. Restricted maximum likelihood estimates of variance parameters were computed. Adjusted regression models were built by introducing all covariates and testing the covariance structure. Covariates included in the model were age, BMI, number o f alcoholic drinks per day, and cigarettes smoked per day.
Other methodological information: A total o f 175 male employees (100 Antwerp and 75 Decatur) who participated in the 2000 surveillance year also participated in at least one previous fluorochemical medical surveillance exam since 1994/95. Therefore, this provided an opportunity to undertake a longitudinal assessment.
RESULTS
Describe results:
PFOS results When mean serum PFOS levels were compared by surveillance year, PFOS levels have been decreasing in the participants in medical surveillance in both plants. W hen the data were analyzed by the 3 subcohorts (those who participated in 2 or more medical exams between 1995 and 2000), Antwerp and Decatur employees in each of the 3 subcohorts had lower mean serum PFOS levels in 2000 than at their year of entry.
When analyzed using mixed model multivariable regression and combining Antwerp and Decatur employees, there was no association between PFOS and serum cholesterol or triglycerides in male participants over time. There were also no significant associations between PFOS and changes over time in FIDL, alkaline phosphatase, GGT, AST, ALT, total bilirubin, and direct bilirubin.
PFOA results When mean serum PFOA levels were compared by surveillance year, PFOA levels in the employees participating in medical surveillance at the Antwerp plant increased between 1994/95 and 1997 and then decreased slightly between 1997 and 2000. At the Decatur plant, PFOA serum levels decreased between 1994/95 and 1997 and then increased between 1997 and 2000. When the data were analyzed by plant and the 3 subcohorts (those who participated in 2 or more medical exams between 1995 and 2000), there were no consistent changes across subcohorts at the Antwerp plant. However, among the 3 Decatur subcohorts, mean PFOA levels tended to increase.
When analyzed using mixed model multivariable regression and combining Antwerp and Decatur employees, there was a statistically significant positive association between PFOA and serum cholesterol (p = .0008) and triglycerides (p = .0002) over time. W hen analyzed by plant and also by subcohort, these associations were limited to the Antwerp employees (p = .005) and, in particular, the 21 Antwerp employees who participated in all 3 surveillance years (p = .001). However, the association between PFOA and triglycerides was also statistically significant (p =
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.02) for subgroup B (employees who participated in biomonitoring in 1994/95 and 2000). There was not a significant association between PFOA and triglycerides among Decatur workers.
There were no significant associations between PFOA and changes over time in HDL, alkaline phosphatase, GGT, AST, ALT, total bilirubin, and direct bilirubin.
Total Organic Fluorine (TOF) results When analyzed using mixed model multivariable regression and combining Antwerp and Decatur employees, there was a statistically significant positive association between TOF and serum cholesterol (p = .007) and triglycerides (p = .008) over time. However, the interaction term with time (years) was not significant. This association was more consistent for Antwerp employees than Decatur employees.
Study strengths and weaknesses: Study limitations include the following: 1. a very small num ber o f employees participated in all 3 study periods (only 24% , n = 41) 2. different labs were used each year for analysis and different analytical techniques for PFOS and PFOA 3. could not analyze female employees due to small numbers 4. PFOS levels in employees are decreasing over time while PFOA levels are increasing 5. PFOS and PFOA serum levels in employees are below those causing effects in laboratory animals 6. mean serum PFOS and PFOA levels were significantly lower in Antwerp employees than Decatur 7. more Antwerp employees than Decatur participating in this study (57% vs.43%) 8. there are several consistent differences betw een the Antwerp and Decatur male populations (eg., statistically significant differences in BM1, age, consumption o f alcohol, and differences in chemistry profiles) 9. only 1 measurement at a certain point in time was collected for each clinical chemistry test 10. other perfluorinated chemicals are present in the plants 11. all participants were volunteers
Research sponsors: 3M
Consistency of results: This is the first longitudinal analysis o f the surveillance data and will probably not be repeated.
The positive association between PFOA and serum cholesterol and triglycerides is not consistent with the hypolipidemia effect observed in rodents (and not observed in primates). In addition, the authors report that this effect has not been observed at 3M 's Cottage Grove facility where PFOA serum levels in workers are much higher than at the Decatur or Antwerp plant. However, the most recent data (2000) have not been submitted to EPA for review.
CONCLUSIONS
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A longitudinal analysis over a six-year period o f 175 Antwerp and Decatur m ale em ployees did not show significant changes, consistent with toxicological data, o f lipid or hepatic clinical chemistry values associated with PFOS. A positive statistically significant association was observed between PFOA and cholesterol and triglycerides. When analyzed by plant and also by subcohort, these associations were limited to the Antwerp employees and, in particular, the 21 Antwerp employees who participated in all 3 surveillance years. The limitations discussed above should be considered when interpreting these results.
REFERENCE Olsen, G.W., Burlew, M.M, Burris, J.M., Mandel, J.H. A Longitudinal Analysis o f Serum Perfluorooctanesulfonate (PFOS) and Perfluorooctanoate (PFOA) Levels in Relation to Lipid and Hepatic Clinical Chemistry Test Results from Male Employee Participants of the 1994/95, 1997, and 2000 Fluorochem ical M edical Surveillance Program. 3M Final Report. O ctober 11, 2001.
OTHER
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GENETIC TOXICITY STUDIES
Title: An Assay of Cell Transformation and Cytotoxicity in C3H 10T '/2Clonal Cell Line for the Test Chemical T-2942 CoC
TEST SUBSTANCE
Identity: T-2942 CoC
Remarks: White powder dissolved in DMSO; composition and purity not indicated
METHOD
Method/Guideline followed: No guideline number specified
Test type: Cytotoxicity and cell transformation
Test system: Mouse embryo fibroblast
GLP: No
Year study performed: 1980
Species/Strain/cell-type/cell line: Cells of the C3H 10T-1/2 clone 8
Metabolic activation: None
Concentrations tested: 0.1, 1.0, 10, 50, 100, 200 i Ig/mL
Statistical methods used: None
Remarks: The test material was dissolved in DMSO and <20pi of the solution was added to the cultures growing in Eagle Basal Medium (BME). Benzo(a)pyrene and di-epoxybutane were used as positive controls and DMSO as a solvent control. Prior to the performance of the transformation assay, dose range data were obtained in the form of cytotoxicity measurements as expressed by plating (cloning) efficiency. The approximate in vitro LD50 cytotoxicity dose was chosen as the median dose for the study of the transformation potential of the test chemical. The transformation assay was performed in two phases: assessment of cell transformation in the colony mode (phase 1) and determination of foci transformation potential (phase 2). Phase 1 was performed in six replicates. The test chemical was removed 24-hours after application and the cultures were re-fed every 3 days for 14 to 17 days. Plates were washed, fixed, and stained at 14 days to score for transformation. Phase 2 was also performed in 6 replicates per dose (1.0, 10, and 100 pg/mL) with butadiene epoxide as a positive control. In phase 2, the cultures were processed at 38 days after removal of the test substance.
RESULTS Overall transformation results: Negative
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Cytotoxic concentration: approximate LD50= 50 pg/mL
Statistical results: None
CONCLUSIONS
There was no evidence of transformation observed at any of the dose levels tested in either the colony or foci assay methods.
REFERENCE
Garry, V.F., and R.L. Nelson. 1981. An Assay of Cell Transformation and Cytotoxicity in C3H 10 V2 Clonal Cell Line for the Test Chemical T-2942 CoC. 3M Company, St. Paul, MN.
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Title: Mutagenicity Test on T-6564 in an In Vivo Mouse Micronucleus Assay
TEST SUBSTANCE
Identity: T-6564
Remarks: clear, colorless liquid; composition and purity not indicated
METHOD
Method/Guideline followed: Protocol No. 455, Edition 17, modified for 3M Corporation
Test type: In vivo mouse micronucleus assay
GLP: Yes
Year study performed: 1996
Species/Strain: Mouse/ Crl:CD-l(ICR)BR
Sex: Male and female
No. animals/sex/dose: Five (micronucleus assay), Three (range-finding studies)
Vehicle (if used): Deionized water
Route of administration: Oral gavage
Doses: Dose Selection Study I: 1000, 1510, 2010, 2540, 3010 mg/kg Dose Selection Study II: 1010, 1520, 2020, 2530 mg/kg Micronucleus Assay: 498, 995, 1990 mg/kg
Frequency of treatment: Once
Statistical methods used: Analysis of variance; Dunnet's t test
Remarks: The initial body weight of the animals was 22.4 - 28.3 g for females and 31.0 - 37.7 g for males; their age was 8 weeks. Dosing was achieved using a volume of 10 mL/kg. Based on the results of the dose selection study, the maximum tolerated dose was estimated as 2000 mg/kg. A vehicle control, using deionized water, and a positive control, using cyclophosphamide at 80 mg/kg, were implemented. Animals were sacrificed at 24, 48, and 72 hours after dosing and bone marrow was extracted, spread on slides and stained with May-Grunwald solution and Giemsa prior to analysis. The slides were coded for analysis, and scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ratio. One thousand PCEs per animal were scored.
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RESULTS
Effect on mitotic index or PCE/NCE ratio by dose level and sex: Negative
Genotoxic effects (unconfirmed, dose-response, equivocal): Negative
Statistical results: Negative
Remarks: All animals in the micronucleus test group appeared normal immediately after dosing; however, one male from a secondary dose group was found dead 22 hours after dosing. All other animals appeared normal. The positive control induced significant increases in micronucleated PCEs as compared to the vehicle control. In the dose selection studies, there were mortalities at the 1510, 1520, and 300 mg/kg dose levels. Clinical signs of toxicity in the dose selection studies were hunched posture, hypoactivity, and rough hair coat.
CONCLUSIONS
The test article did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes and is considered negative in the mouse micronucleus assay.
REFERENCE
Murli, H. 1996. Mutagenicity test on T-6564 in an in vivo mouse micronucleus assay. Study number 17750-0-455. 3M Corporation, St. Paul, MN.
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Title: Mutagenicity Test on T-6564 Measuring Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells: with a Confirmatory Assay with Multiple Harvests
TEST SUBSTANCE
Identity: T-6564, also referred to as L-13167 and FC-1015-X.
Remarks: The specific gravity of the test substance was 1.22 g/mL. Substance was a clear colorless liquid.
METHOD
Method/Guideline followed: Protocol No. 437C, Edition 4 was modified for 3M Corporation
Test type: In vitro cytogenetics
Test system: Chinese hamster ovary cells in culture
GLP: Y
Year study performed: 1996
Species/Strain/cell-type/cell line: Chinese hamster ovary cells/permanent cell line supplied by Dr. S. Wolff. University of California, San Francisco. Cell line had an average cycle time of 12 to 14 hours. Modal chromosome number was 21.
Type of metabolic activation used: Aroclor 1254 induced rat liver S9 homogenate, 15.0 pL/ml, plus NADP at 1.5 mg/ml and isocitric acid at 2.7 mg/ml.
Concentrations tested: Range finding -
0.169, 0.508, 1.69,5.08, 16.9, 50.8, 169, 508, 1690, 5080 pg/ml (both with and without activation) Initial study Without activation: 62.5, 125, 250, 500, 1000, 1500, 2000 pg/ml With activation: 250, 500, 1000, 2000, 3000, and 4000 pg/ml Confirmatory study Without activation/20.1 hours: 100, 200, 400, 600, 800, 1000, 1200 jig/ml Without activation/44.2 hours: 50, 100, 200,400, 600, 800, 1000, and 1200 pg/ml With activation/20.1 + 44.2 hrs: 500, 1000, 1500, 2000, 2250, 2500, 2750, and 3000 pg/ml
Test conditions:
Number of cells: In the main studies (both with and without activation), cells were cultured for about 24 hours before treatment by seeding about 1.2 x 106cells for the 17.8 hour assay and 0.8 x 106 cells for the 44.2 hour assay. The culture medium used was McCoy's 5a culture medium supplemented with nutrients and antibiotics.
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Negative Controls: In the nonactivation assays, negative controls were cultures with cells and culture medium only. Solvent controls contained only the solvent for the test article, sterile deionized water at 10.0 pl/ml. In the activation studies, negative and solvent controls were the same as those in the nonactivation studies but also included the S9 activation mix.
Positive Controls: Mitomycin C was used for nonactivation studies. Cyclophosphamide was used in the activation studies.
Only cells with 21 + 1 centromeres were analyzed. One hundred cells from each replicate culture of treatment groups and negative and solvent control groups were analyzed. At least 25 cells from the positive control cultures were analyzed.
In the main study (after range-finding study was completed), both initial and confirmatory studies were done.
Statistical methods used: Fisher's Exact Test (Sokal and Rohlf 1981) was used to compare the percent of cells with aberrations in each treatment group with the control group. Linear trend tests of increasing numbers of cells with aberrations with increasing dose were also done. Tests were considered to be significant at a p value of < 0.01.
RESULTS
Overall results:
(1) Without activation -
Initial trial - negative (no significant increases in cells with chromosomal aberrations observed at concentrations tested.)
20.1- hour confirmatory study - negative (no significant increases in cells with chromosomal aberrations observed at concentrations tested.)
44.2- hour confirmatory study - negative (no significant increases in cells with chromosomal aberrations or polyploidy were observed at concentrations tested.)
(2) With activation -
Initial trial - negative (no significant increases in cells with chromosomal aberrations observed at concentrations tested.)
20.1 -hour confirmatory study - positive (a significant increase in cells with chromosomal aberrations was observed at 2500 pg/ml.)
44.2-hour confirmatory study - positive (a significant increase in cells with chromosomal aberrations was observed at 2750 iig/ml. Also, a significant increase in polyploidy was observed in cultures dosed with 2250, 2500, and 2750 pg/ml.)
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Genotoxic effects: Positive results for chromosomal aberrations at two concentrations (2500 and 2750 pg/ml) in the presence of activation. Positive for polyploidy and endoreduplication in the presence of activation.
Cytotoxic concentrations: In the range-finding study, complete cytotoxicity was observed in cultures
dosed with 5080 pg/ml (both with and without activation). The lowest doses at which cytotoxic effects
were seen in the main study were:
Without activation
Initial - 500 pg/ml
20.1- hr confirmatory - 600 pg/ml
44.2-
hr confirmatory - 400 pg/ml
With activation
Initial - 2000 pg/ml
20.1- hr confirmatory - 2000 pg/ml
44.2- hr confirmatory - 2250 pg/ml
Statistical results: Two concentrations in the main study (2500 and 2750 pg/ml) showed significantly greater numbers of chromosomal aberrations.
Remarks: Statistical significance was judged using a rule that was stricter than usually used to judge statistical significance (i.e. .P < 0.01 was used rather than p < 0.05.) No information was available to determine whether chromosomal aberrations were significant at p < 0.05.
In the initial trial without activation, mitotic index reductions were 25%, 30%, 87%, and 96% at doses of 125, 250, 1000, and 1500 pg/ml, respectively when compared with solvent controls. Chromosomal aberrations were analyzed from cultures dosed with 125, 250, 500, and 1000 pg/ml. In the initial trial with activation, a mitotic index reduction of 2% was seen at 250 pg/ml when compared with solvent controls. Chromosomal aberrations were analyzed from cultures dosed with 250, 500, 1000, and 2000 pg/ml.
In the 20.1-hour confirmatory trial without activation, mitotic index reductions were 73%, 84%, and 95% at doses of 800, 1000, and 1200 pg/ml, respectively when compared with solvent controls. Chromosomal aberrations were analyzed from cultures dosed with 200, 400, 600, and 800 pg/ml.
In the 44.2-hour confirmatory trial without activation, mitotic index reductions were 12%, 61%, 63%, and 92% at doses of 50, 400, 600, and 800 pg/ml when compared with solvent controls. Chromosomal aberrations were analyzed from cultures dosed with 100, 200, 400, and 600 pg/ml.
In the 20.1-hour confirmatory trial with activation, mitotic index reductions were 13%, 38%, 15%, 11%, and 30% at doses of 1000, 1500, 2000, 2250, and 2500 pg/ml, respectively when compared with solvent controls. Chromosomal aberrations were analyzed from cultures dosed with 1500, 2000, 2250, and 2500 pg/ml.
In the 44.2-hour confirmatory trial with activation, mitotic index reductions were 14%, 17%, 56%, and 53% at doses of 1000, 2250, 2500, and 2750 pg/ml when compared with solvent controls. Chromosomal aberrations were analyzed from cultures dosed with 2000, 2250, 2500, and 2750 pg/ml.
The authors describe the following deviations from the protocol - endoreduplication and polyploidy were analyzed separately because some dose levels showed increased endoreduplication and some showed increased polyploidy.
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CONCLUSIONS
The authors state that T-6564 was considered negative for inducing chromosomal aberrations in Chinese hamster ovary cells expect at a single dose level (with activation) that induced significant toxicity.
Rem arks: Significant toxicity was observed in the 20.1 and 44.2 h confirmatory trials with activation at the highest concentrations tested. These two concentrations were also positive for chromosomal aberrations. The significance of a positive response in the presence of excessive toxicity is questionable, however.
REFERENCE
Murli, H. 1996. Mutagenicity Test on T-6564 Measuring Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells: with a Confirmatory Assay with Multiple Harvests. Final Report. Corning Hazleton Inc. CHV Study No.: 17750-0-437CO. September 16.
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Title: Mutagenicity Test with T-6564 in the Salmonella - Escherichia Co/i/Mammalian-microsome Reverse Mutation Assay with a Confirmatory Assay
TEST SUBSTANCE
Identity: T-6564, also referred to as L-13167 and FC1015-X
Remarks: The substance was a clear, colorless liquid. No other information was provided on the test substance.
METHOD
Method/Guideline followed: CHV Protocol 409R, Edition 4. Experimental methods and materials were based on Ames et al. (1975) and Green and Muriel (1976).
Test type: Salmonella - Escherichia Col//Mamma 1ian-microsomc Reverse Mutation Assay with a Confirmatory Assay
Test system: Salmonella typhimurium and Escherichia coli strains
GLP: Y
Year study performed: 1996
Species/Strain/cell-type/cell line:
Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley.
Escherichia coli strain WP2wwA from National Collection of Industrial Bacteria, Torrey Research Station, Scotland.
Metabolic activation: Aroclor 1254-induced rat liver S9 homogenate, plus NADP, H20, NaH2P 0 4/Na2HP04, glucose-6-phosphate, KCl/MgCl2.
Concentrations tested: Rangefinding -
10 doses from 6.67 to 5000 pg/plate Main study -
LOO, 333, 1000, 3330, 5000 pg /plate (with and without activation)
Test conditions:
Number of cells: Density of tester strain cultures were > 0.5 x 109 bacteria per ml.
Negative (vehicle) controls: These were plated for all tester strains both in the presence and absence of S9 mix. Positive controls: These were used both in the presence and absence of the S9 mix; different chemicals were used depending on the strain being tested.
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During the range-finding study, the growth inhibitory effect (cytotoxicity) of the test article to the test system was determined using S. typhimurium strain TA100. Cytotoxicity was determined to be a decrease in the number of revertant colonies per plate or thinning of the background bacterial lawn. No cytotoxicity was observed in the range-finding study.
During the main study, both initial and confirmatory studies were conducted.
The authors' criteria for a positive test result was to produce at least a 2- or 3-fold increase (the amount depends on the strain tested) in mean revertants per plate compared with the vehicle control. The increase had to be accompanied by a dose response for increasing concentrations of the test article.
Statistical methods used: None indicated
Remarks:
RESULTS
Overall results: In the initial assay and confirmatory assay, all data were acceptable and there were no positive increases in numbers of revertants per plate both with and without activation. In the confirmatory assay, however, a non-dose responsive 5-fold increase in number of revertants was observed in S. typhimurium strain TA1537 in the absence of S9 mix. This strain was retested and all data were acceptable and no positive increases in numbers of revertants per plate were observed.
Genotoxic effects: A 5-fold increase in revertants occurred in one strain (see Overall Results), but was not confirmed upon retesting.
Cytotoxic concentration: No cytotoxicity observed in the range-finding test, and none was noted in the main study.
Statistical results: NA
Remarks:
Since the positive response seen with S. typhimurium TA1537 occurred at only one dose at the beginning of the dose response curve and was not repeatable, the test agent, T-6564 is considered negative in this assay.
CONCLUSIONS
The authors conclude that under the conditions of the study, T-6564 did not cause a positive increase in the number of revertants per plate of any tester strains, either in the absence or presence of the S9 mix.
REFERENCE
Lawlor, T.E. 1996. Mutagenicity Test with T-6564 in the Salmonella - Escherichia Co///Mammalianmicrosome Reverse Mutation Assay with a Confirmatory Assay. Coming Hazleton Inc. Final Report. CHV Study No: 17750-0-409R. September 13.
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Title: Mutagenicity Test on T-6342, Measuring Chromosomal Aberrations in Human Whole Blood Lymphocytes With a Confirmatory Assay With Multiple Harvests
TEST SUBSTANCE
Identity: T-6342; clear, colorless liquid. No other information was given on solubility or purity.
Remarks:
METHOD
Method/Guideline followed: Protocol No.: 449CO, Edition No.: 2, Modified for 3M Corporation
Test type: In vitro mutagenicity assay
Test system: Human whole blood lymphocytes
GLP: Yes
Year study performed: 1996
Species/Strain/cell-type/cell line: Human/whole blood lymphocytes
Metabolic activation: With and without metabolic activation from the S9 supernatant fraction of homogenates from livers of rats pretreated with Aroclor 1254 for the nonspecific induction of metabolizing enzymes.
Concentrations tested: Range-finding assay--0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 167, 500, 1670, 5000 pg/mL; Initial trial (without activation)-- 127, 253, 505, 1010, 1510, 2010, 2510 pg/mL; Initial trial (with activation)-- 253, 505, 1010, 1510, 2010, 2510, 3010, 4010 pg/mL; Confirmatory trial (replicate cultures, without activation, 22.1 hour assay)-- 125, 250, 500, 900, 1200, 1600, 2000 pg/mL; Confirmatory trial (replicate cultures, without activation, 46.0 hour assay)--62.5, 125, 250, 500, 900, 1200, 1600, 2000 pg/mL; Confirmatory trial (replicate cultures, with activation, 22.1 hour assay and 46.0 hour assay)-- 250, 500, 1000, 1500, 2000, 2500, 3000 pg/mL
Statistical methods used: Statistical analysis employed a Cochran-Armitage test for linear trend and Fisher's Exact Test (Thakur et al., 1985) to compare the percentage of cells with aberrations (and, if applicable, the percentage of cells with more than one aberration), polyploidy, and endoreduplication in treated cells with results from vehicle controls. Test article significance was established where p<0.01. All factors as stated previously were taken into account and the final evaluation of the test article was based upon scientific judgement.
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Remarks: Experimental Design Human venous blood from a single, normal, healthy male donor was drawn into sterile, heparinized Vacutainers. Cultures were initiated with 0.3 mL of blood/5 mL culture (dose range finding assay) or 0.6 mL of blood/10.0 mL culture (chromosomal aberrations assays) in 15 mL centrifuge tubes. The cells were incubated at approximately 37C on a slope, with loose caps, in an atmosphere of about 5% C 0 2 in air. The culture medium used was RPMI 1640 (JRH Biosciences) supplemented with 15% fetal bovine serum (FBS; Biochemed, Lot# E5331, dose range-finding assay; Lot No.: T06024, chromosomal aberrations assay), 1% phytohemagglutinin (PHA-M; Gibco), penicillin (100 units/mL; Quality Biologicals) and streptomycin (100 ug/mL; Quality Biologicals), and 2mM L-glutamine (Quality Biologicals). Deionized water (Prepared at CHV, Lot# 20) was the solvent of choice for this assay. The test article was dissolved in deionized water at a concentration of 500 mg/mL. The test article solutions and the vehicle control, deionized water, were dosed with a dosing volume of 1% (10 uL/mL) for this assay.
Negative and Solvent Controls In the nonactivation assays, negative controls were cultures that contained only cells and culture medium. Solvent controls were cultures that contained deionized water at the highest concentration used in test cultures (1% or 10.0 pL/mL). In the activation assays, the negative and solvent controls were the same as described in the nonactivation assays, but with the S9 activation mix included.
Positive Control Agents The positive control agents, which were used in the assays, were mitomycin C (MMC; CAS# 5007-7, Sigma, Lot# 40H2508) for the nonactivation series and cyclophosphamide (CP; CAS# 6055-19-2, Sigma, Lot# 43H0269) in the metabolic activation series. In the chromosomal aberrations assays, three concentrations of MMC (0.08 and 0.10 pg/mL, initial and confirmatory trials; 0.1, 0.2, and 0.3 pg/'mL, third trial) and CP (20, 30, and 50 pg/mL) were used to induce chromosomal aberrations. One of the dose levels was analyzed in each of the aberration assays. Both MMC and CP were dissolved in water.
Range-finding Assays In these assays, cultures were initiated with 0.3 mL of blood/5 mL culture and were incubated for 2 days prior to treatment.
Assay Without Metabolic Activation The lymphocytes were incubated with the test article for 19.5 hours at 37C. The test article was washed from the cells with phosphate-buffered saline and fresh complete medium containing Colcemid (final concentration = 0.1 pg/mL) was added. The cultures were harvested 2.0 hours later.
Assay With Metabolic Activation In this assay, the lymphocytes were incubated with the test article for 3 hours at =37C in the presence of a rat liver S9 reaction mixture (S9=15 pl/ml, NADP = 1.5 mg/mL, and isocitric acid = 2.7 mg/mL). The S9 fraction (Molecular Toxicology, Inc., Lot# 0667) was derived from the liver of male Sprague-Dawley rats, which had been previously treated with Aroclor 1254. In order to avoid possible inactivation of short lived and highly reactive intermediates produced by the S9 enzymes through binding to serum proteins, the medium did not have FBS during the exposure period. After the exposure period, the cells were washed twice with buffered saline.
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Complete RPMI culture medium was added to the cultures, which were incubated for 18.5 hours. Colcemid (final concentration = 0.1 pg/mL), which was added for the last 2.0 hours to collect metaphase cells.
Assay Evaluation Mitotic index was analyzed from the surviving dose levels by analyzing the number of metaphases present in 1000 consecutive cells.
Chromosomal Aberration Assay--With and Without Metabolic Activation In the chromosomal aberration assays, replicate cultures were used at each dose level and for negative controls, positive controls, and solvent controls. The aberration assays were conducted with a 22.0-hour harvest time in the initial trial and with 22.1- and 46.0-hour harvest times in the confirmatory trials. Chromosomal aberrations were analyzed from the cultures treated at 4 dose levels and from one of the positive control doses.
Aberration Assay Without Metabolic Activation Cultures were initiated 2 days prior to treatment with 0.6 mL of whole blood/10.0 mL culture in 15 mL centrifuge tubes. Two days after culture initiation, cells were treated with the test article at predetermined concentrations for approximately 19.3 and 43.3 hours. The cultures were washed with buffered saline. Complete RPMI 1640 medium, containing 0.1 pg/mL Colcemid, was placed back onto the cells. Two hours later, the cells were harvested and air-dried slides were made. The slides were stained in 5% Giemsa solution for the analysis of chromosomal aberrations. Table 1 contains a summary of the treatment schedule of the aberrations assay without metabolic activation.
Aberration Assay With Metabolic Activation Cultures were initiated 2 days prior to treatment with 0.6 mL of whole blood/10.0 mL culture in 15 mL centrifuge tubes. Two days after culture initiation, the cultures were incubated at 37C for 3 hours in the presence of the test article and the S9 reaction mixture. After the 3-hour exposure period, the cells were washed twice with buffered saline and the cells were refed with complete RPMI 1640 medium. One-tenth pg/mL Colcemid was added to the cultures during the last 2.0 hours of incubation. The metaphase cells were harvested and prepared for cytogenetic analysis.
Harvest Procedure The cell suspension was centrifuged, the supernatant was discarded, and the cells were treated with hypotonic KC1 (0.075 M) for 10 minutes. After centrifugation and removal of the KC1, the cells were washed 3 times with freshly prepared fixative (absolute methanol:glacial acetic acid, 3:1, v:v). Air-dried slides were prepared from the harvested cells.
Slide Preparation and Staining Slides were prepared by dropping the harvested cultures on clean slides. The slides were stained with 5% Giemsa solution for the analysis of mitotic index and chromosomal aberrations. All slides were air-dried and cover slipped.
Aberration Analysis and Assay Evaluation Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 46 were analyzed. One hundred cells, if possible, from each replicate culture at 4 dose levels of the test article, the negative control, the solvent control, and positive control
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cultures were analyzed for the different types of chromosomal aberrations (Evans, 1962). At least 25 cells were analyzed for chromosomal aberrations from those cultures with >25% cells with chromosomal aberrations. Cells with aberrations were recorded. Mitotic index was assessed by analyzing the number of mitotic cells/1000 cells; the ratio was expressed as a percentage of mitotic cells. Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of the test article to induce chromosomal aberrations. Percent polyploidy and endoreduplication were analyzed and the results were tabulated.
RESULTS
Overall results: positive, negative, ambiguous: Negative
Genotoxic effects (unconfirmed, dose-response, equivocal - with/without activation): Negative
Cytotoxic concentration:
Range-finding Assay Without Metabolic Activation Hemolysis was observed prior to wash in the cultures dosed with 5000 pg/mL. Reductions of 35%, 6%, 38%, 15%, 18%, 57%, and 100% were observed in the mitotic indices of the cultures dosed with 0.500, 1.67, 5.00, 16.7, 500, 1670, and 5000 pg/mL.
Range-finding Assay With Metabolic Activation Hemolysis was observed prior to wash in the cultures dosed with 5000 iig/mL. Reductions of 8%, 5%, 4%, 1%, and 100% were observed in mitotic indices of the cultures dosed with 1.67, 50.0, 500, 1670, and 5000 pg/mL, as compared with the solvent control culture.
Chromosomal Aberration Assay Without Metabolic Activation Initial Trial--Hemolysis was observed prior to wash in the cultures dosed with 2510 pg/mL. Reductions of 38%, 18%, 18%, 50%, 55%, and 95% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures treated with 127, 253, 505, 1010, 1510, and 2010 pg/mL, respectively.
Confirmatory Trial--In the 22.1-hour confirmatory trial, hemolysis was observed prior to wash of the cultures dosed with 1600 and 2000 pg/mL, and a slight evidence of hemolysis was evident at harvest of the cultures dosed with 1600 pg/mL. Reductions of 2%, 14%, 64%, 74%, 93%, and 93% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures dosed with 125, 500, 900, 1200, 1600, and 2000 pg/mL, respectively. In the 46.0-hour confirmatory trial, hemolysis was observed prior to wash of the cultures dosed with 1600 and 2000 pg/mL. Reductions of 59%, 93%, 98%, and 98% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures dosed with 900, 1200, 1600, and 2000 pg/mL, respectively.
Chromosomal Aberration Assay With Metabolic Activation Initial Trial--Hemolysis was observed prior to wash in the cultures dosed with 2010, 2510, 3010, and 4010 pg/mL. No cells were visible prior to the addition of Colcemid to the cultures dosed with 4010 pg/mL. Hemolysis was observed prior to the addition of Colcemid to the cultures dosed with 3010 pg/mL. Reductions of 15%, 20%, 15%, 43%, 77%, and 95% in the mitotic
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indices, as compared with the solvent control cultures, were observed in the cultures treated with 253, 505, 1010, 1510, 2010, and 2510 pg/mL, respectively.
Confirmatory Trial--In the 22.1-hour confirmatory trial, hemolysis was observed prior to wash and prior to harvest of the cultures dosed with 2000, 2500, and 3000 pg/mL. Reductions of 15%, 5%, 69%, 82%, 97%, and 100% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures treated with 500, 1000, 1500, 2000, 2500, and 3000 pg/mL, respectively. In the 46.0-hour confirmatory trial, hemolysis was observed prior to wash and prior to harvest of the cultures dosed with 2000, 2500, and 3000 pg/mL. Reductions of 15%, 80%, 100%, and 100% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures treated with 1500, 2000, 2500, and 3000 pg/mL, respectively.
Statistical results: The test substance did not significantly increase chromosomal aberrations with or without metabolic activation.
Remarks:
Chromosomal Aberration Assay Without Metabolic Activation Initial Trial--No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (253, 505, 1010, 1510 pg/mL).
Confirmatory Trial--In the 22.1-hour confirmatory trial, no significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (125, 250, 500, and 900 pg/mL). In the 46.0-hour confirmatory trial, no significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (125, 250, 500, and 900 pg/mL). The sensitivity of the cell culture for induction of chromosomal aberrations was shown by the increased frequency of aberrations in the cells exposed to MMC, the positive control agent.
Chromosomal Aberrations Assay With Metabolic Activation Initial Trial-- No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (505, 1010, 1510, and 2010 pg/mL).
Confirmatory Trial--In the 22.1 -hour confirmatory trial, no significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (250, 500, 1000, and 1500 pg/mL). In the 46.0-hour confirmatory trial, due to toxicity, only 52 metaphases were available for analysis in one of the cultures dosed with 2000 pg/mL. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (500, 1000, 1500, 2000 pg/mL), except for a weak increase in endoreduplication at 2000 pg/mL. The successful activation of the metabolic system was illustrated by the increased incidence of cells with chromosomal aberrations in the cultures treated with cyclophosphamide, the positive control agent.
CONCLUSIONS
The test article, T-6342, was considered negative for inducing chromosomal aberrations in cultured whole blood human lymphocytes cells with and without metabolic activation. These results were verified in independently conducted confirmatory trials.
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REFERENCE
Murli, Hemalatha. 1996. Mutagenicity Test on T-6342, Measuring Chromosomal Aberrations in Whole Blood Lymphocytes With a Confirmatory Assay With Multiple Harvests. Corning-Hazelton, Inc. (CHV). Vienna, VA. CHV Study No.: 17073-0-449CO.
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Title: Mutagenicity Test on T-6342, Measuring Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells With a Confirmatory Assay With Multiple Harvests
TEST SUBSTANCE
Identity: T-6342 C; clear, colorless liquid.
Remarks: T-6342 was soluble in sterile, deionized water >4.980 g/L (the test substance at a concentration of 4980 ug/mL in culture medium remained completely soluble at a pH of 7.5).
METHOD
Method/Guideline followed: Protocol No.: 437CO, Edition No.: 4, Modified for 3M Corporation
Test type: In vitro mutagenicity assay
Test system: Chinese hamster ovary (CHO) cells
GLP: Yes
Year study performed: 1996
Species/Strain/cell-type/cell line: Chinese hamster ovary cells (CHO-WBL) from a permanent cell line, originally obtained from the laboratory of Dr. S. Wolff, University of California, San Francisco. The cells used in this study had been recloned to maintain karyotypic stability. This cell line had an average cycle time of 12-14 hours with a modal chromosome number of 21. The CHO cells were grown in McCoy's 5a culture medium, which was supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin and streptomycin, at approximately 37C, in an atmosphere of about 5% C 0 2 in air.
Metabolic activation: With and without rat liver S-9
Concentrations tested: Range-finding assay--0.166, 0.498, 1.66, 4.98, 16.6, 49.8, 166, 498, 1660, 4980 pg/mL; Initial trial (without activation)-- 125, 250, 500, 750, 1000, 1500, 2000 pg/mL; initial trial (with activation)--250, 500, 1250, 2500, 3750, 5000 pg/mL; Confirmatory trial (without activation)-- 125, 249,498, 746, 995, 1490, 1990 pg/mL; Confirmatory trial (with activation)-- 249, 498, 1250, 2490, 3730, 4970 pg/mL; Repeat trial (without activation)--250, 500, 1250, 2490, 3740, 4980 pg/mL
Statistical methods used: The Fisher's Exact Test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) was employed to compare the percentage of cells with aberrations in each treatment group with the results from the solvent controls. A linear trend test of increasing number of cells with aberrations with increasing dose (Armitage, 1971) was also performed. Test article significance was established where p<0.01.
Remarks: Experimental Design In the chromosomal aberrations assays, replicate cultures were used at each dose level and for negative and solvent controls. Single cultures were used for each of two doses of the positive
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control. The aberrations assays were conducted with a 20.0-hour harvest time in the initial trial and with 20.0- and 44.1-hour harvest times in the confirmatory trials. Chromosomal aberrations were analyzed from the cultures treated at 4 dose levels and from only one of the positive control doses.
Negative and Solvent Controls In the nonactivation assays, negative controls were cultures that contained only cells and culture medium. Solvent controls were cultures containing the solvent for the test article at the highest concentration used in test cultures. In the activation assays, the negative and solvent controls were the same as described in the nonactivation assays, but with the S9 activation mix included.
Positive Control Agents The positive control agents, which were used in the assays, were mitomycin C (MMC; CAS# 5007-7, Sigma, Lot# 25H0619) for the nonactivation series and cyclophosphamide (CP; CAS# 6055-19-2, Sigma, Lot#67F0155) in the metabolic activation series. In the chromosomal aberrations assays, two concentrations of MMC (0.08 and 0.10 pg/mL, initial and confirmatory trials; 0.50 and 1.0 pg/mL, third trial) and CP (5.0 and 10.0 pg/mL) were used to induce chromosomal aberrations in the CHO cells. One of the dose levels was analyzed in each of the aberration assays. Both MMC and CP were dissolved in water.
Range-finding Assays In these assays, the cells were cultured for approximately (~) 24 hours prior to treatment by seeding 0.3 x 106cells/25 cm2flask into 5 mL of complete McCoy's 5a culture medium. Table 1 contains a summary of the treatment schedule of the range-finding assay. All dosing was achieved with a 1% (10 pL/mL) dosing of each stock solution and the solvent control culture was dosed with 10 pL/mL of sterile deionized water
Assay Without Metabolic Activation The cultures were incubated with the test article for 17.8 hours at 37C. The test article was washed from the cells with phosphate-buffered saline and fresh complete medium containing Colcemid (final concentration = 0.1 pg/mL) was added. The cultures were then trypsinized and harvested 2.0 hours later.
Assay With Metabolic Activation In this assay, the CHO cells were exposed to the test article for 3 hours at =37C in the presence of a rat liver S9 reaction mixture (S9=15 pl/ml, NADP = 1.5 mg/mL, and isocitric acid = 2.7 mg/mL). The S9 fraction (Molecular Toxicology, Inc., Lot# 0583) was derived from the liver of male Sprague-Dawley rats, which had been previously treated with Aroclor 1254. In order to avoid possible inactivation of short lived and highly reactive intermediates produced by the S9 enzymes through binding to serum proteins, the medium did not have FBS during the exposure period. After the exposure period, the cells were washed twice with buffered saline. Complete McCoy's 5a medium was added to the cultures, which were incubated for 16.8 hours, with Colcemid (final concentration = 0.1 pg/mL) which was added for the last 2.0 hours to collect metaphase cells. The cultures were trypsinized, harvested, fixed, and slides were prepared and stained as described for the nonactivation range-finding assay.
Assay Evaluation Mitotic index was analyzed from the highest five surviving dose levels by analyzing the number of metaphases present in 1000 consecutive cells.
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Aberration Assay Without Metabolic Activation Cultures were initiated by seeding approximately 1.2 x 106cells (20.0 hour assay) and 0.8 x 106 cells (44.0 hour assay) per 75 cm2flask into 10 mL of complete McCoy's 5a medium. One day after culture initiation, for the initial and confirmatory trials, the cells were incubated at ~37C with the test article at predetermined doses for about 17.7 (20.0 hour assay) and 41.8 (44.1 hour assay) hours. All dosing was achieved with a 1% (10 uL/rnL) dosing of each stock solution and the solvent control culture was dosed with 10 pL/mL of sterile deionized water. The cultures were washed with buffered saline. Complete McCoy's 5a medium, containing 0.1 pg/mL Colcemid, was placed back onto the cells. Approximately 2 hours later, the cells were harvested and air-dried slides were made. The slides were stained in 5% Giemsa solution for the analysis of chromosomal aberrations. For the third trial, one day after culture initiation, the cultures were incubated at 37C for 3 hours in the presence of the test article in McCoy's 5a medium without FBS. After the 3-hour exposure period, the cells were washed twice with buffered saline and the cells were refed with complete McCoy's 5a medium. One-tenth pg/mL Colcemid was added during the last 2.0 hours of incubation. The metaphase cells were harvested and prepared for cytogenetic analysis.
Aberration Assays With Metabolic Activation Cultures were initiated by seeding approximately 1.2 x 106 cells (20.0 hour assay) and 0.8 x 106 cells (44.1 hour assay) per 75 cm2 flask into 10 mL of complete McCoy's 5a medium. One day after culture initiation, the cultures that were treated under the conditions of metabolic activation were incubated at 37C for 3 hours in the presence of the test article and the S9 reaction mixture in McCoy's 5a medium without FBS. All dosing was achieved with a 1% (10 pL/mL) dosing of each stock solution and the solvent control culture was dosed with 10 pL/mL of sterile deionized water. After the 3-hour exposure period, the cells were washed twice with buffered saline and the cells were refed with complete McCoy's 5a medium. One-tenth pg/mL Colcemid was added to the culture medium during the last 2.0 hours of incubation. The metaphase cells were harvested and prepared for cytogenetic analysis.
Harvest Procedure Prior to the harvest of the cultures, visual observations of toxicity were made. These observations included an assessment of the percent confluence of the cell monolayer within the culture flasks. The cultures were also evaluated for the presence of mitotic or dead cells floating in the medium. The cultures from the dose range-finding assay were trypsinized first to collect mitotic and interphase cells and were treated with 0.075 M KC1 hypotonic solution. The cultures were fixed with an absolute methanokglacial acetic acid (3:1, v:v) fixative and washed several times before air-dried slides were prepared.
Slide Preparation and Staining Slides were prepared by dropping the harvested cultures on clean slides. The slides were stained with 5% Giemsa solution for the analysis of mitotic index and chromosomal aberrations. All slides were air-dried and cover slipped using Depex mounting medium.
Analysis of Aberrations and Assay Evaluation Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 1 (range = 20-22) were analyzed. One hundred cells, if possible, from each replicate culture at 4 dose levels of the test article and from the negative and solvent control cultures were analyzed for the different types of chromosomal aberrations (Evans, 1962). At least 189
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25 cells were analyzed for chromosomal aberrations from one of the positive control cultures. Cells with aberrations were recorded. Mitotic index was assessed by analyzing the number of mitotic cells/1000 cells; the ratio was expressed as a percentage of mitotic cells. Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of the test article to induce chromosomal aberrations. Percent polyploidy was analyzed from the 44.0-hour assay.
RESULTS
Overall results: positive, negative, ambiguous: Positive
Genotoxic effects (unconfirmed, dose-response, equivocal - with/without activation): The tested substance was positive for clastogenic activity for highly toxic treatments of short duration (3 hours) both in the presence and absence of an S9 metabolic activation system. The tested substance was also positive for polyploidy for the highly toxic treatments.
Cytotoxic concentration: This section only describes the cytoxicity observed at concentrations at which statistically significant increases in chromosomal aberrations occurred.
Chromosomal Aberrations Assay With Metabolic Activation Initial Trial--The treatment with 3750 ug/mL was only slightly toxic, but the 5000 ug/mL treatment was highly toxic, causing about 95% reduction in monolayer confluency for one culture and about 75% reduction for the replicate culture. Floating dead cells and debris were abundant, and only 94 metaphases were analyzable from the culture with greater toxicity (5000 ug/mL).
Confirmatory Trial-- For the 20 hour harvest, the confluency of the cultures treated with 4970 ug/mL was reduced by about 70% and the mitotic index was reduced by 43%. For the cultures treated with 3730 ug/mL (20 hour harvest), there was approximately a 30% reduction in confluency and an 18% reduction in mitotic index.
Aberration Assays Without Metabolic Activation; 3-Hour Treatment 20 hour harvest--A 30% reduction in confluency occurred in the cultures treated with 2490 ug/mL. The next higher dose, 3740 ug/mL, was so toxic that only a small number of metaphases could be examined. 44 hour harvest--A 55% reduction in monolayer confluency occurred in the cultures treated with 3740 ug/mL and the 2490 ug/mL treatment resulted in a 30% reduction. The mitotic index was significantly reduced (57%) for the high dose (3740 ug/mL).
Statistical results: Chromosomal Aberration Assays With Metabolic Activation Initial Trial-- Significant increases in cells with chromosomal aberrations were observed in the cultures dosed with 3750 and 5000 pg/mL. Confirmatory trial--For the 20 hour harvest, a significant increase in cells with chromosomal aberrations (simple and complex) was observed for the cultures dosed with 4970 pg/mL. Although the 4970 pg/mL treatment was too toxic to yield sufficient metaphases for analysis, it was, nevertheless, noted that the percent polyploid cells increased significantly.
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Chromosomal Aberration Assays Without Metabolic Activation; 3-Hour Treatment A significant increase in the number of cells with chromosomal aberrations was observed in the cultures dosed with 3740 pg/mL (7.5%). A significant increase in percent polyploidy was also observed in the cultures dosed with 3740 pg/mL. The mitotic index was significantly reduced (57%) for the 3740 pg/mL dose.
Remarks: Range-finding Assay Without Metabolic Activation In the culture treated with 4980 pg/mL, no visible mitotic cells were observed; the cell monolayer was <5% confluent and the culture consisted only of debris and dead cells, floating or attached. A severe reduction in the number of visible mitotic cells was observed in the culture dosed with 1660 pg/mL. The cell monolayer looked unhealthy and the degree of confluency was reduced about 15%; floating dead cells and debris were noted. Floating debris was also observed in the culture dosed with 498 pg/mL, but otherwise the culture was similar to the negative control. Mitotic indices were analyzed from the cultures dosed with 16.6, 49.8, 166, 498, and 1660 pg/mL and compared to the solvent control. At 1660 pg/mL, the mitotic index was essentially zero. However, little or no reduction in mitotic index was observed at the next lower dose of 498 pg/mL.
Range-finding Assay With Metabolic Activation An unhealthy cell monolayer was observed in the culture dosed with 4980 pg/mL. The cell monolayer confluence was reduced by approximately 85% and a severe reduction in the number of visible mitotic cells occurred (about 63%). At the next lower dose of 1660 pg/mL, the monolayer confluence was the same as the solvent control and the mitotic index was only somewhat reduced (about 27%).
Chromosomal Aberration Assays Without Metabolic Activation Initial Trial--No significant increases in cells with chromosomal aberrations were observed for the cultures analyzed (750, 1000, 1500, and 2000 pg/mL). Unhealthy cell monolayers, floating dead cells and debris, severe loss of visible mitotic cells, and about 25% reduction in cell monolayer confluence were observed in the cultures dosed with 1500 and 2000 pg/mL. Slightly unhealthy cell monolayers, floating dead cells and debris, a reduction in the number of visible mitotic cells, and about 15% reduction in cell monolayer confluence were observed in the cultures dosed with 1000 pg/mL. At the lowest analyzed dose of 750 pg/mL, the cell monolayers appeared to be almost normal, with only a slight reduction in the number of visible mitotic cells and about a 15% reduction in the degree of confluence. Severe reductions in mitotic index, relative to the solvent control cultures, occurred at all dose levels (95%, 86%, 67%, and 57% at 2000, 1500, 1000, and 750 pg/mL, respectively).
Confirmatory Trial--For the 20-hour harvest, no significant increases in cells with chromosomal aberrations were observed for any of the cultures analyzed (746, 995, 1490, and 1990 pg/mL). A 95% reduction in mitotic index was observed at 1990 pg/mL; whereas, a 25% reduction in mitotic index was observed at 746 pg/mL. The cell monolayer confluence was reduced by about 25% at 1990 pg/mL and only 83 metaphases could be scored from one of the replicate cultures. To compensate, 117 metaphases were scored from the replicate culture, in order to score a total of 200 metaphases for this dose. For the 44.1-hour harvest, chromosomal aberrations were analyzed from the cultures dosed with 249, 498, 746, and 995 pg/mL. No significant increases in cells with chromosomal aberrations were observed for any of the cultures analyzed. Percent polyploidy was
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scored at this harvest time and no significant increases were noted. The two highest applied doses of 1990 and 1490 pg/mL were lethal. Treatment with 995 pg/mL was severely toxic, resulting in about 40% reductions in monolayer confluence and 79% reduction in mitotic index. At 746 pg/mL, however, the toxicity was much less (about 15% reduction in confluency and 18% reduction in mitotic index, compared to the solvent control cultures).
Chromosomal Aberration Assays With Metabolic Activation Initial Trial--Chromosomal aberrations were analyzed from the cultures dosed with 1250, 2500, 3750, and 5000 pg/mL in the presence of the rat liver S9 metabolic activation system. Significant increases in cells with chromosomal aberrations were observed in the cultures dosed with 3750 and 5000 pg/mL. The treatment with 3750 pg/mL was slightly toxic, but the 5000 pg/mL treatment was highly toxic, causing about 95% reduction in monolayer confluency for one culture and about 75% reduction for the replicate culture. Floating dead cells and debris were abundant and only 94 metaphases were analyzable from the culture with greater toxicity. However, the mitotic index was reduced by only 13%, compared to the solvent control cultures.
Confirmatory Trial-- For the 20 hour harvest, a significant increase in cells with chromosomal aberrations (simple and complex) was observed for the cultures dosed with 4970 pg/mL. This toxic treatment reduced the confluency by about 70% and caused a 43% reduction in the mitotic index. The treatment with 3730 pg/mL caused about 30% reduction in the confluency and 18% reduction in the mitotic index. There was no clastogenic activity observed at 3730 pg/mL. For the 44.1-hour harvest, no significant increases in cells with chromosomal aberrations were observed for any of the cultures analyzed (498, 1250, 2490, 3730 pg/mL). Although the 4970 pg/mL treatment was too toxic to yield sufficient metaphases for analysis, it was, nevertheless, noted that the percent polyploid cells increased significantly. At 3730 pg/mL, there was little o r n e f no toxicity evident. The successful activation by the metabolic system was illustrated by the large increase in percent cells with chromosomal aberrations (at 20 hours harvest) in the cultures exposed to cyclophosphamide.
Chromosomal Aberration Assays Without Metabolic Activation; 3-Hour Treatment For the 20-hour harvest, no significant increases in cells with chromosomal aberrations were observed in the cultures analyzed (250, 500, 1250, 2490 pg/mL). At 2490 pg/mL, a 30% reduction in confluency was observed. However, the next highest dose, 3740 pg/mL, was so toxic that only a small number of metaphases could be examined. For the 44-hour harvest, chromosomal aberrations were analyzed from the cultures dosed with 500, 1250, 2490, and 3740 pg/mL. A significant increase in the number of cells with chromosomal aberrations was observed in the cultures dosed with 3740 pg/mL (7.5%). A significant increase in percent polyploidy was also observed in the cultures dosed with 3740 pg/mL. Relative to the solvent controls, the 3740 pg/mL treatment resulted in about 55% reduction in monolayer confluency and the 2490 pg/mL treatment resulted in about 30% reduction. The mitotic index was also significantly reduced (57%) for the high dose.
CONCLUSIONS
The test article, T-6342, was evaluated as being clastogenic to cultured CHO cells for highly toxic treatments of short duration (eg., 3 hours) both in the presence and absence of an S9 metabolic activation system. Polyploidy was also induced for the highly toxic treatments.
REFERENCE
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Murli, Hemalatha. 1996. Mutagenicity Test on T-6342, Measuring Chromosomal Aberrations in Chinese
Hamster Ovary (CHO) Cells: with a Confirmatory Assay with Multiple Harvests. Corning-Hazelton Inc (CHY). Vienna, VA. CHV Study No.: 17073-0-437CO.
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Title: Mutagenicity Test on T-6342, Measuring Chromosomal Aberrations in Human Whole Blood Lymphocytes With a Confirmatory Assay With Multiple Harvests
TEST SUBSTANCE
Identity: T-6342; clear, colorless liquid,
Remarks: No other information was given about purity or solubility
METHOD
Method/Guideline followed: Protocol No.: 449CO, Edition No.: 2, Modified for 3M Corporation
Test type: In vitro mutagenicity assay
Test system: Human whole blood lymphocytes
GLP: Yes
Year study performed: 1996
Species/Strain/cell-type/cell line: Human/whole blood lymphocytes
Metabolic activation: With and without metabolic activation from S9 supernatant fraction of livers of rats pretreated with Aroclor 1254 for the non-specific induction of metabolizing enzymes.
Concentrations tested: Range-finding assay--0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 167, 500, 1670, 5000 pg/mL; Initial trial (without activation)-- 127, 253, 505, 1010, 1510, 2010, 2510 pg/mL; Initial trial (with activation)-- 253, 505, 1010, 1510, 2010, 2510, 3010, 4010 pg/mL; Confirmatory trial (replicate cultures, without activation, 22.1 hour assay)-- 125, 250, 500, 900, 1200, 1600, 2000 pg/mL; Confirmatory trial (replicate cultures, without activation, 46.0 hour assay)--62.5, 125, 250, 500, 900, 1200, 1600, 2000 pg/mL; Confirmatory trial (replicate cultures, with activation, 22.1 hour assay and 46.0 hour assay)--250, 500, 1000, 1500, 2000, 2500, 3000 pg/mL
Statistical methods used: Statistical analysis employed a Cochran-Armitage test for linear trend and Fisher's Exact Test (Thakur et al., 1985) to compare the percentage of cells with aberrations (and, if applicable, the percentage of cells with more than one aberration), polyploidy, and endoreduplication in treated cells with results from vehicle controls. Test article significance was established where p<0.01. All factors as stated previously were taken into account and the final evaluation of the test article was based upon scientific judgement.
Remarks: Experimental Design Human venous blood from a single, normal, healthy male donor was drawn into sterile, heparinized Vacutainers. Cultures were initiated with 0.3 mL of blood/5 mL culture (dose rangefinding assay) or 0.6 mL of blood/10.0 mL culture (chromosomal aberrations assays) in 15 mL
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centrifuge tubes. The cells were incubated at approximately 37C on a slope, with loose caps, in an atmosphere of about 5% C 0 2 in air. The culture medium used was RPMI 1640 (JRH Biosciences) supplemented with 15% fetal bovine serum (FBS; Biochemed, Lot# E5331, dose range-finding assay; Lot No.: T06024, chromosomal aberrations assay), 1% phytohemagglutinin (PHA-M; Gibco), penicillin (100 units/mL; Quality Biologicals) and streptomycin (100 ug/mL; Quality Biologicals), and 2mM L-glutamine (Quality Biologicals). Deionized water (Prepared at CHV, Lot# 20) was the solvent of choice for this assay. The test article was dissolved in deionized water at a concentration of 500 mg/mL. The test article solutions and the vehicle control, deionized water, were dosed with a dosing volume of 1% (10 uL/mL) for this assay.
Negative and Solvent Controls In the nonactivation assays, negative controls were cultures that contained only cells and culture medium. Solvent controls were cultures that contained deionized water at the highest concentration used in test cultures (1% or 10.0 pL/mL). In the activation assays, the negative and solvent controls were the same as described in the nonactivation assays, but with the S9 activation mix included.
Positive Control Agents The positive control agents, which were used in the assays, were mitomycin C (MMC; CAS# 5007-7, Sigma, Lot# 40H2508) for the nonactivation series and cyclophosphamide (CP; CAS# 6055-19-2, Sigma, Lot# 43H0269) in the metabolic activation series. In the chromosomal aberrations assays, three concentrations of MMC (0.08 and 0.10 pg/mL, initial and confirmatory trials; 0.1, 0.2, and 0.3 pg/mL, third trial) and CP (20, 30, and 50 pg/mL) were used to induce chromosomal aberrations. One of the dose levels was analyzed in each of the aberration assays. Both MMC and CP were dissolved in water.
Range-finding Assays In these assays, cultures were initiated with 0.3 mL of blood/5 mL culture and were incubated for 2 days prior to treatment.
Assay Without Metabolic Activation: The lymphocytes were incubated with the test article for 19.5 hours at ~37C. The test article was washed from the cells with phosphate-buffered saline and fresh complete medium containing Colcemid (final concentration = 0.1 pg/mL) was added. The cultures were and harvested 2.0 hours later.
Assay With Metabolic Activation In this assay, the lymphocytes were incubated with the test article for 3 hours at 37C in the presence of a rat liver S9 reaction mixture (S9=15 pl/ml, NADP = 1.5 mg/mL, and isocitric acid = 2.7 mg/mL). The S9 fraction (Molecular Toxicology, Inc., Lot# 0667) was derived from the liver of male Sprague-Dawley rats, which had been previously treated with Aroclor 1254. In order to avoid possible inactivation of short lived and highly reactive intermediates produced by the S9 enzymes through binding to serum proteins, the medium did not have FBS during the exposure period. After the exposure period, the cells were washed twice with buffered saline. Complete RPMI culture medium was added to the cultures, which were incubated for 18.5 hours. Colcemid (final concentration = 0.1 pg/mL), which was added for the last 2.0 hours to collect metaphase cells.
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Assay Evaluation Mitotic index was analyzed from the surviving dose levels by analyzing the number of metaphases present in 1000 consecutive cells.
Chromosome Aberration Assay--With and Without Metabolic Activation In the chromosomal aberration assays, replicate cultures were used at each dose level and for negative controls, positive controls, and solvent controls. The aberration assays were conducted with a 22.0-hour harvest time in the initial trial and with 22.1- and 46.0-hour harvest times in the confirmatory trials. Chromosomal aberrations were analyzed from the cultures treated at 4 dose levels and from one of the positive control doses.
Chromosome Aberration Assay Without Metabolic Activation Cultures were initiated 2 days prior to treatment with 0.6 mL of whole blood/10.0 mL culture in 15 mL
centrifuge tubes. Two days after culture initiation, cells were treated with the test article at predetermined concentrations for approximately 19.3 and 43.3 hours. The cultures were washed with buffered saline. Complete RPMI 1640 medium, containing 0.1 pg/mL Colcemid, was placed back onto the cells. Two hours later, the cells were harvested and air-dried slides were made. The slides were stained in 5% Giemsa solution for the analysis of chromosomal aberrations. Table 1 contains a summary of the treatment schedule of the aberrations assay without metabolic activation.
Chromosome Aberration Assay With Metabolic Activation Cultures were initiated 2 days prior to treatment with 0.6 mL of whole blood/10.0 mL culture in 15 mL centrifuge tubes. Two days after culture initiation, the cultures were incubated at 37C for 3 hours in the presence of the test article and the S9 reaction mixture. After the 3-hour exposure period, the cells were washed twice with buffered saline and the cells were refed with complete RPMI 1640 medium. One-tenth pg/mL Colcemid was added to the cultures during the last 2.0 hours of incubation. The metaphase cells were harvested and prepared for cytogenetic analysis. Table 1 contains a summary of the treatment schedule of the aberrations assay with metabolic activation.
Harvest Procedure The cell suspension was centrifuged, the supernatant was discarded, and the cells were treated with hypotonic KC1 (0.075
M) for 10 minutes. After centrifugation and removal of the KC1, the cells were washed 3 times with freshly prepared fixative (absolute methanokglacial acetic acid, 3:1, v:v). Air-dried slides were prepared from the harvested cells.
Slide Preparation and Staining Slides were prepared by dropping the harvested cultures on clean slides. The slides were stained with 5% Giemsa solution for the analysis of mitotic index and chromosomal aberrations. All slides were air-dried and cover slipped.
Aberrations Analysis and Assay Evaluation Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 46 were analyzed. One hundred cells, if possible, from each replicate culture at 4 dose levels of the test article, the negative control, the solvent control, and positive control cultures were analyzed for the different types of chromosomal aberrations (Evans, 1962). At least 25 cells were analyzed for chromosomal aberrations from those cultures with >25% cells with chromosomal aberrations. Cells with aberrations were recorded. Mitotic index was assessed by analyzing the number of mitotic cells/1000 cells; the ratio was expressed as a percentage of mitotic cells. Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however,
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considered in the evaluation of the ability of the test article to induce chromosomal aberrations. Percent polyploidy and endoreduplication were analyzed and the results were tabulated.
RESULTS
Overall results: positive, negative, ambiguous: Negative
Genotoxic effects (unconfirmed, dose-response, equivocal - with/without activation): Negative.
Cytotoxic concentration:
Range-finding Assay Without Metabolic Activation Hemolysis was observed prior to wash in the cultures dosed with 5000 pg/mL. Reductions of 35%, 6%, 38%, 15%, 18%, 57%, and 100% were observed in the mitotic indices of the cultures dosed with 0.500, 1.67, 5.00, 16.7, 500, 1670, and 5000 pg/mL.
Range-finding Assay With Metabolic Activation Hemolysis was observed prior to wash in the cultures dosed with 5000 pg/mL. Reductions of 8%, 5%, 4%, 1%, and 100% were observed in mitotic indices of the cultures dosed with 1.67, 50.0, 500, 1670, and 5000 pg/mL, as compared with the solvent control culture.
Chromosomal Aberration Assays Without Metabolic Activation Initial Trial--Hemolysis was observed prior to wash in the cultures dosed with 2510 pg/mL. Reductions of 38%, 18%, 18%, 50%, 55%, and 95% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures treated with 127, 253, 505, 1010, 1510, and 2010 pg/mL, respectively.
Confirmatory Trial-- In the 22.1 -hour confirmatory trial, hemolysis was observed prior to wash of the cultures dosed with 1600 and 2000 pg/mL, and a slight evidence of hemolysis was evident at harvest of the cultures dosed with 1600 pg/mL. Reductions of 2%, 14%, 64%, 74%, 93%, and 93% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures dosed with 125, 500, 900, 1200, 1600, and 2000 pg/mL, respectively. In the 46.0-hour confirmatory trial, hemolysis was observed prior to wash of the cultures dosed with 1600 and 2000 pg/mL. Reductions of 59%, 93%, 98%, and 98% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures dosed with 900, 1200, 1600, and 2000 pg/mL, respectively.
Chromosomal Aberration Assays With Metabolic Activation Initial Trial--Hemolysis was observed prior to wash in the cultures dosed with 2010, 2510, 3010, and 4010 pg/mL. No cells were visible prior to the addition of Colcemid to the cultures dosed with 4010 pg/mL. Hemolysis was observed prior to the addition of Colcemid to the cultures dosed with 3010 pg/mL. Reductions of 15%, 20%, 15%, 43%, 77%, and 95% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures treated with 253, 505, 1010, 1510, 2010, and 2510 pg/mL, respectively.
Confirmatory Trial--In the 22.1-hour confirmatory trial, hemolysis was observed prior to wash and prior to harvest of the cultures dosed with 2000, 2500, and 3000 pg/mL. Reductions of 15%,
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5%, 69%, 82%, 97%, and 100% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures treated with 500, 1000, 1500, 2000, 2500, and 3000 pg/mL, respectively. In the 46.0-hour confirmatory trial, hemolysis was observed prior to wash and prior to harvest of the cultures dosed with 2000, 2500, and 3000 pg/mL, Reductions of 15%, 80%, 100%, and 100% in the mitotic indices, as compared with the solvent control cultures, were observed in the cultures treated with 1500, 2000, 2500, and 3000 pg/mL, respectively.
Statistical results: The test substance did not significantly increase chromosomal aberrations with or without metabolic activation.
Remarks:
Chromosomal Aberration Assays Without Metabolic Activation Initial Trial--No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (253, 505, 1010, 1510 pg/mL).
Confirmatory Trial-- In the 22.1-hour confirmatory trial, no significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (125, 250, 500, and 900 pg/mL). In the 46.0-hour confirmatory trial, no significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (125, 250, 500, and 900 pg/mL). The sensitivity of the cell culture for induction of chromosomal aberrations was shown by the increased frequency of aberrations in the cells exposed to MMC, the positive control agent.
Chromosomal Aberration Assays With Metabolic Activation Initial Trial--No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (505, 1010, 1510, and 2010 ug/mL).
Confirmatory Trial--In the 22.1-hour confirmatory trial, no significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (250, 500, 1000, and 1500 pg/mL). In the 46.0-hour confirmatory trial, due to toxicity, only 52 metaphases were available for analysis in one of the cultures dosed with 2000 pg/mL. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed (500, 1000, 1500, 2000 pg/mL), except for a weak increase in endoreduplication at 2000 pg/mL. The successful activation of the metabolic system was illustrated by the increased incidence of cells with chromosomal aberrations in the cultures treated with cyclophosphamide, the positive control agent.
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CONCLUSIONS
The test article, T-6342, was considered negative for inducing chromosomal aberrations in cultured whole blood human lymphocytes cells with and without metabolic activation. These results were verified in independently conducted confirmatory trials.
REFERENCE
Murli, Hemalatha. 1996. Mutagenicity Test on T-6342, Measuring Chromosomal Aberrations in Whole Blood Lymphocytes With a Confirmatory Assay With Multiple Harvests. Coming-Hazelton, Inc. (CHV). Vienna, VA. CHV Study No.: 17073-0-449CO.
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GENETIC TOXICITY (IN VIVO)
Title: Mutagenicity test on T-6342 in an in vivo mouse micronucleus assay
TEST SUBSTANCE
Identity: T-6342
Remarks: It is a clear, colorless liquid. The purity and chemical identity of the test substance were not indicated.
METHOD
Method/Guideline followed: This study was conducted using modifications of the procedures suggested by Heddle et al. (1983). Protocol No. 455, Edition 17 (no reference given), modified for 3M Corporation.
Test type: Mouse micronucleus assay
GLP: Yes
Year study performed: 1995
Species/Strain: Mouse/CrkCD-1(ICR)BR
Sex: Male and female
No. animals/sex/dose: 15 for treatment groups, 5 for control groups
Vehicle (if used): Deionized water
Route of administration: Oral gavage
Doses: 1250, 2500, and 5000 mg/kg; Doses selected on the basis of a previous study.
Frequency of treatment: Single dose
Statistical methods used: The analysis of the data was performed using ANOVA (Winer, 1971) on either untransformed (when variances were homogeneous) or rank transformed (when variances were heterogeneous) proportions of cells with micronuclei per animal. If the ANOVA was significant (P<0.05), a Dunnetf s t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were significantly different from the negative control. Analyses were performed separately for each harvest time and sex combination.
Remarks: The animals were dosed with the appropriate amount of the test substance and euthanized approximately 24, 48, or 72 hours after dosing for extraction of the bone marrow. Ten animals (5 males and 5 females) were randomly assigned to each dose/harvest time group. Ten animals (5 males and 5 females) were assigned to the control groups. The negative control animals were dosed with the vehicle, deionized water. The positive control animals were dosed with 80 mg/kg cyclophosphamide (CP). Both control groups were euthanized after 24 hours. After the bone marrow was collected, it was spread onto
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slides, dried, and stained with May-Grunwald solution and Giemsa. The slides were scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ratio. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this strain is about 0.0-0.4%.
RESULTS
Effect on mitotic index or PCE/NCE ratio by dose level and sex:
Treatment
Harvest
% Micronucleated PCEs
Ratio PCE:NCE
time Males
Females
Males
Females
Vehicle control:
24h
0.14 ! 0.02
0.02 ! 0.02
0.64 ! 0.07
0.63 ! 0.05
Positive control
24 h
5.44 ! 0.37*
2.50 ! 0.33*
0.51 ! 0.06
0.69 ! 0.11
(CP):
1250 mg/kg:
24h
0.28 ! 0.05
0.00 ! 0.00
0.69 ! 0.09
0.75 ! 0.17
48h
0.00 ! 0.00
0.02 ! 0.02
0.68 ! 0.08
0.74 ! 0.06
72h
0.06 ! 0.02
0.02 ! 0.02
0.36 ! 0.08
0.59 ! 0.06
2500 mg/kg:
24h
0.12 ! 0.04
0.04 ! 0.02
0.69 ! 0.07
0.64 ! 0.05
48h
0.02 ! 0.02
0.02 ! 0.02
0.79 ! 0.07
0.65 ! 0.01
72h
0.20 ! 0.07
0.06 ! 0.02
0.47 ! 0.07
0.64 ! 0.07
5000 mg/kg:
24h
0.06 ! 0.04
0.10 ! 0.04
0.80 ! 0.08
0.79 ! 0.09
48h
0.15 ! 0.09
0.06 ! 0.02
0.56 ! 0.10
0.63 ! 0.06
72h
0.17 ! 0.03
0.03 ! 0.03
0.17 ! 0.04* 0.24 ! 0.05*
*Significantly different from the corresponding vehicle control (p < 0.05).
Genotoxic effects (unconfirmed, dose-response, equivocal): Negative
Statistical results: No significant increases in micronucleated polychromatic erythrocytes were observed.
Remarks: Due to toxicity, the PCE/NCE ratios of the males and females from the 5000 mg/kg dose group at the 72 hour harvest group were significantly lower than the vehicle control animals.
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CONCLUSIONS
The test material, T-6342, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay.
REFERENCE
Murli, H. 1995. Mutagenicity test on T-6342 in an in vivo mouse micronucleus assay. Corning Hazleton Inc. Vienna, Virginia. 3M Corporation. St. Paul, MN.
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GENETIC TOXICITY (IN VITRO)
Title: Evaluation of the Ability of T-7524 to Induce Chromosome Aberrations in Cultured Peripheral Human Lymphocytes
TEST SUBSTANCE
Identity: T-7524
Remarks: The specific gravity of the test substance was 1.23. Substance was a brown paste of 98% purity; the remaining 2% was l-methyl-20pyrrolidinone.
METHOD
Method/Guideline followed: OECD Guideline 473; EEC Directive 67/548/EEC; OPPTS Guideline 870.5375; Japanese Ministry of Health and Welfare and Japanese Ministry of International Trade and Industry.
Test type: In vitro cytogenetics
Test system: Cultured human lymphocytes
GLP: Y
Year study performed: 2000
Species/Strain/cell-type/cell line: cells obtained from healthy adult male volunteers
Type of metabolic activation used: rat liver microsomal enzymes routinely prepared from adult male Wistar rats, obtained from Charles River, Sulzfeld, Germany. Animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the Standard Operating Procedures (SOP's). Rats were injected intraperitoneally with Aroclor 1254 induced in com oil. Livers were washed and minced and supernatant was used for activation.
Concentrations tested:
Without S9 First Experiment: 1000, 3330 and 5000 pg/ml
Second Experiment: 333, 560, 1000, 1300, 1800, 2400, and 3330 pg/ml. 333, 420, 560, 750, 1000, 1300, and 1800 pg/ml.
With S9 (1.8% v/v) Experiment 1: 1000, 3333 and 5000 pg/ Experiment 2-1000, 3333, and 5000 pg /ml
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Test conditions:
Range-Finding Test: Lymphocyte cultures (0.4 ml blood of a healthy male donor plus 5 ml or 4.8 ml culture medium, with and without S9 respectively and 0.1 ml (9 mg/ml) phytohaemagglutinin) were cultured for 48 hours and thereafter exposed to selected doses of T-7524 for 3 h, 24 h, and 48 h in the absence of S9-mix or for 3 h in the presence of S9 mix. After 3 hrs, the cells exposed to T-7524 were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 20-22 h (24 h fixation time). The cells treated for 24 hr and 48 hr in absence of S9-mix were not rinsed after treatment but were fixed immediately after 24 h and 48 h. Based on the results of dose range-finding test, an appropriate range of dose levels was chosen for the cyotgenetic assay.
Experiment 1: Lymphocytes were cultured for 48 hours and then exposed in duplicate to selected doses of T-7524 for 3 hours with and without S9-mix. After 3 hrs, the cells were rinsed with 5 ml HBSS and incubated in 5 ml culture medium for another 20-22 hr (24 h fixation time). Based on the mitotic index of the dose range finding test and the first cytogenetic assay, appropriate dose levels were selected for the second cytogenetic assay - the highest dose level tested inhibited the mitotic index by approximately 50% or more; the mitotic index of the lowest dose tested was approximately the same as the solvent control.
Experiment 2: Cells were cultured for 48 h and thereafter exposed in duplicate to selected doses of T7524 for 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. After 3 h treatment, cells in the presence of S9-mix were rinsed once with 5 ml HBSS and incubated in 5 ml culture medium for 44-46 h (48 h fixation time). The cells treated for 24 h and 48 h without S9-mix were not rinsed after treatment but were harvested immediately after 24 h and 48 h fixation time.
Chromosome preparation: During the last 3 h of culture, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 ug/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm and the supernatant was removed. Cells in the remaining cell pellet were swollen by 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37C. After hypotonic treatment, the cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).
Slide preparation: Fixed cells were dropped on cleaned slides that were immersed for 24 hr in a 1:1 mix of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. Two slides were prepared/culture. Slides were set to dry and then stained for 10-30 min with 5% (v/v) Giemsa solution in tap water. Slides were then rinsed in tapwater and set to dry. The dry slides were cleared by dipping in xylene before they were embedded in MicroMount and mounted with coverslip.
Mitotic index scoring: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least 3 analysable concentrations were used; the highest concentrations analyzed were those cultures where the mitotic index was inhibited approximately 50% or more compared to the solvent control. The mitotic index of the lowest dose level analyzed was about the same as the mitotic index of the solvent. Cultures treated with an intermediate dose were also examined for chromosome aberrations.
Counting chromosome aberrations: To prevent bias, slides were randomly coded prior to examination of chromosome aberrations and scored. An adhesive label with the NOTOX study ID number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. If the number of aberrant cells (gaps excluded) were 25 or more in 50 metaphases, no more metaphases were examined. Only metaphases with 46 chromosomes were analyzed. Numbers of cells with aberrations and numbers of aberrations were calculated.
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RESULTS
Genotoxic effects: T-7524 did not induce a statistically or biologically significant increase in numbers of cells with chromosome aberrations.
Cytotoxic concentrations: Without S-9:
3 hr treatment; 24 hr fixation: no toxic dose; highest dose tested 5000 pg/ml. 24 h treatment; 24 h fixation: 2400 and 3330 pg/ml 48 h treatment; 48 h fixation: 1300 and 1800 pg/ml
With S-9 3 hr treatment; 24 hr fixation: no toxic dose; highest dose tested 5000 pg/ml. 3 hr treatment; 48 hr fixation: no toxic dose; highest dose tested 5000 ug/ml.
CONCLUSIONS
When tested without S-9 and with 24 h continuous treatment, T-7524, induced a statistically significant increase in the number of chromosome aberrations at 1000 pg/ml when gaps were included in the total number of aberrations. However, when gaps are excluded the number of aberrations are within the historical control range of the laboratory. This increase was not seen at 1000 pg/ml with 48 hour continuous treatment. Because this increase occurred at the middle dose only, included gaps, and the chromosome aberrations seen, excluding gaps are within the historical control range of the performing laboratory and no increase occurred with 48 h continuous treatment, the increase is not considered to constitute a positive response.
Based on the above results, T-7524 is not clastogenic in human lymphocytes under the experimental conditions of the study.
Remarks: none
REFERENCE
NOTOX. 2000. Evaluation of the Ability of T-7524 to Induce Chromosome Aberrations in Cultured Peripheral Human Lymphocytes. NOTOX Project Number 292062. Hertogenbosch, The Netherlands.
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Title: CHO/HGPRT FORWARD MUTATION ASSAY - ISO (T-6889.7) Test Substance: Ammonium Perfluorooctanoate (FL-143)
Identity: CAS#: 3825-26-1; T6889.7
Remarks: The chemical was a white, crystalline, solid; Lot # 332: at pH 4 - 7; it was soluble at >lg/mL.
METHOD:
Method/Guideline followed:
Biological Evaluation of Medical Devices - Part 3: Tests
for Genotoxicity Carcinogenicity and Reproductive toxicity,
ANSO/AAMI/ISO 109933: 1993
OECD Guideline #476
Hsie, 1981
Test type: In vitro gene mutation in mammalian cells in culture
Test system: Chinese hamster ovary (CHO) cells
GLP: Y
Year study performed: 2002
Species/Strain/cell-type/cell line: CHO-K1 line of CHO cells
M etabolic activation: Aroclor 1254 induced rat liver S9 homogenate plus NADP and isocitric acid.
Concentrations tested: Test article was dissolved in culture medium at a top dose of 5 mg/mL. 1:128, 1:256, and 1:512 dilutions of the 5 mg/mL solution were tested both with and without activation.
Statistical m ethods used: Statistical program, Tallarida, R.S. and R.B. Murray's Pharmacological Calculations Procedure; ANOVA and Newman-Keuls Test for Confirmation.
Remarks: There were no significant protocol deviations. Duplicates of each concentration were tested. The negative control for both the activation and non-activation assays was culture medium; the positive control was ethylmethanesulfoxide for the non-activation assay and demethylbenzathracene for the activation assay. Cultures were exposed to chemical for 17 hours for the non-activation-assay and for 5 hours in the activation assay. After exposure, cells were washed 2X and supplemented with complete medium. Forty-eight hours and 6414 after exposure the cells from the non-activation and activation assays respectively were trypsinized, counted, and plated a 1 x 106cells per 100 mm dish. The cells were passed every 48-72 hours to maintain exponential growth during phenotypic expression for 6 days. Following phenotypic expression
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cells were grown in hypoxanthine-free Ham's F-12 medium with 10 pM 6-thioguanine, fetal bovine serum and antibiotics at a density of 2 x 105/l 00 mm Petri dish for 9 days to allow mutant colonies to develop. Cloning efficiency was determined at the same time by plating cells in selective medium without 6thioguanine. Both sets of cultures were incubated for 9 days to allow colony formation. At the end of the incubation period, plates were fixed in methanol and stained with Giemsa. Only colonies with 50 or more cells were counted.
RESULTS
O verall results: positive, negative, ambiguous: Negative
Genotoxic effects (unconfirmed, dose-response, equivocal - with/without activation): Negative both with and without activation.
Cytotoxic concentration: Both with and without metabolic activation cytotoxicity ranged from 50% cell survival at a dilution of 1:128 to 80% or greater at a dilution of 1:512. These doses were chosen for treatment and are adequate.
Statistical results: Negative
Remarks: The test article did not induce a statistically significant increase in the number of mutant colonies in treated cultures vs those in the control cultures. Positive and negative controls reacted as expected indicating that the test system was behaving properly.
CONCLUSIONS
Author's conclusions are that the test agent does not induce gene mutation in Chinese hamster ovary iCHO-Kfl cells in culture: this is correct as stated.
REFERENCE
Sadhu, D. 2002 CHO/HGPRT FORWARD MUTATION ASSAY - ISO (T6.889.7) Toxicon Corporation, Bedford, MA, Toxicon Final Report: 01-7019-G1 Submitted to: 3M, St. Paul Minnesota 55144-1000.
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ACUTE TOXICITY
Title: An Acute Inhalation Toxicity Study of T-2305 CoC in the Rat TEST SUBSTANCE
Identity: Ammonium Perfluorooctanoate; CAS#: 3825-26-1
Remarks: Fine white powder, received from 3M Company; composition and purity not indicated.
METHOD
Method/guideline followed: Guideline number not stated.
GLP: No
Year study performed: 1979
Species/Strain: Rat/Sprague-Dawley (211 to 264 grams)
Sex (Males/females/both): Both
No. of animals/sex/dose: 5/sex/group
Route of Administration: Inhalation
Remarks: The nominal test concentration was 18.6 mg/L, with a total of 17.90 grams of test material delivered in a total volume of 960 liters of dry air. The test material was sieved and packed into the cylinder of a Wright dust-feed mechanism. The flow rate of dry air was 16 liters/minute. The resulting dust-laden air was passed into the 32.3 liter glass exposure chamber housing the test animals for an exposure period of one hour. The animals were observed for abnormal signs at 15-minute intervals during the exposure, upon removal from the chamber, hourly for 4 hours after removal from test chamber, and daily thereafter for 14 days prior to sacrifice.
RESULTS
Number of deaths at each dose level (by sex): There were no deaths.
Remarks: During the exposure period, the study animals exhibited excessive lacrimation and salivation, decreased activity, labored breathing, gasping, closed eyes, mucoid nasal discharge, and irregular breathing. After removal from the exposure chamber, red nasal discharge (10/10), yellow staining of the anogenital fur (9/10), dry rales (6/10), red material around the eyes (5/10), excessive salivation (4/10), excessive lacrimation (1/10), and body tremors (1/10) were observed in the rats. Excessive salivation,
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lacrimation and body tremors had abated by the one-hour post-exposure observation and there was no red material observed around the eyes at the two hour post exposure observation. Red nasal discharge (10/10) yellow staining of the anogenital for (6/10) and dry rales (5/10) were still seen at the one hour post exposure observation. During the 14-day observation period, excessive lacrimation (6/10) and salivation (3/10), mucoid nasal discharge (10/10), dry rales (8/10), dry red material around the nose (2/10), and moist rales (1/10) were observed in the rats. Weight gains appeared normal. Necropsy findings showed lung discoloration in 8/10 rats (a higher than normal incidence).
CONCLUSIONS
It was concluded from the study results that the test substance is not fatal to rats at a nominal exposure of 18.6 mg/L for one hour. Immediate effects of treatment were excessive lacrimation, salivation, decreased activity. Labored breathing, gasping, closing of the eyes, mucoid nasal discharge, irregular breathing, yellow staining of the ano-genital for, and dry rales. The persistence of lacrimation, salivation, mucoid nasal discharge, dry rales, dry red material around the nose and moist rales along with lung discoloration seen at necropsy indicate that the material may have a prolonged or residual effect at 14 days.
REFERENCE
Bio/dynamics Inc. 1979. An Acute Inhalation Study of T-2305 CoC in the Rat. 3M Company, St. Paul, MN. Project No. 78-7184.
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ACUTE TOXICITY
Title: Acute Oral Toxicity (LD50) Study in Rats
TEST SUBSTANCE
Identity: Ammonium Perfluorooctanoate; CAS#: 3825-26-1
Remarks: White powder, 3M Lot No. 340; composition and purity not indicated
METHOD
Method/guideline followed: Guideline number not stated
GLP (Y/N): No
Year study performed: 1978
Species/Strain: Rat/Charles River CD
Sex (Males/females/both): both
Number of animals/sex/dose: 5/sex/dose
Vehicle: 40% acetone/60% corn oil
Route of Administration: Oral gavage
Remarks: The test material was administered at the following dose levels: 100, 215, 464, 1000, and 2150 mg/kg in a volume of 10 mL/kg. The rats weighed 180-218 g at study initiation. Animals were observed for mortality and pharmcotoxic signs during the first four hours after dosing, at 24 hours and daily thereafter for a total of 14 days. Body weights were recorded immediately prior to dosing and at 7 and 14 days. Animals that died on study were subjected to gross necropsy as were all survivors at the end of the 14 day observation period.
RESULTS
The following clinical signs were observed: ptosis, piloerection, hypoactivity, decreased limb tone, ataxia, corneal opacity, hypothermic to touch, and death. Signs observed at necropsy of animals which were found dead or sacrificed moribund included: 1) congestion, pitting and red foci in the lungs; 2) distended stomachs; or stomachs filled with fluid including dark red or red fluid, stomachs containing dark red particulate material; stomachs with hyperemic or thickened mucosa or erosion of the glandular mucosa; 3) intestines filled with red fluid; and 4) pale discoloration of the liver;5) red staining around the nose and mouth. Animals that survived to day 14 had thickened stomach mucosa, hyperemic glandular stomach mucosa, hydrometra of the uterus and mottled coloration of the kidneys. Signs were observed at all
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treatment levels. Treatment did not appear to have an effect upon weight of the animals that survived to terminal sacrifice.
Number of deaths at each dose level (by sex): 100 mg/kg: 1/5 males, 0/5 females 215 mg/kg: 0/5 males, 0/5 females 464 mg/kg: 2/5 males, 3/5 females 1000 mg/kg: 3/5 males, 5/5 females 2150 mg/kg: 5/5 males, 5/5 females
Remarks:
CONCLUSIONS LD50 (male rats) = 680 mg/kg, with 399 - 1157 mg/kg 95% confidence limit LDy, (female rats) = 430 mg/kg, with 295 - 626 mg/kg 95% confidence limit LD50 (combined sexes) = 540 mg/kg, with 389 - 749 mg/kg 95% confidence limit
REFERENCE Fluorad Fluorochemical FC-143 Acute Oral Toxicity (LD50) Study in Rats. 1978. Study No. 137-091, International Research and Development Corporation. Sponsored by 3M Company.
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ACUTE TOXICITY
Title: Acute Oral Toxicity Study of T-6669 in Rats (OECD Guidelines) TEST SUBSTANCE
Identity: Ammonium Perfluorooctanoate; CAS#: 3825-26-1
Remarks: Purity not stated
METHOD
Method/guideline followed: OECD Guidelines
GLP (Y/N): Y
Year study performed: 1997
Species/Strain: albino rats of Crl:CD (SD)BR strain
Sex (Males/females/both): both
Number of animals/sex/dose: 5/sex/dose
Vehicle: distilled water
Route of Administration: gavage
Remarks: Doses of 250 and 500 mg/kg were tested. All dose levels were administered as volumes of 10 ml/kg body weight. The rats weighed 208-269 g and were 8-12 weeks old. Clinical observations were conducted at 1, 2.5, and 4 hours after test material administration and each day for 14 days. Mortality checks were conducted twice a day (morning and afternoon) for 13 days and on the morning of Day 14. Body weights were determined on Days 0, 7, and 14 or at death if survival exceeded 1 day. All animals were subject of an abbreviated gross necropsy.
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RESULTS
LD50: Males: > 500 mg/kg Females: between 250 and 500 mg/kg
Number of deaths at each dose level (by sex): 250 mg/kg - none; 500 mg/kg - 2/5 males and 5/5 females.
Remarks: All animals exhibited body weight gain throughout the study. All animals treated at 250 mg/kg appeared normal during the study except for two females that exhibited red-stained faces and/or wet urogenital area within 24 hours of test material administration. Clinical signs of toxicity observed the animals treated with 500 mg/kg were: red-stained face, yellow-stained or wet urogenital area, hypoactivity, hunched posture, staggered gait, and excessive salivation. There were no test-material related lesions observed at necropsy, although at 250 mg/kg one female had large pelvises in both kidneys. At 500 mg/kg, one male had a cannibalised right flank, one female had multiple dark brown areas in the glandular mucosa of the stomach, and a second female had a clear fluid in the lumen of the bilateral horns of the uterus.
CONCLUSIONS
None were specified beyond the results.
REFERENCE
Glaza, S.M. 1997. Acute Oral Toxicity Study of T-6669 in Rats. Corning Hazleton Inc. CHW 61001760. January 10. Sponsored by 3M, St. Paul, Minnesota.
OTHER
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ACUTE - SKIN IRRITATION
Title: Primary Skin Irritation Study - Rabbits
TEST SUBSTANCE
Identity: T-1395
Remarks: no other identifying information was given.
METHOD
Note pH of test material: Not specified
Method/Guideline followed: Similar to test in Section 1500.41 - Hazardous Substances and Articles, Administration and Enforcement Regulations, Federal Register, Vol. 38, No. 187, p. 27019+, 27 September 1973.
Test Type: in vivo
Species/strain/cell type: albino rabbits
Sex (males/females/both): Not specified
Number of animals/sex/dose: 6 total
Total dose: Doses of 0.5g were placed on an intact site and an abraded site. Sites were examined while dry and after application of water at the end of the experiment.
Vehicle: Not specified
Length of time test material is in contact with animal/cell: Wrappings were removed after 24 hours but there was no indication about whether the site was washed. Readings were also made at 72 hours.
Grading scale: Scores for erythema and eschar formation as well as edema formation were presented.
Remarks: Six albino rabbits had their hair clipped from their backs and flanks, and five tenths of one gram (0.5 g) of test material was placed on abraded-dry or intact-dry prepared test sites, then covered with gauze patches and an impervious material was wrapped around the site to keep the bandage in place. After 24 hours and 72 hours the coverings were removed and the degree of erythema and edema was recorded according to a standardized scale (the Draize method).
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RESULTS
In all cases it is reported the primary skin irritation scores were 0, indicating no reddening or swelling detected. No clinical signs were reported. Primary irritation score: zero Remarks: There was no indication of reliability and no QA/QC. CONCLUSIONS Based on the results, the substance was not classified as a primary irritant. REFERENCE
Gabriel, Karl. Summary of: Primary Skin Irritation Study - Rabbits. Performed by: Biosearch. Submitted to 3M Company, 3M Center, St. Paul, MN.
OTHER
General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
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ACUTE - EYE IRRITATION Title: Primary Eye Irritation Study - Rabbits TEST SUBSTANCE Identity: T-1395
Remarks: no other identifying information was given. METHOD
Note pH of test material: Not specified
Method/Guideline followed: Similar to test in Section 1500.42 - Hazardous Substances and Articles, Administration and Enforcement Regulations, Federal Register, Vol. 38, No. 187, p. 27019+, 27 September 1973.
Test Type: in vivo Species/strain/cell type or line: adult albino rabbits
Sex (males/females/both): Not specified Number of animals/sex/dose: 6/single dose
Total dose: 0.1 gram per eye. Length of time test material is in contact with animal/cell: 5 seconds in 3 animals; 30 seconds in 3 animals Observation period: 1 hr, 24 hr, 48 hr, 72 hr, and 5 and 7 days
Remarks: The test material was washed from the eyes in 3 animals after 5 seconds with 200 cc of water and after 30 seconds in 3 animals with 200 cc of water. No other information on the methods was given. RESULTS
Corrosive: Not specified
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Irritation score:
After 5 sec: All scores for Cornea and Iris were zeros (for all rabbits). In two rabbits, scores for conjunctivae were 4, and in 1 rabbit, the score was 2, throughout the observation period.
After 30 sec: All scores for Cornea and Iris were zeros (for all rabbits). In one rabbit, scores for Conjunctivae were 4 at 1-48 hrs, and increased to 6 at 72 hrs and at 5 and 7 days. In 1 rabbit, the score was 4 throughout the observation period and in 1 rabbit the score was 6 throughout the observation period.
Tool used to assess score: Scoring was done according to the Illustrated Guide for Grading Eve Irritation By Hazardous Substances.
Description of lesions: None
Remarks: None
CONCLUSIONS
The only conclusion provided in study was that the test substance was not an ocular irritant.
Remarks: This conclusion is suspect due to the positive scores observed during the study. However, without additional information on the scoring method, there is no way to provide an informed judgment about the results.
REFERENCE
Gabriel, Karl. Summary of: Primary Eye Irritation Study - Rabbits. Performed by: Biosearch. Submitted to 3M Company, 3M Center, St. Paul, MN.
OTHER
General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
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ACUTE - EYE IRRITATION Title: Primary Eye Irritation-Rabbit TEST SUBSTANCE Identity: 3M identified the test substance as T-1395. Remarks: The test substance was not further characterized. METHOD Note pH of test material: Not specified Method/Guideline followed: The methods employed in the testing, evaluation, and grading of the test material are those described in Section 1500.42-Hazardous Substances and Articles, Administration and Enforcement Regulations, Federal Register, Vol. 38, No. 187, p. 27019, 27 Sept 1973.
Test Type: in vivo
Species/strain/cell type or line: Albino rabbit/Not specified
Sex (males/females/both): Not specified
Number of animals/sex/dose: 6/single dose
Total dose: 0.1 gram
Test conditions: The test substance was instilled into the right eyes of the test animals; the left eyes were not treated and served as controls. The test substance was not washed from the treated eyes. Length of time test material is in contact with animal/cell: The exposed eye was not washed following administration of the test substance. Observation period: 1, 24, 48, and 72 hours post-exposure; 5 and 7 days post-exposure
Scoring method used: Interpretation of the results was made in accordance with the grading system outlined in "Illustrated Guide for Grading Eye Irritation By Hazardous Substances". Remarks: Healthy young adult animals were used in this study. RESULTS Corrosive: Not specified Irritation score: No overall irritation score was given. Individually, 1 hour after application of the test substance, 6/6 animals exhibited iris irritation scores of 5 and 4/6 animals exhibited conjunctivae irritation
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scores of 10; the remaining two animals exhibited conjunctivae irritation scores of 6 and 8. 6/6 animals continued to display irritation of the iris and conjunctivae (irritation scores: 5 and 2-6, respectively) up to 48 hours after administration of the test substance. Irritation scores and incidence of irritation had decreased by day 7 of the study, at which time 2/6 animals exhibited iris irritation scores of 5 and 1/6 animals exhibited a conjunctivae irritation score of 2. No corneal irritation was observed in any of the animals throughout the study.
Tool used to assess score: Interpretation of the results was made in accordance with the grading system outlined in "Illustrated Guide for Grading Eye Irritation By Hazardous Substances".
Description of lesions: Not specified
Remarks: None
CONCLUSIONS
Remarks: Based on the results of this study and the source used for interpreting the results, the authors considered the test substance to be a primary ocular irritant; I agree with this conclusion.
REFERENCE
3M Company. 1976. Primary Eye Irritation Study-Rabbits.
OTHER
Remarks: This summary was based on a summary report; therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
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ACUTE - SKIN IRRITATION
Title: Primary Skin Irritation Test with T-3371 in Albino Rabbits
TEST SUBSTANCE
Identity: Riker Laboratories, Inc. identified the test substance as T-3371.
Remarks: The test substance was not further characterized.
METHOD
Note pH of test material: Not specified
iVlethod/Guideline followed: ICAO
Test Type: in vivo
Species/strain/cell type: Rabbit/New Zealand White Albino
Sex (males/females/both): Female
Number of animals/sex/dose: 3/exposure period
Total dose: 0.5 gram
Vehicle: None
Length of time test material is in contact with animal/cell: 3 minutes, 1 hour, or 4 hours
Test conditions: One day prior to application of the test substance, the hair was shaved from the back and flanks of each rabbit. For each rabbit, two test sites were selected. The test sites were lateral to the midline of the back and approximately 10 centimeters apart. The test substance was applied to each of the test sites and occluded with 2-inch square gauze patches. The patches were secured with gauze wrap; then, the trunk of each animal was wrapped with impervious plastic sheeting for the duration of the exposure period. After the exposure period, all wrappings were removed and the test sites were examined for irreversible damage and scored for erythema and edema.
Grading scale: The authors presented separate irritation scores for erythema and edema, each based on a grading scale of 0 - 4. Test sites were also observed for the presence or absence of chemical bum, eschar, necrosis, and epithelial sloughing.
Remarks: Animals were housed in standard wire-mesh cages in temperature and humidity controlled rooms with food and water offered ad libitum.
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RESULTS
Results: The test substance produced irreversible tissue damage following a 3-minute, 1- hour, and 4bour contact period. Moderate erythema and edema, as well as chemical bum, eschar, and necrosis were produced following all three contact periods. An endpoint was not achieved due to the extreme irritation following each contact period.
Primary irritation score: Not reported
Remarks:
CONCLUSIONS
The chemical produced irreversible tissue damage to the skin of female albino rabbits; including moderate erythema and edema, chemical bum, eschar, and necrosis. Inadequate information was presented in the report to evaluate quality of the study and validity of the conclusion.
REFERENCE
Primary Skin Irritation Test with T-3371 in Albino Rabbits. 1983. Safety Evaluation Laboratory, Riker Laboratories, Inc. St. Paul, Minnesota. Experiment #0883EB0079
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ACUTE TOXICITY Title: Acute Oral Toxicity Study-Rats TEST SUBSTANCE Identity: 3M identified the test substance as T-1585. Remarks: The test substance was not further characterized. METHOD Method/guideline followed: Not specified GLP (Y/N): No
Year study performed: 1976
Species/Strain: Rat/Sherman-Wistar, 200-300 grams
Sex (Males/females/both): Male and female Number of animals/sex/dose: 5/sex/single dose Vehicle: 50% water Route of Administration: Oral
Test conditions: Each animal was weighed and dosed by direct administration of the test substance into the stomach by means of a syringe and dosing needle. The dosage-level was 1,000 mg/kg and the test substance was dosed as a 50% w/v suspension in water. Following administration of the test substance, the animals were allowed food and water ad libitum for the 14-day observation period, during which time the rats were observed for signs of toxicity and mortalities. Remarks: The test animals were deprived of food (not water) for 24 hours prior to dosing. RESULTS LD50: <1,000 mg/kg Number of deaths at each dose level (by sex): 1,000 mg/kg-3/5 males, 4/5 females Remarks: 60% of males (3/5) and 80% of females (4/5) died following administration of the test substance; therefore, the LD50 was determined to be < 1,000 mg/kg. Signs of toxicity included an eroded
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gastric mucosa and writhing; the number of animals, which exhibited these signs, was not specified. The three surviving animals exhibited no change in initial and final body weight.
CONCLUSIONS
Remarks: The authors concluded the LD50to be < 1,000 mg/kg; I agree with this conclusion.
REFERENCE Acute Oral Toxicity in Rats-T-1585. 1976. 3M Company.
OTHER Remarks: This summary was based on a summary report submitted by 3M; therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
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ACUTE - EYE IRRITATION Title: Primary Eye Irritation Study - Rabbits TEST SUBSTANCE Identity: T-1585 Remarks: No other specifications regarding the test substance were noted. METHOD Note pH of test material: Not specified Method/Guideline followed: Section 1500.42 - Hazardous Substances and Articles, Administration and Enforcement Regulations, Federal Register, Vol. 38, No. 187, p.27019, September 27, 1973. Test Type: in vivo Species/strain/cell type or line: Rabbit/Albino Sex (males/females/both): Not specified Number of animals/sex/dose: 6/single dose Total dose: 0.1 g Length of time test material is in contact with animal/cell: 7 days Observation period: lh, 24 h, 48 h, 72 h, 5 d, and 7 d Scoring method used: Used grading system outlined in "Illustrated Guide for Grading Eye Irritation by Hazardous Substances" Remarks: The test substance (0.1 g) was administered to the right eye of each rabbit, while the untreated eye served as a control. The test material was not washed from the eyes. At each observation, the treated eyes were scored for comeal opacity and area, iris, and conjunctival redness, chemosis, and discharge. RESULTS
Corrosive: Not specified Irritation score: Corneal opacity and area = 4; Iris = 2; Conjunctival redness = 2; Conjunctival chemosis = 4; Conjunctival discharge = 3 Tool used to assess score: "Illustrated Guide for Grading Eve Irritation By Hazardous Substances." Description of lesions: None
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Remarks: The scores for all the rabbits were the same for each category and observation. The scores remained the same through day 7. Only a summary table of scores was given, with no description of the effects.
CONCLUSIONS
The test substance is a primary ocular irritant and requires cautionary labeling. Inadequate information is presented in the report to evaluate the quality of the study and the validity of the conclusions.
REFERENCE
Biosearch, Inc. 1976. Primary Eye Irritation Study - Rabbits. Philadelphia, PA. 3M Company. St. Paul, MN.
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ACUTE TOXICITY
Title: Acute Dermal Toxicity Study of T-6342 in Rabbits
TEST SUBSTANCE
identity: T-6342; a clear, colorless liquid.
Remarks: None
METHOD
Method/Guideline followed: OECD Guideline TP2071. Most likely this should be OECD Guideline 404 for Dermal Toxicity. OECD Guidelines do not begin with letters and do not go as high as 4 numbers.
GLP: Yes
Year study performed: 1995
Species/strain : Rabbits/Hra(NZW) SPF
Sex (males/females/both): Both
Number of animals/sex/dose: 5 males, 5 females/single dose
Total dose: 2000 mg/kg
Vehicle: None
Route of administration: Dermal
Remarks: The rabbits had the hair clipped from their backs before the appropriate amount of the test substance was applied to the intact skin. The area of application was covered with a gauze patch and an occlusive dressing. Collars were used to restrain the animals. After the 24 hour exposure period, the collars and dressings were removed. The test sites were washed with tap water and disposable paper towels. Clinical observations and mortality checks were made at approximately 1, 2.5, and 4 hours after test material administration and twice daily thereafter for 14 days. Body weights were determined before test material was applied, at Day 7 and on Day 14. The initial dermal irritation reading was made 30 minutes after removal of the test material and on Days 3, 7, 10, and 14. At the end of the study, the animals were euthanized and subjected to necropsy.
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RESULTS
Number of deaths at each dose level (by sex): none
Remarks: All animals appeared normal and exhibited body weight gains throughout the study, with the exception of one male that lost weight during the first week. Dermal irritation consisted of slight to moderate erythema, edema, and atonia, and slight desquamation, coriaceousness, and Assuring. No visible lesions were observed at necropsy. The dermal LD50 in rabbits was greater than 2000 mg/kg.
CONCLUSIONS
The dermal LD50 of T-6342 was greater than 2000 mg/kg in male and female rabbits. The test substance produced slight to moderate dermal irritation. No clinical signs during the study or visible lesions at necropsy were noted.
REFERENCE
Glaza, S. 1995. Acute dermal toxicity study of T-6342 in rabbits. Coming Hazelton, Inc. Madison, WI. Project ID: HWI 50800374. 3M Company. St. Paul, MN.
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SKIN SENSITIZATION DATA Title: Assessment of Contact Hypersensitivity to T-7524 in the Albino Guinea Pig (Maximisation-Test) TEST SUBSTANCE
Identity: T-7524
Remarks: Purity was 98%; the remaining 2% was I-methyl-2-pyrrolidinone. Substance was a brown paste, with a specific gravity of 1.23. METHOD
Method/guideline followed: EC - methods for the determination of toxicity, as last amended by Commission Directive 96/54/EC; OECD Guideline Number 406; Magnusson and Kligman 1970.
Study duration: about 90 days
GLP (Y/N): Y
Year study performed: 2001 Species/strain: Guinea pig; Dunkin Hartley strain Sex: Females Number of animals per dose group: 10/treatment group; 5/control group Route of administration: Intradermal and epidermal administration Range-finding study:
Concentrations: Starting and subsequent concentrations were taken from the following series: 1%, 2%, 5%, 10%, 20%, 50%, and 100%, and doses lower than 1% if needed. There was no indication about which doses were used for induction or challenge. Vehicle not indicated. Vehicle: not indicated Induction method: Intradermal injections of four concentrations were used. Dermal reactions were assessed at 24 and 48 hours.
Challenge method: epidermal application was done using 4 test substance concentrations. Dermal reactions were assessed 24 and 48 hours after exposure.
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Main study:
Induction (concentration and method):
Day 1: Three injections were made at a clipped scapular region: 1:1 w/w mixture of Freund's Complete Adjuvant with water, test substance at 5% concentration, and a 1:1 w/w mixture of 10% test substance with Freund's Adjuvant.
The vehicle was not indicated.
Day 7: Ten percent sodium-dodecyl-sulfate (SDS) was applied epidermally to a clipped area between injection sites; this provoked a mild inflammatory reaction.
Day 8: Fifty percent test substance concentration was applied epidermally to the SDS-treated area and held in place with Micropore tape. The dressing was removed after 48 hrs.
Induction vehicle: Not indicated.
Challenge (concentration and method):
Day 22: A fifty percent concentration of the test substance was applied epidermally to the clipped flank. Vehicle was not indicated.
Challenge vehicle: Not indicated.
Post-observation period:
Sites were assessed 24 and 48 hours after induction and challenge phases.
Grading scale: The source of the scale was not indicated, but both irritation and challenge reactions were graded. Results were evaluated according to EC criteria for classification and labeling requirements for dangerous substances and preparations.
Statistical methods used: None
Remarks: A reliability check of the test (study date not indicated) was done using alphahexylcinnamicaldehyde as a positive control. Skin reactions were indicative of a sensitisation rate of 100 percent for this compound.
RESULTS
Toxic response/effects by dose level: No mortality occurred and no systemic toxicity was observed. Body weights and body weight gain were similar between controls and treated groups.
Induction and challenge scores: During induction, skin effects were enhanced by the 10% SDS treatment. During challenge, no skin reactions were evident after challenge in treated groups and controls. Light yellow staining was seen at the test substance-treated skin sites (24 and 48 hours post challenge). The staining did not hamper scoring of the skin reactions.
Sensitization: A sensitization rate of 0 percent was determined. 229
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Remarks: none. CONCLUSIONS There was no evidence that T-7524 caused skin hypersensitivity in guinea pigs. Remarks: none. REFERENCE NOTOX. 2001. Assessment of Contact Hypersensitivity to T-7524 in the Albino Guinea Pig (Maximisation-Test). NOTOX Project number 292027. Hertogenbosch, The Netherlands.
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REPEAT DOSE DATA
Title: 5 Daily Dose Oral Toxicity Study with T-6669 in Rats
TEST SUBSTANCE
Identity: The test material was perfluorooctanoic acid, ammonium salt, T-6669 (FC-143), Lot No. 235., CASRN 3825-26-1.
Remarks: It is described as a white powder that is 93-97% ammonium perfluorooctanoate.
METHOD
Method/guideline followed: The study was conducted in accordance with a company protocol TP6785 dated December 20, 1996 included in the report.
Study duration: 20 days
GLP (Y/N): Y
Year study performed: 1997
Species/strain: Rat/Crl:CD(SD)BR VAF/Plus
Sex: Male
Number of animals per dose group: 10
Route of administration: Oral gavage
Doses tested and frequency: 0, 5.0, 14.0, or 42.0 mg/kg/day. Doses were administered once daily for 5 consecutive days.
Post-treatment observation period: 15 days
Statistical methods used: Methods are outlined in the report and include ANOVA (Winer, 1971), Levene's Test (Levene, 1960), and Dunnett's multiple comparison t-test (Dunnett, 1964).
Remarks: In a deviation from the protocol, the animals weighed between 303 and 395 g at study initiation; the protocol stated weights between 210 and 250 g. The animals were provided food and water ad libitum. Reverse osmosis water was used as the vehicle and the dose volume was 5 mL/kg. The animals were observed twice a day for mortality and moribundity. Each animal was removed from its cage and examined for clinical signs before dosing and at approximately 1, 2.5, and 4 hours after each dose administration and daily thereafter. Bodyweights were measured daily. Blood samples were collected on Days -2 (predose, though the protocol states that the predose sample is collected on Day -1), 6, 9, 15. and 20. On Day 6, five animals per group were sacrificed, and the liver was removed from each animal and weighed. The right lateral lobe of the liver was collected from each animal, weighed, and
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analyzed for palmitoyl CoA oxidase activity. The remaining liver tissue was also collected and weighed. On Day 20, the remaining animals were sacrificed and their livers were collected and weighed.
RESULTS
NOAEL (dose and effect): Greater than 42 mg/kg , the highest dose tested. A NOEL of < 5.0 mg/kg, the lowest dose tested based on higher hepatic palmitoyl CoA oxidase activity was determined by the study author.
LOAEL (dose and effect): Greater than 42 mg/kg , the highest dose tested. A LOEL of 5.0 mg/kg, based on higher hepatic palmitoyl CoA oxidase activity reported in study.
Toxic response/effects by dose level: All animals survived to scheduled sacrifice. No treatment-related clinical signs were observed in any groups.
5.0 mg/kg: higher hepatic palmitoyl CoA oxidase activity (24 IU/G compared to 5 IU/G for controls)
14.0 mg/kg: significantly reduced mean body weight gains during dosing period, higher hepatic palmitoyl CoA oxidase activity (39 IU/G compared to 5 IU/G for controls)
42.0 mg/kg: significantly reduced mean body weight gains during dosing period, higher hepatic palmitoyl CoA oxidase activity (39 IU/G compared to 5 IU/G for controls)
Statistical results: The lower mean body weight gains for the mid- and high-dose groups were statistically significant compared to controls (p < 0.05).
Remarks: Though the mid- and high-dose level groups exhibited lower body weight gains during the dosing period, they showed recovery usually within the first 3 days after dosing was completed. The overall mean body weight gains were not significantly different between any groups.
CONCLUSIONS
The author stated that based on significant increases in the levels of palmitoyl CoA oxidase activity, an indication of peroxisome proliferation, at all dose levels tested, the no effect level of T-6669 is less than 5.0 mg/kg when administered to male Crl:CD(SD)BR VAF/Plusrats for 5 consecutive days.
Remarks: The author designated the no effect level based on significant increases in the levels of palmitoyl CoA oxidase activity. However, enzyme induction may be transient and in the absence of other changes is not clearly indicative of a toxic effect, therefore no LOAEL was determined by this study.
REFERENCE
Henwood, S. 1997. 5 Daily Dose Oral Toxicity Study with T-6669 in Rats. Corning Hazleton, Inc., Madison, WI. Laboratory Project Identification: CHW 6329-197. 3M, St. Paul, MN.
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REPEAT DOSE DATA
Title: 28-Day Oral Toxicity Study with FC-143 in Albino Mice; IBT NO. 8532-10655
TEST SUBSTANCE
Identity: Fluorad Fluorochemical FC-143, also referred to as PFOA ammonium salt, ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143, or as a major component of FX-1003 (octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1), T-1742CoC, Lot No. 269.
Remarks: The purity of the test substance was not indicated.
METHOD
Method/guideline followed: Not stated
Study duration: 28 days
GLP (Y/N): N
Year study performed: 1977
Species/strain: Charles River CD albino mice
Sex: male and female
Number of animals per dose group: 10 (5 males and 5 females)
Route of administration: Diet - Purina rat chow
Doses tested and frequency: 0, 30, 100, 300, 1000, 3000, 10000, 30000 ppm
Post-observation period:
None
Statistical methods used: A test was used to compare mean body weights, liver weights, and liver to body weight ratios among groups, but the type of test was not noted.
Remarks: Daily observations for behavioral reactions or mortalities were made. Weekly body weight measurements were taken. Gross pathologic examination was conducted at final sacrifice on all surviving mice. It was stated that "a representative set of organs and other tissues was removed and preserved in neutral buffered formalin for future histopathologic examination." However, organs examined were not specified. Liver weights and liver to body weight ratios were calculated. Microscopic examination was conducted on livers from all sacrificed mice. Samples of livers and blood were sent to 3M for analysis, but the results, if the analyses were completed, are not included.
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RESULTS
NOAEL (dose and effect): none; effects at all doses
LOAEL (dose and effect): 30 ppm (hepatocellular hypertrophy; hepatocellular degeneration and/or necrosis; cytoplasmic vacuoles; bile duct proliferation; increased liver weight; increased liver to body weight ratios; body weight loss).
Toxic response/effects by dose level:
30 ppm: [see LOAEL] 100 ppm: increased liver weight; cyanosis; hepatocellular degeneration and/or necrosis; bile duct proliferation; hepatocellular hypertrophy; cytoplasmic lipid vacuoles; body weight loss 300 ppm: muscular weakness; roughed fur; cyanosis; hepatocellular degeneration and/or necrosis; bile duct proliferation; body weight loss 1000 ppm: muscular weakness; roughed fur; cyanosis; hepatocellular degeneration and/or necrosis; hepatocellular hypertrophy; cytoplasmic vacuoles (before death by day 9) 3000 ppm: muscular weakness; roughed fur (before death during week 1) 10000 ppm: muscular weakness; roughed fur (before death during week 1) 30000 ppm: muscular weakness; roughed fur (before death during week 1)
Statistical results:
The following parameters were significantly different from controls (at a level of at least p < 0.05): (A) Body weight loss:
30 ppm: females at week 4 100 ppm: males at weeks 1-4; females at weeks 2-4 300 ppm: males at weeks 1-4; females at weeks 1-3 1000 ppm: males and females at week 1 (animals died after week l)
(B) Absolute liver weight increase: 30 ppm: males and females 100 ppm: males and females
Not reported at other doses because mice died before day 28.
(C) Relative liver weight increase: 30 ppm: females 100 ppm: males
Not reported at other doses because mice died before day 28.
Remarks: Body weight was suppressed in a dose-related manner; increased amounts of food were consumed although food waste was also seen. 1000 ppm and higher: all males and females died within the first 9 days of testing. 300 ppm: all mice except 1 male died within 26 days of testing. 30 and 100 ppm test groups: one animal died in each group. No other deaths occurred. Gross pathology showed enlargement and/or discoloration of 1 or more liver lobules in all animals sacrificed after 28 days.
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CONCLUSIONS
No conclusions were given by the study authors.
Remarks: All mice fed APFO lost weight. Reductions in body weight gain were followed by weight losses in mice fed 30, 100, or 300 ppm. A dose-related pattern was seen in the depressed body weights. Relative and absolute liver weights were increased in mice fed 30 ppm or more APFO. Gross pathological examination of kidneys or other organs besides livers is not discussed. Treatment-related changes were observed in the livers among all APFO treated animals including enlargement and/or discoloration of 1 or more liver lobes. Histopathologic examination of all APFO treated mice revealed diffuse cytoplasmic enlargement of hepatocytes throughout the liver (panlobular hypertrophy) accompanied by focal to multifocal cytoplasmic vacuoles. Degeneration and /or necrosis of hepatocytes and focal bile duct proliferation was also noted in mice within all groups.
REFERENCE
Christopher, B. and Marias, A.J. 1977. 28-Day Oral Toxicity Study with FC-143 in Albino Mice, Final Report, Industrial Bio-Test Laboratories, Inc. Study No. 8532-10655, 3M Reference No. T-1742CoC, Lot 269.
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REPEAT DOSE DATA
Title: 28-Day Oral Toxicity Study with FC-143 in Albino Rats
TEST SUBSTANCE
Identity: Fluorad Fluorochemical FC-143, also referred to as PFOA ammonium salt, ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143, or as a major component of FX-1003 (octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1), T-1742CoC, Lot No. 269.
Remarks: Purity of the test substance was not indicated
METHOD
Method/guideline followed: Not stated
Study duration: 28 days
GLP (Y/N): N
Year study performed: 1977
Species/strain: Charles River CD albino rats
Sex: male and female
Number of animals per dose group: 10 (5 males and 5 females)
Route of administration: Diet - Purina rat chow
Doses tested and frequency: 0, 30, 100, 300, 1000, 3000, 10000, 30000 ppm
Post-observation period: None
Statistical methods used: A test was used to compare mean body weights, liver weights, and liver to body weight ratios among groups, but the type of test was not specified.
Remarks: Animals were observed daily for clinical signs of toxicity. Body weight and food consumption were determined weekly. Liver weights, body weights, and liver weight to body weight ratios were determined at terminal sacrifice. Gross pathological examination was conducted on all surviving rats. It is stated that the study included a complete examination of gross pathology and Aa complete set@ of tissues and organs were examined, but the specific list is not supplied. Livers were weighed to determine relative organ weight then stained for histopathologic examination. Samples of livers and blood were sent to 3M for analysis, but the results, if the analyses were completed, are not included.
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RESULTS
NOAEL (dose and effect): none; effects observed at lowest test dose
LOAEL (dose and effect): 30 ppm (increased liver weight and hepatocyte hypertrophy)
Toxic response/effects by dose level: All animals given 10000 or 30000 ppm died within the first week of testing. There were no premature deaths or other clinical signs of toxicity in the other groups. Males at 300 ppm showed slightly reduced body weight gains. Body weight gains among animals fed 1000 ppm or more were significantly reduced in a dose-dependent manner. Reduced food intake was evident among rats fed either 1000 or 3000 ppm. Treatment-related morphologic changes in the liver were observed among all male and female test animals. The primary lesion consisted of focal to multifocal cytoplasmic enlargement (hypertrophy) of hepatocytes among animals in test groups 0, 30, and 100 ppm and multifocal to diffuse enlargement of hepatocytes was seen among animals in test groups 300, 1000, and 3000 ppm
Statistical results: Differences in body weight between the control and treatment groups were statistically significant at 1000 and 3000 ppm. Absolute liver weights were significantly increased for males at 300 ppm and in females at 1000 and 3000 ppm. Relative liver weight was significantly increased for males at 3000 ppm.
Rem arks: Gross pathological exam did not reveal treatment-related effects in kidneys or other organs besides livers. The main effect was focal to multifocal cytoplasmic enlargement of hepatocytes among animals fed 300 ppm. In animals fed 1000 ppm or more the effect was multifocal to diffuse enlargement of hepatocytes. The severity/degree of tissue involvement of these lesions was more pronounced in male test animals. Absolute liver weights in males at 1000 and 3000 ppm are not significantly different than controls only because of body weight reduction in the higher dose animals.
CONCLUSIONS
The authors gave no conclusion.
Rem arks: All animals in the 10,000 and 30,000 ppm groups died before the end of the first week. Body weight gains were reduced in the groups receiving 1000 or more ppm. Reduced food intake was observed in rats fed 1000 ppm or higher in a dose-related manner.
Relative liver weights were increased in males fed 30 ppm or more and females fed 300 ppm or more. Treatment-related changes were observed in the livers among all APFO treated animals. The severity of effects was more pronounced in male test animals
REFERENCE Metrick, M. and Marias, A.J. 1977. 28-Day Oral Toxicity Study with FC-143 in Albino Rats, Final Report, Industrial Bio-Test Laboratories, Inc. Study No. 8532-10654, 3M Reference No. T-1742CoC, Lot 269, September 29, 1977.
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REPEAT DOSE DATA
Title: Ninety-Day Subacute Rhesus Monkey Toxicity Study
TEST SUBSTANCE
Identity: Fluorad Fluorochemical FC-143, also referred to as PFOA ammonium salt, ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143, or as a major component of FX-1003 (octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1)
Remarks: The test substance used in this study was obtained from the sponsor, 3M. Company, St. Paul, MN. The lot number of FC-143 used in this study was 340. The test substance was a white powder.
METHOD
Method/guideline followed: Not stated
Study duration: 90-day
GLP (Y/N): No
Year study performed: 1978
Species/strain: Rhesus monkey
Sex: Male and female
Number of animals per dose group: Two per sex per group
Route of administration: Oral gavage
Doses tested and frequency: 0 (control group), 3, 10, 30, and 100 mg/kg/day, 7 days/week, for 90 days
Post-observation period: None
Statistical methods used: All statistical analyses compared the treatment groups with the control group, by sex. The tests were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie (1960) using Dunnetf s multiple comparison tables to judge significance of differences.
Remarks: Ten male rhesus monkeys (2.60 - 3.90 kg) and 10 female rhesus monkeys (2.95 - 3.80 kg) were used in this study. The monkeys were housed individually in hanging wire mesh, "squeeze type" cages and maintained in a temperature, humidity, and light controlled environment. Purina Monkey Chow was fed twice/day and fresh apples were fed 3 times/week. Water was available ad libitum. Prior to test substance administration, the staff veterinarian conducted a complete physical examination. Only monkeys in good health were selected for the study. The test compound, suspended in 0.5% Methocel, was administered by gavage at a frequency of 7 days/week for 90 days. All doses were given in a constant volume. The same volume of 0.5% Methocel was given to the vehicle control group. Individual daily doses were based upon the body weights obtained weekly. The monkeys were observed twice daily for
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general physical appearance, behavior, and pharmacotoxic signs. General physical examinations were conducted in the control period and monthly during the study. Blood and urine samples were obtained for analysis from all monkeys once during the control period, then again 1 and 3 months after study initiation. After completion of the test substance administration period, all surviving monkeys were anesthetized with Sernylan (Phencyclidine HCl), exsanguinated, and necropsied. At necropsy, the heart, liver, adrenals, spleen, pituitary, kidneys, testes/ovaries, and brain were weighed; representative tissues were collected in buffered neutral 10% formalin. Eyes were fixed in Russell's fixative. The thyroid/parathyroid was weighed after fixation. Histopathology was performed on the following organs from all monkeys in the control and treatment groups: adrenals, aorta, bone, brain, esophagus, eyes, gallbladder, heart (with coronary vessels), duodenum, ileum, jejunum, cecum, colon, rectum, kidneys, liver, lung, skin, mesenteric lymph node, retropharyngeal lymph node, mammary gland, nerve (with muscle), spleen, pancreas, prostate/uterus, rib junction (bone marrow), salivary gland, lumbar spinal cord, pituitary, stomach, testes/ovaries, thyroid, parathyroid, thymus, trachea, tonsil, tongue, urinary bladder, vagina, identifying tattoo, and any tissues(s) with lesions. Histopathology was performed on the following organs from all monkeys in the control and treatment groups: adrenals, aorta, bone, brain, esophagus, eyes, gallbladder, heart (with coronary vessels), duodenum, ileum, jejunum, cecum, colon, rectum, kidneys, liver, lung, skin, mesenteric lymph node, retropharyngeal lymph node, mammary gland, nerve (with muscle), spleen, pancreas, prostate/uterus, rib junction (bone marrow), salivary gland, lumbar spinal cord, pituitary, stomach, testes/ovaries, thyroid, parathyroid, thymus, trachea, tonsil, tongue, urinary bladder, vagina, identifying tattoo, and any tissues(s) with lesions.
RESULTS
NOAEL (dose and effect): 0 (control) and 3 mg/kg/day-- Soft stool, moderate to marked diarrhea, and frothy emesis were occasionally observed at both of these dosage levels.
LOAEL (dose and effect): 30 mg/kg/day--Clinical signs of toxicity, adverse histologic changes in the adrenals, bone marrow, spleen, and lymph nodes, and deaths.
Toxic response/effects by dose level: There was no mortality at the 0, 3, and 10 mg/kg/day dosage levels. Three monkeys in the 30 mg/kg/day dosage group, and all 4 monkeys in the 100 mg/kg/day dosage group, died during this study.
3 mg/kg/day-- Soft stool and/or moderate to marked diarrhea, frothy emesis were noted at this dosage level.
10 mg/kg/day--The following symptoms were noted in one monkey: anorexia during week 4, pale and swollen face during week 7, and black stools during week 12.
30 mg/kg/day--Three monkeys died during weeks 7, 12, and 13 of the study. From week 4, these 3 monkeys were anorexic. All 4 monkeys in this group showed slight to moderate, and sometimes severe, decreased activity. In addition, swollen face, eyes, and vulva, as well as pallor of the face and gums were noted in all 4 monkeys. Other symptoms observed in some/all monkeys included the following: emesis, ataxia, black stools, dehydration, ptosis of the eyelids, and loss of body weight. The surviving male had decreased numbers of erythrocytes, decreased hemoglobin, decreased hematocrit, and increased platelets. Prothrombin time and activated prothrombin time were also increased. These increases were apparent at 1 month but were much more marked at three months. There was a decrease in alkaline phosphatase levels at one month (365 vs 597 in the control) which persisted in the one surviving male (360 vs 851 IU/1 in the control) at 3 months. SGOT levels were reduced at one
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month (59 vs 29 IU/1 in the control) and in the one surviving male at 3 months (88 vs 45 IU/1 in the control). SGPT was elevated at 1 month (44 vs a control value of 15 IU/1) and at 3 months (46 vs 31 IU/1 in the control group). Cholesterol in the one surviving male was elevated (240 vs 165 mg/100 ml). Total protein and albumin in this animal were reduced; total protein was 5.52 vs a control level of 8.21 g/100 ml and total albumin was 2.00 vs a control level of 4.82 g/100 ml. During the pathological studies, the following compound-related symptoms were observed in male and female monkeys: marked lipid depletion of the adrenals, slight to moderate hypocellularity of the bone marrow, and moderate atrophy of lymphoid follicles of the spleen and lymph nodes.
100 mg/kg/day-- All monkeys from the 100 mg/kg/day dosage level died during weeks 2 - 5 of the study. In addition, the monkeys in this group showed the same signs of toxicity as the monkeys dosed 30 mg/kg/day. However, the signs of toxicity observed in the 100 mg/kg/day dosage group (anorexia, frothy emesis, pale face, pale gums, swollen face and eyes, decreased activity, prostration, body weight loss, and body trembling) appeared earlier in the study than was observed in the 30 mg/kg/day dosage group. During the pathological studies, the following compound related symptoms were observed in male and female monkeys: marked diffuse lipid depletion of the adrenals, slight to moderate hypocellularity of the bone marrow, and moderate atrophy of lymphoid follicles of the spleen and lymph nodes.
Statistical results: There were statistically significant decreases in body weight for the male monkeys at the 30 mg/kg/day dosage level during week 13 of the study. The female monkeys of the 30 mg/kg/day dosage level and the monkeys of the 100 mg/kg/day dosage level were already dead at this time. Statistically significant variations in sex-group mean weights of a few organs occurred between the control and experimental groups. The statistically significant variations were the following: decreased absolute and relative heart weight for females in the 10 mg/kg/day dosage group (p < 0.05, p < 0.01, respectively), decreased absolute brain weight for females in the 10 mg/kg/day dosage group (p < 0.01), and increased relative pituitary weight for males in the 3 mg/kg/day dosage group (p < 0.05). These variations were of unknown biological significance and were not accompanied by morphologic alterations.
Remarks: Following one month of treatment, glucose was significantly elevated in the 3 mg/kg/day group (117 vs 89 mg/100 ml in the control). The authors of the report attribute this to a single high value for male #7366 who had a value of 131. The other three monkeys in the 3 mg/kg/day group had levels of 112, 105, and 120 mg/100 ml. Glucose levels in the 10 and 30 mg/kg/day groups were 104 and 122 mg/100 ml, respectively, after one month of treatment. At three months of treatment, glucose levels were 81, 96, 88, and 66 mg/100 ml in the control, 3, 10 and 30 mg/kg/day groups respectively. SGPT was elevated in the 10 and 30 mg/kg/day dose groups at 1 month, but were comparable to control at 3 months. There were no treatment related changes in urinalysis studies at any time period studied.
CONCLUSIONS
The test substance caused clinical signs of toxicity, adverse histologic changes in the adrenals, bone marrow, spleen, and lymph nodes, and deaths in male and female rhesus monkeys when administered at dosage levels of 30 and 100 mg/kg/day.
Remarks: The test substance was not sufficiently characterized.
REFERENCE
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3M Company, International Research and Development Corporation. 1978. Ninety-Day Subacute Rhesus Monkey Toxicity Study. Laboratory study number 137-090.
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Title: 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys.
TEST SUBSTANCE
Identity: Ammonium Perfluoroctanoate (APFO); Perfluorooctanoate (PFOA)
Remarks: APFO a White Powder; Lot 332; 95.2% pure
METHOD
Method/guideline followed: No guideline followed.
Study duration: 40 weeks, 26-week treatment period, followed by 13 weeks recovery.
GLP (Y/N): Y
Year study performed: 1998/1999
Species/strain: Cynomolgus monkeys
Sex: Males
Number of animals per dose group: 6 animals/group for groups 1, 3, and 4; 4 animals/group for group 2.
Route of administration: Oral capsule
Doses tested and frequency of administration: 0 mg/kg/day, 3 mg/kg/day, 10 mg/kg/ or 30/20 mg/kg/day for 26 weeks
Post-observation period: 13 weeks
Statistical methods used: Levene's test for variance homogeneity: ANOVA, Dunnett's t-test ANCOVA, covariate-adjusted means, 5% two-tailed probability level.
Remarks: Dosing of animals in the 30 mg/kg/day dose group was stopped from days 11-21 because of toxicity. When dosing was resumed on Day 22, animals received 20 mg/kg/day and this group was designated the 30/20 mg/kg/day.
Animals were observed twice daily for mortality and moribundity and were examined at least once daily for signs of poor health or abnormal behavior; food consumption was assessed qualitatively. Ophthalmic examinations were done before initiation of treatment and during weeks 26 and 40. Body weight data were recorded weekly before the start of treatment, on Day 1 of treatment and weekly thereafter. Blood and urine samples were collected for clinical hematology, clinical chemistry, and urinalysis before the start of treatment and at specified intervals during treatment and recovery. Blood samples were also taken for hormone determinations. The following organs were weighed at scheduled and unscheduled sacrifices; paired organs were weighed separately: adrenal (2), brain, epididymis (2), kidney (2), liver, pancreas, testis (2), and thyroid (2) with parathyroid. Organ to body weight percentages and organ to brain weight ratios were calculated.
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The following tissues were collected for histopathology: adrenal (2), aorta, brain, cecum, colon, duodenum, epididymis (2), esophagus, eyes [preserved in Davidson's fixative (2)], femur with bone marrow (articular surface of the distal end), gallbladder, heart, ileum, jejunum, kidneys (2), lesions, liver, lung, mesenteric lymph node, mammary gland, pancreas, pituitary, prostate, rectum, salivary gland [mandibular (2)], sciatic nerve, seminal vesicle (2), skeletal muscle (thigh), skin, spinal cord (cervical, thoracic, and lumbar), spleen, sternum with bone marrow, stomach, testis [(2) preserved in Bouin's solution, thymus, thyroid (2) with parathyroid, trachea and urinary bladder.
Blood, urine and feces were collected during Week 2 and every 2 weeks thereafter during treatment and recovery for PFOA concentration analyses. In addition, blood was collected from the animal in Group 4 that was sacrificed in moribund condition. Animals were not fasted before collections. Samples were collected without anticoagulant, maintained at room temperature, and allowed to clot. Samples were centrifuged within an hour of collection, serum was harvested and stored at 10C to -30C until packed on dry icc and shipped to 3M for analysis.
Whole blood was collected from the vena cava of each animal at both scheduled and unscheduled sacrifice, divided into approximately equal sized samples of serum (collected without anticoagulant), whole blood and plasma using potassium EDTA as the anticoagulant. Samples were stored a t -10 to -30 C. Whole blood was collected from the animals in the control group at terminal sacrifice using sodium heparin as an anticoagulant. One-half was transferred into cryotubes and pooled. The remaining one-half was centrifuged and separated into plasma and red blood cells. The plasma and red blood cells were pooled and transferred into cryotubes. All samples were stored frozen at -60C to -80C until shipped frozen on dry ice to 3M for possible future analysis.
At least 2mL of urine and at least 5 g of feces were collected at the same time as blood for serum collection for PFOA determinations. Animals were not fasted before collections. Urine was collected on wet ice; both urine and feces were collected overnight; stored at -10C to -30C until shipped on dry ice to 3M for analysis.
A non-formalin liver sample was collected from each animal at both scheduled and unscheduled sacrifice. The sample was weighed, flash-frozen in liquid nitrogen, stored at -60C to -80C and shipped frozen on dry icc to 3M for analysis.
The right lateral lobe of the liver was collected from each animal at necropsy for palmitoyl CoA oxidase activity analyses. Representative samples of liver, right and left testes, and pancreas were collected from each animal for cell proliferation evaluation using proliferation cell nuclear antigen (PCNA).
All available bile (up to 5 mL) was collected at both scheduled and unscheduled sacrifices, flash-frozen in liquid nitrogen and stored at -60C to 80C until shipped to the University of Dundee for bile acid determination.
The decision to analyze blood for testosterone was made after terminal sacrifice. Aliquots of plasma from the prestudy time period, collection Days 35, 66, 94, and 183 and recovery Days 220, 248 and 276 were sent to Ani Lytics Inc. where they were analyzed for testosterone.
Results: NOAEL (dose and effect level): 10 mg/kg/day, death; hypoactivity; weight loss; few or no feces; low or no food consumption, mildly increased triglycerides; and moderate to marked increases in serum enzyme concentrations (i.e. aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, and creatine kinase) and a mildly increased serum bile acid concentration.
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LOAEL (dose and effect level): 30 mg/kg/day, death; hypoactivity; weight loss; few or no feces; low or no food consumption; mildly increased triglycerides; moderate to marked increases in serum enzyme concentrations (i.e. aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, and creatine kinase) and a mildly increased serum bile acid concentration.
Toxic response by dose level: 30/20 mg/kg/day: death; hypoactivity, weight loss, few or no feces, low or no food consumption; mildly increased triglycerides; and mildly to moderately decreased absolute neutrophil count, total protein concentration and albumin concentration. Two animals in this dose group, one tenninated in moribund condition and one for whom treatment was stopped because of toxicity had mildly increased serum bile acid concentrations.
Statistical results: Statistically significant lower body weight during Week 2 for Group 4 animals; mean body weight changes for Group 4 were significantly lower than controls during Weeks 7, 9 and 24. Statistically significant increases in mean absolute liver weight and mean liver-to-body weight percentages in animals from all dose groups at terminal sacrifice. Mean liver to brain weight was significantly increased in animals in the 10 mg/kg/day dose group. No effects were noted at recovery sacrifice.
Remarks:
Two animals, one male from the 30/20 mg/kg/day dose group and one male from the 3 mg/kg/day dose group were sacrificed in moribund condition during the study. The male in the 30/20 mg/kg/day dose group was sacrificed on Day 29. This animal exhibited signs of hypoactivity, weight loss, few or no feces, low or no food consumption and the entire body was cold to the touch before death. The animal from the 3 mg/kg/day dose group was sacrificed on Day 137. This animal showed clinical signs of limited use and paralysis of the hind limbs, ataxia and hypoactive behavior, few feces and no food consumption.
Males given 30 mg/kg/day from Days 1-11 had clinical signs of few feces and low food consumption and they lost weight during week 1 of treatment. Based on these signs, treatment was stopped on Day 11 and was not resumed until Day 22. When treatment was resumed, the dose was lowered to 20 mg/kg/day; this group was then designated the 30/20 mg/kg/day dose group. Of the remaining animals in this group, only 2 tolerated this dose level for the remaining 23 weeks of treatment. Treatment of three males given 30/20 mg/kg/day was halted on Days 43 (Week 7), 66 (Week 10), and 81 (Week 12) respectively. Clinical signs in these animals included thin appearance, few or no feces, low or no food consumption, and weight loss. The animals appeared to recover from compound-related effects within 3 weeks after cessation of treatment.
Mean body weight changes were notably lower during Weeks 1 and 2 for males receiving 30 mg/kg/day. During week 2, this change was statistically significant. Treatment was stopped on Day 11 and when it was resumed at 20 mg/kg/day on Day 21, mean body weight changes were significantly lower than controls during Weeks 7, 9 and 24. Overall mean body weight changes through Week 27 were notably lower for the males in the 30/20 mg/kg/day dose group.
There was an increased incidence of low or no food consumption for animals in the 30/20 mg/kg/day dose group that was considered to be treatment related.
There were no consistent or clearly dose-related effects on estrone, estradiol, estriol, thyroid stimulating hormone, or testosterone that were seen in any treatment group over time. In general, thyroid hormones
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were decreased beginning on Day 35 in animals in the 10 or 30/20 mg/kg/day dose groups. Triiodothyronine remained depressed through Day 183 in the 30/20 mg/kg dose group. Total thyroxin was decreased beginning on Day 35 in animals administered 10 or 30/20 mg/kg APFO per day. The effect on thyroxin was most pronounced at Day 35 in animals administered 10 mg/kg/day and Day 66 in animals administered 30/20 mg/kg/day. Thereafter, the effect began to diminish and recovery was observed either in the last 3 months of dosing or during the recovery phase of the experiment. No changes in chloecstokinin concentrations were seen over time in either dose group.
Two males from the 30/20 mg/kg/day dose group, the one sacrificed on Day 29 and one for whom treatment was stopped because of poor health, had moderate to marked increased serum enzyme concentrations (i.e. aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, and creatine kinase) and a mildly increased serum bile acid concentration.
At 3 mg/kg/day or 10 mg/kg/day, APFO had no effects on hematology, coagulation, clinical chemistry, or urinalysis. At 30/20 mg/kg/day there were slight increases in triglyceride concentrations and mild to moderate decreases in absolute neutrophil count, total protein concentration and albumin concentration. Two animals in the 30/20 mg/kg/day dose group had markedly increased serum enzyme activities and mildly increased serum bile acid concentrations.
There was no evidence of persistent or delayed toxic effects on clinical pathology test results during the recovery period.
Enhanced cell proliferation was equivocal in the livers of treated monkeys; it was not seen in the pancreas or testes of treated animals.
There were no treatment-related macroscopic or microscopic changes in any organs at the terminal sacrifice, including liver, adrenal, spleen, pancreas, and testis. There were no treatment-related effects on terminal body weights or on absolute or relative organ weights at recovery sacrifice indicating that the liver weight changes seen at terminal sacrifice were reversible over time. There no treatment-related macroscopic or microscopic findings at recovery sacrifice.
Of the two males sacrificed in a moribund condition, the male from the 30/20 mg/kg/day dose group had esophageal and gastric lesions that were indicative of an injury that occurred during dosing and liver lesions that were presumed to be due to the test material. The cause of death in the male from the 3 mg/kg/day dose group was not determined.
Nine hundred forty seven liver, sera, urine and feces samples were collected from monkeys in treatment groups 1, 2, 3 and 4 and sent to 3M for analysis during the treatment phase of the study. Of the 947 samples, 202 were serum, 19 were liver, 272 were urine and 272 were feces specimens. During the recovery phase of the study, a total of 96 samples from Groups 1 and 3 were sent to 3 M for analysis. Of these, 32 were serum specimens, 4 were liver, 28 were urine and 28 were feces specimens. Serum, liver and urine samples were analyzed at 3M; fecal samples were shipped to Centre Analytical Laboratories for analysis. Samples were analyzed for PFOA.
Sera, liver, and urine were extracted using an ion-pairing reagent and methyl-tert-butyl ether (MtBE) or ethyl acetate. Extracts were analyzed using high-performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ES/MS/MS) in the multiple response monitoring mode. PFOA levels in sera and liver were quantitated by external calibration (without an internal standard reference), while the urine analysis was performed by internal calibration (with an internal standard reference). All blanks in the liver assays were below the lower limit of quantitation (LOQ) for the compound of interest; in some instances
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the blanks were above the LOQ for sera and urine samples. Because there were endogenous levels of fluorochemicals in unexposed monkey sera and liver, rabbit sera and liver was used as a surrogate matrix. Monkey urine was used for standardization and QC samples in the urine analysis. Fecal samples were extracted with acetonitrile, fdtered through a glass acrodisc filter, passed through a carbon SPE column and collected. The columns were eluted with acetonitrile followed by acetonitrile:ascorbic acid in methanol. The combined extracts were evaporated to almost dryness and reconstituted with methanol to a final volume of 2 mL. The samples were analyzed using electrospray LC/MS/MS. THPFOS was used as a surrogate standard but was used only to monitor the efficiency of the extraction procedure and was not used to calculate the amount of PFOA in the test samples.
Matrix spike samples were prepared from control samples of sera, liver and urine with each batch. Sera samples were spiked with either monkey and/or rabbit sera at a final concentration of 250-500 ng/mL. Liver samples were spiked at a level of approximately 250 ng/g and urine samples were spiked at approximately 60 ng/mL. These levels approximate the background levels in the sera, liver and urine of the Group 1 (0 mg/kg/day) monkeys.
Low levels of PFOA were often detected in the sera, liver, urine and feces of the Group 1 animals. Flowever, these levels were significantly lower than those found in the low (3 mg/kg/day) dose animals. PFOA was found in the sera, liver urine and feces of treated animals; levels increased with dose.
Although PFOA was detected in the serum, liver, urine and feces of control animals, levels in tissues from treated animals were significantly higher than those seen in the controls. PFOA levels in the sera of test animals increased with dose but decreased over time during treatment. Serum levels went from 126 36.1 pg/mL in Group 2 animals and 1597 2392 pg/mL in Group 4 animals on Day 9 to 52.5 9.14 pg/mL in Group 2 and 51.5 77.6 pg/mL in Group 4 during Weeks 26/27. This seems to be the case for urine and feces also.
Levels in the urine were 73.5 38.1, 221 124, and 909 209 pg/mL in Groups 2, 3, and 4 respectively on Day 9 and 51.6 13.7, 109 75.2, and 19.2 27.0 pg/g respectively in these same groups During Week 26. In the feces, PFOA levels were less than the limit of quantitation in the control animals, and 7.43 6.54, 15.4 10.2 and 56.6 73.7 ug/g in Groups 2, 3, and 4 respectively during Week 2 and were 2.92 1.35, 43.0 36.9 and 10.3 20.8 pg/g during Week 26. As might be expected, once treatment stopped, PFOA levels in both urine and feces fell to levels that were comparable to control levels. During Weeks 28-34, levels in the feces of the control animals were 0.279 0.732 pg/g and 0.387 0.372 in Group 3 samples. There is no explanation for the high levels of PFOA seen in the feces of the control animals during Week 22. PFOA appears to be preferentially excreted in the urine rather than the feces although it is obviously excreted in both urine and feces throughout treatment.
PFOA levels in serum also decreased over time during recovery until they reached 1.18 0.827 pg/mL serum in Group 3 as compared to 0.0738 0.00256 in the controls during Week 40. It is difficult to draw any firm conclusions about comparative levels in liver and serum since there is only one time period, Week 27, for which data for liver and serum can be compared. At this point, levels in serum appear to be higher than those in the liver. This is not a definitive statement however because there is only this one time point available for comparison and because dosing of three animals in the 30/20 mg/kg dose group had stopped by Week 12 and this may have affected levels in these animals. At week 27, serum and liver levels in the Group 4 animals are similar.
During the later periods of treatment, it appears that levels at 10 mg/kg/day in serum, urine and feces are higher than they are at 30/20 mg/kg/day. This may be because dosing for three animals in the 30/20 mg/kg dose group was stopped during treatment, on Days 43, 66, and 81 respectively. This may have
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affected PFOA levels in tissues from animals in Group 4. However, in the one comparative sample that is available for the liver, levels in Group 4 are higher at Week 27 than they are in Groups 2 and 3. At Week 27, PFOA levels in the liver are 15.3 3.02, 14.0 7.55, and 42.8 63.3 for Groups 2, 3, and 4 respectively. The PFOA level in the control liver at Week 27 is 0.177 0.0730. By Week 40, levels in the livers from Group 3 animals are 0.114 0.0441 and the levels in the control are below the limits of detection.
PFOA levels in serum are shown in Tables 1 and 1A; levels in the liver are shown in Tables 2 and 2A; levels in the urine are shown in Tables 3 and 3A and levels in the feces are shown in Tables 4 and 4a. Individual animal values are presented in Tables 1, 2, 3, and 4 respectively. Values in Tables 1A, 2A, 3A and 4A are group averages the standard deviation associated that group at each time period.
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000146
247
Table 1 Individual PFOA Levels in Serum (ug/mL)
Group 1 0.0
mg/kg/day Control
Group 1 0.0
mg/kg/day Control
Group 2 3
mg/kg/day
Animal ID
105709M 105714M 105715M 105718M 105720M 105725M Animal ID
105709M 105714M 105715M 105718M 105720M 105725M Animal ID
105702M 105706M 105717M 105721M 105723M (105721M replacement)
Day 9
0.645 0.137 <LOQ 0.0889 <LOQ 0.0499 Week
26 0.471 0.108 0.101 0.206 0.308 0.0623 Day 9
91.6 177 116
119
Week 4
0.299 0.230 0.0785 0.347 0.167 0.114 Week 26 & 27 0.377 0.219 0.0945 0.206 0.308 0.136 Week 4
rA O O rA
oO
Week 6
0.125 <LOQ <LOQ <LOQ <LOQ
Week 28
0.153 0.209
Week 6
a o_i
V
Week 8 <LOQ <LOQ
<LOQ <LOQ Week
30
0.127 0.161
Week 8
Week 10
0.191 0.105 0.0919 0.129 0.128 0.110 Week 32
0.0943 0.125
Week 10
Week 12
0.207 0.0994 0.064 0.139 0.140 0.0869 Week
34
0.0680 0.104
Week 12
Week 14 Week 16
0.275 0.156 0.0981 0.153 0.182 0.105 Week 36
0.275 <LOQ <LOQ <LOQ <LOQ <LOQ Week 38
0.0795 0.142
0.0711 0.117
Week 14 Week 16
Week 18
0.263 0.244 0.0950 0.165 0.193 0.138 Week
40
0.0719 0.756
Week 18
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000147
Table 1A PFOA Concentrations in the Serum (pg/mL) of Treated Animals
Time Point
______ 22X9______ Week 4 Week 6 Week 8 Week 10 Week 12 Week 14 Week 16 Week 18 Week 20 Week 22 Week 24 Week 26
Week 26/27 Week 28 Week 30 Week 32 Week 34 Week 36 Week 38 Week 40
rA O O
Group 1 0.0 mg/kg/day Average SD
0.0613 0.0472 0.0206 0.0105 0.103 0.0113
0.126 0.0348 0.123 0.0507 0.162 0.0643 0.128 0.0721 0.183 0.0637 0.224 0.0730 0.232 0.131
<LOQ 0.209 0.156 0.223 0.105 0.181 0.0391 0.144 0.0238 0.110 0.0216 0.0861 0.0256 0.111 0.0445 0.0941 0.0324 0.0738 0.00256
Group 2 3 mg/kg/day Average SD
126 36.1 98.4 42.7 102 33.6 94.7 27.3 105 37.7 79.6 25.9 90.0 28.9 68.6 26.0 29.4 25.4 33.0 26.0 77.6 24.9 72.2 68.8 118 27.5 52.5 9.14
NS NS NS NS NS NS NS
Group 3 10 mg/kg/day Average SD
189 48.9 172 71.9 95.8 20.6 97.6 23.5 93.6 13.7 90.6 24.5 92.2 27.6 98.5 42.3 17.4 14.3 96.6 26.6 105 36.3 90.9 21.2 77.4 16.9 74.1 33.1 25.9 7.07 11.3 5.11 7.60 3.90 3.97 2.09 2.75 1.88 1.84 1.40 1.18 0.827
Group 4 30/20 mg/kg/day
Average SD
1597 2392 10841839 145 21.6 166 98.9 2 3 7 158 140 77.5 79.4 28.3 83.9 58.5 36.2 21.2 97.7 129 58.6 45.6 62.7 74.3 77.8 126 51.5 77.6
NS NS NS NS NS NS NS
LOQ = Limit of Quantitation NS = No Sample Results are expressed as group averages the standard deviation associated with that group. Data arc accurate to within one SD of the average fortified sample recovery. The average fortified sample recovery was 94% with an SD of 11%.
Table 2 PFOA Concentrations in the Liver (pg/g) of Treated Animals
Time Point
Week 20 Week 27 Week 40
Group 1 0.0 mg/kg/day Average SD
NS 0.117 0.0730
<LOQ
Group 2 3 mg/kg/day Average SD
18.3 15.3 3.02
NS
Group 3 10 mg/kg/day Average SD
NS 14.0 7.55 0.114 0.0441
Group 4 30/20 mg/kg/day
Average SD
NS 42.8 63.3
NS
LOQ = Limit of Quantitation NS = No Sample Results arc expressed as group averages the standard deviation associated with that group.
Data are accurate to within one SD of the average fortified sample recovery. The average fortified sample recovery was 90% with an SD of 26%.
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000148
Table 3 PFOA Concentrations in the Urine (pg/mL) of Treated Animals
Time Point Week 2 Week 4 Week 6 Week 8 Week 10 Week 12 Week 14 Week 16 Week 18 Week 20 Week 22 Week 24 Week 26 Week 28 Week 30 Week 32 Week 34 Week 36 Week 38 Week 40
,oo
V
Group 1 0.0 mg/kg/day Average SD
0.152 0.337 0.0587 0.0716 0.0161 0.00940 0.0177 0.00114 0.0141 0.00648
7.96 19.5 0.0299 0.0339 0.0256 0.0248 0.0211 0.0130 0.0231 0.00688 0.0125 0.00749 0.0268 00.0265
0.118 0.142 <LOQ <LOQ <LOQ
0.0117 0.00841 <LOQ <LOQ
Group 2 3 mg/kg/day Average SD
73.5 38.1 54.9 4.62 65.7 46.9 47.6 20.6 39.9 18.7 48.8 18.8 63.1 47.2 50.2 21.0 37.7 19.3 52.1 9.63 95.8 80.8 46.3 8.52 51.6 13.7
NS NS NS NS NS NS NS
Group 3 10 mg/kg/day Average SD
221 124 190 91.6 128 50.0 206 73.1 175 92.3 201 92.7 139 52.5 139 57.0 186 63.9 144 135 158 78.4 157 63.3 109 75.2 0.327 0.0182 0.361 0.118 0.114 0.0608 0.121 0.0305 0.0502 0.0166 0.0284 0.0102 0.0254 0.0101
Group 4 30/20 mg/kg/day
Average SD
909 269 2 4 0 161 2 7 2 140 180 109 359 449 118 111 72.9 84.0 50.2 67.9 43.1 84.6 44.0 59.9 98.5 134 56.0 77.2 19.2 27.0
NS NS NS NS NS NS NS
LOQ = Limit of Quantitation NS = No Sample Results arc expressed as group averages the standard deviation associated with that group.
Data are accurate to within one SD of the average fortified sample recovery. The average fortified sample recovery was 88% with an SD of 17%.
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000149
Table 4 PFOA Concentrations in the Feces (pg/g) of Treated Animals
Time Point Week 2 Week 4
Week 6
Week 8 Week 10 Week 12
Week 14 Week 16
Week 18 Week 20 Week 22 Week 24 Week 26
Weeks 28-34 Weeks 36-40
Group 1 0.0 mg/kg/day
Average SD
Group 2 3 mg/kg/day
Average SD
<LOQ 0.0214 0.0178 0.108 0.00192 0.0782 0.103
<LOQ 0.0498 0.0894 0.139 0.308 0.0572 0.0762 0.258 0.654 0.405 1.08 15.5 36.9
0.517 1.13 0.0172 0.00892
0.279 0.732 0.0103 0.00684
7.43 6.54 10.4 12.0
12.1 14.1
9.46 9.21 3.96 3.68 7.15 5.65
7.50 2.43 6.88 2.62
5.72 7.15 6.81 4.89 13.8 5.22 6.22 5.45 2.92 1.35
NS NS
Group 3 10 mg/kg/day Average SD
15.4 10.2 23.4 10.6
23.3 8.46
41.0 25.0 26.0 17.4 10.3 6.07
27.2 29.4 31.4 23.3
17.3 13.3 52.4 39.5 39.5 21.0 40.5 21.8 43.0 36.9
0.387 0.372 0.0336 0.0313
Group 4
30/20 mg/kg/day Average SD
56.6 73.7 22.0 6.23
101 86.7
36.7 34.2 48.0 34.0 32.0 42.9
19.2 25.2 18.2 28.8
22.1 31.7 37.8 58.1 25.2 36.0 34.6 47.7 10.3 20.8
NS NS
LOQ = Limit of Quantitation NS = No Sample Results are expressed as group averages the standard deviation associated with that group. Data are accurate to within one SD of the average fortified sample recovery. The average fortified sample recovery was 117% with an SD o f 22%.
Reference
Thomford, PJ (2001) 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys. Study performed by Covance Laboratories Inc., M adison Wisconsin 53704-2592 for APME Ad-hoc APFO Toxicology Working Group. Study No. Covance 6329-231, Com pletion Date Decem ber 18, 2001, 463 pp.
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000150
REPEAT DOSE DATA
Title: Ninety Day Subacute Rat Toxicity Study
TEST SUBSTANCE
Identity: Fluorad Fluorochemical FC-I43. Also referred to as PFOA ammonium salt, ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143, or as a major component ofFX-1003 (octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1)
Remarks: White powder, 3M stock no. 98-0211-0008-0 Lot 340; purity of the test substance was not indicated.
METHOD
Method/guideline followed: No guideline number stated
Study duration: 90 days
GLP (Y/N): No
Year study performed: 1977-78
Species/strain: Charles River CD rat
Sex: Male and female
Number of animals per dose group: 5 animals/sex/group
Route of administration: Dietary
Doses tested and frequency: 0, 10, 30, 100, 300, and 1,000 ppm
Post-treatment observation period: None
Statistical methods used: Statistical analysis comparing results of the treatment groups with the control group was performed by analysis of variance (one-way), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) using Dunnett's multiple comparison tables to judge significance of differences.
Remarks: Initial age of the test animals was not given; however, the mean initial body weight of the males was 222-254 g and the initial weight of the females was 151-179 g. The test chemical was mixed with 500 g of feed at weekly intervals. The treated animals were observed twice daily for clinical signs of toxicity and for mortality. Individual body weights were recorded weekly. Blood and urine samples were collected prior to study initiation and at 1 and 3 months of treatment to evaluate hematology, biochemistry, urinalysis, and serum samples. Food consumption was recorded. At 90 days, the animals were sacrificed and necropsied for macroscopic and microscopic examination. Histopathology was performed on the following organs from rats from the control, 100, 300, and 1,000 ppm dose groups: brain with cervical cord, lumbar spinal cord, peripheral nerve, eyes, pituitary, thyroid with parathyroid, adrenals, lung, heart with coronary vessels, aorta, spleen, mesenteric lymph node, thymus, bone with
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000151
marrow (sternum), salivary gland, small intestines (duodenum, jejunum, ileum) colon, pancreas, liver, kidneys, urinary bladder, testes, ovaries, prostate, uterus, skin (mammary gland), any tissue(s) with gross lesions. Livers from rats from the 10 and 30 ppm dose groups were also examined microscopically and liver samples from all dose groups were frozen and sent to the sponsor for analysis.
RESULTS
NOAEL Males: 30 ppm Females: 300 ppm
LOAEL Males: 100 ppm (decrease in food consumption, liver lesions) Females: 1,000 ppm (liver lesions, increased liver weight)
Remarks: One female in the 100 and one female in the 300 ppm group died during collection of blood. These deaths were not considered to be treatment related. All other animals survived until scheduled sacrifice.
There was a significant reduction in mean body weight in males in the 1,000 ppm group (362 g vs 466 g in the control group). Food consumption was reduced in males in the 100, 300 and 1000 ppm groups, but the differences were not statistically significant.
Males in the 30, 100, 300 and 1,000 ppm groups had significantly reduced numbers of erythrocytes at the end of the treatment period. The values were 7.95, 7.05, 7.16, 6.72, and 6.94 in the control, 30, 100, 300 and 1000 ppm groups, respectively. Males had reduced leukocyte values compared to the controls in all dose groups, but were statistically significant at the 300 ppm group only; leukocyte values were 10.64, 8.88, 9.33, 9.35, 7.63, and 8.06 in the control, 10, 30, 100, 300 and 1,000 ppm groups, respectively. A similar phenomenon was seen with hemoglobin values which were reduced at all dose levels but were significant at the 10 ppm dose level only. Hemoglobin values were 16.2, 14.7, 15.0, 15.4, 14.9, 13.1 in the control, 10, 30, 100, 300 and 1000 ppm groups, respectively. There was no similar effect upon the hematological parameters of female rats in the study.
Males at the 30, 100, 300, and 1,000 ppm dose levels had increased glucose levels (mg/100 ml) which were statistically significant at all but the 100 ppm dose level. Reported glucose levels were 121, 120, 136, 134, 143 and 135 mg/100 ml for the 0, 10, 30 100, 300 and 1,000 ppm groups, respectively. B.U.N. levels were elevated in males at the 100, 300, and 1,000 ppm dose levels; mean values at 90 days were 20.4, 23.9 and 35.1 mg/100 ml for the three dose groups, respectively, compared to 16.2 mg/100 ml for the controls. Alkaline phosphatase was elevated in males in the 100, 300, and 1,000 ppm groups; the levels were 147, 204 and 212 IU/1 for the three groups, respectively, compared to 104 IU/1 for the controls. Females showed no similar changes in biochemical measurements.
Neither males nor females showed any treatment related changes in urinalysis parameters although females from all groups showed a higher frequency of occult blood in the urine than did males.
The only gross necropsy observation was noted in males at the 1,000 ppm dose level. These animals had enlarged livers which showed varying degrees of surface discoloration. Neither females from the 1,000 ppm dose level nor males or females from the lower dose levels showed such effects.
253
000152
Both absolute and relative liver weights were significantly increased in males in the 30, 300 and 1,000 ppm groups and in one female in the 1,000 ppm group. Compound-related liver lesions occurred in all male rats in the 100, 300 and 1,000 ppm groups. These lesions consisted of focal to multifocal, very slight to slight hypertrophy of hepatocytes in centrilobular to midzonal regions of the affected liver lobules. In some instances these lesions were accompanied by increased amount of yellowish-brown pigment resembling lipofuscin in the cytoplasm of hepatocytes and occasionally in sinusoidal lining cells. The incidence and severity of the lesions was more pronounced among male rats at the 1,000 ppm dietary level. CONCLUSIONS The test substance resulted in a significant reduction in mean body weight in males at 1000 ppm, a significant reduction in the number of erythrocytes in males at 30, 100, 300 and 1000 ppm, and liver lesions in males at 100, 300 and 1000 ppm. REFERENCE Goldenthal, E., D. Jessup, R. Geil, N. Jefferson, and R. Arceo. 1978. Ninety day subacute toxicity study: Fluorad Fluorochemical FC-143. 3M Company. Study no. 137-089.
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000153
REPEAT DOSE DATA
Title: 13-Week Dietary Toxicity Study with T-5180, Ammonium Perfluorooctanoate (CAS No. 3825-26-1) in Male Rats
TEST SUBSTANCE
Identity: T-5180, Ammonium perfluorooctanoate (APFO), CAS No. 3825-26-1, Lot No. 115. It is a lightly colored powder.
Remarks: Purity of the test substance was not indicated.
METHOD
Method/guideline followed: Guideline 82-1 (source not specified). The study was conducted in compliance with Hazleton Wisconsin, Inc. (HWI) protocol TP9321 dated November 30, 1990.
Study duration: 13 weeks
GLP (Y/N): Y
Year study performed: 1993
Species/strain: Rat/Sprague-Dawley, Crl:CDBR
Sex: Male
Number of animals per dose group: 55 in each group, except pair-fed controls, which had 45 animals
Route of administration: Dietary
Doses tested and frequency: 0 (pair-fed and nonpair-fed controls), 1,10, 30, or 100 ppm (approximate mean compound consumption at week 13 of 0.05, 0.47, 1.44, and 4.97 mg/kg/day) fed ad libitum. Fifteen animals per group were sacrificed at 4, 7, and 13 weeks. The remaining 10 animals per group (all groups except pair-fed controls) were sacrificed after 13 weeks of treatment and 8 weeks without treatment.
Post-treatment observation period: 8 weeks
Statistical methods used: Levene's test was used to test for variance homogeneity. In cases of heterogeneity of variance at p < 0.05, transformations were used to stabilize the variance. ANOVA was performed on the homogeneous or transformed data. If the ANOVA was significant, Games and Howell Modified Tukey-Kramer test was used for pairwise comparisons between groups. One-way ANOVA was used to analyze body weights, cumulative body weight gains, food consumption, clinical chemistry, hormone values, organ weights, organ-to-body weight percentages, and organ-to-brain weight ratios. Body weights, cumulative body weight gains, and food consumption values were analyzed for all groups (except pair-fed controls) using the SAS program according to HWI methods. Group comparisons were evaluated at the 5.0% two-tailed probability level. In the analysis of the data, animals in groups exposed to 1, 10, 30, and 100 ppm APFO were compared to the control animals in the nonpair-fed group, while the data from the pair-fed control animals were compared to animals exposed to 100 ppm APFO.
255
000154
Remarks: Male rats were used to characterize the effects of the test substance on testicular physiology. At study initiation, the animals were approximately 41 days old and weighed 181 to 229 g. The appropriate amount of the test substance was thoroughly mixed with rodent chow before providing it ad libitum to the animals. Control groups of pair-fed and nonpair-fed rats were maintained on a basal diet not containing the test substance. All diets were assayed weekly for the first four weeks to determine the dietary concentration of the test substance; thereafter, weekly analyses were performed on the control diet and one test diet, selected sequentially.
Throughout the study, animals were observed twice daily for signs of toxicity. Individual body weight data were recorded on the first day, weekly thereafter, and on the day of necropsy. Food consumption data were collected daily for pair-fed groups and weekly for nonpair-fed groups. Serum samples collected from 10 animals/group at each scheduled sacrifice were analyzed for estradiol, total testosterone, luteinizing hormones, and test material content. At necropsy, samples of liver, testes, lungs, and subcutaneous adipose tissue were collected from each animal and frozen for possible test material residue analysis. A section of liver was obtained from all animals at each scheduled sacrifice. For 5 animals/group at each scheduled sacrifice, the liver was assayed for the level of palmitoyl CoA oxidase as an indicator of peroxisome proliferation. Fifteen animals/group were necropsied after 4, 7, and 13 weeks of treatment, as well as 10 animals/group at the end of the 8-week recovery period. The macroscopic examinations included the external surface of the body, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the nasal cavity and paranasal sinuses, and the thoracic, abdominal, and pelvic cavities and viscera. The brain, liver, lungs, testes, and accessory sex organs (seminal vesicle, prostate, coagulating gland, urethra) were weighed. Organ-to-body weight percentages and organ-tobrain weight ratios were calculated. The following were examined microscopically (when present): lesions, brain, liver, lungs, testes, and accessory sex organs. Electron microscopy was also used to evaluate tissues from the brain, liver, lungs, testes, and accessory sex organs.
RESULTS
NOAEL (dose and effect): 1.0 ppm
LOAEL (dose and effect): 10 ppm
Toxic response/effects by dose level: One animal at the 100 ppm dose level was sacrificed during week 4 due to severe neck sores, but all other animals survived until scheduled sacrifice. No clinical signs of toxicity were observed in any groups during treatment or recovery.
10 ppm -- higher hepatic palmitoyl CoA oxidase activity, decreased mean body weight gains, increased absolute and relative liver weights, and hepatocellular hypertrophy
30 ppm -- higher hepatic palmitoyl CoA oxidase activity, decreased mean body weight gains, increased absolute and relative liver weights, and hepatocellular hypertrophy
100 ppm -- lower body weights and cumulative body weight gains, lower food consumption, higher hepatic palmitoyl CoA oxidase activity, increased absolute and relative liver weights, hepatocellular hypertrophy, and elevated estradiol levels
Statistical results: High dose animals exhibited consistently significantly lower body weights and cumulative body weight gains compared to those of the nonpair-fed group. They also consumed significantly less food than the nonpair-fed controls at weeks 1 and 2. Animals at dose levels of 30 and 100 ppm exhibited higher hepatic palmitoyl CoA oxidase activities that were statistically significant at
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000155
weeks 5, 8, and 14. Animals fed 10 ppm had transiently higher hepatic palmitoyl CoA oxidase activity that was statistically significant at week 5. Absolute and relative liver weights were significantly higher in the animals of the high dose group than the pair-fed controls at weeks 4, 7, and 13. Rem arks: Though statistically significant increases in hepatic palmitoyl CoA oxidase activities were reported during treatment, no difference was seen after the 8-week recovery period. The effect was dosedependent and reversible. Increased absolute and relative liver weights and hepatocellular hypertrophy were observed in animals fed 10, 30, or 100 ppm; absolute and relative liver weights were significantly higher in the animals of the high dose group than the pair-fed controls at weeks 4, 7, and 13. The progression of hepatocellular hypertrophy did not appear to be affected by the length of treatment. The changes observed are suggestive of a test material effect on intracellular metabolism and may be associated with peroxisome proliferation. There was no evidence of increased liver weights of animals after the recovery period, which indicates that the effects were reversible. Animals in the high dose group exhibited consistently significantly lower body weights and cumulative body weight gains than those of the nonpair-fed control group. They also consumed significantly less food than the nonpair-fed controls at weeks 1 and 2. Overall, no significant difference in mean food consumption between nonpair-fed and pair-fed groups was noted. Though there was no statistically significant difference, the estradiol levels in the high dose animals appeared to be elevated at week 5. CONCLUSIONS The study author concluded that the no observed adverse effect level (NOAEL) for the test substance when fed ad libitum to rats for at least 13 weeks was 100 ppm and that the no observed effect level (NOEL) was 1.0 ppm. Remarks: In animals fed 10, 30, and 100 ppm, the report describes treatment-related liver effects that may be considered adverse, including increased absolute and relative liver weights, hepatocellular hypertrophy, and significantly increased hepatic palmitoyl CoA oxidase activities. On the basis of these findings, the LOAEL was 10 ppm and the NOAEL was 1.0 ppm. REFERENCE Palazzolo, M. 1993. 13-Week dietary toxicity study with T-5180, ammonium perfluorooctanoate (CAS No. 3826-1) in male rats. Hazleton Wisconsin, Inc. Madison, WI. 3M Company. St. Paul, MN.
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000156
REPEAT DOSE DATA
Title: Two Year Oral (Diet) Toxicity/Carcinogenicity Study of Fluorochemical FC-143 in Rats
TEST SUBSTANCE
Identity: Fluorad Fluorochemical FC-143, also referred to as PFOA ammonium salt, ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143, or as a major component of FX-1003 (octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1)
Remarks: The test substance, a white powder, was analyzed prior to the start of the study, after approximately one year from the start of the study, and at the termination of the dosing period. No detectable changes were found. The composition and purity of the test substance were not indicated in the main body of the study report.
METHOD
Method/guideline followed: Guideline number not stated
Study duration: Two years
GLP (Y/N): Yes
Year study performed: 1981 - 1983
Species/strain: Sprague-Dawley rat [Crl:COBSRCD(SD)BR]
Sex: Male/female
Number of animals per dose group: The control and high-dose groups contained 65 rats/sex and the low-dose group contained 50 rats/sex.
Route of administration: Diet
Doses tested and frequency: Low-dose: 1.3 mg/kg/day (males), 1.6 mg/kg/day (females) High-dose: 14.2 mg/kg/day (males), 16.1 mg/kg/day (females)
Post-treatment observation period: None
Statistical methods used: Bartlett's test for homogeneity of variance was used to analyze the test data. If this test was not significant at alpha = 0.001, the data were further analyzed by comparing each treated group to the control group using a two-tailed Dunnett's test at the alpha = 0.05 significance level.
Remarks: Test animals were 39 to 41 days of age when treatment began. An interim termination at one year included 15 rats/sex from both the control and high-dose groups. All animals were observed daily throughout the dosing period. Weekly physical examinations included palpation for any masses present and pharmacotoxic observations. Body weights and feed consumption were recorded weekly or bi weekly. Eye examinations using indirect ophthalmoscopy and/or slit lamp biomicroscopy were performed at the one-year period. Clinical pathology determinations included hematology, clinical (serum) chemistry and urinalysis. Tests were conducted on samples obtained at 3, 6, 12, 18, and 24
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000157
months from randomly selected animals of each dose group. Hematologic tests included total red and white blood cell counts, hemoglobin, hematocrit, and a differential white blood cell count. Clinical chemistry parameters included total bilirubin, total protein, albumin, blood urea nitrogen (BUN), glucose, alkaline phosphatase (AP), creatine phosphokinase (CPK), aspartate aminotransferase, and calcium. Urine tests included pH, specific gravity, albumin, glucose, bilirubin, occult blood and ketones. Metabolic examinations involved collection of urine and fecal samples. Post mortem examinations were performed on all animals and the weights of the adrenal glands, brain, testes, heart, kidneys, liver, spleen, and uterus were recorded from 15 randomly selected rats/sex/group. Samples of many different tissues were collected and observed microscopically from these animals.
RESULTS
Survival rates: -Generally, survival rates for the FC-143-treated rats were good during the full two years of the study. Fewer deaths were seen in high-dose males and females than in the controls.
Neoplastic effects:
Percent Neoplastic Lesions in Males
Control
Adrenal
Pheochromocytoma, benign
4
Phcochromocytoma, malig.
0
Liver
Hepatocellular carcinoma
6
Pituitary
Adenoma
35
Testes/Epididymis
Leydig cell adenoma
0
Thyroid
C-cel 1adenoma
0
C-ccl 1carcinoma
5
Source: Table 19
*Significantly different (p <0.05) from controls
Low
8 2 2 36 4 4 0
High
8 0 10 28 14* 9 0
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000158
Percent N eoplastic Lesions in Fem ales
Control
Adrenal
Pheochromocytoma, benign
4
Pheochromocytoma, malig.
0
Liver
Hepatocellular carcinoma
0
Mammary gland
Adenocarcinoma
15
Adenoma
7
Carcinoma
2
Fibroadenoma
22
Lymphangiosarcoma
0
Pituitary
Adenoma
72
Thyroid
C-ccll adenoma
2
C-cell carcinoma
0
Source: Table 19
*Significantly different (p <0.05) from controls
Low High
0 10
02
02
31 11 00 00 42 48* 02
83 72
00 00
Statistical analysis of neoplastic effects (i.e., percent that was statistically significantly different from controls; p < 0.05):
Females (16.1 mg/kg): Mammary gland fibroadenomas Males (14.2 mg/kg): Leydig cell adenomas in testis
Nonneoplastic effects:
NOAEL (dose and effect): none
LOAEL (dose and effect):
1.3 mg/kg/day (males) - based upon salivary gland sialadenitis (note that the study authors implied an association of this lesion with a suspected outbreak of sialodacryoadenitis viral infection; however, the presence of a virus was not confirmed)
1.6 mg/kg/day (females) - based upon ovarian tubular hyperplasia (and ataxia, a clinical sign).
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000159
Percent N on-neoplastic Lesions in M ales
Control
Adrenal
Nodular hyperplasia
4
Sinusoidal ectasis
22
Heart
Myocarditis, chronic
28
Liver
Cystoid degeneration
8
Hepatocellular alt. basophil.
4
Hyperplastic nodule
0
Megalocytosis
0
Portal mononuclear cell infil.
74
Necrosis
6
Lung
Alveolar macrophages
20
Hemorrhage
20
Perivas. mono, infil.
42
Vascular mineralization
86
Pneumonia, interstitial
32
Testis/epididymis
Tubular atrophy
14
Vascular min.
0
Thyroid
C-cell hyperlasia
2
Pancreas
Acinar atrophy
13
Salivary gland
Sialadenitis, chronic
2
Spleen
Hemosiderosis
32
Source: Table 20
*Significantly different (p <0.05) from controls
1
Low
2 26
36
14 2 0 12 64 10
32 28 6* 86 10*
20 6
13
20
27*
8*
n 1
High
18 32
34
56* 12 6 80* 96* 10
62* 44* 14* 94 14
22 18*
2
22
30*
44
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000160
Percent N on-neoplastic Lesions in Fem ales
Control
Adrenal
Nodular hyperplasia
0
Sinusoidal ectasis
84
Heart
Myocarditis, chronic
32
Liver
Cystoid degeneration
0
Hepatocellular alt. basoph.
16
Hyperplastic nodule
2
Megalocytosis
0
Portal mono, cell inf'll.
38
Necrosis
10
Lung
Alveolar macrophages
28
Hemorrhage
28
Perivas. mono, infil.
26
Vascular mineralization
44
Pneumonia, interstitial
14
Testis/epididymis
Tubular atrophy
Vascular min.
Ovaiy
Cyst 13
Tubular hyperplasia
0
Thyroid
C-cell hyperlasia
0
Uterus
Cystic glands
14
Pancreas
Acinar atrophy
12
Salivary Gland
Sialadenitis, chronic
2
Spleen
Hemosiderosis
50
Source: Table 20
*Significantly different (p <0.05) from controls
Low
6 86
10*
2 16 0 2 22 12
20 26 4* 76* 6
18 14*
2
24
12
2
6*
High
2 82
20
2 4 4 16* 38 4
38 38 28 52 18
11 32*
7
10
9
5
24*
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List of statistically different non-neoplastic effects (increased compared with controls, unless indicated; p < 0.05):
Males (1.3 mg/kg): Chronic sialadenitis (salivary gland) Perivascular mono, infil. (lung)3 Interstitial pneumonia (lung)3 Hemosiderosis (spleen)3
Males (14.2 mg/kg): Cystoid degeneration (liver) Megalocytosis (liver) Portal mononuclear cell infiltration (liver) Alveolar macrophages (lung) Hemorrhage (lung) Vascular mineralization (testis/epididymis) Chronic sialadenitis (salivary gland) Perivascular mono, infil. (lung)3
Females (1.6 mg/kg): Vascular mineralization (lung) Tubular hyperplasia (ovary) Chronic myocarditis3 Perivascular mono, infil. (lung)3 Hemosiderosis (spleen)3
Females (16.1 mg/kg): Megalocytosis (liver) Tubular hyperplasia (ovary) Hemosiderosis (spleen)3
"Decreased incidence relative to controls
Genetic toxicity studies (study type and results):
None
Remarks:
-Dose-related decreased in mean body weights in excess of 10% was observed in high-dose males and females. -Mean feed consumption (as grams diet/kg bw) was increased in all of the FC-143 treated males throughout the study when compared to male control feed consumption. Overall, the variations were related to the variation in body weight among groups. Actual mean feed consumption was decreased in high-dose males relative to controls for the first year of the study. -Dose-related occurrence of ataxia in females was the only clinical sign observed. -A statistically significant (p<0.05) decrease in red blood cell parameters was noted in the high-dose males as compared to the controls. -A statistically significant (p<0.05) increase in relative liver and kidney weights was found in high-dose males and an increase in relative kidney weights was found in high-dose females.
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-Histopathological effects were noted in the liver of high-dose males and females. -Urinary findings included increased incidence and severity of albumin and occult blood in all male and female control and FC-143-treated groups at 12, 18, and 24 months. These findings were more pronounced in males than in females at the termination of the study. -Rats given the test article experienced a suspected outbreak of sialodacryoadenitis (SDA) viral infection between the first and second months of the study; however, the presence of a virus was not confirmed.
CONCLUSIONS
The study results are summarized as follows: Treatment-related changes were found more commonly in males than in females of each of the two treatment groups, which were supported by earlier pharmacokinetic studies demonstrating a higher retention of FC-143 by males than females. The test material was considered to be carcinogenic in the rat, inducing testicular/Leydig cell tumors in the males and mammary gland tumors in females. Based on decreases in body weight gain, increase in liver and kidney weights and toxicity in the hematological and hepatic systems, the LOAEL for male and female rats is 300 ppm (male: 14.2 mg/kg/day ; female: 16.1 mg/kg/day). [The LOAEL for male rats is 1.3 mg/kg/day if salivary gland sialadenitis is based upon; the LOAEL for female rats is 1.6 mg/kg/day if increases in the incidences of ataxia (a clinical sign) and of ovarian tubular hyperplasia (may be reversible) are based upon]. The dose-dependent increases in neoplastic and non-neoplastic lesions were as follows: testicular Leydig cell adenoma (p <0.05 at high dose) and vascular mineralization of the testes (p <0.05 at high dose) thyroid C-cell adenomas in low-dose males thyroid C-cell hyperplasia in high-dose females mammary gland fibroadenomas in females (p <0.05 at high dose) lung lesions in males (p <0.05 at high dose) salivary gland sialadenitis in males (p <0.05 at low and high doses) ovarian tubular hyperplasia in females (p <0.05 at low and high doses) megalocytosis in the liver of males and females (p < 0.05 at high dose) with increases in relative liver weight and elevations of serum enzyme activities indicative of liver toxicity cystoid degeneration and portal mononuclear cell infiltration in the liver of males (p <0.05 at high dose)
Remarks: Influence of potential viral infection in male Sprague-Dawley rats at both doses on the response to the test substance is not clear. Sialodacryoadenitis virus (SDAV) is a common viral infection of F344 rats; evaluation of 29 diet control rat groups at 5 different laboratories with and without viral infection found no consistent influence of viral infection on body weight, survival, or tumor prevalence (Rao, ct.al., 1988).
REFERENCE
3M Company/Riker Laboratories, Inc. Two Year Oral (Diet) Toxicity/ Carcinogenicity Study of Fluorochemical FC-143 in Rats. Experiment No. 0281CR0012. St. Paul, MN.; 8EHQ-1087-0394, Oct. 16, 1987.
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Rao, G.N. , Edmondson, J. and Haseman J.K. Influence of viral infections on tumor incidences, body weight and survival of Fischer 344 rats. Toxicologist. 8:166, 1988,
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REPEAT DOSE STUDIES Title: Mechanisms of Extrahepatic Tumor Induction by Peroxisome Proliferators in Male CD Rats TEST SUBSTANCE Identity: Ammonium perfluorooctanoate
Remarks: The substance was 98-100% pure METHOD
Method/guideline followed: None Study duration: 2 years GLP (Y/N): Unknown Year study performed: 2001 (publication date) Species/strain: Crl: CD BR rats from Charles River Breeding Laboratories (Raleigh, NC). Sex: Males Number of animals per dose group: 156 Route of administration: diet Doses tested and frequency: 0, 300 ppm Post-observation period: None Statistical methods used: One-way analysis of variance. When corresponding F-test for differences among groups was significant, pairwise comparisons were made with Dunnett's test. The Bartlett's test for homogeneity of variance was also performed. Nonparametric procedures included Kruskal-Wallis test for equal medians and Mann-Whitney U test for pairwise comparisons. Remarks: Hormonal analysis was conducted in 10 rats. Blood was collected from the tail vein about 1,3, 6, 9, 12, 15, 18, and 21 months after initiation of the study. Serum was prepared and frozen and then analyzed for testosterone, estradiol, luteinizing hormone, follicle stimulating hormone, and prolactin concentrations. All samples were analyzed simultaneously in duplicate. Rats were euthanized at interim time periods (1, 3, 6, 9, 12, 15, 18, and 21 months). Testes, epididymides, accessory sex gland (ASG) unit with fluid, coagulating gland/seminal vesicle (with fluid removed), prostate, and liver were weighed.
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At 24 months, surviving rats were necropsied. Brain, heart, liver, spleen, kidneys, ASG unit, coagulating gland/seminal vesicles with fluid removed, prostate, epididymides, and testes were weighted at necropsy. Liver, testes, epididymides, pancreas, and organs with gross lesions were examined microscopically for lesions.
Six rats/group were selected for evaluations of cell proliferation. For each tissue type, 1000 cells were scored.
Six rats/group were selected for evaluation of peroxisome proliferation. P-oxidation activity from liver and Leydig cell peroxisomes was measured at all interim time points. P-oxidation activity was determined using the method of Lazarow (1981).
RESULTS
Toxic responses and effects:
Body weight, food consumption, and survival: From test days 8 to 630, body weight was significantly decreased in the C8 group versus the ad libitum control group. The decreased body weight was primarily due to reduced `food efficiency'. On day 714 of the test, survival in the C8 group was increased compared with the control group (statistical significance not indicated). [Flematological changes were discussed in a separate article.]
Liver: Relative liver weights and hepatic P-oxidation activity were significantly increased at all times (p < 0.05) when compared with one or more control groups. C8 produced a statistically significant increase (p < 0.05) in incidence of hepatocellular adenomas (10/76, 13% vs. 2/80, 3% in controls).
Testis: Testis weights were statistically significantly increased (p < 0.05) at 24 months in C8-treated rats. Incidences of Leydig cell hyperplasia and adenomas were also statistically significantly increased (p < 0.05); the incidence of Leydig cell tumors was 8/76(11%) as compared to 0/80 (0%) in controls.
Pancreas: Pancreatic acinar cell proliferation was statistically significantly increased (p < 0.05) at 15, 18, and 21 months. Incidence of acinar cell hyperlpasia and adenomas was significantly increased in C8 rats (p < 0.05). Carcinoma was observed in one C8-treated rat (not statistically significant). The combined incidence of acinar cell adenoma and carcinoma was 8/76 (11%) whereas that of the control was 0/80 (0%).
Serum hormone measurements: Serum estradiol concentrations were significantly elevated (p < 0.05) at 1,3, 6, 9, and 12 months compared to control groups. There were no consistent differences in serum testosterone, FSFI, prolactin, or LF1 concentrations in the treated rats when compared to the controls.
Statistical results: Statistically significant results are reported above.
Remarks: A full range of organs and tissues was not examined histologically. Therefore, it is unknown whether the mammary gland tumors observed in the earlier multi-dose study (3M, 1987) was induced in this study.
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CONCLUSIONS The study demonstrate that C8, a peroxisome proliferator, induces hepatic as well as extrahepatic tumors (testis and pancreas) in CD rats. Data from this study also suggest that the induction of Leydig cell tumors by C8 is a result of a sustained increase in serum estradiol concentration. Rem arks: none Last Modified: 7/03/01 REFERENCE Biegel, L.B., Hurtt, M.E., Frame, S.R., O'Connor, J.C., and J.C. Cook. 2001. Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats. Toxicological Sciences. 60: 44-55. 3M. Final report o f " Two year oral (diet) toxicity and carcinogenicity study of fluorochemical FC-143 (perfluorooctanane ammonium carboxylate) in rats. Vol. 1-4, #M/RIKER Exp. No. 0281Crool2; 8EHQ1087-0394, Oct. 16, 1987.
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DEVELOPMENTAL TOXICITY
Title: Oral Teratology Study of T-2998CoC in Rats
TEST SUBSTANCE
Identity: Ammonium Perfluorooctanoate (APFO) or T-2998CoC (FC-143)
Remarks: Purity of the test substance was not indicated.
METHOD
Method/Guideline followed: The procedure complies with the general recommendations of the FDA issued in January, 1966 ("Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use"). The study was conducted according to the 1978 Good Laboratory Practice Regulations and Safety Evaluation Laboratory's Standard Operating Procedures.
GLP (Y/N): Yes
Year study performed: 1981
Species/Strain: Rat/Sprague Dawley-derived CD
Number of animals per dose: 22
Route of administration: Oral gavage
Dosing regimen: Five groups of 22 time-mated Sprague-Dawley rats were administered 0, 0.05, 1.5, 5, and 150 mg/kg/day APFO in water by gavage on gestation days (GD) 6-15. A constant dose volume of 5 ml/kg was administered.
Doses: 0, 0.05, 1.5, 5, and 150 mg/kg/day
Statistical methods used: Dunnett's t test for dam and pup weights, number of fetuses, number of resorption sites, number of implantation sites and number of corpora lutea; Chi square for percent abnormalities.
Remarks - Detail and discuss any significant protocol parameters and deviations: Based on the results of a range-finding study, an upper dose level of 150 mg/kg/day was set for the definitive study in which five groups of 22 time-mated Sprague-Dawley rats were administered 0, 0.05, 1.5, 5, and 150 mg/kg/day APFO in distilled water by gavage on gestation days (GD) 6-15. Doses were adjusted according to body weight. Dams were monitored on GD 3-20 for clinical signs of toxicity. Individual body weights were recorded on GD 3, 6, 9, 12, 15, and 20. Animals were sacrificed on GD 20 by cervical dislocation and the ovaries, uteri, and contents were examined for the number of corpora lutea, number of viable and non-viable fetuses, number of resorption sites, and number of implantation sites. Fetuses were weighed and sexed and subjected to external gross necropsy. Approximately one-third of the fetuses were fixed in Bouin=s solution and examined for visceral
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abnormalities by free-hand sectioning. The remaining fetuses were subjected to skeletal examination using alizarin red.
RESULTS
NOAEL (dose and effect) - maternal and developmental: The NOAEL for maternal toxicity is 5 mg/kg/day. The NOAEL for developmental toxicity is 150 mg/kg/day, the highest dose tested.
LOAEL (dose and effect) - maternal and developmental: The LOAEL for maternal toxicity is 150 mg/kg/day, based on statistically significant reductions in mean maternal body weight, ataxia, and death. No signs of developmental toxicity were observed at any dose level.
Toxic response/effects by dose level - maternal: Signs of maternal toxicity consisted of statistically significant reductions in mean maternal body weights on GD 9, 12, and 15; and ataxia and death, all at the high-dose group of 150 mg/kg/day.
Toxic response/effects by dose level - developmental: No statistically significant signs of developmental toxicity were seen at any dose level.
Statistical results:
Maternal data: Statistically significant reductions (Dunnetf s t test, p<0.05) in mean maternal body weight were observed on gestation days 9, 12, and 15. No statistical information was available for other signs of maternal toxicity (ataxia and death).
Fetal data: A statistically significant increase (Chi-square, p<0.05) in one sternebrae missing was observed at the highest dosed-group of 150 mg/kg/day.
Remarks - Additional information to adequately assess the data:
Signs of maternal toxicity consisted of statistically significant reductions in mean maternal body weights on GD 9, 12, and 15 at the high-dose group of 150 mg/kg/day. Mean maternal body weight on GD 20 continued to remain lower than controls, although the difference was not statistically significant. Other signs of maternal toxicity occurring only at the high-dose group included ataxia and death observed in three rat dams. No other effects were reported. Administration of APFO during gestation did not appear to affect the ovaries or reproductive tract contact of the dams.
A significantly higher incidence in fetuses with one missing sternebrae was observed at the high-dose group of 150 mg/kg/day; however this skeletal variation also occurred in the controls and the other three dose groups (at similar incidence but lower than the high-dose group) and therefore was not considered to be treatment-related. No significant differences between treated and control groups were noted for other developmental parameters that included the mean number of males and females, total and dead fetuses, the mean number of resorption sites, implantation sites, corpora lutea and mean fetus weights. Likewise, a fetal lens finding initially described as a variety of abnormal morphological changes localized to the area of the embryonal nucleus, was later determined to be an artifact of the free-hand sectioning technique and therefore not considered to be treatment-related.
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CONCLUSIONS Comment on author's conclusions and whether you agree: Conclusions are summarized above and this reviewer agrees. REFERENCE Gortner, E. 1981. Oral Teratology Study of T-2998CoC in Rats. Riker Laboratories, Inc., St. Paul, MN. Experiment #0681TRO110, December 1981.
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DEVELOPMENTAL TOXICITY
Title: Oral Teratology Study of T-3141CoC in Rabbits
TEST SUBSTANCE
Identity: Ammonium Perfluorooctanoate (APFO) or T-3141CoC (FC-143).
Remarks: According to the study authors, the analytical report (Appendix IV) demonstrated that T3141CoC has an analysis within specifications, is stable and is representative of commercial material. By this analysis, the C acid was 97.6% and 98.4% of the test substance pre-study and post-study, respectively.
METHOD
Method/Guideline followed: The procedure complies with the general recommendations of the FDA issued in January, 1966: AGuidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Usc.@
Type of study: Developmental Toxicity
GLP (Y/N): Yes
Year study performed: 1981
Specles/Strain: New Zealand White/Minikin rabbits
Number of animals per dose: 18
Route of administration: Oral gavage
Dosing regimen: Four groups of 18 pregnant New Zealand White rabbits were administered 0, 1.5, 5, and 50 mg/kg/day APFO in distilled water by gavage on gestation days (GD) 6-18. A constant dose volume of 1 ml/kg was administered.
Doses: 0, 1.5, 5, and 50 mg/kg/day
Statistical methods used: Dunnett=s t test for dam and pup weights, number of fetuses, number of resorption sites, number of implantation sites and number of corpora lutea; Chi-square test with Yates correction for percent abnormalities.
Remarks - Detail and discuss any significant protocol parameters and deviations: Based on the results of a range-finding study, an upper dose level of 50 mg/kg/day was set for the definitive study in which four groups of 18 pregnant New Zealand White rabbits were administered 0, 1.5, 5, and 50 mg/kg/day APFO in distilled water by gavage on gestation days (GD) 6-18. Pregnancy was established in each sexually mature female by i.v. injection of pituitary lutenizing hormone in order to induce ovulation, followed by artificial insemination with 0.5 ml of pooled semen collected from male rabbits; the day of insemination was designated as day 0 of gestation. A constant dose volume of 1 ml/kg was administered.
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Individual body weights were measured on GD 3, 6, 9, 12, 15, 18, and 29. The does were observed daily on GD 3-29 for abnormal clinical signs. On GD 29, the does were euthanized and the ovaries, uterus and contents examined for the number of corpora ltea, live and dead fetuses, resorptions and implantation sites. Fetuses were examined for gross abnormalities and placed in a 37 C incubator for a 24 hour survival check. Pups were subsequently euthanized and examined for visceral and skeletal abnormalities. A blood sample was taken from six does prior to dosing and then on GD 18 and 29; a liver sample was taken from the same animals on GD 29. All samples were sent to the sponsor for analysis. This information was unavailable at the time of this review.
RESULTS
NOAEL (dose and effect) - maternal and developmental: A NOAEL of 50 mg/kg/day, the highest dose tested, for maternal toxicity was indicated. A NOAEL for developmental toxicity could not be established since signs of developmental toxicity were seen at all doses, with statistical significance at the highest dose.
LOAEL (dose and effect) - maternal and developmental: No signs of maternal toxicity were observed at any dose level. The LOAEL for developmental toxicity is 50 mg/kg/day, based on dose-related increases in a skeletal variation, with statistical significance at the high-dose group.
Toxic response/effects by dose level B maternal: Signs of maternal toxicity consisted of statistically significant transient reductions in body weight gain on GD 6-9 when compared to controls; body weight gains returned to control levels on GD 12-29.
Toxic response/effects by dose level B developmental: Signs of developmental toxicity consisted of a dose-related increase in a skeletal variation, extra ribs or 13lh rib, with statistical significance at the highdose group of 50 mg/kg/day.
Statistical results:
Maternal data: Statistically significant reductions in mean body weight gains (Dunnett=s test, p<0.05) between gestation day 6-9 were observed in the highest dosed-group of 50 mg/kg/day. After gestation 9, mean body weight gains were comparable to control animals for all dosed-groups.
Fetal data: Dose-related increases in a skeletal variation, extra ribs or 13thrib, with statistical significance (Dunnett=s test, p,0.05) at the high-dose group (38% at 50 mg/kg/day, 30% at 5 mg/kg/day, 20% at 1.5 mg/kg/day, and 16 % at 0 mg/kg/day). A statistically significant increase (Dunnett=s test, p,0.05) in 13th ribs-spurred occurred in the mid-dose group of 5 mg/kg/day.
Remarks - Additional information to adequately assess the data:
Signs of maternal toxicity consisted of statistically significant transient reductions in body weight gain on GD 6-9 when compared to controls; body weight gains returned to control levels on G D I2-29. Administration of APFO during gestation did not appear to affect the ovaries or reproductive tract contents of the does. Six deaths occurred during the study; however, five of the six deaths were attributed to gavage errors. No clinical or other treatment-related signs were reported.
No significant differences were noted between controls and treated groups for the number of males and females, dead or live fetuses, and fetal weights. Likewise, there were no significant differences reported for the number of resorption and implantation sites, corpora ltea, the conception incidence, abortion rate,
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or the 24 hour mortality incidence of the fetuses. Gross necropsy and skeletal/visceral examinations were unremarkable. The only sign of developmental toxicity consisted of a dose-related increase in a skeletal variation, extra ribs or 13thrib, with statistical significance at the high-dose group (38% at 50 mg/kg/day, 30% at 5 mg/kg/day, 20% at 1.5 mg/kg/day, and 16 % at 0 mg/kg/day). A statistically significant increase in 13th ribs-spurred occurred in the mid-dose group of 5 mg/kg/day; however, the biological significance of this effect is uncertain since in both the high- and low-dose groups, this effect occurred at the same rate and was not statistically significantly different from controls. CONCLUSIONS Comment on author=s conclusions and whether you agree: This reviewer does not agree with the conclusions of the authors that the finding of extra ribs, or 13thrib, in this particular study are not a sign of developmental toxicity. While it is agreed that the biological significance of an altered incidence of anatomical variations is difficult to assess, the incidence of 13th rib in this study showed a dose-related increase with statistical significance at the highest dosed-group, in the absence of maternal toxicity, and therefore is evaluated as a possible indication of developmental toxicity.
REFERENCE Gortner, E.G., E.G. Lamprecht, M.T. Case. 1982. Oral teratology study of T-3141CoC in_rabbits. 3M Company. Riker Laboratories, Inc., St. Paul, MN. Experiment No. 0681TB0398,.February 1982.
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DEVELOPMENTAL TOXICITY
Title: The Embryo-Fetal Toxicity and Teratogenic Potential of Ammonium Perfluorooctanoate in the Rat
TEST SUBSTANCE
Identity: Ammonium perfluorooctanoate (APFO), pentadecafluorooctanoic acid ammonium salt, ammonium perfluorooctanoate, ammonium perfluorocaprylate, PFO, FC-116, FC-126, FC-169, FC-143, or as a major component of FX-1003 (CASRN 3825-26-1)
Remarks: The test substance, APFO [CF3(CF2)6COONH4], was obtained from 3M (St. Paul, MN 55144). Its purity was >95%; the contaminants present were [CF3(CF2)4COONH4] and PFOA isomers. No inhibitor, carriers, or additives were present. Degradation of APFO was considered insignificant unless the temperature was to exceed 250C (unpublished Du Pont data).
METHOD
Method/Guideline followed: Not specified
GLP (Y/N): Not specified
Year study performed: 1984
Species/Strain: Rat/Sprague-Dawley derived, Crl .CD (SD)BR strain.
Number of animals per dose: For the inhalation portion of the study, two trials with 12 mated female rats/group/trial. Two additional, pair-fed groups (6 dams/group), were added to Experiment 1 (teratology), and two groups (6 dams/group) were added to Experiment 2 (dams allowed to litter). For the gavagecxposurc portion of the study, 25 and 12 mated female rats at each exposure concentration were included in Experiment 1(teratology) and Experiment 2 (dams allowed to litter), respectively.
Route of administration: Inhalation and oral
Dosing regimen (list all with units): For the inhalation portion of the study, the two trials consisted of 12 pregnant Sprague-Dawley rats per group exposed to APFO by whole-body vapor inhalation to 0, 0.1, 1,10, and 25 mg/m36 hours/day, on GD 6-15; two additional groups (6 dams per group) that were pairfed to the 10 and 25 mg/m3groups, were added to each trail. In the oral portion of the study, 25 and 12 Sprague-Dawley rats for the first and second trials, respectively, were administered 0 and 100 mg/kg/day APFO in com oil by gavage on GD 6-15.
Doses: For inhalation exposures: 0, 0.1, 1, 10, and 25 mg/m3, with two additional groups pair-fed to the 10 and 25 mg/m3 groups; for oral exposures: 0 and 100 mg/kg/day.
Statistical methods used: The litter was used as the experimental unit for the purpose of statistical evaluation (Staples and Haseman, 1974; Haseman and Hogan, 1975). The significance of differences in the incidence of pregnancy, clinical signs, and maternal death was determined by use of the Fisher-exact probability test (Siegel, 1956). A two-way analysis of variance was used to detect differences in feed consumption among breeding lots and between groups. Dunnett's test (Steel and Torrie, 1960) was used to test the statistical significance of differences between the control and APFO groups in maternal body
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weight, in body weight gain, and in feed consumption when the one-way analysis of variance was significant. The presence of concentration-related responses for the inhalation portion of the study was determined by Jonckheere's test (Jonckheere, 1954). The significance of differences in incidence of structural alterations between the control group and the APFO group was determined by application of the Mann-Whitney U test (Mann and Whitney, 1947). When more than 75% ties occurred in the data, the Fisher's exact probability test was applied (Haseman and Hoel, 1974). The level of significance selected was p [ 0.05. Variability about means was expressed as standard error of the mean (SE). In addition, several reproductive indices were calculated for some results from Experiment 2 (dams allowed to litter).
Remarks - Detail and discuss any significant protocol parameters and deviations: The study design consisted of an inhalation and an oral portion, each with two trials or experiments. The first trial was the teratology portion of the study, in which the dams were sacrificed on GD 21; while in the second trial, the dams were allowed to litter and the pups were sacrificed on day 35 post-partum. For the inhalation portion of the study, the two trials consisted of 12 pregnant Sprague-Dawley rats per group exposed to APFO by whole-body vapor inhalation to 0, 0.1, 1, 10, and 25 mg/m36 hours/day, on GD 6-15. In the oral portion of the study, 25 and 12 Sprague-Dawley rats for the first and second trials, respectively, were administered 0 and 100 mg/kg/day APFO in corn oil by gavage on GD 6-15. For both routes of administration, females were mated on an as-needed basis and when the number of mated females were bred, they were ranked within breeding days by body weight and assigned to groups by rotation in order of rank. Finally, two additional groups (six dams per group) that were pair-fed to the 10 and 25 mg/m3 groups, were added to each trail.
For the teratology portion of the study (trial one), dams were weighed on GD 1, 6, 9, 13, 16, and 21 and observed daily for abnormal clinical signs. On GD 21, the dams were sacrificed by cervical dislocation and examined for any gross abnormalities, liver weights were recorded and the reproductive status of each animal was evaluated. The ovaries, uterus and contents were examined for the number of corpora lutea, live and dead fetuses, resorptions and implantation sites. Pups (live and dead) were counted, weighed and sexed and examined for external, visceral, and skeletal alterations. The heads of all control and high-dose group fetuses were examined for visceral alterations as well as macro- and microscopic evaluation of the eyes.
For trial two, in which the dams were allowed to litter, the procedure was the same as that for trial one up to GD 21. Two days before the expected day of parturition, each dam was housed in an individual cage. The date of parturition was noted and designated Day 1 post-partum (PP). Dams were weighed and examined for clinical signs on Days 1, 7, 14, and 22 PP. On Day 23 PP all dams were sacrificed. Pups were counted, weighed, and examined for external alterations. Each pup was subsequently weighed and inspected for adverse clinical signs on Days 4, 7, 14, and 22 PP. The eyes of the pups were also examined on Days 15 and 17 PP for the inhalation portion and on Days 27 and 31 PP for the gavage portion of the study. Pups were sacrificed on Day 35 PP and examined for visceral and skeletal alterations.
RESULTS
NOAEL (dose and effect) - maternal and developmental:
Inhalation: For trial one - the NOAEL for maternal toxicity is 1 mg/m3; the NOAEL for developmental toxicity is 10 mg/m3. For trial two - the NOAEL for maternal toxicity is lmg/m3; the NOAEL for developmental toxicity is 10 mg/m3.
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Oral: A NOAEL could not be determined for either maternal or developmental toxicity since this portion of the study used only one dose level.
LOAEL (dose and effect) - maternal and developmental: Inhalation:
For trial one - the LOAEL for maternal toxicity is 10 mg/m3, based on treatment-related clinical signs (consisting of wet abdomens, chromodacryorrhea, chromorhinorrhea, and a general unkept appearance), and significant reductions in food consumption and body weight; the LOAEL for developmental toxicity is 25 mg/m3, based on reductions in mean fetal body weights and a statistically significant increased incidence of fetuses with partially ossified stemebrae. For trial two - the LOAEL for maternal toxicity is 10 mg/m3, based on treatment-related clinical signs consisting of wet abdomens, chromodacryorrhea, chromorhinorrhea, and a general unkept appearance; the LOAEL for developmental toxicity is 25 mg/m3, based on statistically significant reductions in pup body weight.
Oral: A LOAEL could not be determined for either maternal or developmental toxicity since this portion of the study used only one dose level.
Toxic response/effects by dose level - maternal: Inhalation At 10 and 25 mg/m3, treatment-related clinical signs of maternal toxicity, and reductions in food consumption and body weight; at 25 mg/m3, statistically significant increases in liver weights; lethargy and death. Oral At 100 mg/kg/day, the only dose tested: clinical signs of toxicity, reductions in food
Consumption, reductions in body weight gains, and deaths.
Toxic response/effects by dose level - developmental: Inhalation At 25 mg/m3, reductions in mean fetal and pup body weights and statistically significant increases in the incidence of fetuses with partially ossified sternebrae. Oral At 100 mg/kg/day, the only dose tested, no signs of developmental toxicity were observed.
Statistical results:
Inhalation - maternal At 10 and 25 mg/m3: treatment-related clinical signs of maternal toxicity (no statistical significance assigned), significant reductions in food consumption (21.8 + 0.46 vs 23.4 + 0.38 in controls), and significant reductions in body weight with statistical significance at 25 mg/m3(Dunnett's test, 0<0.05); at 25 mg/m3: statistically significant increases in mean liver weights (two-tailed Mann-Whitney U test, p<0.05); lethargy (4 out of 12) and death (3 out of 12 dams). Inhalation - developmental
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At 25 mg/m3, reductions in mean fetal (p = 0.002) and pup body weights (p = 0.02), and statistically significant increases (p = 0.04 by the two-tailed Mann-Whitney U test) in the incidence of fetuses with partially ossified sternebrae.
Oral - maternal At 100 mg/kg/day, the only dose tested: clinical signs of toxicity (no statistical
significance assigned), reductions in food consumption (no statistical significance assigned), reductions in body weights gains (p < 0.05), and deaths (3 out of 12 dams).
Oral - developmental No statistically significant differences were noted between treated and control groups for any of the parameters measured.
Remarks - Additional information to adequately assess the data:
Inhalation Exposure
Trial One:
Treatment-related clinical signs of maternal toxicity for trial one (teratology) occurred at 10 and 25 mg/m ' and consisted of wet abdomens, chromodacryorrhea, chromorhinorrhea, a general unkept appearance, and lethargy in four dams at the end of the exposure period (high-concentration group only). Three out of 12 dams died during treatment at 25 mg/m3 (on GD 12, 13, and 17). Food consumption was significantly reduced at both 10 and 25 mg/m3; however, no significant differences were noted between treated and pair-fed groups. Significant reductions in body weight were also observed at these concentrations, with statistical significance at the high-concentration only. Likewise, statistically significant increases in mean liver weights were seen at the high-concentration group. Under the conditions of the study, a NOAEL and LOAEL for maternal toxicity of 1 and 10 mg/m3, respectively, was indicated.
No effects were observed on the maintenance of pregnancy or the incidence of resorptions. Mean fetal body weights were significantly decreased in the 25 mg/m3 group and in the control group pair-fed 25 mg/m'. A detailed microscopic visceral and eye examination of the fetuses did not reveal any treatmentrelated effects; however in the control group that was pair-fed 25 mg/m3, a statistically significant increased incidence of fetuses with partially ossified sternebrae was observed. Under the conditions of the study, a NOAEL and LOAEL for developmental toxicity of 10 and 25 mg/m3, respectively, was indicated.
Trial Two:
Clinical signs of maternal toxicity seen at 10 and 25 mg/m3were similar in type and incidence as those described for trial one. Maternal body weight gain during treatment at 25 mg/m3was less than controls, although the difference was not statistically significant. In addition, 2 out of 12 dams died during treatment at 25 mg/m3. No other treatment-related effects were reported, nor were any adverse effects noted for any of the measurements of reproductive performance. Under the conditions of the study, a NOAEL and LOAEL for maternal toxicity of 1 and 10 mg/m3, respectively, were indicated.
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Signs of developmental toxicity in this group consisted of statistically significant reductions in pup body weight on Day 1PP (6.1 g at 25 mg/m3vs. 6.8 g in controls). On Days 4 and 22 PP, pup body weights continued to remain lower than controls, although the difference was not statistically significant (Day 4 PP: 9.7 g at 25 mg/m3vs. 10.3 in controls; Day 22 PP: 49.0 g at 25 mg/m3vs. 50.1 in controls). No significant effects were reported following external examination of the pups or with ophthalmoscopic examination of the eyes. Under the conditions of the study, a NOAEL and LOAEL for developmental toxicity of 10 and 25 mg/m3, respectively, were indicated. Oral Exposure Trial One: Three out of 25 dams died during treatment of 100 mg/kg APFO during gestation (one death on GD 11; two on GD 12). Clinical signs of maternal toxicity in the dams that died were similar to those seen with inhalation exposure. Food consumption and body weights were reduced in treated animals compared to controls. No adverse signs of toxicity were noted for any of the reproductive parameters such as maintenance of pregnancy or incidence of resorptions. Likewise, no significant differences between treated and control groups were noted for fetal weights, or in the incidences of malformations and variations; nor were there any effects noted following microscopic examination of the eyes. Trial Two: Similar observations for clinical signs were noted for the dams as in trial one. Likewise, no adverse effects on reproductive performance or in any of the fetal observations were noted. CONCLUSIONS Comment on author's conclusions and whether you agree: The author's conclusions appear to be supported by the data. REFERENCE Provide full citation of study reviewed: Staples, R.E. et al. 1984. The Embryo-Fetal Toxicity and Teratogenic Potential of Ammonium Perfluorooctanoate (PFOA) in the Rat. Fundamental and Applied Toxicology. 4:429-440. This study was performed at Haskell Laboratory for Toxicology and Industrial Medicine, Newark, DE 19711.
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REPRODUCTIVE TOXICITY STUDIES
Title: ORAL (GAVAGE) TWO-GENERATION (ONE LITTER PER GENERATOIN) REPRODUCTION STUDY OF AMMONIUM PERFLUOROOCTANOATE (APFO) IN RATS - ARGUS RESEARCH LABORATORIES STUDY NUMBER: T-6889.6, 2002.
TEST SUBSTANCE
Identity: Ammonium Perfluorooctanoate, CAS No. 3825-26-1
Remarks: The test substance was received on November 7, 2000 and stored at room temperature. Solutions of the test substance were prepared weekly at the testing facility and prepared formulations were stored refrigerated, protected from light. Information regarding the purity, identity, strength and composition of the test article is on file with the Sponsor.
METHOD
Method/Guideline followed (i.e., OECD 414, etc.): The requirements of the U.S. Environmental Protection Agency (EPA) were used as a basis for the study design.
Type of study (one-generation, two-generation, etc.): Two-generation reproductive toxicity
GLP (Y/N): The study was conducted in compliance with the Good Laboratory Practice (GLP) regulations of the U.S. EPA and the Organization for Economic Co-Operation and Development (OECD). There were no deviations from the GLP regulations that affected the quality or integrity of the study. Quality Assurance Unit findings derived from the inspections during the conduct of this study have been documented.
Year study performed: 2001
Species/Strain: Sprague Dawley rats
Sex (males/females/both): Both
Number of animals per dose: 30
Route of administration: Gavage
Dosing regimen (list all with units): Five groups of 30 rats per sex per dose group were administered PFOS by gavage for six weeks prior to and during mating. Treatment of the F0 male rats continued until mating was confirmed, and treatment of the F0 female rats continued throughout gestation, parturition, and lactation.
Doses: 0, 1,3, 10, and 30 mg/kg/day
Premating exposure period for males/females (P and FI, if appropriate): Male and female P generation rats were given test substance once daily, beginning at approximately six weeks of age
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(at least 70 days before cohabitation) and continuing until the day before sacrifice. FI generation males and females were given dosages once daily, beginning at weaning (approximately 70 days before cohabitation) and continuing until the day before sacrifice.
Statistical methods used: Continuous data (body weights, body weight changes, feed consumption data, durations of gestation and delivery, litter averages for pup body weights and percent male fetuses or pups and mortality, and cumulative survival) were analyzed using Bartlett's Test of Flomogeneity of Variance and Analysis of Variance (ANOVA) when appropriate (i.e., Bartlett's Test was not significant or p>0.001). If the ANOVA was significant (p < 0.05), Dunnetf s Test was used to identify the statistical significance of the individual groups. If the ANOVA was not appropriate, i.e., p< 0.001, the Kruskal-Wallis Test was used. In cases where the Kruskal-Wallis Test was statistically significant (p < 0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test was used. All other natural delivery data involving discrete data were evaluated using Kruskal-Wallis Test procedures previously described.
Remarks - Detail and discuss any significant protocol parameters and deviations:
F0 Generation:
The F0 animals were examined twice daily for clinical signs, abortions, premature deliveries, and deaths. Body weights of F0 male rats were recorded weekly during the dosage period and then on the day of sacrifice. Body weights of F0 female rats were recorded weekly during the pre- and cohabitation periods and then on gestation days (GD) 0, 7, 10, 14, 18, 21, and 25 (if necessary) and on lactation days (LD) 1, 5, 8, 11, 15, and 22 (terminal body weight). Food consumption values in F0 male rats were recorded weekly during the treatment period, while in F0 female rats, values were recorded weekly during the precohabitation period, on GDs 0, 7, 10, 14, 18, 21, and 25 and on LDs 1, 5, 8, 11, and 15.
Estrous cycling was evaluated daily by examination of vaginal cytology beginning 21 days before the scheduled cohabitation period and continuing until confirmation of mating by the presence of sperm in a vaginal smear or confirmation of a copulatory plug. On the day of scheduled sacrifice, the stage of the estrous cycle was assessed.
Within each dosage group, consecutive order was used to assign parental generation rats to cohabitation, one male rat per female rat. The cohabitation period consisted of a maximum of 14 days. Female rats with evidence of sperm in a vaginal smear or copulatory plug were designated as GD 0 and assigned to individual housing. Parental females were evaluated for length of gestation, fertility index, gestation index, number and sex of offspring per litter, number of implantation sites, general condition of the dam and litter during the postpartum period, litter size and viability, viability index, lactation index, percent survival, and sex ratio. Maternal behavior of the dams was recorded on LDs 1 ,5 ,8 , 15, and 22.
F0 generation animals were sacrificed by carbon dioxide asphyxiation (day 106 to 110 of the study for male rats, i.e., after completion of the cohabitation period; and LD 22 for female rats), necropsied, and examined for gross lesions. Gross necropsy included examination of external surfaces and orifices, as well as internal examination of tissues and organs. Individual organs were weighed and organ-to-body weight and organ-to-brain weight ratios were calculated for the brain, kidneys, spleen, ovaries, testes, thymus, liver, adrenal glands, pituitary, uterus with oviducts and cervix, left epididymis (whole and cauda), right epididymis, prostate and seminal vesicles, (with coagulating glands and with and without fluid). Tissues retained in neutral buffered 10% formalin for possible histological evaluation included the
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pituitary, adrenal glands, vagina, uterus, with oviducts, cervix and ovaries, right testis, seminal vesicles, right epididymis, and prostate. Histological examination was performed on tissues from 10 randomly selected rats per sex from the control and high dosage groups. All gross lesions were examined histologically. All F0 generation rats that died or appeared moribund were also examined.
Histological examination of the reproductive organs in the low- and mid-dose groups was conducted in rats that exhibited reduced fertility by either failing to mate, conceive, sire, or deliver healthy offspring; or for which estrous cyclicity or sperm number, motility, or morphology were altered. Sperm number, motility, and morphology were evaluated in the left cauda epididymis of F0 generation male rats; testicular spermatid concentrations were evaluated in the left testis. The number and distribution of implantation sites were recorded in F0 generation female rats. Rats that did not deliver a litter were sacrificed on GD 25 and examined for pregnancy status. Uteri of apparently nonpregnant rats were examined to confirm the absence of implantation sites. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Female rats without a confirmed mating date that did not deliver a litter were sacrificed on an estimated day 25 of gestation.
At scheduled sacrifice, after completion of the cohabitation period in F0 male rats and on LD 22 in F0 female rats, blood samples (10 males and 10 females each for the 10 and 30 mg/kg/day dose groups; 3 males and 3 females for the control group) were collected and frozen for future analysis. The methods section cites that liver samples were also collected, but no other details were provided and the results did not appear to be available at the time of the report.
FI Generation:
The F 1 generation pups in each litter were counted once daily. Physical signs (including variations from expected lactation behavior and gross external physical anomalies) were recorded for the pups each day. Pup body weights were recorded on LDs 1, 5, 8, 15 and 22. On LD 12, all FI generation male pups were examined for the presence of nipples. Pups that died before examination of the litter for pup viability on LD 1 were evaluated for vital status at birth. Pups found dead on LDs 2 to 22 were examined for gross lesions and for the cause of death. All FI generation rats were weaned on LD 22 based on observed growth and viability of these pups.
At weaning (LD 22), two FI generation pups per sex per litter per group (60 male and 60 female pups per group) were selected for continued evaluation, resulting in 600 total rats (300 rats per sex) assigned to the five dosage groups. At least two male pups and two female pups per litter, when possible, were selected. F1 generation pups not selected for continued observation for sexual maturation were sacrificed. Three pups per sex per litter were examined for gross lesions. Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. The brain, spleen and thymus from one of the three selected pups per sex per litter were weighed and the brain, spleen, and thymus from the three selected pups per sex per litter were retained for possible histological evaluation. All remaining pups were discarded without further examination.
The F 1 generation rats were given the same dosage level of the test substance and in the same manner as their respective F0 generation sires and dams. Dosages were given once daily, beginning at weaning and continuing until the day before sacrifice. FI generation female rats were examined for age of vaginal patency, beginning on day 28 postpartum (LD 28). FI generation male rats were evaluated for age of preputial separation, beginning on day 39 postpartum (LD 39). Body weights were recorded when rats reached sexual maturation. A table of random units was used to assign FI generation rats to cohabitation, one male rat per female rat. If random assignment to cohabitation resulted in the pairing of FI generation siblings, an alternate assignment was made. The cohabitation period consisted of a maximum of 14 days.
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Body weights o f the FI generation male rats were recorded weekly during the postweaning period and on the day o f sacrifice. Body weights o f the FI generation female rats were recorded weekly during the postw eaning period to cohabitation, and on DGs 0, 7, 10, 14, 18, 21 and 25 (if necessary) and on LDs 1, 5, 8, 11, 15 and 22. Food consum ption values for the F I generation male rats were recorded weekly during the dosage period. Food consumption values for the FI generation female rats were recorded weekly during the postweaning period to cohabitation, on GDs 0, 7, 10, 14, 18, 21 and 25 and on LDs 1,5, 8, 11 and 15. Because pups begin to consume m aternal food on or about LD 15, food consumption values were not tabulated after LD 15.
At scheduled sacrifice, after completion of the cohabitation period in male rats and on LD 22 in female rats, blood samples (10 males and 10 females each for the 10 and 30 mg/kg/day dose groups; 3 males and 3 females for the control group) were collected and frozen for future analysis, but only the serum analyses for the F0 generation were presented in the report. The methods section cites that liver samples were also collected, but no other details were provided and the results did not appear to be available at the time of the report.
F2 generation litters were examined after delivery to identify the number and sex of pups, stillbirths, live births and gross alterations. Each litter was evaluated for viability at least twice each day of the 22-day postpartum period. Dead pups observed at these times were removed from the nesting box. Anogenital distance was measured for all live F2 generation pups on LDs 1 and 22.
RESULTS
NOAEL (dose and effect) - for F0, FI, and F2 (as appropriate): A NOAEL for the F0 parental males could not be determined since treatment-related effects were seen at all doses tested. The NOAEL for F0 parental females = 10 mg/kg/day. A NOAEL for the FI males could not be determined since treatment-related effects were seen at all doses tested. The NOAEL for FI generation females = 10 mg/kg/day. The NOAEL for the F2 generation offspring = 30 mg/kg/day; the highest dose tested.
LOAEL (dose and effect) - for F0, FI, and F2 (as appropriate): The LOAEL for F0 parental males is considered to be 1 mg/kg/day, the lowest dose tested, based on significant increases in the liver and kidney weights-to-terminal body weight and to brain weight ratios. The LOAEL for F0 parental females is considered to be 30 mg/kg/day, based on significant reductions in kidney weight and kidney weight-toterminal body weight and to brain weight ratios observed at the highest dose. The LOAEL for FI generation males is considered to be 1 mg/kg/day, based on significant decreases in body weights and body weight gains, and in terminal body weights; and significant changes in absolute liver and spleen weights and in the ratios of liver, kidney, and spleen weights-to-brain weights; and based on significant, dose-related reductions in body weights and body weight gains observed prior to and during cohabitation and during the entire dosing period. The LOAEL for FI generation females is considered to be 30 mg/kg/day, based on statistically significant increases in postweaning mortality, delays in sexual maturation, decreases in body weight and body weight gains, and decreases in absolute food consumption, all observed at the highest dose tested. A LOAEL for the F2 generation could not be determined; under the conditions of the study, no treatment-related effects were observed at any doses tested.
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Toxic effects in FO generation animals: FO males; Statistically significant increases in clinical signs were observed in the high-dose group. Significant reductions in body weight and body weight gain were reported for most of the dosage period and continuing until termination of the study in the 3, 10, and 30 mg/kg/day dose groups. Absolute food consumption values were also significantly reduced during these periods at the 30 mg/kg/day dose group, while significant increases in relative food consumption values were observed in the 3, 10, and 30 mg/kg/day within those same periods. At necropsy, statistically significant reductions in terminal body weights were seen at 3, 10, and 30 mg/kg/day. Absolute weights of the left and right epididymides, left cauda epididymis, seminal vesicles (with and without fluid), prostate, pituitary, left and right adrenals, spleen, and thymus were also significantly reduced at 30 mg/kg/day. The absolute weight of the liver was significantly increased in all dose-groups. Kidney weights were significantly increased in the 1, 3, and 10 mg/kg/day dose groups, but significantly decreased in the 30 mg/kg/day group. All organ weight-to-terminal body weight and ratios were significantly increased in all treated groups. Organ weight-to-brain weight ratios were significantly reduced for some organs at the high dose group, and significantly increased for other organs among all treated groups. Increased thickness and prominence of the zona glomerulosa and vacuolation of the cells of the adrenal cortex were observed in the 10 and 30 mg/kg/day dose groups. F0 females; The weights of the left and right kidney, and the ratios of these organ weights-to-terminal body weight and of the left kidney weight-to-brain weight were significantly reduced at the highest dose of 30 mg/kg/day.
Toxic effects in the FI generation animals: FI males; Necroscopic examination revealed statistically significant treatment-related effects at 3, 10, and 30 mg/kg/day ranging from tan areas in the lateral and median lobes of the liver to moderate to slight dilation of the pelvis of one or both kidneys. Statistically significant, dose-related decreases in terminal body weights were observed. The absolute weights of the liver and spleen were significantly decreased at all treated groups. The absolute weights of the left and/or right kidneys were significantly decreased in the 30 mg/kg/day dose group. The absolute weight of the thymus was also significantly decreased in the 10 and 30 mg/kg/day dose groups. The absolute weight of the prostate, brain and left adrenal gland were significantly decreased in the 30 mg/kg/day dosage group. The ratios of the weights of the seminal vesicles, with and without fluid, liver and left and right kidneys to the terminal body weights were significantly increased in all treated groups. The ratios of the weights of the left testis, with and without the tunica albuginea and the right testis to the terminal body weight, were significantly increased at 3 mg/kg/day and higher. The ratios of the weights of the left epididymis, left cauda epididymis, right epididymis and brain to the terminal body weight were significantly increased at 10 mg/kg/day and higher. The ratios of the weight of the seminal vesicles with fluid to the brain weight were increased at 1 mg/kg/day and higher, with statistical significance at 1 and 10 mg/kg/day. The ratios of the liver weight-to-brain weight were significantly increased in the 1 mg/kg/day and higher dosage groups, and the ratios of the left and right kidney weights-to-brain weight were significantly increased in all treated groups. The ratios of the spleen weight-to-brain weight were significantly decreased at 1 mg/kg/day and higher, and the ratios of the thymus weight-to-brain weight were significantly decreased at 10 and 30 mg/kg/day. The ratios of the left and right testes weight-to-brain weight were increased in the 3 mg/kg/day and higher dosage groups. These ratios were significantly increased at 10 mg/kg/day (right testis only) and 30 mg/kg/day. Treatment-related microscopic changes were observed in the adrenal glands of high-dose animals (cytoplasmic hypertrophy and vacuolation of the cells of the adrenal cortex) and in the liver of animals treated with 3, 10, and 30 mg/kg/day (hepatocellular hypertrophy). FI females; Statistically significant increases in the average numbers of estrous stages per 21 days were observed in high-dose animals (4.7 versus 5.4 in controls). Statistically significant decreases in body weights and body weight gains were observed in high-dose animals on days 50 and 57 postweaning, during precohabitation (recorded on the day cohabitation began, when FI generation rats were 92-106 days of age), and during gestation and lactation. Decreases in absolute food consumption were observed during during precohabitation and during gestation and lactation in animals treated with 30 mg/kg/day.
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Toxic response/effects by dose level - offspring (F1/F2): Toxic effects in F 1 generation male pups consisted of significant reductions in pup body weight on a per litter basis in the 30 mg/kg/day group on days 1,5, and 8 of lactation. Significant increases in treatment-related deaths (7 animals total) were reported in FI males in the high dose group of 30 mg/kg/day. One rat was moribund sacrificed on day 39 postweaning and another was found dead on day 107 postweaning, but the majority of the FI male rats were found dead on days 2-4 postweaning. Body weights and body weight gains were statistically significantly reduced prior to and during cohabitation and during the entire dosing period in all treated groups. Statistically significant reductions in body weights were observed at 10 and 30 mg/kg/day during days 8-15, 22-29, 29-36, 43-50, and 50-57 postweaning. Body weight gains were also significantly reduced in the 30 mg/kg/day group on days 1-8, 15-22, 36-43, 57-64, and 64-70 postweaning. Statistically significant, dose-related reductions in body weight gains were observed for the entire dosage period (days 1-113 postweaning). Absolute food consumption values were significantly reduced at 10 and 30 mg/kg/day during the entire precohabitation period (days 1-70 postweaning), while relative food consumption values were significantly increased. Statistically significant delays in sexual maturation (the average day of preputial separation) were observed in high-dose animals versus concurrent controls (52.2 days of age versus 48.5 days of age, respectively). Toxic effects in FI generation female pups consisted of significant reductions in pup body weight on a per litter basis in the 30 mg/kg/day group on days 1,5, and 8 of lactation. An increase in treatment-related mortality (6 animals total) was observed in FI females on postweaning days 2-8 at the highest dose of 30 mg/kg/day. Statistically significant decreases in body weights and body weight gains were observed in high-dose animals on days 8, 15, 22, and 29 postweaning, and during lactation. Decreases in absolute food consumption were observed during days 1-8, 8-15 postweaning and during lactation in animals treated with 30 mg/kg/day. Statistically significant delays in sexual maturation (the average day of vaginal patency) were observed in high-dose animals versus concurrent controls (36.6 days of age versus 34.9 days of age, respectively).
Statistical results:
F0 generation male animals: Statistically significant increases (p< 0.01) in clinical signs were observed in the high-dose group. Significant reductions (p< 0.05 or p< 0.01) in body weight and body weight gain were reported for most of the dosage period and continuing until termination of the study in the 3,10, and 30 mg/kg/day dose groups. Absolute food consumption values were also significantly reduced (p< 0.01) during these periods at the 30 mg/kg/day dose group, while significant increases (p< 0.05 or p< 0.01) in relative food consumption values were observed in the 3, 10, and 30 mg/kg/day within those same periods. At necropsy, statistically significant reductions (p< 0.01) in terminal body weights were seen at 3, 10, and 30 mg/kg/day. Absolute weights of the left and right epididymides, left cauda epididymis, seminal vesicles (with and without fluid), prostate, pituitary, left and right adrenals, spleen, and thymus were also significantly reduced (p< 0.05 or p< 0.01) at 30 mg/kg/day. The absolute weight of the liver was significantly increased (p< 0.01) in all dose-groups. Kidney weights were significantly increased (p< 0.05 or p< 0.01) in the 1, 3, and 10 mg/kg/day dose groups, but significantly decreased (p< 0.05 or p< 0.01) in the 30 mg/kg/day group. All organ weight-to-terminal body weight and ratios were significantly increased (p< 0.05 or p< 0.01) in all treated groups. Organ weight-to-brain weight ratios were significantly reduced for some organs at the high dose group, and significantly increased for other organs among all treated groups.
F0 generation female animals: The weights of the left and right kidney, and the ratios of these organ weights-to-terminal body weight and of the left kidney weight-to-brain weight were significantly reduced (p< 0.05 or p< 0.01) at the highest dose of 30 mg/kg/day.
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FI generation offspring: Males: Significant reductions (p< 0.01) in pup body weight on a per litter basis was observed in the 30 mg/kg/day group on days 1, 5, and 8 of lactation. Significant increases in treatment-related deaths (7 animals total) were reported in FI males in the high dose group of 30 mg/kg/day. One rat was moribund sacrificed on day 39 postweaning and another was found dead on day 107 postweaning, but the majority of the FI male rats were found dead on days 2-4 postweaning. Body weights and body weight gains were statistically significantly reduced prior to and during cohabitation and during the entire dosing period in all treated groups. Statistically significant reductions (p< 0.05 or p< 0.01) in body weights were observed at 10 and 30 mg/kg/day during days 8-15, 22-29, 29-36, 43-50, and 50-57 postweaning. Body weight gains were also significantly reduced (p< 0.05 or p< 0.01) in the 30 mg/kg/day group on days 1-8, 15-22, 36-43, 57-64, and 64-70 postweaning. Statistically significant (p< 0.05 or p< 0.01), dose-related reductions in body weight gains were observed for the entire dosage period (days 1-113 postweaning). Absolute food consumption values were significantly reduced (p< 0.01) at 10 and 30 mg/kg/day during the entire precohabitation period (days 1-70 postweaning), while relative food consumption values were significantly increased (p< 0.05 or p< 0.01). Statistically significant delays (p< 0.01) in sexual maturation (the average day of preputial separation) were observed in high-dose animals versus concurrent controls (52.2 days of age versus 48.5 days of age, respectively). Females: significant reductions (p< 0.01) in pup body weight on a per litter basis in the 30 mg/kg/day group on days 1,5, and 8 of lactation. An increase in treatment-related mortality (6 animals total) was observed in FI females on postweaning days 2-8 at the highest dose of 30 mg/kg/day. Statistically significant decreases (p< 0.05 or p< 0.01) in body weights and body weight gains were observed in high-dose animals on days 8, 15, 22, and 29 postweaning, and during lactation. Significant decreases (p< 0.05 or p< 0.01) in absolute food consumption were observed during days 1-8, 8-15 postweaning and during 0.02) lactation in animals treated with 30 mg/kg/day. Statistically significant delays (p< 0.01) in 0.03) sexual maturation (the average day of vaginal patency) were observed in high-dose animals 0.04) versus concurrent controls (36.6 days of age versus 34.9 days of age, respectively).
FI generation adult animals: Males; Necroscopic examination revealed statistically significant (p< 0.05 or p< 0.01) treatment-related effects at 3, 10, and 30 mg/kg/day ranging from tan areas in the lateral and median lobes of the liver to moderate to slight dilation of the pelvis of one or both kidneys. Statistically significant (p< 0.05 or p< 0.01), dose-related decreases in terminal body weights were observed. The absolute weights of the liver (p< 0.01) and spleen (p< 0.05 or p< 0.01) were significantly decreased at all treated groups. The absolute weights of the left and/or right kidneys were significantly decreased (p< 0.01) in the 30 mg/kg/day dose group. The absolute weight of the thymus was also significantly decreased (p< 0.01) in the 10 and 30 mg/kg/day dose groups. The absolute weight of the prostate, brain and left adrenal gland were significantly decreased (p< 0.05 or p< 0.01) in the 30 mg/kg/day dosage group. The ratios of the weights of the seminal vesicles, with and without fluid, liver and left and right kidneys to the terminal body weights were significantly increased (p< 0.05 or p< 0.01) in all treated groups. The ratios of the weights of the left testis, with and without the tunica albuginea and the right testis to the terminal body weight, were significantly increased (p< 0.05 or p< 0.01) at 3 mg/kg/day and higher. The ratios of the weights of the left epididymis, left cauda epididymis, right epididymis and brain to the terminal body weight were significantly increased (p < 0.05 or p < 0.01) at 10 mg/kg/day and higher. The ratios of the weight of the seminal vesicles with fluid to the brain weight were increased at 1 mg/kg/day and higher, with statistical significance (p < 0.05) at 1 and 10 mg/kg/day. The ratios of the liver weight-to-brain weight were significantly increased (p < 0.01 jin the 1 mg/kg/day and higher dosage groups, and the ratios of the left and right kidney weights-to-brain weight were significantly increased (p < 0.05 or p < 0.01) in all treated groups. The ratios of the spleen weight-to-brain weight were significantly decreased (p < 0.05 or p < 0.01) at 1 mg/kg/day and higher, and the ratios of the thymus weight-to-brain weight were significantly decreased (p < 0.05 or p < 0.01) at 10 and 30 mg/kg/day. The ratios of the left
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and right testes weight-to-brain weight were increased in the 3 mg/kg/day and higher dosage groups. These ratios were significantly increased (p < 0.05 or p < 0.01) at 10 mg/kg/day (right testis only) and 30 mg/kg/day. Females: Statistically significant increases (p <0.01) in the average numbers of estrous stages per 21 days were observed in high-dose animals (4.7 versus 5.4 in controls). Statistically significant decreases (p < 0.05 or p < 0.01) in body weights and body weight gains were observed in highdose animals on days 50 and 57 postweaning, during precohabitation (recorded on the day cohabitation began, when FI generation rats were 92-106 days of age), and during gestation and lactation. Significant decreases (p < 0.05 or p < 0.01) in absolute food consumption were observed during during precohabitation and during gestation and lactation in animals treated with 30 mg/kg/day.
F2 generation offspring: Under the conditions of the study, no statistically significant, treatmentrelated effects on any of the observed parameters were noted in the F2 generation offspring.
Remarks:
Prior to mating, the study authors noted a statistically significant increase in the average numbers of estrus stages per 21 days in high-dose animals (5.4 versus 4.7 in controls). For this calculation, the number of independent occurrences of estrus in the 21 days of observation was determined. This type of calculation can be used as a screen for effects on the estrus cycle, but a more detailed analysis should then be conducted to determine whether there is truly an effect. 3M Company (2002) recently completed an analysis that showed there were no effects on the estrus cycle; there were no differences in the number of females with > 3 dyas of estrus or with > 4 days of diestrus in the control and high dose groups. Analyses conducted by the US EPA (2002) also demonstrated that there were no differences in the estrus cycle among the control and high dose groups. The cycles were evaluated as having either regular 4-5 day cycles (R), uneven cycling (IR; defined as brief periods with irregular pattern) or periods of prolonged diestrus (defined as 4-6 day diestrus periods) extended estrus (defined as 3 or 4 days of cornified smears), possibly pseudopregnant, (PSP; defined as 6-greater days of leukocytes) or persistent estrus (PE; defined as 5-or greater days of cornified smears). The data are summarized in below. The two groups were not different in any of the parameters measured.
Ovarian Pattern R Ri
(i
Cl
cc
PSP PE
Type Irregularity
Total 3 day Estrus 4 day diestrus no pattern
Control 18 10 2 2 6 1 1
APFO (30m g /kg ) 20 9 1 2 6
1
Thus, the increase in the number of estrus stages per 21 days that was noted by the study authors is due to the way in which the calculation was done, and is not biologically meaningful. Parental Males (F0)
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One FO male rat in the 30 mg/kg/day dose group was sacrificed on day 45 of the study due to adverse clinical signs (emaciation, cold-to-touch, and decreased motor activity). Necroscopic examination in that animal revealed a pale and tan liver, and red testes. All other FO generation male rats survived to scheduled sacrifice. Statistically significant increases in clinical signs were also observed in male rats in the high-dose group that included dehydration, urine-stained abdominal fur, and ungroomed coat.
Significant reductions in body weight and body weight gain were reported for most of the dosage period and continuing until termination of the study in the 3,10, and 30 mg/kg/day dose groups. Absolute food consumption values were also significantly reduced during these periods at the 30 mg/kg/day dose group, while significant increases in relative food consumption values were observed in the 3, 10, and 30 mg/kg/day within those same periods.
No treatment-related effects were reported at any dose level for any of the mating and fertility parameters assessed including, numbers of days to inseminate, numbers of rats that mated, fertility index, numbers of rats with confirmed mating dates during the first and second week of cohabitation, and numbers of pregnant rats per rats in cohabitation. At necropsy, none of the sperm parameters evaluated (sperm number, motility, or morphology) were affected by treatment at any dose level.
At necropsy, statistically significant reductions in terminal body weights were seen at 3, 10, and 30 mg/kg/day. Absolute weights of the left and right epididymides, left cauda epididymis, seminal vesicles (with and without fluid), prostate, pituitary, left and right adrenals, spleen, and thymus were also significantly reduced at 30 mg/kg/day. The absolute weight of the seminal vesicles without fluid was significantly reduced in the 10 mg/kg/day dose group. The absolute weight of the liver was significantly increased in all dose-groups. Kidney weights were significantly increased in the 1, 3, and 10 mg/kg/day dose groups, but significantly decreased in the 30 mg/kg/day group. All organ weight-to-terminal body weight and ratios were significantly increased in all treated groups. Organ weight-to-brain weight ratios were significantly reduced for some organs at the high dose group, and significantly increased for other organs among all treated groups.
No treatment-related effects were seen at necropsy or upon microscopic examination of the reproductive organs, with the exception of increased thickness and prominence of the zona glomerulosa and vacuolation of the cells of the adrenal cortex in the 10 and 30 mg/kg/day dose groups.
Serum analysis for the F0 generation males sampled at the end of cohabitation showed that PFOA was present in all samples tested, including controls. Control males had an average concentration of 0.0344+ 0.0148 ug/1 PFOA. Treated males had 51.1+9.30 and 45.3+12.6 ug/1, respectively for the 10 and 30 mg/kg/day dose groups.
Parental Females (F0)
No treatment-related deaths or adverse clinical signs were reported in parental females at any dose level. No treatment-related effects were reported for body weights, body weight gains, and absolute and relative food consumption values.
There were no treatment-related effects on estrous cyclicity, mating or fertility parameters. None of the natural delivery and litter observations were affected by treatment, that is, the numbers of dams delivering litters, the duration of gestation, the averages for implantation sites per delivered litter, the gestation index (number of dams with one or more livebom pups/number of pregnant rats), the numbers of dams with
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stillborn pups, dams with all pups dying, liveborn and stillborn pups viability index, pup sex ratios, and mean birth weights were comparable to controls among all treated groups.
Necropsy and histopathological evaluation were also unremarkable. Terminal body weights, organ weights, and organ-to-terminal body weight ratios were comparable to control values for all treated groups, except for kidney and liver weights. The weights of the left and right kidney, and the ratios of these organ weights-to-terminal body weight and of the left kidney weight-to-brain weight were significantly reduced at the highest dose of 30 mg/kg/day. The ratio of liver weights-to-terminal body weight was also significantly reduced at 3 and 10 mg/kg/day.
Results of the serum analysis in F0 generation females sampled on LD 22 showed that PFOA was present in all samples tested, except in controls where the level was below the limits of quantitation (0.00528ug/l). Treated females had an average concentration of 0.370.0805 and 1.02+0.425 ug/1, respectively for the 10 and 30 mg/kg/day dose groups.
FI Generation - Males
No effects were reported at any dose level for the viability and lactation indices. No differences between treated and control groups were noted for the numbers of pups surviving per litter, the percentage male pups, litter size and average pup body weight per litter at birth. Pup body weight on a per litter basis was significantly reduced in the 30 mg/kg/day group on days 1,5, and 8 of lactation. Of the pups necropsied at weaning, no statistically significant, treatment-related differences were observed for the weights of the brain, spleen and thymus and the ratios of these organ weights to the terminal body weight and brain weight.
Significant increases in treatment-related deaths (7 animals total) were reported in F 1 males in the high dose group of 30 mg/kg/day. One rat was moribund sacrificed on day 39 postweaning and another was found dead on day 107 postweaning, but the majority of the FI male rats were found dead on days 2-4 postweaning.
Statistically significant increases in clinical signs of toxicity were also observed in F 1 males during most of entire postweaning period. These signs included an increased incidence of annular constriction of the tail at all doses, with statistical significance at the 1,10, and 30 mg/kg/day; a significant increase at 10 and 30 mg/kg/day in the number of male rats that were emaciated; and a significant increase in the incidence of urine-stained abdominal fur, decreased motor activity, and abdominal distention at 30 mg/kg/day.
Body weights and body weight gains were statistically significantly reduced prior to and during cohabitation and during the entire dosing period in all treated groups. Statistically significant reductions in body weights were observed at 10 and 30 mg/kg/day during days 8-15, 22-29, 29-36, 43-50, and 50-57 postweaning. Body weight gains were also significantly reduced in the 30 mg/kg/day group on days 1-8, 15-22, 36-43, 57-64, and 64-70 postweaning. Statistically significant, dose-related reductions in body weight gains were observed for the entire dosage period (days 1-113 postweaning). Absolute food consumption values were significantly reduced at 10 and 30 mg/kg/day during the entire precohabitation period (days 1-70 postweaning), while relative food consumption values were significantly increased.
Statistically significant (p< 0.01) delays in sexual maturation (the average day of preputial separation) were observed in high-dose animals versus concurrent controls (52.2 days of age versus 48.5 days of age, respectively).
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No apparent effects were observed on any of the mating or fertility parameters including fertility and pregnancy indices (number of pregnancies per number of rats that mated and rats in cohabitation, respectively), the number of days to inseminate, the number of rats that mated, and the number of rats with confirmed mating dates during the first week. No statistically significant, treatment-related effects were observed on any of the sperm parameters (motility, concentration, or morphology).
Necroscopic examination revealed statistically significant treatment-related effects at 3, 10, and 30 mg/kg/day ranging from tan areas in the lateral and median lobes of the liver to moderate to slight dilation of the pelvis of one or both kidneys.
Statistically significant, dose-related decreases in terminal body weights of parental FI males were observed. The absolute weights of the liver were significantly increased and the absolute weights of the spleen were significantly decreased at all treated groups. The absolute weights of the left and/or right kidneys were significantly increased in the 1 and 3 mg/kg/day dose groups and significantly decreased in the 30 mg/kg/day dose group. The absolute weight of the thymus was also significantly decreased in the 10 and 30 mg/kg/day dose groups. The absolute weight of the prostate, brain and left adrenal gland were significantly decreased in the 30 mg/kg/day dosage group. The ratios of the weights of the seminal vesicles, with and without fluid, liver and left and right kidneys to the terminal body weights were significantly increased in all treated groups. The ratios of the weights of the left testis, with and without the tunica albuginea and the right testis to the terminal body weight, were significantly increased at 3 mg/kg/day and higher. The ratios of the weights of the left epididymis, left cauda epididymis, right epididymis and brain to the terminal body weight were significantly increased at 10 mg/kg/day and higher. The ratios of the weight of the seminal vesicles with fluid to the brain weight were increased at 1 mg/kg/day and higher, with statistical significance at 1 and 10 mg/kg/day. The ratios of the liver weightto-brain weight were significantly increased in the 1 mg/kg/day and higher dosage groups, and the ratios of the left and right kidney weights-to-brain weight were significantly increased in all treated groups. The ratios of the spleen weight-to-brain weight were significantly decreased at 1 mg/kg/day and higher, and the ratios of the thymus weight-to-brain weight were significantly decreased at 10 and 30 mg/kg/day. The ratios of the left and right testes weight-to-brain weight were increased in the 3 mg/kg/day and higher dosage groups. These ratios were significantly increased at 10 mg/kg/day (right testis only) and 30 mg/kg/day.
Histopathologic examination of the reproductive organs was unremarkable; however, treatment-related microscopic changes were observed in the adrenal glands of high-dose animals (cytoplasmic hypertrophy and vacuolation of the cells of the adrenal cortex) and in the liver (hepatocellular hypertrophy) of animals treated with 3,10, and 30 mg/kg/day. No other treatment-related effects were reported.
FI Generation - Females
No effects were reported at any dose level for the viability and lactation indices. No differences between treated and control groups were noted for the numbers of pups surviving per litter, the percentage male pups, litter size and average pup body weight per litter at birth. Pup body weight on a per litter basis was also significantly reduced in the 30 mg/kg/day group on days 1, 5, and 8 of lactation. Of the pups necropsied at weaning, no statistically significant, treatment-related differences were observed for the weights of the brain, spleen and thymus and the ratios of these organ weights to the terminal body weight and brain weight.
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An increase in treatment-related mortality (6 animals total) was observed in FI females on postweaning days 2-8 at the highest dose of 30 mg/kg/day. No adverse clinical signs of treatment-related toxicity were reported for any dose level during any time of the study period.
Statistically significant decreases in body weights and body weight gains were observed in high-dose animals on days 8, 15, 22, 29, 50, and 57 postweaning, during precohabitation (recorded on the day cohabitation began, when FI generation rats were 92-106 days of age), and during gestation and lactation. Decreases in absolute food consumption were observed during days 1-8, 8-15 postweaning during precohabitation and during gestation and lactation in animals treated with 30 mg/kg/day. Relative food consumption values were comparable across all treated groups.
Statistically significant (p< 0.01) delays in sexual maturation (the average day of vaginal patency) were observed in high-dose animals versus concurrent controls (36.6 days of age versus 34.9 days of age, respectively).
No effects were observed on estrous cyclicity, or on any of the mating and fertility parameters (numbers of days in cohabitation, numbers of rats that mated, fertility index, rats with confirmed mating dates during the first week of cohabitation and number of rats pregnant per rats in cohabitation). There were however, statistically significant increases in the average numbers of estrous stages per 21 days in highdose animals (4.7 versus 5.4 in controls).
All natural delivery observations were also apparently unaffected by treatment at any dose level. Numbers of dams delivering litters, the duration of gestation, averages for implantation sites per delivered litter, the gestation index (number of dams with one or more liveborn pups/number of pregnant rats), the numbers of dams with stillborn pups, dams with all pups dying and liveborn and stillborn pups were comparable among treated and control groups.
No treatment-related effects were observed on terminal body weights. The absolute weight of the pituitary and the ratios of the pituitary weight-to-terminal body weight and to the brain weight were significantly decreased at 3 mg/kg/day and higher, but did not show a dose-response. No other differences were reported for the absolute weights or ratios for other organs evaluated. No treatmentrelated effects were reported following necroscopic and histopathologic examinations.
F2 Generation Offspring
No treatment-related adverse clinical signs were observed at any dose level. Likewise, no treatmentrelated effects were reported following necroscopic examination, with the exception of no milk in stomach in pups that were found dead. The numbers of pups found either dead or stillborn did not show a dose-response (3/28, 6/28, 10/28, 10/28, and 6/28 in 0, 1, 3, 10, and 30 mg/kg/day dose groups, respectively) and therefore were unlikely related to treatment. Terminal body weights in F2 pups were not significantly different from controls. Absolute weights of the brain, spleen and thymus and the ratios of these organ weights-to-terminal body weight and to brain weight were also comparable among treated and control groups.
No effects were reported at any dose level for the viability and lactation indices. No differences between treated and control groups were noted for the numbers of pups surviving per litter, the percentage male pups, litter size and average pup body weight per litter when measured on LDs 1, 5, 8, 15,or 22. Anogenital distances measured for F2 male and female pups on LDs 1 and 22 were also comparable among the five dosage groups and did not differ significantly.
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CONCLUSIONS
Dosing with APFO at 30 mg/kg/day appeared to delay the onset of sexual maturation in both male and female F 1 offspring. The authors of the study contend that the delays in sexual maturation (preputial separation or vaginal patency) observed in high-dose animals are due to the fact that these animals have a decreased gestational age, a variable which they have defined as the time in days from evidence of mating in the F0 generation until evidence of sexual maturation in the FI generation. The authors state that gestational age appeared to be decreased in high-dose animals at the time of acquisition (the time when sexual maturation was reached), which they believe meant the animals in that group were younger and more immature than the control group, in which there was no significant difference in sexual maturation.
In order to test this hypothesis, the authors covaried the decreases in body weight and in gestational age with the delays in sexual maturation in order to determine whether or not body weights and gestational age were a contributing factor. When the delays in sexual maturation were covaried with the significantly reduced body weight at the time of acquisition, the difference was still significant, but at the p< 0.05 versus p< 0.01. This indicates that the delay in sexual maturation was partly related to body weight, but not entirely. When the delays in sexual maturation observed at the high dose group were covered with gestational age at the time of sexual maturation, there was no significant difference in the time of onset of sexual maturation between controls and high-dose animals. This indicates that the effect of delayed sexual maturation could possibly be attributed to decreased gestational age.
The authors also covaried the decreases in both body weight and in gestational age with the significant increases in the average number of estrous stages per 21 days that were observed at the high dose of 30 mg/kg/day and found that the difference between treated and control animals was still significant.
While it is known and commonly accepted that changes in the body weights of offspring can affect the time to sexual maturation, whether or not gestational age, as defined by the authors, also affects the time of acquisition is purely speculative, especially since there was no data provided by the authors to support this relationship. Additionally, covaring the delay in sexual maturation with gestational age is problematic from a statistical standpoint. Since there was no significant change in the length of gestation at 30 mg/kg/day, based on the authors' definition of `gestational age', the decreases in gestational age would have to be due mostly to changes in time to sexual maturation. Therefore, sexual maturation is essentially being covaried with itself. Still, even if a relationship between gestational age and time to sexual maturation were shown, it merely offers an explanation for the observed delays in sexual maturation in high-dose animals, but does not diminish its significance.
REFERENCE
York, R.G. 2002. Oral (Gavage) Two-Generation (One Litter Per Generation) Reproduction Study of Ammonium Perfluorooctanoic (APFO) in Rats. Argus Research Laboratories, Inc. Protocol Number: 418-020, Sponsor Study Number: T-6889.6, March 26, 2002.
3M Company, 2002. Submission dated September 17, 2002 to USEPA, AR 226.
USEPA 2002. Memorandum from Dr. Ralph Cooper, NHEERL, to Dr. Jennifer Seed, dated October 2, 2002.
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000191
Ecotoxicity Study
TOXICITY TO AQUATIC PLANTS (SELENASTRUM CAPRICORNUTUM) TEST SUBSTANCE Identity: Perfluorooctanoic acid, tetrabutylammonium salt; may also be referred to as PFOA tetrabutylammonium salt, tetrabutylammonium perfluorooctanoate, N2803-2, or as a major component of L-13492. (Octanoic acid, pentadecafluoro-, tetrabutylammonium salt, CAS # 95658-53-0) Remarks: The 3M production lot number was 2327. The test sample is referred to by the testing laboratory as L-13492. The T.R. Wilbury study number is 841-TH. The purity of the sample was not sufficiently characterized, although current information indicates it is a solution of 44.9% tetrabutylammonium perfluorooctanoate, 27.9% water, and 27.2% isopropanol. The following summary applies to the test sample as a mixture o f the test substance in an isopropanol/ water solution with incompletely characterized concentrations o f impurities. Data may not accurately relate toxicity o f the test sample with that o f the test substance. METHOD Method: U.S. EPA-TSCA Guideline 797.1050 Test: Acute static GLP: Yes Year completed: 1995 Species: Selenastrum capricornutum Source: Originally from The Culture Collection of Algae at the University of Texas at Austin, maintained in culture medium at T.R. Wilbury, Inc., Marblehead, MA. Element basis: growth rate. Exposure period: 96-hours Test organisms laboratory culture: Algae cultures were growing in U.S. EPA-rccommended sterile enriched medium for at least 14 days prior to test initiation. Statistical methods: The average specific growth rate was calculated as the natural log of the number of cells/mL at the exposure period minus the natural log of the number of cells/mL at 0 hours divided by the exposure period. The percent change from the control was calculated by subtracting the treatment average specific growth rate from the control average specific growth rate, dividing the difference by the average specific growth rate in the control, and multiplying that value by 100. The EC50 values were calculated based on a nonlinear regression estimation procedure (Bruce and Versteeg, 1992). The NOEC was determined using a parametric one-way analysis of variance and the average specific growth in each test vessel at the end of the test. Test Conditions: Dilution water source: The algae medium was prepared to U.S. EPA recommended concentrations by spiking deionized water with nutrient stocks. The pH of the synthetic algal medium at test initiation was 7.5. Stock and test solutions preparation: A 16 mg/L primary stock
293
solution was prepared in sterile enriched media. Appropriate amounts of this stock solution were added directly to dilution water to formulate the test media. A 1,000 mg/L isopropyl alcohol stock solution was also prepared and evaluated. Exposure vessels: 250 mL glass Erlenmeyer flasks containing 100 mL of test solution. Agitation: Continuous at 100 rpm Number of replicates: 3 Initial algal cell loading: 1.0 X 10 4 cells/mL Number of concentrations: Five plus a negative control Lighting: -400 ft-c from continuous cool-white fluorescent lighting Water chemistry: PH range: (0 - 96 hours) 7.5 - 10.4 (control exposure) 7.4 - 7.6 (16 mg/L exposure) 7.4 - 10.4 (1,000 mg/L isopropyl alcohol exposure) Test temperature range: (0 - 96 hours) 23.4 - 23.7C RESULTS Nominal concentrations: Blank control, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L. A test was performed simultaneously with isopropyl alcohol, a component that represents 27% of L-13492, at 4.4 and 1,000 mg/L. Element value and 95% confidence interval: 24-hour ErCIO (growth rate) = <1.0 mg/L (Cl not calculable) 24-hour ErC50 (growth rate) = 15 (9.8 - >16) mg/L 24-hour ErC90 (growth rate) = >16 (7.7 - >16) mg/L 48-hour ErCIO (growth rate) = <1.0 mg/L (Cl not calculable) 48-hour ErC50 (growth rate) = 14 (8.2 - >16) mg/L 48-hour ErC90 (growth rate) = >16 mg/L (Cl not calculable) 72-hour ErCIO (growth rate) = <1.0 mg/L (Cl not calculable) 72-hour ErC50 (growth rate) = 7.1 (4.1 - 11) mg/L 72-hour ErC90 (growth rate) = >16 mg/L (Cl not calculable) 96-hour ErCIO (growth rate) = <1.0 mg/L (Cl not calculable) 96-hour ErC50 (growth rate) = 4.9 (3.5 - 6.7) mg/L 96-hour ErC90 (growth rate) = >16 mg/L (Cl not calculable) 96-hour NOEC: 1.0 mg/L Algal growth was not affected by isopropyl alcohol concentrations of 4.4 or 1,000 mg/L. Element values are based on nominal concentrations.
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Biological observations after 96-hours:
Nominal Concentration, mg/L
Control 1.0 2.0 4.0 8.0 16
Mean Number of Cells per mL 1,249,000 1,311,000 275,000 113,000 84,000 27,000
Percent
Percent
Inhibition via Inhibition via
Density
Growth Rate
--
-5 0 88 30 91 50 93 56 98 80
Control response. Satisfactory Biological observations after 96-hours for isopropyl alcohol:
Nominal Concentration, mg/L
Mean Number of Cells per mL
Percent Inhibition via Density
Control 4.4 1,000
1,249,000 1,190,000 1,347,000
-
5 -8
Observations: Algal cell counts in each test vessel were determined by means of direct microscope counts with a hemocytometer. After 96 hours of exposure, there were no signs of aggregation, flocculation or adherence of the algae to the flasks in the control or any test treatment group. In addition, there were no noticeable changes in cell size, color or morphology when compared to the control. Reversibility of Growth Inhibition: Effect of the test substance was determined to be algistatic based on the results of the post-definitive test exposure. Remarks: Testing was conducted on the mixture as described in the Test Substance Remarks field. The values reported apply to that mixture and not the fluorochemical component alone. CONCLUSIONS The test sample 96-hour EC50 and 95% confidence interval for Selenastrum capricornutum was determined to be 4.9 mg/L with a 95% confidence interval of 3.5 - 6.7 mg/L. The 96-hour no observed effect concentration (NOEC) for the test substance in solution was 1.0 mg/L. Algae growth in the vessels containing isopropyl alcohol was not affected at 4.4 or 1,000 mg/L, indicating that the concentration of isopropyl alcohol in L -13492 can not, by itself, account for the toxicity of L-13492 to algae. No signs of aggregation, flocculation, or adherence were noted in any of the test solutions. This test substance was determined to be algistatic. Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133
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DATA QUALITY Reliability: Klimisch ranking 2. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized.
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REFERENCES This study was conducted at T.R. Wilbury Laboratories, Inc., Marblehead, MA, at the request of the 3M Company, Lab Request number N2332, 1995. OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations. Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that 100% test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity o f the test sample with that o f the test substance." I agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
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Ecotoxicity Study
TOXICITY TO AQUATIC PLANTS (SELENASTRUM CAPRICORNUTUM) TEST SUBSTANCE Identity: Perfluorooctanoic acid, tetrabutylammonium salt; may also be referred to as PFOA tetrabutylammonium salt, tetrabutylammonium perfluorooctanoate, N2803-2, or as a major component of L-13492. (Octanoic acid, pentadecafluoro-, tetrabutylammonium salt, CAS # 95658-53-0) Remarks: The 3M production lot number was 2327. The test sample is referred to by the testing laboratory as N2803-2. The T.R. Wilbury study number is 890-TH. The purity of the sample was not sufficiently characterized, although current information indicates it is a solution of 44.9% tetrabutylammonium perfluorooctanoate, 27.9% water, and 27.2% isopropanol Thefollowing summary applies to the test sample as a mixture o f the test substance in an isopropanol/water solution with incompletely characterized concentrations o f impurities. Data may not accurately relate toxicity o f the test sample with that o f the test substance. METHOD Method: U.S. EPA-TSCA Guideline 797.1050 Test: Acute static GLP: Yes Year completed: 1995 Species: Selenastrum capricornutum Source: Originally from The Culture Collection of Algae at the University of Texas at Austin, maintained in culture medium at T.R. Wilbury, Inc., Marblehead, MA. Element basis: Algal cell count (cells/mL), and specific growth rate. Exposure period: 96-hours Test organisms laboratory culture: Algae cultures were growing in U.S. EPA-recommended sterile enriched medium for at least 14 days prior to test initiation. Statistical methods: The average specific growth rate was calculated as the natural log of the number of cells/mL at the exposure period minus the natural log of the number of cells/mL at 0 hours divided by the exposure period. The percent change from the control was calculated by subtracting the treatment average specific growth rate from the control average specific growth rate, dividing the difference by the average specific growth rate in the control, and multiplying that value by 100. The EC50 values were calculated by probit analysis. The NOEC was determined using a parametric one -way analysis of variance and the average specific growth rate and the number of cells/mL in each test vessel at the end of the test. Test Conditions: Dilution water source: The algae medium was prepared to U.S. EPA recommended concentrations by spiking deionized water with nutrient stocks. The pH of the synthetic algal medium at test initiation was 7.5. Stock and test solutions preparation: A 160 mg/L primary stock
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solution was prepared in sterile enriched media. Appropriate amounts of this stock solution were added directly to dilution water to formulate the test media. Exposure vessels: 250 mL glass Erlenmeyer flasks containing 50 mL of test solution. Agitation: Continuously at 100 rpm Number of replicates: 3 Initial algal cell loading: 1.0 X 104 cells/mL Number of concentrations: Five plus a negative control Lighting: Continuous lighting at -380 ft-c using cool-white fluorescent lamps Water chemistry: pH range: (0 - 96 hours) 7.5 - 10.8 (control exposure) 7 .5 -8 .4 (16 mg/L exposure) Test temperature range: (0 - 96 hours) 23.4 - 23.7C RESULTS Nominal concentrations: Blank control, 0.99, 2.0, 4.0, 8.0, 16.0 mg/L. Element value and 95% confidence interval: 24-hour EC 10 (cell density) = 4.1 (0 - 9.0) mg/L 24-hour EC50 (cell density) = >16 (7.7 - >16) mg/L 24-hour ErC50 (growth rate) = >16 mg/L (Cl not calculable) 24-hour EC90 (cell density) = >16 mg/L (Cl not calculable) 48-hour EC 10 (cell density) = 1.2 (<0.99 - 1.6) mg/L 48-hour EC50 (cell density) = 7.1 (6.0 - 8.6) mg/L 48-hour ErC50 (growth rate) = >16 mg/L (Cl not calculable) 48-hour EC90 (cell density) = >16 mg/L (Cl not calculable) 72-hour EC 10 (cell density) = <0.99 (<0.99 - 1.9) mg/L 72-hour ErCIO (growth rate) = 2.2 (1.6 - 2.8) mg/L 72-hour EC50 (cell density) = 2.8 (1.1 - 5.8) mg/L 72-hour ErC50 (growth rate) =11 (9.4 - 15) mg/L 72-hour EC90 (cell density) = 8.4 (4.5 - >16) mg/L 72-hour ErC90 (growth rate) = >16 mg/L (Cl not calculable) 96-hour EC10 (cell density) = 1.4 (<0.99 - 2.4) mg/L 96-hour ErCIO (growth rate) = 2.3 (<0.99 - 3.6) mg/L 96-hour EC50 (cell density) = 2.9 (1.0 - 7.7) mg/L 96-hour ErC50 (growth rate) = 8.4 (5.9 - 14) mg/L 96-hour EC90 (cell density) = 6.0 (3.5 - >16) mg/L 96-hour ErC90 (growth rate) = >16 mg/L (Cl not calculable) 96-hour NOEC (cell density): 0.99 mg/L 96-hour NOEC (growth rate): 2.0 mg/L Element values were based on nominal concentrations. Control response: Satisfactory
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Biological observations after 96-hours:
Nominal Concentration, mg/L
Control 0.99 2.0 4.0 8.0 16
Mean Number of Cells per mL 3,227,000 3,633,000 2,440,000 581,000 169,000 61,000
Percent
Percent
Inhibition via Inhibition via
Density
Growth Rate
--
-13 -2 24 5 82 30 95 52 98 68
Observations: Algal cell counts in each test vessel were determined by means of direct microscope counts with a hemocytometer. After 96 hours of exposure, there were no signs of aggregation, flocculation or adherence of the algae to the flasks in the control or any test treatment group. In addition, there were no noticeable changes in cell size, color or morphology when compared to the control. Reversibility of Growth Inhibition: Effect of the test substance was determined to be algistatic based on the results of the post-definitive test exposure. Remarks: Testing was conducted on the mixture as described in the Test Substance Remarks field. The values reported apply to that mixture and not the fluorochemical component alone.
CONCLUSIONS The test sample 96-hour EC50 and 95% confidence interval for Selenastrum capricornutum was determined using two calculation methods. The test substance 96-hour EC50 for Selenastrum capricornutum was determined to be 2.9 mg/L with a 95% confidence interval of 1.0 - 7.7 mg/L, when calculated using cell density. The 96-hour EC50 for Selenastrum capricornutum was determined to be 8.4 mg/L with a 95% confidence interval of 5.9 - 14 mg/L using growth rate. No signs of aggregation, flocculation, or adherence were noted in any of the test solutions. This test substance was determined to be algistatic. Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 2. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized. REFERENCES This study was conducted at T.R. Wilbury Laboratories, Inc., Marblehead, MA, at the request of the 3M Company, Lab Request number N2803-2, 1995. OTHER
300
000199
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations. Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that 100% test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity o f the test sample with that o f the test substance. " I agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
301
000200
Ecotoxicity Study
TOXICITY TO AQUATIC PLANTS (SELENASTRUM CAPRICORNUTUM) TEST SUBSTANCE Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, orFC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1) Remarks: The 3M production lot number was 427. The test sample is FC-143, referred to by the test laboratory as N2803-4. The T.R. Wilbury study number is 895-TH. The purity of the sample was not sufficiently characterized, although current information indicates it is a mixture of 96.5 - 100% test substance and 0 3.5% C6, C7, and C9 perfluoro analogue compounds. METHOD Method: U.S. EPA-TSCA Guideline 797.1050 Test: Acute static GLP: Yes Year completed: 1996. Species: Selenastrum capricornutum Source: Originally from The Culture Collection of Algae at the University of Texas at Austin, maintained in culture medium at T.R. Wilbury, Inc., Marblehead, MA. Element basis: Algal cell counts (cells/ml), and specific growth rates. Exposure period: 96-hours Statistical methods: Cell densities, growth rates and percent inhibition values used to estimate the EC10, EC50, and EC90 values and 95% confidence limits were calculated using the computer software of C.E. Stephan. The no observed effect concentration (NOEC) was calculated using one-way analysis of variance (ANOVA). Analytical monitoring: pH and temperature Test Conditions: Algal nutrient medium: U.S. EPA-recommended sterile enriched medium, prepared by spiking deionized water with nutrient stocks. The pH of the synthetic algal medium at test initiation was 7.5. Stock and test solutions preparation: A 1,000 mg/L primary stock solution was prepared in sterile enriched media. Appropriate amounts of this stock solution were added directly to dilution water to formulate the test media. Exposure vessels: 250 mL glass Erlenmeyer flasks containing 50 mL of test solution. Agitation: Shaken continuously at 100 rpm Number of replicates: 3 Initial algal cell loading: 1.0 X 10 4 cells/mL Number of concentrations: Five plus a negative control Lighting: ~400 ft-c from continuous cool-white fluorescent lighting Water chemistry: pH range: (0 - 96 hours) 7.5 - 9.6 (control exposure) 5.4 - 7.4 (1,000 mg/L exposure)
302
Test temperature range: (0 - 96 hours) 23.5 -23.7C RESULTS Nominal concentrations: Bk control, 62, 130, 250, 500, 1,000 mg/L. Element value and 95% confidence interval: 72-hour EC 10 (cell density) = 310 (110-440) mg/L 72-hour ErCIO (growth rate) = 470 (380 - 550) mg/L 72-hour EC50 (cell density) = 520 (250 - 1,000) mg/L 72-hour ErC50 (growth rate) = >1,000 mg/L (C.I. not calculable) 72-hour EC90 (cell density) = >1,000 mg/L (C.I. not calculable) 72-hour ErC90 (growth rate) = >1,000 mg/L (C.I. not calculable) 96-hour EC 10 (cell density) = 97 (77 - 120) mg/L 96-hour ErCIO (growth rate) = 220 (160 - 280) mg/L 96-hour EC50 (cell density) = 310 (280 - 350) mg/L 96-hour ErC50 (growth rate) = >1,000 mg/L (C.I. not calculable) 96-hour EC90 (cell density) = 1,000 (830 - >1,000) mg/L 96-hour ErC90 (growth rate) = >1,000 mg/L (C.I. not calculable) 96-hour NOEC (cell density): 62 mg/L 96-hour NOEC (growth rate): 500 mg/L Element values based on nominal concentrations
Biological observations after 96-hours:
Nominal
Mean
Percent
Concentration, Number
Inhibition via
mg/L
of Cells per Density
mL
Control
1,407,000
-
62
1,445,000
-3
130
1,129,000
20
250
753,000
46
500
443,000
69
1,000
181,000
87
Percent Inhibition via Growth Rate
-
0 6 13 25 42
Control response: Satisfactory Observations: Algal cell counts in each test vessel were determined by means of direct microscope counts with a hemocytometer. After 96 hours of exposure, there were no signs of aggregation, flocculation or adherence of the algae to the flasks in the control or any test treatment group. In addition, there were no noticeable changes in cell size, color or morphology when compared to the control. Reversibility of Growth Inhibition: Effect of the test substance was determined to be algistatic based on the results of the post-definitive test exposure. CONCLUSIONS The test sample 96-hour EC50 and 95% confidence interval for Selenastrum capricornutum was determined using two calculation methods. By cell density, it was 310 (280 - 350) mg/L, and by growth rate
303
000202
>1,000 rag/L. The 96-hour NOEC was determined to be 62 mg/L using cell density and 500 mg/L using growth rate. No signs of aggregation, flocculation, or adherence were noted in any of the test solutions. This test substance was determined to be algistatic. Subm itter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133. DATA QUALITY Reliability: Klimisch ranking = 2. The study lacks analytical measurement of test substance concentrations in the test solutions and the sample purity is not sufficiently characterized. REFERENCES This study was conducted at T.R. Wilbury Laboratories, Inc., Marblehead, MA, at the request of the 3M Company, Lab Request number N2803-4. OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
304
000203
Ecotoxicity Study Title: Multi-Phase Exposure/Recovery Algal Assay Test Method TEST SUBSTANCE Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1) Remarks: The 3M production lot number was 37. The test sample was FC-143. The purity was not completely characterized, although information indicates it is a mixture of 96.5-100 percent test substance and 0-3.5% C6, C7, and C9 perfluoro analogue compounds. The chemical is soluble in water at ambient room temperature. METHOD Method/guideline followed: Modified from and modeled after ASTM-E-35.23 Draft No. 2, OECD; A.G. Payne, (as described inUSEPA 600/9-78-018) Test type: Static GLP (Y/N): No Year study performed: 1981 Species: Selenastrum capricornutum (7 day old stock) Supplier: USEPA - ERL in Corvallis, Oregon. Measure of growth used: biomass in cell dry-weight (mg/L); cell count (no./mL) Concentrations used: Range-finding: 0, 100, 250, 500, 750, 1000, 1500 mg/L; Main study: 0, 100, 180, 320, 560, 1000, 1800 mg/L (nominal values). Exposure period: 4, 7, 10, and 14 days Analytical monitoring: There was no information on the measurement of the chemical during the test. There was no information on detection limits of the chemical or impurities. Statistical methods: EC50 values and 95% confidence limits were calculated using the linear regression model 3M Sixcur
305
000204
Test conditions: - The algal culture was stored in the dark at 4C before use. The initial algal cell count in the stock culture was 277,000 cells/mL. - Mineral (inorganic) standard nutrient medium was used for culturing/testing algae. This was prepared with all mineral nutrients essential for algal growth. The pH was adjusted to 7.5 +_0.1 prior to use in the assays. This nutrient medium was the diluent for all operations that used algae including the preparation of stock solutions. - Exposure vessels were 250 mL Erlenmeyer flasks with 50 mL of test solution and stoppered with autoclaved foam plugs. - During the test, the temperature was 23 + 2C. The light was by fluorescent illumination of 400 ft candles + 10%, and cultures were agitated with a continuous shaking platform at 100 rpm. - Initial algal loading was 1.0 x 104 cells/mL - Algal recovery response was evaluated following the exposure periods. - There was no information on dilution water source, contaminants, or chemistry of the water. -Three replicates were taken at each dose.
Remarks: pH values were acceptable according to OPPTS Harmonized Guidelines. Water hardness was not presented.
RESULTS
Dose associated with each endpoint (as mg/L):
EC50s
Number of Days Exposed Cell Dry Weight (mg/L) Cell-Count (no.cells/mL)
4 7 10 14 '95% Confidence Limits
Number of Days Exposed
149 (57-340)' 70 (34-118) 49 (15-96) 73 (25-147)
EC 10s Cell-Count (no. cells/mL)
49 (28-75) 30 (21-40) 27 (8-50) 43 (14-81)
4 7
10
'95% Confidence Limits
Number of Days Exposed
5.3 (3-7) 3.3 (2-4) 2.9 (1-5) 5 (2-8)
EC90s
Cell-Count (no. cells/mL)
4 624 (C.I. not calculated)1 306
000205
7 283 (150-590) 10 386 (C.I. not calculated) 14 307 (C.I. not calculated)
'95% Confidence Limits Was control response satisfactory (yes/no/unknown): unknown
Statistical results, as appropriate: No p-values were reported Remarks: After exposure ceased, the algal cells recovered and resumed logarithmic growth when resuspended in fresh nutrient medium in the absence of the test substance.
CONCLUSIONS Ammonium perfluorooctanoate exhibits a 14-day EC50 (cell count) value of 43 mg/L with a 95% confidence interval of 14 to 81 mg/L.
Submitters' remarks: The authors indicate a Klimisch ranking of 2. The study meets the criteria for quality testing at the time it was conducted. However, the study lacked information on test substance purity and actual measurements of the test substance in solution.
REFERENCE
Elnabarawy, M.T. 1981. 3M Technical Report Summary, Multi-Phase Exposure/Recovery Algal Assay Test Method. Report Number 006. Project Number 9970030000. October 16.
OTHER General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited. The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations. As it is we are operating in the dark on this issue.
307
000206
INVERTEBRATE TOXICITY Title: Acute Toxicity to Aquatic Invertebrates (Summary of three 48-hour studies in diet) TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; also referred to as PFOA ammonium salt, ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS# 3825-26-1) Remarks: The 3M product lot number used was 37. The test sample was FC-143. It's purity was not completely characterized, although information indicated it was a mixture of 96.5-100% test substance and 0-3.5% C6, C7, and C9 perfluoro-analogue compounds. METHODS
Method/guideline followed: Not stated
Test type: Static
GLP (Y/N): No Year study performed: 1982 Species: Daphnia magna
age = First-Instar young length = 0.5 - 1.5 mm Supplier: USEPA-ERL Duluth, MN Concentrations tested: 0, 10, 30, 100, 300, 1000 mg/L in two tests; 0, 10, 100, 500, 1000 in third study; nominal concentrations Exposure period: 48 hours Analytical monitoring: No monitoring of the test substance concentrations was done Statistical methods: Chi-square test was used Test conditions: -Dilution water used was carbon-filtered well water (all three tests), with a temperature of 24C in two tests (the temp, in the third test was not given). -Test solutions were made from a common stock solution with a concentration of 5g/L. -Two replicates were taken
308
000207
-Number of Daphnia were 10-20 per replicate -Exposure vessels were 250 mL glass beakers containing 200 mL test solution (6 cm in depth)
-Water chemistry during the test was as follows: Temp ranged from 22-24C pH was 8.7 for the tests in which it was reported (measured at 48 hours) DO was 8.2-8.5 ppm for tests in which it was reported and for which it could be read (measured at 48 hours)
Remarks: pH and hardness measurements were not presented for dilution water; hardness during the test was not presented. Information on diet was also not presented.
RESULTS
Dose of each endpoint (as mg/L): 48-hour EC50 ranged from 126 mg/L (with C.I. 86 to 183 mg/L) to > 1000 mg/L
Remarks: - Lowest test substance concentration causing 100% mortality was 500 mg/L (from both replicates of a single test)
- Mortality of controls was 10% in one replicate of one test - Abnormal responses included marked reduction in locomotion at 1000 mg/L in one test
Was control response satisfactory (yes/no/unknown): It appears that the control response was satisfactory, given the information provided.
Statistical results, as appropriate: Some statistically significant results are presented (p values associated with Chi-square tests), but it is unclear what they refer to.
CONCLUSIONS
The test sample results (i.e., the large range in EC50 values) were inconsistent. The authors suggest that this difference may be due to differences in diet.
Submitters' remarks: The Klimisch ranking for the study was 3. Several reasons were indicated, including: the method was not described, sample purity was not properly characterized and lacked analytical confirmation of test substance concentrations. They also note the values from multiple tests contradict each other. The authors also note that they conducted the studies specifically to evaluate diet.
Reviewers' remarks: Although the authors indicate that diet may be the cause of the differences in EC50s, no information on diets is presented.
309
000208
REFERENCE Not enough information except what submitters include in their reference section, which states: "These studies were conducted by the 3M Company, Environmental Laboratory, St. Paul, MN, completed from May to June, 1982" OTHER
General remarks: This summary was based on a report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited. The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
310
000209
INVERTEBRATE TOXICITY Title: Chronic Toxicity to Freshwater Invertebrates (Daphnia magna) TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; also referred to as PFOA ammonium salt, ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS# 3825-26-1) Remarks: The 3M product lot number used was 264. The test sample was FC-143. It's purity was not completely characterized, although information indicated it was a mixture of 96.5-100% test substance and 0-3.5% C6, C7, and C9 perfluoro analogue compounds. METHODS
Method/guideline followed: USEPA-1982, OECD 1981
Test type: Semi-static life-cycle toxicity
GLP (Y/N): No
Year study performed: 1984
Species: Daphnia magna Age = < 24-hour neonates
Supplier: 3M Environmental Laboratory St. Paul, MN Concentrations tested: Acute test: 25, 40, 63, 100, 160, 250, 400, 630 mg/L Chronic test: 0, 5, 8, 13, 22, 36, and 60 mg/L, nominal concentrations. (Results, however, were based on mean measured concentrations - see below.) Exposure period: 21 days Analytical monitoring: Not stated Statistical methods: Acute test: Probit analysis. Chronic test: moving average angle method. Asci Corp, Duluth, MN recalculated statistics in 1998. NOECs and LOECs were generated using Toxstat. Reproduction was normal and homogenous therefore Dunnett's Test was used. Fisher's Test was used for survival. IC50s (for reproduction) were generated using ICp program. Survival EC50s were generated using `Trimmed Spearman-Karber'.
311
000210
Test conditions:
-Dilution water source was aerated carbon-filtered well water -Dilution water chemistry was:
hardness: 240 mg/L as CaC03 alkalinity: 230 mg/L as CaC03 pH: 7.8 COD: <0.4 mg/L
-Media renewal information/rationale: medium was changed once every two days -Exposure vessels were 250 ml glass beakers (containing 200 mL of solution to a depth of about 5 cm; 1 animal per 40 ml, no aeration during the test) -Ambient laboratory lighting was used (cool-white fluorescent, at ambient levels, 16 hours per day) -Four replicate beakers were used -There were 20 animals per concentration (5 per replicate) -Water temperature during the test was 22 + 2C -A suspension of fish food and yeast containing 5 mg dry solids per 1 ml mixture was fed on a daily basis
Remarks: pH and hardness measurements of water during the test were not presented. Also, the OPPTS harmonized guidelines state that hardness should be a maximum of 180 mg/L as CaC03 during the test; if chemistry prior to the test was indicative of during the test, the hardness value of 240 mg/L of the dilution water was too high.
RESULTS
Dose of each endpoint (as mg/L)*:
14 day NOEC (survival) = 60 mg/L 14 day NOEC (reproduction) = 8 mg/L 21 day NOEC (survival) = 22 mg/L 21 day NOEC (reproduction) = 22 mg/L 14 day IC50 (reproduction) = 40 (28-48) mg/L 21 day IC50 (reproduction) = 43 (35-46) mg/L
*A11 element concentrations were based on mean measured concentrations and the reported endpoint values are from the reanalysis of the data by AscI Corp.
Remar ks:
- All surviving first generation daphnids appeared normal at the end of the test. In the 36 and 60 mg/L treatments, survival was statistically different from the negative control group. - Neonates started to be produced on Day 7. Reproduction was statistically significantly different from the control (Dunnetf s) at the 13, 22, 36, and 60 mg/L concentrations after 14 days, and in the 36 and 60 mg/L test solutions at 21 days. - 100% mortality was not observed at any dose -N o mortality was seen in the controls
312
000211
Was control response satisfactory (yes/no/unknown): Yes, based on the fact that there was no mortality. Statistical results, as appropriate: It appears some results are statistically significant, based on computer printouts. However, lack of information made these difficult to interpret. CONCLUSIONS There were no adverse effects on survival or reproduction at concentrations < 22 mg/L for 21 days. Submitters' remarks: The Klimisch ranking was 3. The study was apparently well conducted according to the methodology available at the time of the start of the test. Reviewers' remarks: none REFERENCE 3M Company. Chronic toxicity to freshwater invertebrates. [No other information available] OTHER
General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited. Also, an acute test was conducted, possibly as a range-finding study. However, the purpose of the acute test was not very clear from the study. The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
313
000212
INVERTEBRATE TOXICITY Title: Acute toxicity to aquatic invertebrates (Daphnia magna) TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; also referred to as PFOA ammonium salt, ammonium perfluorooctanoate, FC-116, FC-126, FC-169, orFC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS# 3825-26-1) Remarks: The 3M production lot number was 390. The test sample was FC-126, a white powdery solid. Its purity ws not sufficiently characterized, although current information indicates it is a mixture of 7893% test substance and 7-22% C5, C6, and C7 perfluoro analogue compounds. METHODS
Method/guideline followed: Not stated
Test type: Static
GLP (Y/N): No
Year study performed: 1987
Species: Daphnia magna Age = < 24 hour neonates
Supplier: 3M Environmental Labs Concentrations tested: 0, 100, 180, 320, 560, and 1000 mg/L, nominal concentrations Exposure period: 48 hours Analytical monitoring: None because concentrations were nominal. Statistical methods: Probit analysis Test conditions: -Dilution water source was carbon-filtered well water with the following chemical characteristics:
Temp was 21C DO was 9.4 ppm pH was 7.9 -Test solutions were prepared by direct weights addition -Stability of the test chemical solution was not noted
314
000213
-Exposure vessel was a 250 mL Pyrex glass beaker containing 200 mL test solution (to a 6 cm depth) -Number of daphnids per replicate was 10 -Water chemistry during test:
DO control: 9.0 mg/L 1000 mg/L exposure: 8.8 mg/L pH control: 8.0 1000 mg/L: 8.1
Temp
21C
Remarks: No other details of the test conditions were presented, including water hardness.
RESULTS
Dose of each endpoint (as mg/L): 48-hour EC50 was 221 mg/L (95% Cl: 186-261)
Remarks: - Lowest test substance concentration causing 100% mortality was 560 mg/L at 48 hours (both replicates)
- There was no mortality in the controls
Was control response satisfactory (yes/no/unknown): Yes, based on the fact that there was no mortality
Statistical results, as appropriate: Not presented
CONCLUSIONS
The 48-hour EC50 was determined to be 221 mg/L (with a 95 % C.I. of 186-261)
Submitters' remarks: The data quality Klimisch ranking was 2. Testing met the criteria for quality testing. However, sample purity was not properly characterized and it lacked analytical confirmation of test substance concentrations.
Reviewers' remarks: none
REFERENCE
3M Company. 1987. [Report title not given.] Lab Request Number E1282-1. Completed on April 30.
OTHER
General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
315
000214
The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
316 000215
TOXICITY TO INVERTEBRATES
Title: Static Acute Toxicity of FX-1003 to the Daphnid, Daphnia magna
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOS ammonium salt, ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143, or as a major component of FX1003. (Octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1)
Remarks: The 3M production lot number was 2327. The purity of the test sample, FX-1003, was not sufficiently characterized, though available information indicated it was a solution of <45% ammonium perfluorooctanoate, 50% water, <3% inert perfluorinated compound, and 1 - 2% C5and C7perfluoroanalogue compounds.
METHODS
Method/guideline followed: OECD 202
Test type: Static
GLP (Y/N): Yes
Year study performed: 1990
Species: Daphnids used in the test were less than 24 hours old at the start of the test. They were produced from an in-house culture that was maintained under test conditions for at least 14 days. Prior to testing, daphnids were maintained in 100% dilution water (collected from wells at EnviroSystems in Hampton, New Hampshire) under static conditions. During acclimation, daphnids were not treated for disease and were free of apparent sickness, injuries, and abnormalities at the beginning of the test. Daphnids were fed yeast, trout chow, and the freshwater alga Selenastrum capricormitum once daily before the test. Daphnids were not fed during the test.
Supplier: EnviroSystems-daphnids, 3M-test substance
Concentrations tested: A screening test was performed prior to the definitive toxicity test. Nominal concentrations of the test substance were 0.1, 1, 100 and 1000 mg/L; the number of replicates was not indicated. For the definitive test, four replicates of each of the following test substance concentrations were used: 0 (blank control), 150, 250, 400, 600, and 1000 mg/L.
Exposure period: 48 hours
Analytical monitoring: Test substance concentrations were not measured during the study. Dissolved oxygen, pH, conductivity, and temperature were measured and recorded daily in each test chamber containing live test animals.
317
000216
Statistical methods: Results of the toxicity test were interpreted by standard statistical techniques (Stephan, 1983). The probit, moving average, or linear interpolation method was used to calculate the 48-hour EC5o (based on immobilization) using nominal concentrations of the test substance.
Test conditions: Water used for acclimation of the test organisms, and for all toxicity testing, was collected from wells at EnviroSystems in Hampton, New Hampshire. Water was adjusted to a hardness of 180 mg/L as CaC03 and aerated in 500-gallon polyethylene tanks prior to test initiation. During the acclimation period (24-hours prior to the test initiation), the dilution water temperature was 19.8C. No stock solution was prepared, as the test substance was added directly to dilution water contained in the test vessels without the use of a solvent. The stability of the test solutions was not indicated. Nominal concentrations of the test substance were 0 (blank control), 150, 250, 400, 600, and 1000 mg/L. Four replicates of each test concentration were used. Twenty daphnids were randomly and equally distributed among four replicates of each treatment. Exposure vessels were 250 mL glass beakers containing 200 mL of the test solution (approximately 7 cm depth). Test vessels were randomly arranged in an incubator during the 48-hour test. During the test, the following water chemistry ranges (0 - 48 hours) were determined: Conductivity 800 - 900 pmhos/cm (control exposure and 1000 mg/L exposure); pH 8.1 8.2 (control exposure), 8.3 - 8.4 (1000 mg/L exposure); Temperature 19.5 - 20.1 C (control exposure and 1000 mg/L exposure); Dissolved 0 2 9.0 - 9.5 mg/L (control exposure), 9.1 - 9.5 mg/L (1000 mg/L exposure). Aeration was not required during the study to maintain dissolved oxygen concentrations above acceptable levels. A 16-hour light and 8-hour dark photoperiod was automatically maintained with coolwhite fluorescent lights that provided an intensity of 45 pEs 'm'2. Loading rate during the toxicity test was approximately 0.018 g/L.
Remarks: No additional comments
RESULTS
Dose of each endpoint (as mg/L): Screening test: LC40= 1000 mg/L, NOEC =100 mg/L
Definitive test (based on immobilization): 24-hour EC50>1000 mg/L, 48-hour EC50 = 584 mg/L (95% confidence level = 400 - 1000 mg/L)
Remarks: For the screening test, after 48 hours of exposure, there was 60% survival at 1000 mg/L and 100% survival at all other tested concentrations. For the definitive test, all test vessels maintained a clear appearance throughout the study. 100% survival occurred in the control exposure. Control daphnids had an average wet weight (blotted dry) of 0.0007 g at the end of the test. Loading rate during the toxicity test was approximately 0.018 g/L. Exposure of daphnids to the reference toxicant, sodium dodecyl sulfate, resulted in a 48-hour LC50of 33 mg/L and a 48-hour EC50 of 16 mg/L.
Was control response satisfactory (yes/no/unknown): yes
Statistical results, as appropriate: No additional comments
CONCLUSIONS The test sample 48-hour EC50 for Daphnia magna was determined to be 584 mg/L, with a 95% confidence interval of 400 - 1000 mg/L.
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Submitters' remarks: Klimisch ranking 2. Testing meets the criteria for quality testing. However, sample purity was not properly characterized and no attempt was made to confirm test substance concentrations. Reviewers' remarks: The submitters' conclusions appear to be accurate, based on the data. REFERENCE
EnviroSystems, Inc. 1990. Static Acute Toxicity of FX-I003 to the Daphnid, Daphnia magna. Hampton, NH. EnviroSystems study number 9013-3. OTHER General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations. Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that 100% test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity of the test sample with that of the test substance. " I agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
319
000218
AQUATIC PLANTS TOXICITY
Title: Growth and Reproduction Toxicity Test with N2803-3 and the Freshwater Alga, Selenastrum capricornutum
TEST SUBSTANCE
Identity: Perfluorooctanoic acid; may also be referred to as PFOA, FC-26, or FX-1001. (Octanoic acid, pentadecafluoro-, CASRN 335-67-1)
Remarks: The 3M production lot number was 269. The test sample was FC-26, referred to by the test laboratory as N2803-3. The purity of the sample was not sufficiently characterized, although current information indicated it was a mixture of 96.5 - 100% test substance and 0 - 3.5% C6, C7, and C9 perfluoro-homologue compounds. The test sample was a white powder. As stated by the submitter, the sample preparation directions given to the laboratory were to dissolve the test material in a 50:50 water:isopropanol solution. In the protocol amendment, it was stated that the sample was combined with isopropanol in a 50:50 ratio prior to use.
METHODS
Method/guideline followed: USEPA-TSCA Guideline 797.1050
Test type: Static
GLP (Y/N): Y
Year study performed: 1995
Species: The algae were acclimated in sterile, enriched media and maintained at test conditions for at least 14 days prior to the definitive test. The sub-sample of algae used to inoculate media at the start of the definitive test was from a 10-day old culture.
Supplier: The supplier of the algae culture was the Culture Collection of Algae at the U of Texas at Austin.
Measure of growth used: number of cells/mL, growth rate
Concentrations tested: For the range-finding test, the following concentrations of test substance were used: 0.050, 0.50, 5.0, and 50 mg/L (the number of replicates was not specified). For the definitive test, one dilution water control and five nominal concentrations were used in the study. The 50:50 mixture of isopropanol and test substance (N2803-3) was considered to be 100% test substance during the performance of the toxicity test. All results were reported both on the basis of test substance as-tested (50% N2803-3 and 50% isopropanol) and test substance as-received (N2803-3 without isopropanol). The as-tested concentrations were: 0, 63, 125, 250, 500, and 1000 mg/L; the as-received concentrations were: 0, 32, 63, 130, 250, and 500 mg/L. Three replicates of each control and test concentration were utilized under static test conditions.
Exposure period: 96 hours
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Analytical monitoring: Concentrations of the test substance were not measured during the study. The pH in each test vessel was measured at the beginning and end of the test. Incubator temperature was measured and recorded daily. The temperature in a representative vessel of water, which was incubated with the test vessels, was continuously recorded.
Statistical methods: Cell densities, growth rate, and percent inhibition values, which were used to estimate 72- and 96-hour EC50 values and 95% confidence limits, were calculated using the binomial/interpolation method (Stephan, 1984). All calculations were performed using the number of cells/mL, the average specific growth rates, and the nominal concentrations of the test substance. The no observed-effect-concentration (NOEC) was calculated using a parametric one-way analysis of variance (ANOVA), the number of cells/mL, and the average specific growth rate in each test vessel at the end of the test.
Test conditions: The algal medium was prepared according to USEPA recommended concentrations by combining de-ionized water and nutrient stocks. Water used for the acclimation of the test organisms and for all toxicity testing was sterile, enriched media, adjusted to a target pH of 7.5 with 0.1 M HC1 prior to use (the pH of the synthetic algal medium at test initiation was 7.4). Algal medium was used for culturing and as the diluent. A chemical characterization of a representative sample of test media, and water used to formulate test media, was performed. Phosphorous, nitrate, and chloride were detected at the following concentrations: 0.46, 0.08, and 14 mg/L, respectively. Heavy metals detected in the diluent and test medium included cadmium (0.0002 mg/L) and lead (0.08 mg/L). Other potential contaminants were at or below the level of detection. The test substance (as-received) was assumed to have a purity of 100% active ingredient and to be stable under storage and testing conditions. A 1000 mg/L primary stock solution of the test substance (as-tested) was prepared by combining N2803-3 (as-tested) and sterile, enriched media. Appropriate amounts of this stock solution were added directly to dilution water to formulate the test media. Algae were distributed among three replicates of each treatment at the rate of 10,000 cells/mL. Exposure vessels consisted of 250 mL glass Erlenmeyer flasks containing 50 mL of test solution. Test vessels were randomly arranged on a rotary shaker adjusted to 100 rpm in an incubator during the test. The pH of the test solutions for the 250, 500, and 1000 mg/L exposure concentrations were in the range of 2.9-4.0 at test initiation. As stated by the submitter, this low pH may have adversely affected the survival and subsequent growth of the algae. The measured water chemistry values (0-96 hours) were as follows: pH range = 7.4 - 10.3 (control exposure), 2.9 - 3.0 (1000 mg/L exposure), mean temperature range = 23.5 - 24C. A 24-hour light and 0-hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of approximately 380 footcandles. Algal cell counts in each test vessel were determined daily by means of direct microscope counts with a hemocytometer.
Remarks: Water hardness was not indicated. The pH for many exposure concentrations was outside the accepted range for S. capricornutum toxicity testing (7.5 0.1). As stated by the
submitter, there appears to be a discrepancy between the sample preparation directions given to the laboratory and the procedure conducted by the laboratory to prepare the test solutions.
RESULTS
Dose of each endpoint (as mg/L): The NOEC for the range-finding study was 50 mg/L. For the definitive study, the 96-hour EC50 (and associated 95% confidence limits) was 180 mg/L (125 -250 mg/L), based on test substance as-tested (50% N2803-3 and 50% isopropanol) and 90 mg/L (63 - 130 mg/L), based on test substance as-received. The 96-hour NOEC and LOEC values were 125 and 250
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mg/L, respectively, based on test substance as-tested. The 96-hour NOEC and LOEC values were 63 and 130 mg/L, respectively, based on the test substance as-received. The cell growth data is presented are table 1.
Table 1: Cell growth data of acute toxicity test with N2803-3 and the freshwater algae, Selenastrum capricornutum
Nominal concentration
(as-tested), mg/L
Hours 0
Control 63 125 250 500
1,000
10 10 10 10 10 10
Number of cells/mL x 103 (hour!
24 48 72
21 156 424 24 149 487 21 109 513 <10 <10 <10 <10 <10 <10 <10 <10 <10
96
1,671 1,732 1,560 <10 <10 <10
Percent Inhibition
via Density
-
-4 7 100 100 100
Percent Inhibition
via Growth
Rate
-
-2 0 100 100 100
Remarks: After 96 hours of exposure, there were no signs of aggregation, flocculation, or adherence of the algae to the flasks in the control or any test treatment group. In addition, there were no noticeable changes in cell size, color, or morphology when compared to the control. The effect of N2803-3 was determined to be algistatic, not algicidal, based on the results of the post-definitive test exposure. As stated by the submitter, biological data generated by a previous exposure of algae to isopropyl alcohol (Ward, et al. 1995) demonstrated that 1000 mg/L of isopropyl alcohol did not inhibit growth. This finding indicated that the concentration of isopropyl alcohol in the test chemical did not account for the toxicity of N2803-3. Test substance concentrations above 125 mg/L reduced pH values to 4.0 and lower; the low pH may have caused inhibition of algae growth at higher concentrations of test substance. The lowest concentration of test substance observed to cause 100% mortality was 250 mg/L.
Was control response satisfactory (yes/no/unknown): yes
Statistical results, as appropriate: No additional comments
CONCLUSIONS
The NOEC for the range-finding study was 50 mg/L. For the definitive study, the 96-hour EC50was 180 mg/L, based on test substance as-tested, and 90 mg/L, based on test substance as-received. The 96-hour NOEC and LOEC values were 125 and 250 mg/L (as-tested) and 63 and 130 mg/L (as-received), respectively.
Submitters' remarks: Klimich ranking 3. The study lacks analytical measurement of test substance concentrations in the test solutions. Sample purity is not sufficiently characterized. Additionally, there appears to be a discrepancy between the sample preparation directions given to the laboratory and the
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procedure conducted by the laboratory to prepare the test solutions. Survival and growth of the algae may have been adversely affected by initial low pH values in the higher test substance concentrations. Reviewers' remarks: The conclusions appear to be supported by the data. The low pH, which may have further increased growth inhibition, was clearly stated as a possible source of error. REFERENCE T.R. Wilbury Laboratories, Inc. 1995. Growth and Reproduction Toxicity Test with N2803-3 and the Freshwater Alga, Selenastrum capricomutum. Marblehead, MA. Study number B93-TH. OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
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000222
TOXICITY TO INVERTEBRATES
Title: Acute Toxicity of N2 803-3 to the Daphnid, Daphnia magnet
TEST SUBSTANCE
Identity: Perfluorooctanoic acid; may also be referred to as PFOA, FC-26, or FX-1001. (Octanoic acid, pentadecafluoro-, CASRN 335-67-1)
Remarks: The 3M production lot number was 269. The test substance was a white powder. The test sample, FC-26, was referred to by the test laboratory as N2803-3. The purity of the sample was not sufficiently characterized, although available information indicated it was a mixture of 96.5 - 100% test substance and 0 - 3.5% Q , C7, and C9perfluoro- homologue compounds.
METHODS
Method/guideline followed: U.S. EPA-TSCA Guideline 797.1300
Test type: Static
GLP (Y/N): Yes, with one exception: the stability of the test substance was assumed, but not verified.
Year study performed: 1996
Species: Water used for acclimation of the test organisms was deionized water collected at T.R. Wilbury Laboratories in Marblehead, MA. Daphnids employed in the study were less than 24 hours old. Test specimens were produced by adult daphnids that were maintained under test conditions for more than 7 days. The original culture was obtained from Aquatic Research Organisms, Hampton, NH. During acclimation, daphnids were not treated for disease, they were free of apparent sickness, injuries, and abnormalities at test initiation. There was no mortality during the 48 hours preceding the start of the test. The culture was supplied with a yeast/trout chow mix and the freshwater alga Selenastrum capricomutum daily during acclimation. Daphnids were not fed during the test.
Supplier: T.R. Wilbury Laboratories supplied the test organisms. 3M, the sponsor, supplied the test substance.
Concentrations tested: For the static screening test, the nominal concentrations of the test substance (as-received) were: 0.050, 0.50, 5.0, 50, and 500 mg/L. For the static definitive test, two replicates of each concentration were used. The following nominal concentrations were utilized: 0 (blank control), 130, 200, 360, 600, and 1000 mg/L (tested as a 50:50 mixture of test substance and isopropanol). The nominal concentration of test substance (as-received) in solution was 0 (blank control), 65, 110, 180, 300, and 500 mg/L.
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Exposure period: 48 hours
Analytical monitoring: Test substance concentrations were not measured during the study. All toxicity tests were based on nominal concentrations of test substance. Dissolved oxygen, pH, conductivity, and temperature were measured and recorded daily in each test chamber that contained live animals. The temperature in a beaker of water incubated among the test vessels was recorded continuously during the test.
Statistical methods: LC50and EC50 values were calculated, when possible, by probit analysis, moving average method, or binomial probability with non-linear interpolation (Stephan, 1983).
Test conditions:
Water used for acclimation of the test organisms and for all toxicity testing was deionized water collected at T.R. Wilbury Laboratories in Marblehead, MA. Water was adjusted to a hardness of 160 - 180 mg/L as CaC 03and stored in 500-gallon polyethylene tanks, where it was aerated and continuously passed through a particle filter, ultraviolet sterilizer, and activated carbon. A chemical characterization of a representative sample of dilution water detected iron at 0.03 mg/L; all other potential contaminants were below the level of detection or not present. The test substance was prepared by combining N2803-3, asreceived from the sponsor, with isopropanol in a 50:50 ratio. However, the test substance preparation directions given to the laboratory were to dissolve the test material in a 50:50 water:isopropanol solution. This 50:50 mixture was then considered to be 100% test substance during the toxicity test, but all results are reported on an active ingredient basis (active ingredient is the test substance as-received minus the isopropanol). The test substance was assumed to have a purity of 100% active ingredient and to be stable under storage and testing conditions. For the test organisms, measurements made during the 7 days prior to the start of the definitive test with N2803-3 indicated a culture temperature range of 19.5 - 20.5C and a dissolved oxygen concentration of at least 9.1 mg/L. A 1000 mg/L stock solution was prepared by combining 2.0 g of the test substance (50:50 mixture of N2803-3 and isopropanol) and dilution water to a final volume of 2000 mL. The test vessels were 300 mL glass beakers that contained 250 mL of test solution (approximate depth was 9 cm) during the test. Appropriate amounts of the stock solution were added to dilution water in test vessels to formulate test media with the use of a solvent. Twenty daphnids were indiscriminately and equally distributed among two replicates of each test concentration. Test vessels were randomly arranged in an incubator and loosely covered during the 48-hour test. During the definitive test, the following ranges were estimated from measurements: dissolved oxygen = 8.7 - 8.8 mg/L, temperature = 19.4 - 20.7C, conductivity = 570 - 610 pmhos/cm, pH = 8.3 - 8.5. A 16-hour light and 8-hour dark photoperiod with a 15-minute transition period was automatically maintained with coolwhite fluorescent lights that provided a light intensity of 58 footcandles. Aeration was not required to maintain dissolved oxygen concentrations above an acceptable level.
A static test was conducted with 390 mg/L isopropanol and a dilution water control. Two replicates of each concentration and 10 daphnids/replicate were used. This test was conducted in a manner similar to the test with N2803-3. Measurements made during the 7 days prior to
initiation of the isopropanol toxicity test. These measurements indicated a culture temperature range of 20.1 - 20.9 and a dissolved oxygen concentration of at least 8.3 mg/L. The test vessels were randomly arranged in a water bath and the light intensity was 1 3 - 2 5 footcandles. The following ranges were estimated from measurements during the test: dissolved 0 2= 8.1 - 8.5 mg/L, temperature = 20.4 20.9C, conductivity = 530 - 540 pmhos/cm, pH = 8.3 - 8.5.
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Remarks: No additional comments
RESULTS
Dose of each endpoint (as mg/L): LC50 and EC50 values and 95% confidence intervals Based on test substance as-received: 24-hour LC50 = 500 (300 - >500) mg/L 24-hour ECS0 = 420 (370 - 490) mg/L 48-hour LC5o= 400 (350 - 460) mg/L 48-hour EC50 = 360 (300 - 500) mg/L 48-hour NOEC = 180 mg/L Based on test substance as-tested (50:50 ratio of N2803-3:isopropanol): 24-hour LC50 = 1 0 0 0 (600 - > 1 0 0 0 ) mg/L 24-hour EC50 = 840 (740 - 970) mg/L 48-hour LC50 = 800 (700 - 920) mg/L 48-hour ECso = 720 (660 - 780) mg/L 48-hour NOEC = 360 mg/L
Remarks: During the toxicity test with 390 mg/L isopropanol, no mortality or sublethal effects were observed and the 48-hour EC5o and LC50values were > 390 mg/L. For the screening test (48-hours post exposure), there was at least 95% survival at 0 (blank control), 0.050, 0.5, and 5.0 mg/L. Also, there was 15% survival at 500 mg/L (surviving daphnids exposed to 500 mg/L were immobilized). During the definitive toxicity test with N2803-3, no insoluble material was noted during the test. After 48 hours of exposure, the control daphnids had an average wet weight (blotted) of 0.53 mg. The test substance (N2803-3) did not cause 100% mortality at any concentration tested. No mortality was observed in the controls during the definitive test or screening test. Table 1 depicts cumulative percent mortality of the definitive test.
Table 1. Cumulative Percent Mortality
Was control satisfactory
Nominal as-tested concentration (mg/L)
Blank control 130
200
360 600
1000
(yes/no/unknown): Yes
24 hours
0 0 0 0 10
50
48 hours
0 0 0 0
15 80
response
Statistical results, as appropriate: No additional comments
CONCLUSIONS The as-tested test substance (N2803-3) 48-hour LC50 for Daphnia magna was determined to be 800 mg/L with a 95% confidence interval of 700 - 920 mg/L, The as-tested test substance 48hour EC50 for Daphnia magna was determined to be 720 mg/L with a 95% confidence interval of
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009225
660 - 780 mg/L. The as-tested test substance 48-hour no-observed-effect-concentration (NOEC) was 360 mg/L. The 48-hour LC50 for Daphnia magna, based on the concentration of test substance in solution was determined to be 400 mg/L, with a 95% confidence interval of 350 460 mg/L. The 48-hour EC50 for Daphnia magna, based on the concentration of test substance in solution was determined to be 360 mg/L, with a 95% confidence interval of 300 - 500 mg/L. The 48-hour no- observed-effect-concentration (NOEC) for the test substance in solution was 180 mg/L.
Submitters' remarks: Klimisch ranking 3. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized. Additionally, there appears to be a discrepancy between the sample preparation directions given to the laboratory and the procedure conducted by the laboratory to prepare the test solutions.
Reviewers' remarks: The author's conclusions appear to be supported by the data.
REFERENCE
T.R. Wilbury Laboratories, Inc. 1996. Acute Toxicity ofN2803-3 to the Daphnid, Daphnia magna. Marblehead, MA. Study number 892-TH.
OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
327
00022G
AQUATIC PLANTS TOXICITY
Title: Growth and reproduction toxicity test with FC-1015 and the freshwater alga, Selenastrum capricornutum
TEST SUBSTANCE
Identity: Perfulorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143 or as the major component of FC-1015. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS #3825-26-1)
Remarks: The test sample is FC-1015. Its purity was not sufficiently characterized, though current information indicates it is a 30% straight carbon chain version of FC-143 in 80% water. The 3M product lot number was "HOGE 205." Data may not accurately relate toxicity of the test sample with that of the test substance. Data were used to compare toxicity of the branched/straight chain ammonium perfluorooctanoate homolog mixture in FC-143 v. what is supposed to be the 100% straight carbon chain ammonium perfluorooctanoate in FC-1015.
METHODS
Method/guideline followed: OECD 201, USEPA-TSCA, Guideline 797.1050
Test type: static
GLP (Y/N): N
Year study performed: 1996
Species: Selenastrum capricornutum
Supplier: University of Texas at Austin
Measure of growth used: algal cell counts (cells/mL), cell dry weights
Concentrations used: 0, 210, 430, 830, 1670, and 3330 mg/L. The concentrations were nominal. Three replicates at each concentration were tested.
Exposure period: 96 hours
Analytical monitoring: none
Statistical methods: Probit analysis
Test Conditions: The algae were grown in sterile enriched nutrient medium per USEPA 1978 guideline and were allowed 14 days for acclimation to the conditions. The nutrient medium provided all mineral nutrients essential for algal growth and also served as the diluent for all algal operations. The pH of this synthetic algal medium was adjusted to 7.5 prior to use in assays. For preparation of the test solution, a 3330 mg/L stock solution was prepared by diluting 3.33 g of test substance in 1 liter of water. Aliquots
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were then added directly to dilution water in test vessels to create test solutions. The exposure vessels used were 250 mL Erlenmeyers containing 100 mL test solution and capped with inverted glass beakers. The vessels were loaded with an initial concentration of 1.0 x 104 cells/mL and shaken continuously at 100 rpm. Four hundred foot-candles of lighting were provided by continuous cool-white fluorescent lighting. The water chemistry parameters measured during the test included: pH = 7.5-10.8 (control) and 7.4-7.6 (3330 mg/L exposure) and temperature = 24.0-24.2 (incubator). Three replicates of the experiment were performed. Measurements of dilution water chemistry were also performed (see Appendix for parameters and detection limits). The only chemical detected was iron at 0.03 mg/L.
Remarks: The pH range for the control was outside acceptable limits, but the other tested sample was within the acceptable range. Water hardness, during the study, was not indicated.
RESULTS
Dose of each endpoint (as mg/L): Values calculated using cell count (cells/mL): 24 h EC50 = 2510 (1340-3330) mg/L 48 h EC50 > 3330 mg/L 72 h EC50 = 2040 (1190-3330) mg/L 96 h EC50 = 1980 (1710-2360) mg/L 96 h NOEC = 210 mg/L 96 h LOEC = 430 mg/L
Values calculated using the average specific growth rate: 24 h EC50 = 1700 (673-3300) mg/L 48 h EC50 > 3330 mg/L 72 h EC50 > 3330 mg/L 96 h EC50 > 3330 mg/L 96 h NOEC = 430 mg/L 96 h LOEC = 830 mg/L
Remarks: Aliquots of the 3330 mg/L test solution were diluted with algal medium and cultured for 72 hours. Based on growth observed in the recovery phase, the effect on algal growth was found to be algistatic.
Was control response satisfactory (yes/no/unknown): yes
Statistical results, as appropriate: none
CONCLUSIONS
FC-1015 exhibits a 96 hour EC50 cell count value of 1980 (1710-2360 mg/L) and a 96 hour EC50 growth rate value of >3330 mg/L. The 96 hour No Observed Effect Concentration (NOEC) is 210 mg/L for cell count and 430 mg/L for growth rate. The Lowest Observed Effect Concentration (LOEC) is 430 mg/L for cell count and 830 mg/L for growth rate. This test substance was determined to be algistatic.
Submitters' remarks: For data reliability, this study was assigned a Klimisch rating of 2. The study meets criteria for quality testing. However, it lacks information on the purity of the test substance and actual measurements of the amount of test substance in solution. Also, no explanation is given as to why the 48 hour values were in excess of 24 hour values.
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Reviewers' remarks: none
REFERENCE
Ward, T., Nevius, J. and R. Boeri. 1996. Growth and reproduction toxicity test with FC-1015 and the freshwater alga, Selenastrum capricornutum. T.R. Wilbury Laboratories, Inc. Lab Request number P I624. 3M Company, St. Paul, MN.
OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that 100% test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity o f the test sample with that o f the test substance." I agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
APPENDIX
Chemical measurements of dilution water
Parameter:
Metals Aluminum Arsenic Boron Cadmium Chromium Cobalt Copper Iron Lead Mercury Nickel Silver Zinc
Detection Limit:
0.1 mg/L 0.01 mg/L 0.5 mg/L 0.0002 mg/L 0.01 mg/L 0.03 mg/L 0.005 mg/L 0.03 mg/L 0.005 mg/L 0.0003 mg/L 0.03 mg/L 0.02 mg/L 0 . 0 2 mg/L
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Nitrate
0.05 mg N/L
Chloride
1 mg/L
Fluoride
0.1 mg/L
Total organic carbon
1 mg/L
Total phosphorous
0.03 mg/L
Organochlorine Pesticides
0.5 pg/L
Toxaphene
2 pg/L
Organophosphorous Pesticides 0.5 pg/L
Dimethoate
2.0 pg/L
TEPP
2.0 pg/L
Monocrotophos
2.0 pg/L
PCBs
0.5 pg/L
331 000230
INVERTEBRATE TOXICITY
Title: Acute toxicity of FC-1015 to Daphnid, Daphnia magna
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143 or as the major component of FC-1015. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS #3825-26-1)
Remarks: The test sample is FC-1015. Its purity was not sufficiently characterized, though current information indicates it is a 30% straight carbon chain version of FC-143 in 80% water. The 3M product lot number was "HOGE 205."
METHODS
Method/guideline followed: USEPA-TSCA, Guideline 797.1300
Test type: static
GLP (Y/N): N
Year study performed: 1996
Species: Daphnia magna, less than 24 hours old, wet weight = 0.35 mg.
Supplier: initial brood stock from Aquatic Biosystems
Concentrations used: 0, 430, 730, 1200, 2000, and 3300 mg/L. The concentrations were nominal. Two replicates at each concentration were tested.
Exposure period: 48 hours
Analytical monitoring: none
Statistical methods: Probit analysis
Test Conditions: The deionized dilution water used in the test was adjusted to a hardness of 160-180 mg/L (as CaC03), which is within the acceptable range. The water was passed through a particulate filter, ultraviolet sterilizer, and activated carbon. Water used for the test had a hardness of 176 mg/L and an alkalinity of 108 mg/L as CaC03, and it contained <0.01 mg/L particulate matter and <1 mg/L total organic carbon. Further measurements of dilution water chemistry were also performed (see Appendix for parameters and detection limits). The only chemical detected was iron at 0.03 mg/L. Test solutions were created by direct weights addition. Glass beakers (300 mL) containing 250 mL test solution (9 cm depth) were used as exposure vessels. They were lightly covered during the experiment. Two replicates, each of 10 daphnids, were tested at each concentration. Cool-white fluorescent lights at 130 foot-candles
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000231
were used for lighting. A daily photoperiod of 16 hours light and 8 hours dark with a 15 minute transition period was maintained throughout the testing period.
The water chemistry parameters measured during the study included: conductivity range = 610-620 pmhos/cm (control) and 710-720 pmhos/cm (2000 mg/L exposure), pH = 8.2-8.3 (control) and 8 .1-8.2 (2000 mg/L exposure), temperature = 20.4-20.7 C (control) and 20.3-20.8 C (2000 mg/L exposure), and dissolved oxygen = 8 .1-9.1 mg/L (control) and 8 .1-9.1 mg/L (2000 mg/L exposure). The 2000 mg/L (second highest) concentration was used because the highest concentration resulted in total mortality by 48 hours.
Remarks: Water hardness, during the study, was not indicated.
RESULTS
Dose of each endpoint (as mg/L): 24 hour EC50 = 1790 (1550-2070) mg/L 48 hour EC50 = 1200 (730-2000) mg/L 48 hour NOEC = 730 mg/L
Remarks: none
Was control response satisfactory (yes/no/unknown): yes
Statistical results, as appropriate: none
CONCLUSIONS
The FC-1015 48 hour EC50 was determined to be 1200 mg/L with a 95% confidence interval of 7302070 mg/L. The 48 hour No Observed Effect Concentration (NOEC) was 730 mg/L.
Submitters' remarks: For data reliability, the study was assigned a Klimisch rating of 2. The study meets the criteria for quality testing. However, the study lacks information on purity of the test substance and actual measurements of the amount of test substance in solution.
Reviewers' remarks: none
REFERENCE Ward, T., Nevius, J., and R. Boeri. 1996. Acute toxicity of FC-1015 to Daphnid, Daphnia magna. T.R. Wilbury Laboratories, Inc. Lab request number P1624. 3M Company, St. Paul, MN.
OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis,
333
000232
volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that 100% test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity o f the test sample with that o f the test substance." I agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
APPENDIX
Chemical measurements of dilution water
Parameter:
Detection Limit:
Metals
Aluminum
0.1 mg/L
Arsenic
0.01 mg/L
Boron
0.5 mg/L
Cadmium
0.0002 mg/L
Chromium
0.01 mg/L
Cobalt
0.03 mg/L
Copper
0.005 mg/L
Iron 0.03 mg/L
Lead
0.005 mg/L
Mercury
0.0003 mg/L
Nickel
0.03 mg/L
Silver
0.02 mg/L
Zinc 0.02 mg/L
Nitrate
0.05 mg N/L
Chloride
1 mg/L
Fluoride
0.1 mg/L
Total organic carbon
1 mg/L
Total phosphorous
0.03 mg/L
Organochlorine Pesticides
0.5 pg/L
Toxaphene
2 pg/L
Organophosphorous Pesticides 0.5 pg/L
Dimethoate
2.0 pg/L
TEPP
2.0 pg/L
Monocrotophos
2.0 pg/L
PCBs
0.5 pg/L
334
000233
Ecotoxicity Study
ACUTE TOXICITY TO AQUATIC INVERTEBRATES (DAPHNIA MAGNA) TEST SUBSTANCE Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1) Remarks: The 3M production lot number was 427. The test sample is FC-143, referred to by the test laboratory as N2803-4. The T.R. Wilbury study number is 895-TH. The purity of the sample was not sufficiently characterized, although current information indicates it is a mixture of 96.5 - 100% test substance and 0 - 3.5% C6 , C l, and C9 perfluoro analogue compounds. METHOD Method: U.S. EPA-TSCA Guideline 797.1300 Test type: Acute static GLP: Yes Year Completed: 1995 Species: Daphnia magna Supplier: Obtained from cultures maintained by T.R. Wilbury Laboratories Inc, Marblehead, MA from an original culture from Aquatic Research Organisms, Hampton, NH. Analytical monitoring: DO, conductivity, pH, and temperature Exposure period: 48-hours Test organism age: < 24-hours Statistical methods: Interpreted by standard statistical techniques. Computer methods (Stephan, 1983) were used to calculate LC50s and EC50s. Test conditions Dilution water: Deionized water adjusted to a hardness of 160-180 mg/L as CaC03/L Dilution water chemistry: Hardness: 164 mg/L as CaC03 Alkalinity: 106 mg/L as CaC03 TOC: < 1.0 mg/L Residual chlorine: <0.1 mg/L Lighting: Cool-white fluorescent bulbs with an intensity of 110 ft-c. Photoperiod of 16-hours light, 8 -hours dark with a 15 minute transition period. Stock and test solutions preparation: A 1,000 mg/L primary stock solution was prepared in dilution water. After mixing, the primary stock was proportionally diluted with dilution water to prepare the test concentrations. No insoluble material was noted during the test. Exposure vessels: 300 mL glass beakers containing 250 mL of test solution. The approximate depth of test solution was 9 cm. Number of replicates: 2 Number of daphnids per replicate: 10 Number of concentrations: five plus a negative control
335
Water chemistry during the study: Dissolved oxygen range (0 - 48 hours): 8.4 - 8.7 mg/L (control exposure) 8.1 - 8.7 mg/L (1,000 mg/L exposure) Conductivity range (0 - 48 hours) 540 - 560 umhos/cm (control exposure) 7 1 0 -7 2 0 pmhos/cm (1,000 mg/L exposure) PH range (0 - 48 hours) 8.0 - 8.3 (control exposure) 8.0 - 8.2 (1,000 mg/L exposure) Test temperature range (0 - 48 hours) 19.1 20.5C (control exposure) 19.4 - 20.5C (1,000 mg/L exposure) RESULTS Element basis: mortality and immobilization Nominal concentrations: Blank control, 130, 220, 360, 600, 1,000 mg/L. Element value and 95% confidence interval: 24-hour EC50 = 780 (600 - 1,000) mg/L 24-hour LC50 = >1,000 mg/L (C.I. not calculable) 48-hour EC50 = 720 (600 - 1,000) mg/L 48-hour LC50 = 720 (600 - 1,000) mg/L 48-hour NOEC = 360 mg/L Element values based on nominal concentrations.
Cumulative percent immobilization (includes mortality)
Nominal Test 24-hours 48-hours
Cone., mg/L
Control
0
0
13 0 0
22 0 0
36 0 0 60 0 15
100
100
100
Note: At 1000 mg/L, all daphnids were immobilized at 24 hours and dead at 48 hours. Control response: satisfactory CONCLUSIONS The test substance 48-hour LC50 for Daphnia magna was determined to be 720 mg/L with a 95% confidence interval of 600 - 1,000 mg/L. The test substance 48-hour EC50 for Daphnia magna was also determined to be 720 mg/L with a 95% confidence interval of 600 - 1,000 mg/L. The 48hour no observed effect concentration (NOEC) was 360 mg/L. Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 2. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized.
336
000235
REFERENCES This study was conducted at T.R. Wilbury Laboratories, Inc., Marblehead, MA, at the request of the 3M Company, Lab Request number N2803-4 OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
337
000236
Ecotoxicity Study
ACUTE TOXICITY TO AQUATIC INVERTEBRATES (DAPHNIA MAGNA) TEST SUBSTANCE Identity: Perfluorooctanoic acid, tetrabutylammonium salt; may also be referred to as PFOA tetrabutylammonium salt, tetrabutylammonium perfluorooctanoate, or as a major component of L -13492. (Octanoic acid, pentadecafluoro-, tetrabutylammonium salt, CAS # 95658-53-0) Remarks: The 3M production lot number was 2327 The test sample is referred to by the testing laboratory as L -13492. The T.R. Wilbury study number is 840-TH. The 3M Environmental Laboratory Request Number is N2332. The purity of the sample was not sufficiently characterized, although current information indicates it is a solution of 44.9% tetrabutylammonium perfluorooctanoate, 27.9% water, and 27.2% isopropanol Thefollowing summary applies to the test sample as a mixture o f the test substance in water solution with incompletely characterized concentrations o f impurities. Data may not accurately relate toxicity o f the test sample with that o f the test substance. METHOD Method: U.S. EPA-TSCA Guideline 797.1300 Test type: Acute static GLP: Yes Year Completed: 1995 Species: Daphnia magna Supplier: Obtained from cultures maintained by T.R. Wilbury Laboratories Inc, Marblehead, MA from an original culture from Aquatic Research Organisms, Hampton, NH. Analytical monitoring: DO, conductivity, pH, and temperature were monitored daily. Exposure period: 48-hours Test organism age: < 24-hours Statistical methods: LC50 and EC50 values calculated, when possible, by probit analysis, moving average method or binomial probability with non-linear interpolation using the computer software of C.E. Stephan. Test conditions Dilution water: Deionized water adjusted to a hardness of 160-180 mg/L as CaC03/L Dilution water chemistry: Not given. BACK TO MAIN.Lighting: Cool-white fluorescent bulbs with an intensity of 20 ft-c. Photoperiod of 16-hours light, 8 -hours dark with a 15 minute transition period. Stock and test solutions preparation: A 100 mg/L primary stock solution was prepared in dilution water. After mixing, the primary stock was proportionally diluted with dilution water to prepare the test concentrations. No insoluble material was noted during the test. Exposure vessels: 300 mL glass beakers containing 250 mL of test solution. The approximate depth of test solution was 9 cm.
338
000237
The vessels were loosely covered during the test. Number of replicates: 2 Number of daphnids per replicate: 10 Number of concentrations: five plus a negative control Water chemistry during the study: Dissolved oxygen range (0 - 48 hours): 8.5 - 8.7 mg/L (control exposure) 8.5 - 8.7 mg/L (100 mg/L exposure cone.) Conductivity range (0 - 48 hours) 620 - 630 umhos/cm (control exposure) 620 - 640 |imhos/cm (100 mg/L exposure cone.) pH range (0 - 48 hours) 8.4- 8 . 6 (control exposure) 8.5 - 8 . 6 (100 mg/L exposure cone.) Test temperature range (0 - 48 hours) 20.5 - 20.7C (control exposure) 20.5 - 20.6C (100 mg/L exposure cone.) Element basis: mortality and immobilization RESULTS Nominal concentrations: Blank control, 13, 22, 36, 60, 100 mg/L. Element value: 24-hour EC50 = 89 (60 - >100) mg/L 24-hour LC50 = >100 mg/L (Cl not calculable) 48-hour EC50 = 34 (30 - 39) mg/L 48-hour LC50 = 77 (60 - 100) mg/L 48-hour NOEC = 13 mg/L Element values based on nominal concentrations. Remarks: Testing was conducted on a mixture as described in the Test Substance Remarks field. The values reported apply to that mixture and not the fluorochemical component alone.
Cumulative percent immobilization (includes mortality):
Nominal Test 24-hours 48-hours
Cone., mg/L
Control
0
0
13 0 0
22 5 5
36 5 5
60 0 100
100 70 100
Control response: satisfactory CONCLUSIONS The test substance 48-hour LC50 for Daphnia magna was determined to be 77 mg/L with a 95% confidence interval of 60 - 100 mg/L. The test substance 48-hour EC50 for Daphnia magna was determined to be 34 mg/L with a 95% confidence interval of 30 - 39 mg/L. The test substance 48-hour no observed effect concentration (NOEC) was 13 mg/L. Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St.
339
000238
Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 2. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized. REFERENCES This study was conducted at T.R. Wilbury Laboratories, Inc., Marblehead, MA, at the request of the 3M Company, Lab Request number N2332, 1995. OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations. Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that 1 0 0 % test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity o f the test sample with that of the test substance." I agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
340
000239
Ecotoxicity Study
ACUTE TOXICITY TO AQUATIC INVERTEBRATES (DAPHNIA MAGNA) TEST SUBSTANCE Identity: Perfluorooctanoic acid, tetrabutylammonium salt; may also be referred to as PFOA tetrabutylammonium salt, tetrabutylammonium perfluorooctanoate, N2803-2 or as a major component of L-13492. (Octanoic acid, pentadecafluoro-, tetrabutylammonium salt, CAS # 95658-53-0) Remarks: The 3M production lot number was 2. The test sample is referred to by the testing laboratory as N2803-2. The T.R. Wilbury study number is 889-TH. The purity of the sample was not sufficiently characterized, although current information indicates it is a solution of 44.9% tetrabutylammonium perfluorooctanoate, 27.9% water, and 27.2% isopropanol. Thefollowing summary applies to the test sample as a mixture o f the test substance in an isopropanol/water solution with incompletely characterized concentrations o f impurities. Data may not accurately relate toxicity o f the test sample with that o f the test substance. METHOD Method: U.S. EPA-TSCA Guideline 797.1300 Test type: Acute static GLP: Yes Year Completed: 1996. Species: Daphnia magna Supplier: Obtained from cultures maintained by T.R. Wilbury Laboratories Inc, Marblehead, MA from an original culture from Aquatic Research Organisms, Hampton, NH. Analytical monitoring: DO, conductivity, pH, and temperature were monitored daily. Exposure Period: 48-hours Test organism age: < 24-hours Statistical methods: LC50 and EC50 values calculated, when possible, by probit analysis, moving average method or binomial probability with non-linear interpolation using the computer software of C.E. Stephan. Test conditions Dilution water: Deionized water adjusted to a hardness of 160-180 mg/L as CaC03/L Dilution water chemistry: Hardness: Not noted Alkalinity: Not noted pH: Not noted Lighting: Cool-white fluorescent bulbs with an intensity of 110 fit-c. Photoperiod of 16-hours light, 8 -hours dark with a 15 minute transition period. Stock and test solutions preparation: A 200 mg/L primary stock solution was prepared in dilution water. After mixing, the primary stock was proportionally diluted with dilution water to prepare the test concentrations. No insoluble material was noted during the test.
341
000240
Exposure vessels: 300 mL glass beakers containing 250 mL of test solution. The approximate depth of test solution was 9 cm. The vessels were loosely covered during the test. Number of replicates: 2 Number of daphnids per replicate: 10 Number of concentrations: five plus a negative control Water chemistry during the study: Dissolved oxygen range (0 - 48 hours): 8 . 6 - 9.0 mg/L (control exposure) 8.7 - 9.0 mg/L (100 mg/L exposure cone.) Conductivity range (0 - 48 hours) 670 - 690 pmhos/cm (control exposure) 670 - 720 pmhos/cm (100 mg/L exposure cone.) pH range (0 - 48 hours) 8.3- 8.5 (control exposure) 8.4 - 8 . 6 (100 mg/L exposure cone.) Test temperature range (0 - 48 hours) 19.3 - 20.2C (control exposure) 19.3 - 20.4C (100 mg/L exposure cone.) Element basis: mortality and immobilization RESULTS Nominal concentrations: Blank control, 13, 22, 36, 60, 100 mg/L. Element value and 95% confidence interval: 24-hour EC50 = 72 (63-83) mg/L 24-hour LC50 = >100 mg/L (Cl not calculable) 48-hour EC50 = 62 (36 - 100) mg/L 48-hour LC50 = 93 (74 - >100) mg/L 48-hour NOEC = 13 mg/L Element values based on nominal concentrations. Remarks: Testing was conducted on a mixture as described in the Test Substance Remarks field. The values reported apply to that mixture and not the fluorochemical component alone.
Cumulative percent immobilization (includes mortality)
Nominal Test Cone., mg/L Control 13
22
36 60
100
24-hours
0 0 0 0
30 85
48-hours
0 0
5 5 45
100
Note: at 48-hours, more organisms than noted above were affected at 22, 36, and 60 mg/L (noted as "less active than control daphnids), but were not considered immobilized. Control response: satisfactory
342
000241
CONCLUSIONS The test substance 48-hour LC50 for Daphnia magna was determined to be 93 mg/L with a 95% confidence interval of 74 - >100 mg/L. The test substance 48-hour EC50 for Daphnia magna was determined to be 62 mg/L with a 95% confidence interval of 36 - 100 mg/L. The test substance 48-hour no observed effect concentration (NOEC) was 13 mg/L. Submitter: 3M Company, Environmental Laboratoiy, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 2. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized. REFERENCES This study was conducted at T.R. Wilbury Laboratories, Inc., Marblehead, MA, at the request of the 3M Company, Lab Request Number N2803-2, 1996. OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that %1 0 0 test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity o f the test sample with that of the test substance." I agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
343
000242
Title: Acute Toxicity to Fish TEST SUBSTANCE
TOXICITY TO FISH
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1)
Remarks: The 3M product lot number was 83. The test sample was FC-143. The purity of the sample was not sufficiently characterized, though current information indicates it is a mixture of 96.5 - 1 0 0 % test substance and 0 - 3.5% C6, C7 and C9 perfluoro analog compounds.
METHODS
Method/guideline followed: Not stated
Test type: Static
GLP (Y/N): No
Year study performed: 1974
Species: Fathead minnow (Pimephales promelas) Average weight = 1.6 g Average length = 2 inches
Supplier: Not stated
Concentrations used: 0, 10, 20, 30, 40, 50 mg/L nominal
Exposure period: 96 hours
Analytical monitoring: The test substance concentrations were not measured.
Statistical methods: There were no statistical methods given by the study authors; the LC50 was determined graphically.
Test Conditions: -One replicate was performed. -Dissolved oxygen range (24-96 hours) = 4.7 - 5.7 mg/L (control) and 4.0 - 4.9 mg/L (50 mg/L) -pH range (24-96 hours) = 7.0 - 7.1 (control) and 6.4 - 6.5 (50 mg/L exposure) -Temperature range = 70 -72 degrees Fahrenheit -Water hardness was not indicated. -Dilution water was carbon-filtered city water from St. Paul, MN.
Remarks: Further details on the test conditions and procedures were not provided.
344
000243
RESULTS
Dose of each endpoint (as mg/L): 96 hour LC50 = 70 mg/L (C.I. not calculated) based on nominal concentrations
Remarks: Observed mortality of the controls was 10% at both 72 and 96 hours. There was no other mortality with the exception of 20% mortality in the 50 mg/L group.
Was control response satisfactory (yes/no/unknown): unknown, see Remarks above
Statistical results, as appropriate: obtain a calculated LC5ovalue.
Percent survival versus concentration (mg/L) was plotted to
CONCLUSIONS
The study authors concluded that the acute LC50 to fathead minnow was equal to 70 mg/L (C.I. not calculated) based on nominal concentrations.
Submitter's Remarks: Reliability - Klimisch ranking 3. There was insufficient documentation of the methodology. The LC50 was extrapolated from an insufficient number of data points. The general pre-test health and age of fish were not noted. Sample purity was not properly characterized and it lacks analytical confirmation of test substance concentrations.
Reviewer's Remarks: The pH levels during the test period were within the acceptable range; however, the water hardness was not given. Testing should have been done with two replicates. The number of organisms per dose and the loading rate were not indicated.
REFERENCE
3M Company. [No title given]. Lab Request number 2340. St. Paul, MN.
OTHER
Remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recoveiy rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations. As it is we are operating in the dark on this issue.
345
000244
TOXICITY TO FISH
Title: Acute Toxicity to Fish TEST SUBSTANCE
Identity: Perfluorooctanoic acid; may also be referred to as PFOA, FC-26, or FX-1001. (Octanoic acid, pentadecafluoro-, CASRN 335-67-1)
Remarks: 3M production lot number 269. The test sample was FC-26. The purity of the sample was not sufficiently characterized, though current information indicates it is a mixture of 96.5-100% test substance and 0-3.5% C6, C7, and Cg perfluoro homologue compounds.
METHODS
Method/guideline followed: Not stated
Test type: Static
GLP (Y/N): No
Year study performed: 1974
Species: Fathead minnow (Pimephalespromelas) Average length = 2 inches Average weight = 1.5 g
Supplier: Not stated
Concentrations used: 0, 50, 125, 250, 375, 500 mg/L nominal
Exposure period: 96 hours
Analytical monitoring: Nominal concentrations were not measured
Statistical methods: There were no statistical methods given by the study authors; the LC50 was detennined graphically.
Test Conditions: -One replicate was performed. -Temperature 69-70 degrees Fahrenheit -Dissolved oxygen range (24-96 hours) was between 4.7 and 5.7 mg/L for the control exposure and
between 3.8 and 5.2 mg/L for the 375 mg/L test exposure. -Water hardness was not given. -pH range (24-96 hours) was 7.0 to 7.2fo r the control exposure and 6.0 to 6.7fo r the 375 mg/L exposure.
346
00024S
Remarks: No further details were provided on the test conditions or methods.
RESULTS
Dose of each endpoint (as mg/L): 96-hour LC50 = 440 mg/L (C.I. not calculated) based on nominal concentrations
Remarks: For concentrations of 0 to 375 mg/L, survival was 100%. For the 500mg/L concentration, survival was 100% at 24 hours, but 0% at 48-96 hours.
Was control response satisfactory (yes/no/unknown): Yes
Statistical results, as appropriate: obtain an LC50.
Concentration (mg/L) versus percent survival was graphed to
CONCLUSIONS
Submitter's Remarks: Reliability - Klimisch ranking of 3. This study lacks documentation and information on the methodology. Sample purity was not properly characterized. Test concentrations were not characterized. Condition of the fish prior to study initiation is not known.
Reviewer's Remarks: Testing should have been performed in two replicates. Water hardness during the study period was not given. The number of organisms per dose and the loading rate were not indicated.
REFERENCE
3M Company. [No title given]. Lab Request Number 2485. St. Paul, MN.
OTHER
Remarks: This summary was based on a summary report submitted by 3M, therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations. As it is we are operating in the dark on this issue.
347 000246
TOXICITY TO FISH
Title: Acute Toxicity to Fish
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1)
Remarks: The 3M production lot number was 83. The test sample was FC-143. The purity of the sample was not sufficiently characterized, though current information indicates it is a mixture of 96.5-100% test substance and 0-3.5% C6, C7, and C9 perfluoro analogue compounds.
METHODS
Method/guideline followed: Not stated
Test type: Static
GLP (Y/N): No
Year study performed: 1978
Species: Bluegill sunfish (Lepomis machrochirus) Average length = 4 cm Average weight = 0.20 g
Supplier: Not stated
Concentrations used: 0, 135, 180, 240, 320, 420 mg/L nominal
Exposure period: 96 hours
Analytical monitoring: Concentrations were not measured.
Statistical methods: Not stated
Test Conditions: -Dilution: carbon-filtered well water, -Dilution water chemistry: temperature = 19 degrees Celsius dissolved oxygen = 9.1 ppm pH = 7.9 -One replicate -Exposure vessels were tanks containing 16 liters of test solution - 2 0 fish per replicate -Loading rate = 0.25 g/L
348
000247
-Water chemistry during study (24-96 hours): dissolved oxygen = 5.3-6.9 mg/L (control), 5.1-7.3 mg/L (420 mg/L exposure) pH range = 7.9 (control), 7.8-8.0 (420 mg/L exposure) test temperature = 18-19 degrees Celsius
-Water hardness was not stated
Remarks: No further details were provided on the test methods or conditions.
RESULTS
Dose of each endpoint (as mg/L): 96-hour LC50 > 420 mg/L based on nominal concentrations
Remarks: No mortalities occurred in the controls or 135-240 mg/L groups. Mortalities in the 320 and 420 mg/L groups both consisted of one in twenty fish dead at 72 hours
Was control response satisfactory (yes/no/unknown): Yes
Statistical results, as appropriate: Not stated
CONCLUSIONS
Submitter's Remarks: Reliability - Klimisch ranking 3. Testing lacks information on the method followed. No information on stock or test solution preparations was provided. Average fish weight is suspect. Sample purity was not properly characterized and it lacks analytical confirmation of test substance concentrations.
Reviewer's Remarks: Only one replicate was performed. Water hardness during the study period was not indicated.
REFERENCE
3M Company. [No title given]. Lab Request number 3844. St. Paul, MN.
OTHER
Remarks: This summary was based on a summary report submitted by 3M, therefore, the contents of this summary, in reference to the protocols and results of the study, are limited. The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
349
000248
TOXICITY TO FISH
Title: Acute Toxicity to Fish
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, orFC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1)
Remarks: The 3M production lot number was 83. The test sample was FC-143. The purity of the sample was not sufficiently characterized, though current information indicates it is a mixture of 96.5 100% test substance and 0 - 3.5% C6, C7, and C9 perfluoro analogue compounds.
METHODS
Method/guideline followed: Not stated
Test type: Static
GLP (Y/N): No
Year study performed: 1978
Species: Bluegill sunfish (Lepomis machrochirus) Average length = 3.0 cm Average weight = 1.2 g
Supplier: Not stated
Concentrations used: 0, 420, 560, 750, 1000, 1350 mg/L nominal
Exposure period: 96 hours
Analytical monitoring: Concentrations were not measured.
Statistical methods: Not stated
Test Conditions: -Dilution water source: carbon-fdtered well water -Dilution water chemistry: temperature = 19 degrees C, dissolved oxygen = 9.9 ppm, pH = 7.6 -One replicate, 20 fish per replicate -Test performed in tanks containing 16 liters test solution -Loading rate =1.5 g/L -Water chemistry during test (24 - 96 hours): Dissolved oxygen range = 5.6 to 6.5 mg/L (control) and 4.9 to 5.9 mg/L (750 mg/L exposure) pH range = 7.8 (control) and 7.6 to 7.8 (750 mg/L exposure)
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000249
Temperature = 19 to 20 degrees C -Water hardness not given Remarks: No further details were provided on the testing methods or conditions. RESULTS Dose of each endpoint (as mg/L): 96-hour LC50 = 569 mg/L, based on nominal concentrations Remarks: The 95% confidence interval was 500 -636 mg/L. 100% mortality occurred by 48 hours in the 1000 and 1350 mg/L concentration groups.
Was control response satisfactory (yes/no/unknown): One out of twenty fish died at 48 hours in the control group. Statistical results, as appropriate: Not stated
CONCLUSIONS Submitter's Remarks: Reliability - Klimish ranking 3. Testing lacks information on the method followed. No information on stock or test solution preparations was provided. Sample purity was not properly characterized and the study lacks analytical confirmation of test substance concentrations. Reviewer's Remarks: Only one replicate was performed and water hardness during the test period was not given. REFERENCE 3M Company. [No title given]. Lab Request number 3844. St. Paul, MN. OTHER Remarks: This summary was based on a summary report submitted by 3M, therefore, the contents of this summary, in reference to the protocols and results of the study, are limited. The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
351
000250
TOXICITY TO FISH
Title: Chronic Toxicity to Fish
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as 78.03, PFOA ammonium salt, ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1)
Remarks: The 3M production lot number was 83. The test sample was FC-143. The testing laboratory refers to it as "78.03." The purity of the sample was not sufficiently characterized, though current information indicates it being a mixture of 96.5% - 100% test substance and 0 - 3.5% Q,, C7, and C9 pefluoro analogue compounds.
METHODS
Method/guideline followed: The methodology for the egg and fry exposure closely followed that presented in "Proposed recommended bioassay procedure for egg and fiy stages of freshwater fish," U.S. EPA, 1972.
Test type: Flow-through
GLP (Y/N): No
Year study performed: 1978
Species: Fathead minnow (Pimephalespromelas) Pre-treatment: Eggs were placed in a 60 mg/L malachite green solution for 15 seconds to eliminate possible fungus growth.
Test fish age: Eggs within 48-hours after fertilization
Supplier: U.S. Environmental Protection Agency's Environmental Research Laboratory in Duluth, MN.
Concentrations tested: Blank control, 6.2, 12.5, 25, 50, 100 mg/L
Exposure period: 30 days post hatch
Analytical monitoring: Temperature, dissolved oxygen concentration, and pH were monitored daily. Weekly samples were taken from each aquarium for determination of ammonium perfluorooctanoate concentration. All samples taken during the test were stored in polyethylene bottles and shipped on May 31,1978 to the 3M Company.
Statistical methods: Means of measured biological parameters from duplicate aquaria were subjected to analysis of variance (Steele and Torrie, 1960, completely randomized block design, P=0.05). Data for percentage survival and percentage hatch were transformed to arc sin square root of percentage prior to analysis.
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000251
Test conditions: -Dilution water source and contaminants: Well water pumped to a concrete reservoir where it was aerated
before flowing to the exposure system through aged PVC pipe -Dilution water chemistry (0-30 days):
total hardness = 31-38 mg/L as CaC03 alkalinity = 26 -32 mg/L as CaCOi pH = 7 .0 -7 .4 Specific conductance = 149 - 170 f Jmhos/cm -Stock and test solution preparation: A modified, proportional diluter with a 0.50 dilution factor was used. The diluter delivered five nominal concentrations ammonium perfluorooctanoate ranging from 100 to 6.2 mg/L and control water to duplicate test aquaria. A 4 liter glass Mariotte bottle toxicant delivery system was used to deliver 6.6 mL of a nominal ammonium perfluorooctanoate stock concentration of 29.4 mg/mL in distilled water to the mixing chamber of the diluter. -Fry exposure vessel type: Glass test aquarium measuring 30.5x30.5x30.5 cm with a 17.5 cm high
standpipe drain, water volume of 16 liters. -Egg cups: Acrylic tubes (3 cm O.D., 7 cm long) with 40 mesh Nitex screen on one end. An egg cup
rocker arm apparatus, as described by Mount (1968), was used to gently oscillate the egg cups in the test water. -Diluter: Delivered 0.50 liters of test water to each aquarium 195 times per day, yielding a 90% test water replacement time of approximately 10 hours. -Feeding: Fry werefed live brine shrimp nauplii three times daily on weekdays and twice daily on weekends throughout the exposure period. -Number o f replicates: Two replicates. There were 60 eggsfor the hatchability test and 40fry from each egg cup. -Water chemistry during test: Dissolved oxygen: >95% saturation pH: 7 .0 -7 .3 Temperature: 25 + 1 degrees Celsius, maintained by water bath
Remarks: At the termination of the test, the fry from the control and the high concentration (100 mg/L) were preserved in 10% buffered formalin while the fry from the other test aquaria were frozen. Ten formalin-preserved fry (5 from each replicate) from the control and the high concentration underwent histopathological examination of a transverse section of the nares and cephalic extension of the lateral line. The remaining preserved fry and frozen fry were analyzed at a later date (by 3M Company) for ammonium perfluorooctanoate concentrations.
RESULTS
Dose of each endpoint (as mg/L): 30-day NOAEL > 100 mg/L
Nominal Concentration (mg/L)
Control
6.2
Replicate
A B A
Hatch %
98 95 95
30 Days Post Hatch
Survival % Mean length Mean weight
in mm
in mg
(SD)
92 20(2) 62 95 21(3) 75
98 20(2) 59
353
000252
B 94 88 22(2) 79
12.5 A 93 95 21(3) 70
B
88
100 21(2)
72
25 A 98 90 21(2) 74
B 100 95 21(2) 70
50 A 90 90 20(2) 60
B 95 98 20(3) 65
100 A 95 88 19(2) 59
B 97 82 20(2) 60
Test Material Control
100 mg/L Ammonium Perfluorooctanoate
Number of Observations
10
10
Histopathological
Findings 3/10 Normal 6/10 Liver fatty change 3/10 Gill hyperplasia (Epithelium) I 5/10 Normal 5/10 Liver fatty change 2/10 Gill hyperplasia (Epithelium) I
Remarks: Submitter's Note - Only those tissues which were missing or contained demonstrable change are listed. The only tissue changes observed were hyperplasia of gill lamellar epithelium and fatty change of the liver. These changes were judged to be minimal and consistent with changes seen routinely in healthy fish. Autolipis of gill tissue was observed in several fish. This change was probably due to the poor penetration of the buffered formalin to the posterior dorsal portion of the gill space.
Was control response satisfactory (yes/no/unknown): Yes
Statistical results, as appropriate: No statistically significant findings were reported.
CONCLUSIONS
Biological data generated in this study indicate that the nominal concentration of 100 mg/L had no adverse effect upon the hatchability or eggs or upon the survival and growth of fathead minnow fry through 30 days post-hatch.
Submitters' remarks: Klimisch ranking 2. This study meets all the criteria for quality testing at the time it was conducted, but has several deficiencies. It lacks information on purity of the test substance, and the production lot number from which the test sample was taken. There is no information available on the analysis of the test solution concentrations or on the preserved fry and frozen fry samples.
Reviewers' remarks: Water hardness was not monitored during the study period or at study completion as part of the analysis of test conditions.
354
000253
REFERENCE The effects of continuous exposure to 78.03 on hactchability of eggs and growth and survival of fry of fathead minnow {Pimephales promelas). 1978. Report #BW-78-6-175. Research report submitted to 3M Company, St. Paul, MN by EG&G Bionomics Aquatic Toxicology Laboratory, Wareham, MA. OTHER General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations. As it is we are operating in the dark on this issue.
355
000254
FISH ECOTOXICITY
T itle: 96-hour acute static toxicity to fathead minnow - FX-1001
TEST SUBSTANCE
Identity: Perfluoroctanoic acid, also referred to as PFOA, FC-26, or FX-1001. (Octanoic acid, pentadecafluoro-, CAS # 335-67-1) Remarks: The 3M production lot number was not noted. The test sample was FX-1001. Its purity was not completely characterized, although information indicated it was a mixture of 95-98 percent test substance and 1-5 percent perfluorochemical inerts. METHODS Method/guideline followed: Not noted Test type: Static GLP (Y/N): No Year study performed: 1985 Species: Pimephalespromelas,
average length = 4.1 cm; average weight = 0.50 g; age not noted Supplier: Dale Fattig of Brady, NB Concentrations used: 0, 690, 750, 810, 870, 930 mg/L (nominal values). Exposure period: 96 hours Analytical monitoring: No measurements were taken. Also, there was no information on detection limits of the chemical or impurities in the sample. Statistical methods: Probit analysis
356
000255
Test Conditions: - Fish were pretreated with 25.0 mg/L tetracycline HC1, 5 months prior to study to fight diseases - Dilution water chemistry:
Carbon-filtered well water DO 9.5 ppm pH 7.8 Temp 19C
- Water chemistry during the test: Dissolved oxygen range (24-96 hours): 7.3 - 7.7 mg/L (control) 6.1 - 6 .7 mg/L (870 mg/L)* pH range (24-96 hours): 7.5 - 7.7 (control) 7.5 - 7.5 (870 mg/L)*
*870 mg/L test group (second highest concentration) data given because total mortality occurred in the highest test concentration by 48 hours.
Test temperature (24-96 hours): 19-20 C
- Stock solution was prepared by dissolving 45 g test substance (neutralized with NaOH to pH 7.5) in 3 liters water. Test solutions were prepared by transferring stock solution aliquots to make 5 liters at the selected test concentrations. - Loading rate: 0.50 g fish/L - Stability of the test chemical solutions was not noted - Exposure vessels were glass beakers with a 24 cm inside diameter and a 26 cm depth, and contained 5 liters test solution. -Two replicates were taken at each dose, with 5 fish per replicate
Remarks: Water hardness was not presented. The pH values are acceptable according to the OPPTS harmonized guidelines.
RESULTS
Dose of each endpoint (as mg/L): 96-hour LC50: 843 mg/L (C.I.: 811-878), based on nominal concentrations.
Remarks: - 930 mg/L was the lowest test substance concentration causing 100% mortality (seen at 48 hours)
- There was no mortality in controls - Surfacing of the fish occurred at doses of 810, 870, and 930 mg/L
Was control response satisfactory (yes/no/unknown): Yes, based on zero mortality.
Statistical results, as appropriate: No p-values were reported.
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000256
CONCLUSIONS The test sample 96-hour LC50 for fathead minnow was determined to be 843 mg/L with a 95% C.I. of 811-878 mg/L. Submitter remarks: The data quality ranking was a Klimisch ranking of 2 because testing met criteria for quality testing. However, sample purity was not properly characterized and it lacked analytical confirmation of test substance concentrations. Reviewer remarks: none REFERENCE
3M Company. 1985. 96-hour acute static toxicity to fathead minnow - FX-1001. Environmental Laboratory, St. Paul, MN. Lab Request Number C1006. February 2. OTHER General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited. The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
358
000257
FISH ECOTOXICITY
Title: Acute Toxicity to Fish
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; also referred to as PFOA ammonium salt, ammonium perfluorooctanoate, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS# 3825-26-1)
Remarks: The 3M product lot number used was 390. The test sample was FC-126, a white powdery solid. It's purity was not completely characterized, although information indicated it was a mixture of 7893% test substance and 7-22% C6, C l, and C9 perfluoro analogue compounds.
METHODS
Method/guideline followed: Not stated
Test type: Static
GLP (Y/N): No
Year study performed: 1987
Species: Pimephales promelas Average length = 3.5 cm Average weight = 0.30 g
Supplier: Dale Fattig of Brady, NB
Concentrations tested: 0, 100, 180, 320, 560, and 1000 mg/L
Exposure period: 96 hours
Analytical monitoring: None because nominal concentrations were used.
Statistical methods: Probit analysis
Test conditions: -Dilution water source carbon-filtered well water -Dilution water chemistry:
Temp of 21C DO of 9.3 ppm pH of 7.9
-Test solutions were prepared by direct weights addition 359
000258
-Stability of the test chemical solutions was not noted -Exposure vessels were 4 Liter Pyrex glass beakers containing 3 liters test solution -Two replicates were used - Six fish per replicate were used -Loading rate was 0.6 g/L -Water chemistry during test:
DO range (24-96 hours): 6.6- 1A mg/L (control) 5.6- 6.6 mg/L (320 mg/L*)
pH range (24-96 hours): 1.1-1.9 (control) 7.7- 8.0 (320 mg/L*)
Temp (24-96 hours): 20C T h is group was used because total mortality occurred at higher doses
Remarks: No other details on test conditions were given, including water hardness. The pH values are within acceptable ranges according to OPPTS Harmonized guidelines.
RESULTS
Dose of each endpoint (as mg/L): The 96-hour LC50 for fathead minnow was determined to be 301 mg/L (95% Cl: 244-370).
Remarks:
- Surfacing of fish was observed at 180 and 320 mg/L (both replicates) - Lowest test substance concentration causing 100% mortality was 560 mg/L - No mortality was observed in the controls
Was control response satisfactory (yes/no/unknown): Yes, based on the fact that there was no mortality
Statistical results, as appropriate: Not presented
CONCLUSIONS
Rounding the results to 2 significant figures, the test sample 96-hour LC50 for fathead minnow was determined to be 300 mg/L with a 95% confidence interval of 240-370 mg/L.
Submitters' remarks: Data quality was given a Klimisch ranking of 2. Testing met the criteria for quality testing. However, the sample purity was not properly characterized and it lacked analytical confirmation of the test substance concentrations.
Reviewers' remarks: none
REFERENCE
3M Company. 1987. St. Paul, MN. Lab Request Number E128201, completed on May 1.
360
000259
OTHER General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited. The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
361
000260
TOXICITY TO FISH
Title: Static Acute Toxicity of FX-1003 to the Fathead Minnow, Pimephales promelas
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143, or as a major component of FX1003. (Octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1)
Remarks: The 3M production lot number was 2327. The test sample was FX-1003. The purity of FX-1003 was not sufficiently characterized; however, available information indicated it was a solution of <45% ammonium perfluorooctanoate, 50% water, <3% inert perfluorinated compound and 1 - 2 % C5 and C7 perfluoro- analogue compounds.
METHODS
Method/guideline followed: OECD 203
Test type: Static
GLP (Y/N): Yes
Year study performed: 1990
Species: Prior to testing, juvenile fathead minnows were acclimated for 63 days in 100% dilution water under flow-through conditions in an all glass aquarium. Fish were acclimated to the target test temperature (23 2C) for 14 days before test initiation. During acclimation, fish were not treated for disease and were free of apparent sickness, injuries, and abnormalities at test initiation. Fish were fed a commercial fish food, once or twice daily, for the acclimation period. The average length and weight values of the fish were 3.8 cm and 0.45 grams (wet), respectively.
Supplier: The fish used in the toxicity test were purchased from a commercial supplier (Aquatic Research Organisms, Hampton, NH). The sponsor, 3M, provided the test substance.
Concentrations tested: A screening toxicity test was performed with the following five nominal concentrations: 0.1, 1, 10, 100, and 1000 mg/L. For the definitive toxicity test, five nominal concentrations and one blank dilution water control were used. Two replicates of each concentration were used in the definitive toxicity test and the nominal concentrations tested were: 0, 150, 250, 400, 600, and 1000 mg/L. Both tests were performed under static conditions.
Exposure period: 96 hours
Analytical monitoring: Dissolved oxygen, pH, conductivity, and temperature were measured and recorded daily in each test chamber that contained live fish. Concentrations of the test substance were not measured during the test.
362
000261
Statistical methods: Non-linear interpolation, moving average, and/or probit analysis; however, results of the toxicity test could not be interpreted by standard statistical techniques due to 100% survival at the highest tested concentration.
Test conditions:
The screening test was performed using nominal concentrations (0.1, 1.0, 10, 100, and 1000 mg/L) of the test substance under similar conditions as those of the definitive test.
Water used for acclimation of the test organisms, and for all toxicity testing, was well water collected from wells at EnviroSystems in Hampton, NH. Water was adjusted to a hardness of 88 mg/L as C aC 03 and stored in tanks, where it was aerated. A chemical characterization of a representative sample of the natural well water used as the dilution water for the toxicity test was performed; the following values were determined: pH = 7.4, conductivity = 1500 pmhos/cm. Organochlorine pesticides, organophosphorous pesticides, and PCBs were below the level of detection, or not present. The test vessels were 19.6 L glass aquaria that contained 15 L of test solution (approximate water depth was 17 cm). Stability of the test substance was not indicated. No stock solution was prepared as test material was added directly to dilution water contained in the test vessels without the use of a solvent. The following nominal concentrations were used in the definitive test: 0, 150, 250, 400, 600, and 1000 mg/L. Twenty fish were randomly and equally distributed among two replicates of each treatment. The loading rate was determined to be approximately 0.30 g/L. Test vessels were randomly arranged in a water bath during the 96-hour test. Static conditions were maintained throughout the study. A 16-hour light and 8-hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of 35 pEs 'm-2. Aeration was employed after 48 hours to maintain dissolved oxygen concentrations above acceptable levels. Fish were not fed during the test. The following water chemistry ranges (0 - 96 hours) were determined: conductivity = 1200 - 1500 pmhos/cm (control exposure), = 1300 - 1600 pmhos/cm (1000 mg/L exposure); pH range = 7.4 - 8.4 (control exposure), = 7.8 - 8.2 (1000 mg/L exposure); temperature range = 21.0 - 22C (control and 1000 mg/L exposure); dissolved 0 2= 6.1 9.2 mg/L (control exposure), = 6.2 - 9.1 (1000 mg/L exposure). The pH and hardness were within the accepted ranges (6.0 < pH <8.5; 40<hardness<180 mg/L) for the duration of the study.
Remarks: No additional comments
RESULTS
Dose of each endpoint (as mg/L): Screening test: 96-hour LC50 > 1000 mg/L, 96-hour NOEC > 1000 mg/L
Definitive test: 96-hour LC50 > 1000 mg/L, 96-hour NOEC > 1000 mg/L
Remarks: All test vessels remained clear throughout the test. 100% survival occurred in the control exposure and all other test exposures in the screening test and in the definitive test.
Was control response satisfactory (yes/no/unknown): Yes
Statistical results, as appropriate: Results of the toxicity test could not be interpreted by standard statistical methods due to 100% survival at the highest tested concentration.
363
000262
CONCLUSIONS The 96-hour LC50 and 96-hour NOEC were determined to be >1000 mg/L. Submitters' remarks: Klimisch ranking 2. Testing meets the criteria for quality testing. However, sample purity was not properly characterized and the test lacked analytical confirmation of test substance concentrations. Reviewers' remarks: The conclusions appear to be supported by the data. REFERENCE
EnviroSystems, Inc. 1990. Static Acute Toxicity of FX-1003 to the Fathead Minnow, Pimephales promelas. Hampton, NH. Study number was 9014-3. OTHER General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
364
00Q263
TOXICITY TO FISH
Title: Acute toxicity of N2803-3 to the Fathead Minnow, Pimephales promelas
TEST SUBSTANCE
Identity: Perfluorooctanoic acid; may also be referred to as PFOA, FC-26, or FX-1001. (Octanoic acid, pentadecafluoro-, CASRN 335-67-1)
Remarks: The 3M production lot number was 269. The test substance was a white powder. The test sample, FC-26, was referred to by the laboratory as N2803-3. The purity of the sample was not sufficiently characterized; however, available information indicated it was a mixture of 96.5 - 100% test substance and 0 - 3.5% C6, C7, and C9perfluoro- homologue compounds. The test substance ("asreceived") was combined with isopropanol in a 50:50 ratio prior to use. The substance resulting from this mixture was referred to as the "as-tested" substance.
METHODS
Method/guideline followed: U.S. EPA-TSCA Guideline 797.1400
Test type: Static
GLP (Y/N): Yes
Year study performed: 1996
Species: Juvenile fathead minnows were acclimated under flow-through conditions in a 270 L fiberglass tank. During acclimation, the fish were not treated for disease. The fish were free of apparent sickness, injuries, and abnormalities at test initiation. Mortality during the final 48 hours of acclimation was <3%. During the 14-day period before test initiation, the acclimation temperature range was 21.0C - 22.3C and the dissolved oxygen concentration was at least 8.0 mg/L. During acclimation, fish were fed daily, except during the 48 hours immediately preceding the test. The average total length and wet weight of the test organisms were 33.4 mm and 0.35 grams, respectively.
Supplier: Juvenile fathead minnows were procured from Aquatic Biosystems, Fort Collins, CO. The sponsor, 3M, supplied the test substance.
Concentrations tested: For the static screening test, the following nominal concentrations, as-received, were used: 0, 0.05, 0.50, 5.0, 50, and 500 mg/L. For the static definitive test, the following nominal concentrations, as-tested, were used: 0, 130, 220, 360, 600, and 1000 mg/L. For the static test with isopropanol, nominal concentrations of 0 (dilution water control) and 500 mg/L isopropanol were used.
Exposure period: 96 hours
Analytical monitoring: Dissolved oxygen, pH, temperature, and conductivity were measured and recorded daily in each test chamber. The temperature in one test vessel was recorded at least hourly during the test. Concentrations of the test substance were not measured during the study.
365
000264
Statistical methods: LC50values were calculated by non-linear interpolation (Stephan, 1983), when possible, using probit analysis, moving average method, or binomial probability.
Test conditions:
For the static screening test, nominal concentrations of the test substance, as-received, were 0 (dilution water control), 0.05, 0.50, 5.0, 50, and 500 mg/L. The screening test was performed under similar conditions as those of the definitive test.
Water used for acclimation of the test organisms, and for all toxicity testing, was deionized water collected at T.R. Wilbury Laboratories in Marblehead, MA. Water was adjusted to a hardness of 40 - 48 mg/L as CaC 03and stored in polyethylene tanks. In the tanks, the water was aerated and continuously passed through a particle filter, ultraviolet sterilizer, and activated carbon. In a chemical characterization of a representative sample of dilution water, iron was detected at 0.03 mg/L. Other metals and potential contaminants were either below the level of detection, or not present. The test substance, as-received, was combined with isopropanol in a 50:50 ratio prior to use. This 50:50 mixture was then considered to be 100% test substance during the toxicity test. The test substance was assumed to have a purity of 100% active ingredient and to be stable under storage and testing conditions. The test vessels were 20 L glass aquaria, which contained 15 L of test solution (approximate water depth was 18 cm). Twenty fathead minnows were indiscriminately and equally distributed among two replicates of each treatment. Appropriate amounts of test substance were added directly to dilution water in the test vessels to formulate the media. The test concentrations, as-tested, for the definitive test were: 0, 130, 220, 360, 600, and 1000 mg/L. The test vessels were loosely covered and randomly arranged in a water bath during the 96-hour test. A 16-hour light and 8-hour dark photoperiod, with a 15-minute transition period, was automatically maintained with cool-white fluorescent lights that provided a light intensity of 31 footcandles. Aeration was initiated after 72 hours to maintain dissolved oxygen concentrations above acceptable levels. Measured water chemistry during the test provided the following ranges (0 - 96 hours): Dissolved 0 2 = 6.4 - 8.7 mg/L (control exposure), = 5.2 - 9.0 mg/L (220 mg/L exposure); Conductivity = 110-370 pmhos/cm (control and 220 mg/L exposure); pH = 7.1 - 7.6 (control exposure), = 3.0 - 7.4 (220 mg/L exposure); Temperature = 21.8 - 22.6C (control and 220 mg/L exposure). The pH values of the test solutions for the 360, 600, and 1000 mg/L exposure concentrations were in the range of 3.0 - 4.3 at test initiation. These low pH values were outside the acceptable range for aquatic toxicity studies (6.0 <pH<8.5). Hardness was within the acceptable range for toxicity studies (40 - 180 mg/L, as C aC 03).
For the static toxicity test performed with isopropanol, a similar protocol was followed as that for the screening and definitive toxicity tests with N2803-3. 0 (dilution water control) and 500 mg/L isopropanol (nominal concentration) were used. Test vessels were randomly arranged in a water bath and the light intensity was 28 footcandles.
Remarks: No additional comments
RESULTS
Dose of each endpoint (as mg/L) in the definitive test: Test substance concentration, as-tested, and associated 95% confidence limits: 24-hour LCjo = 280 (220 - 360) mg/L 48-hour LC50 = 280 (220 - 360) mg/L 72-hour LC50 = 280 (220 - 360) mg/L 96-hour LCjo = 280 (220 - 360) mg/L 96-hour NOEC = 220 mg/L 366
000265
Test substance concentration, as-received, and associated 95% confidence limits: 24-hour LCS0= 140 (110 - 180) mg/L 48-hour LC50 = 140 (110 - 180) mg/L 72-hour LC50 = 140 (110 - 180) mg/L 96-hour LC50 = 140 (110 - 180) mg/L 96-hour NOEC =110 mg/L
Remarks: For the screening test, the following survival rates were observed: 100% survival in the control, 90% survival at 0.05 mg/L, 100% survival at 0.50, 5.0, and 50 mg/L, and 0% survival at 500 mg/L. For the definitive test, no insoluble material was observed in any of the test vessels during the study. 100% survival occurred in the control exposure and these fathead minnows did not exhibit any sublethal effects. Fish in the 130 and 220 mg/L exposure concentrations appeared normal. Total mortality was observed within 24 hours in the 360, 600, and 1000 mg/L exposure concentrations; therefore, the lowest concentration, which caused 100% mortality, was 360 mg/L. During the toxicity test with 500 mg/L isopropanol, no mortality or sublethal effects were observed; the 96-hour LC50value was >500 mg/L.
Was control response satisfactory (yes/no/unknown): Yes
Statistical results, as appropriate: No additional comments
CONCLUSIONS The as-tested 96-hour LC50 was determined to be 280 mg/L, with a 95% confidence interval of 220 - 360 mg/L. The as-tested 96-hour no-oberved-effect-concentration (NOEC) was 220 mg/L. The 96-hour LC50, based on the test substance as received, was determined to be 140 mg/L, with a 95% confidence interval of 110 - 180 mg/L. The 96-hour NOEC for the test substance, as-received, was 110 mg/L.
Submitters' remarks: Klimisch ranking 3. The study lacked analytical measurement of test substance concentrations in the test solutions and sample purity was not sufficiently characterized. Additionally, there appeared to be a discrepancy between the sample preparation directions given to the laboratory and the procedure conducted by the laboratory to prepare the test solutions. The absence of partial mortality at intermediate doses resulted in a sharp dose-response curve. As a result, the LC50 values determined in this study may not accurately reflect the true toxicity of the solution. The low pH values observed in the high concentrations tested may have had an adverse effect on survival.
Reviewers' remarks: The conclusions appear to be supported by the data.
REFERENCE T.R. Wilbury Laboratories, Inc. 1996. Acute toxicity of N2803-3 to the Fathead Minnow, Pimephales promelas. Marblehead, MA. Study number 891-TH.
OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis,
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volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations. Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that 100% test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity o f the test sample with that o f the test substance. " I agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
368
000267
FISH ECOTOXICITY
Title: Acute toxicity of FC-1015 to the fathead minnow, Pimephales promelas
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143 or as the major component of FC-1015. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS #3825-26-1)
Remarks: The test sample is FC-1015. Its purity was not sufficiently characterized, though current information indicates it is a 30% straight carbon chain version of FC-143 in 80% water. The 3M product lot number was "HOGE 205." Data may not accurately relate toxicity of the test sample with that of the test substance. Data were used to compare toxicity of the branched/straight chain ammonium perfluorooctanoate homolog mixture in FC-143 vs. what is supposed to be the 100% straight carbon chain ammonium perfluorooctanoate in FC-1015.
METHODS
Method/guideline followed: USEPA-TSCA 1993. 797.1400
Test type: static
GLP (Y/N): N
Year study performed: 1996
Species: Pimephales promelas. The fish were juveniles with an average length of 35 mm and average weight of 0.36g (wet).
Supplier: Aquatic Biosystems
Concentrations used: 0, 530, 830, 1330, 2100, and 3300 mg/L. The concentrations were nominal. Two replicates at each concentration were performed.
Exposure period: 96 hours
Analytical monitoring: none
Statistical methods: LC50 values were calculated using the Stephan computer program, 1983
Test Conditions: The dilution water used was deionized water adjusted for hardness and passed through a particulate filter, ultraviolet sterilizer, and activated carbon. The dilution water chemistry was measured as follows: hardness = 44 mg/L as CaC03, alkalinity = 29-30 mg/L as CaCOi, and TOC < 1 mg/L. For lighting, cool-white fluorescent lights at 30 foot-candles were used. A daily photoperiod of 16 hours light and 8 hours dark with a 15 minute transition period was maintained throughout the testing period. The test solutions were created by direct individual weight additions. After a 14 day acclimation period, the fish were exposed, at a loading of 0.24g fish/L, in 20 L glass aquaria containing 15 liters test solution at
369
000268
an approximate depth of 18 cm. Two replicates were tested at each concentration. Twenty fish were used in each replicate.
The water chemistry parameters measured during the study included: conductivity range = 180-190 pmhos/cm (control) and 300-320 umhos/cm (2100 mg/L exposure), pH = 7.3-7.9 (control) and 7.2-7.7 (2100 mg/L exposure), temperature = 22.0-22.4 C (control) and 21.8-22.5 C (2100 mg/L exposure), and dissolved oxygen = 5.8-9.2 mg/L (control) and 5.7-9.2 mg/L (2100 mg/L exposure). The 2100 mg/L (second highest) concentration was used because the highest concentration resulted in total mortality at 72 hours. The pH range was within the acceptable range. Measurements of dilution water chemistry were also performed (see Appendix for parameters and detection limits). The only chemical detected was iron at 0.03 mg/L.
Remarks: Water hardness, during the study, was not indicated.
RESULTS
Dose of each endpoint (as mg/L): 96 hr LC50 = 2470 (2100-3330) mg/L 96 hr NOEC = 830 mg/L
Remarks: none
Was control response satisfactory (yes/no/unknown): yes
Statistical results, as appropriate: none
CONCLUSIONS
The test sample 96 hour LC50 for fathead minnow was determined to be 2470 mg/L with a 95% confidence interval of 2100-3330 mg/L.
Submitters' remarks: For data reliability, the study was assigned a Klimisch rating of 2. Testing meets the criteria for quality testing. However, sample purity was not properly characterized and it lacks analytical confirmation of test substance concentrations.
Reviewers' remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that 100% test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity o f the test sample with that o f the test substance. " 1agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would
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reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
REFERENCE
Ward, T., Nevius, J. and R. Boeri. 1996. Acute toxicity of FC-1015 to the fathead minnow, Pimephales promelas. T.R. Wilbury Laboratories, Inc. Lab Request number P I624. 3M Company, St. Paul, MN. OTHER General remarks: none APPENDIX
Chemical measurements of dilution water
Parameter:
Detection Limit:
Metals
Aluminum
0.1 mg/L
Arsenic
0.01 mg/L
Boron
0.5 mg/L
Cadmium
0.0002 mg/L
Chromium
0.01 mg/L
Cobalt
0.03 mg/L
Copper
0.005 mg/L
Iron 0.03 mg/L
Lead
0.005 mg/L
Mercury
0.0003 mg/L
Nickel
0.03 mg/L
Silver
0.02 mg/L
Zinc 0.02 mg/L
Nitrate
0.05 mg N/L
Chloride
1 mg/L
Fluoride
0.1 mg/L
Total organic carbon
1 mg/L
Total phosphorous
0.03 mg/L
Organochlorine Pesticides
0.5 pg/L
Toxaphene
2 pg/L
Organophosphorous Pesticides 0.5 pg/L
Dimethoate
2.0 pg/L
TEPP
2.0 pg/L
Monocrotophos
2.0 pg/L
PCBs
0.5 pg/L
371
000270
ACUTE TOXICITY TO FISH (FATHEAD MINNOW) TEST SUBSTANCE Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1) Remarks: The 3M production lot number was 427. The test sample is FC-143, referred to by the test laboratory as N2803-4. The T.R. Wilbury study number is 895-TH. The purity of the sample was not sufficiently characterized, although current information indicates it is a mixture of 96.5 - 100% test substance and 0 3.5% C6, C7, and C9 perfluoro analogue compounds. METHOD Method: U.S. EPA-TSCA Guideline 797.1400 Type: Acute static GLP: Yes Year completed: 1995 Species: Pimephales promelas Supplier: Aquatic Biosystems, Fort Collins, CO Analytical monitoring: DO, conductivity, pH and temperature Exposure period: 96-hours Statistical methods: LC50 values calculated, when possible, by probit analysis, moving average method or binomial probability with non-linear interpolation using the computer software of C.E. Stephan. Test fish age: Not noted. Length and weight: Average length = 28.6 cm Average weight = 0.19g (wet) Loading: 0.13 g/L Pretreatment: None Test conditions: Dilution Water: Deionized water adjusted to a hardness of 40-48 mg/L as CaC03/L Dilution water chemistry: Not noted. Lighting: Cool-white fluorescent bulbs with an intensity of 30 ft-c. Photoperiod of 16-hours light, 8 -hours dark with a 15 minute transition period. Stock and test solutions preparation: Test substance added directly to the dilution water in the test vessels. Concentrations dosing rate: Once Stability of the test chemical solutions: No insoluble material was noted during the test. Exposure vessels: 20 L glass aquaria containing approximately 15 L of test solution, water depth approximately 18 cm. Number of replicates: 2 Number offish per replicate: 10 Number of concentrations: five plus a negative control Water chemistry during the study: Dissolved oxygen range (0 - 96 hours): 7.2 - 8.5 mg/L (control exposure) 6.7 - 8.5 mg/L (1,000 mg/L exposure)
372
Conductivity range (0 - 96 hours) 1 4 0 - 180 pmhos/cm (control exposure) 280 - 380 pmhos/cm (1,000 mg/L exposure) pH range (0 - 96 hours) 7.3 - 7.6 (control exposure) 7.4 - 7.5 (1,000 mg/L exposure) Test temperature range (0 - 96 hours) 22.0 - 22.6C (control exposure) 21.8 - 22.5C (1000 mg/L exposure) RESULTS Nominal concentrations: Bk control, 160, 250, 400, 630, 1,000 mg/L Element value and 95% confidence interval: 24-hour LC50 = > 1,000 mg/L (C.I. not calculable) 48-hour LC50 = 790 (630 - 1,000) mg/L 72-hour LC50 = 760 (630 - 1,000) mg/L 96-hour LC50 = 740 (660 - 830) mg/L 96-hour NOEC = 400 mg/L Element values based on nominal concentrations Statistical Evaluation of Mortality: The 48 and 72-hour LC50 values were determined by binomial interpolation. Probit was used to calculate the 96-hour LC50. Biological observations after 96-hours: Fish in the control and the 160, 250, and 400 mg/L exposure concentrations appeared normal. Dark discoloration and erratic swimming of two fish was observed at the 24-hour observation period in the 1,000 mg/L exposure concentration.
Cumulative percent mortality:
Nominal Test Cone., mg/L Control 160 250 400 630 1,000
24hours
48hours
72- 96hours hours
0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 15 90 90 90
Lowest concentration causing 100% mortality: None Mortality of controls: None
373 000272
CONCLUSIONS The test sample 96-hour LC50 for fathead minnow was determined to be 740 mg/L with a 95% confidence interval of 660 -830 mg/L. The 96-hour no observed effects concentration (NOEC) was 400 mg/L. Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking = 2. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized. REFERENCES This study was conducted at T.R. Wilbury Laboratories, Inc., Marblehead, MA, at the request of the 3M Company, Lab Request number N2803-4. OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
374
000273
ACUTE TOXICITY TO FISH (FATHEAD MINNOW)
TEST SUBSTANCE Identity: Perfluorooctanoic acid, tetrabutylammonium salt; may also be referred to as PFOA tetrabutylammonium salt, tetrabutylammonium perfluorooctanoate, N2803-2, or as a major component of L-13492. (Octanoic acid, pentadecafluoro-, tetrabutylammonium salt, CAS # 95658-53-0) Remarks: The 3M production lot number was 2. The test sample is referred to by the testing laboratory as L-13492. The T.R. Wilbury study number is 839-TH. The 3M Environmental Laboratory Request Number is N2332. The purity of the sample was not sufficiently characterized, although current information indicates it is a solution of 44.9% tetrabutylammonium perfluorooctanoate, 27.9% water, and 27.2% isopropanol Thefollowing summary applies to the test sample as a mixture o f the test substance in an isopropanol/water solution with incompletely characterized concentrations o f impurities. Data may not accurately relate toxicity o f the test sample with that o f the test substance. METHOD Method: U.S. EPA-TSCA Guideline 797.1400 Type: Acute static GLP: Yes Year completed: 1995 Species: Pimephales promelas Supplier: Aquatic Biosystems, Fort Collins, CO Analytical monitoring: DO, conductivity, pH and temperature were monitored daily. Exposure period: 96-hours Statistical methods: LC50 values calculated, when possible, by probit analysis, moving average method or binomial probability with non-linear interpolation using the computer software of C.E. Stephan. Test fish age: Not noted. Length and weight of fish in control: Average length = 32.8 mm Average weight = 0.28 g Loading: 0.19 g fish/L Pretreatment: None BACK TO MAIN Test conditions: Dilution Water: Deionized water adjusted to a hardness of 40 - 48 mg/L as CaC03/L Dilution water chemistry: Not noted. Lighting: Cool-white fluorescent bulbs with an intensity of 28 ft-c. Photoperiod of 16-hours light, 8 -hours dark with a 15 minute transition period. Aeration: Initiated after the 72-hour DO measurements. Stock and test solutions preparation: Test substance was added directly to the dilution water in the test vessels on a weight/'volume basis. No insoluble material was noted during the
375
test. Concentrations dosing rate: Once Exposure vessels: 20 L glass aquaria containing approximately 15 L of test solution, water depth approximately 18 cm. Vessels were loosely covered during the test. Number of replicates: 2 Number of fish per replicate: 10 Number of concentrations: five plus a negative control Water chemistry during the study (from a representative sample of dilution water): Hardness: 44 mg/L as CaC03 Alkalinity: 38 mg/L as CaC03 pH 7.9 Dissolved oxygen range (0 - 96 hours): 6.1 - 8.8 mg/L (control exposure) 5.1 -8.9 mg/L (1,000 mg/L exposure) Note: aeration was supplied to test vessels at 72hours Conductivity range (0 - 96 hours) 170 - 180 pmhos/cm (control exposure) 200 - 210 pmhos/cm (1,000 mg/L exposure) pH range (0 - 96 hours) 7.2 - 7.9 (control exposure) 7.3 - 7.8 (1,000 mg/L exposure) Test temperature range (0 - 96 hours) 22.1 - 22.2C (control exposure) 21.9 - 22.2C (1,000 mg/L exposure) BACK TO MAIN
RESULTS Nominal concentrations: Blank control, 130, 220, 360, 600, 1,000 mg/L. Element value and 95% confidence interval: 24-hour LC50 = 1,000 (600 - >1,000) mg/L 48-hour LC50 = 940 (600 - >1,000) mg/L 72-hour LC50 = 890 (600 - >1,000) mg/L 96-hour LC50 = 890 (600 - >1,000) mg/L 96-hour NOEC = 600 mg/L Element values based on nominal concentrations. Remarks: Testing was conducted on the mixture as described in the Test Substance Remarks field. The values reported apply to that mixture and not the fluorochemical component alone.
Cumulative percent mortality:
Nominal
24-
48-
Test hours hours
Cone.,
mg/L
Control
0
0
130 0 0
72hours
0 0
96hours
0 0
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000275
220 360 600 1,000
0 0 00 0 0 00 0 0 00 50 60 70 70
Lowest concentration causing 100% mortality: None Mortality of controls: None CONCLUSIONS The test substance 96-hour LC50 for fathead minnow was determined to be 890 mg/L with a 95% confidence interval of 600 - >1,000 mg/L. The test substance 96-hour no observed effect concentration (NOEC) was 600 mg/L. BACK TO MAIN Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 2. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized. REFERENCES This study was conducted at T.R. Wilbury Laboratories, Inc., Marblehead, MA, at the request of the 3M Company, Lab Request number N2332, 1995. OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that 100% test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity o f the test sample with that o f the test substance." I agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
377
000276
ACUTE TOXICITY TO FISH (FATHEAD MINNOW) TEST SUBSTANCE Identity: Perfluorooctanoic acid, tetrabutylammonium salt; may also be referred to as PFOA tetrabutylammonium salt, tetrabutylammonium perfluorooctanoate, N2803-2, or as a major component of L -13492. (Octanoic acid, pentadecafluoro-, tetrabutylammonium salt, CAS # 95658-53-0) Remarks: The 3M production lot number was 2. The test sample is referred to by the testing laboratory as N2803-2. The T.R. Wilbury study number is 888-TH. The purity of the sample was not sufficiently characterized, although current information indicates it is a solution of 44.9% tetrabutylammonium perfluorooctanoate, 27.9% water, and 27.2% isopropanol. Thefollowing summary applies to the test sample as a mixture o f the test substance in an isopropanol/water solution with incompletely characterized concentrations o f impurities. Data may not accurately relate toxicity o f the test sample with that o f the test substance. METHOD Method: U.S. EPA-TSCA Guideline 797.1400 Type: Acute static GLP: Yes Year completed: 1995 Species: Pimephales promelas Supplier: Aquatic Biosystems, Fort Collins, CO Analytical monitoring: DO, conductivity, pH and temperature were monitored daily. Exposure period: 96-hours Statistical methods: LC50 values calculated, when possible, by probit analysis, moving average method or binomial probability with non-linear interpolation using the computer software of C.E. Stephan. Test fish age: Not noted. Length and weight of fish in control: Average length = 34.9 mm Average weight = 0.36 g Loading: 0.24 g fish/L Pretreatment: None Test conditions. Dilution Water: Deionized water adjusted to a hardness of 40-48 mg/L as CaC03/L Dilution water chemistry: Not noted. Lighting: Cool-white fluorescent bulbs with an intensity of 25 ft-c. Photoperiod of 16-hours light, 8 -hours dark with a 15 minute transition period. Aeration: Initiated after the 72-hour DO measurements. Stock and test solutions preparation: Test substance was added directly to the dilution water in the test vessels on a weight/volume basis. Concentrations dosing rate: Once Exposure vessels: 20 L glass aquaria containing approximately 15 L of test solution, water depth approximately 18 cm. Vessels were loosely covered during the test.
378
Number of replicates: 2 Number of fish per replicate: 10 Number of concentrations: five plus a negative control Water chemistry during the study: Dissolved oxygen range (0 - 96 hours): 6.3 - 8.6 mg/L (control exposure) 4.6 -8.4 mg/L (1,000 mg/L exposure) Note: aeration was supplied to test vessels at 72hours Conductivity range (0 - 96 hours) 140 - 150 pmhos/cm (control exposure) 170 - 180 pmhos/cm (1,000 mg/L exposure) pH range (0 - 96 hours) 7.4 - 7.7 (control exposure) 7.5 - 7.7 (1,000 mg/L exposure) Test temperature range (0 - 96 hours) 2 1 .6 - 21.9C (control exposure) 2 1 .6 - 21.9C (1,000 mg/L exposure) RESULTS Nominal concentrations: Blank control, 160, 250, 400, 630, 1,000 mg/L. Element value and 95% confidence interval: 24-hour LC50 = >1,000 mg/L (Cl not calculable) 48-hour LC50 = >1,000 mg/L (Cl not calculable) 72-hour LC50 = 970 (630 - >1,000) mg/L 96-hour LC50 = 960 (830 - >1,000) mg/L 96-hour NOEC = 630 mg/L Element values were based on nominal concentrations.
Remarks: Testing was conducted on the mixture as described in the Test Substance Remarks field. The values reported apply to that mixture and not to the fluorochemical component alone. Cumulative percent mortality:
Nominal Test Cone., mg/L Control 160 250 400 630 1,000
24hours
48hours
72hours
96hours
0 0 00 0 0 00 0 0 00 0 0 00 0 0 00 30 45 55 55
Lowest concentration causing 100% mortality: None Mortality of controls: None CONCLUSIONS
379
The test substance 96-hour LC50 for fathead minnow was determined to be 960 mg/L with a 95% confidence interval of 830 - >1,000 mg/L. The test substance 96-hour no observed effect concentration (NOEC) was 630 mg/L. Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 2. The study lacks analytical measurement of test substance concentrations in thetest solutions and sample purity is not sufficiently characterized. REFERENCES This study was conducted at T.R. Wilbury Laboratories, Inc., Marblehead, MA, at the request of the 3M Company, Lab Request number N2803-2, 1995. OTHER
General remarks: The major concern for trying to determine the validity for this test is that ONLY NOMINAL TEST CHEMICAL CONCENTRATIONS were used. OPPT recommends that measured test chemical concentrations be used, so that one can accurately determine the test chemical concentration to which to the test organisms are exposed. If it is determined that the nominal concentrations are only, for example 50% of the measured concentrations, the toxicity values can be and must be adjusted downward by 50% . If analytical measurements of some sort had been furnished, we could calculate chemical recovery rates, and take into account hydrolysis, volatility, and other physicochemical processes that might lower the actual test organism exposure concentrations.
Purity of the test material also is a major concern and was not sufficiently characterized in this test. In some tests it appeared that 100% test chemical was used, for others a chemical of lesser purity (approximately 85%) was used. Water, a solvent (isopropanol) or a combination of both was used in other tests, for no obvious stated reason. In fact, 3M in their summary of each test state: "Data may not accurately relate toxicity of the test sample with that o f the test substance." I agree with this concern. In addition, if this was a "typical" TSCA section 4 review, I would reject these studies, pending receipt of additional information on purity and studies on analytical measurements of the test chemical in the test medium.
380
000279
TOXICITY TO MICROORGANISMS
Title: Microtox Toxicity Test
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; also referred to as PFOA ammonium salt, ammonium perfluorooctanoate, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS# 3825-26-1)
Remarks: The 3M production lot number was 390. The test sample was FC-126, a white powdery solid. Its purity was not sufficiently characterized, although information indicated it was a mixture of 78-93 percent test substance and 7-22 percent C5, C6, and C7 perfluoro analogue compounds.
METHOD
Method/guideline followed: Beckman's Microtox "BASIC" Procedure
Test type: Static
GLP (Y/N): No
Year study performed: 1987
Species/strain: Photobacterium phosphoreum
Supplier: Not noted
Concentrations tested: 0, 420, 560, 750, and 1000 mg/L as nominal concentrations.
Exposure period: 30 minutes
Analytical monitoring: No measurements of the test substance were taken throughout the test.
Statistical methods: Not noted
Test conditions: -Diluent was 2% NaCl Microbic's Reagent -Deionized water pH was 6.5 - A 2000 mg/L stock solution was prepared by dissolving 200 mg test solution in 100 mL diluent. - Test solutions were prepared using aliquots of the stock solution. - Media was not renewed -Stability of the test chemical solutions was not noted. -Exposure vessels were cuvettes. -Two replicates were taken.
Remarks: none
RESULTS
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000280
Dose of each endpoint (as mg/L):
5 minute EC20 = 810 mg/L 5 minute EC50 = >1000 mg/L 5 minute EC80 = > 1000 mg/L 15 min EC20 = 520 mg/L 15 min EC50 = >1000 mg/L 15 min EC80 = >1000 mg/L 30 min EC20 = 420 mg/L 30 min EC50 = 870 (810-930) mg/L 30 min EC80 = > 1000 mg/L
Was control response satisfactory (yes/no/unknown): unknown
Statistical results, as appropriate: Not noted
Remarks: none
CONCLUSIONS
The 30-minute EC50 was determined to be 870 mg/L with 95% confidence intervals of 810 to 930 mg/L
Submitters' remarks: A Klimisch data quality ranking of 2 was given. Testing meets all criteria for quality testing, but lacks analytical confirmation of test substance concentrations. Characterization of the test sample is also lacking.
Reviewers' remarks: none
REFERENCE
3M Company. [No title given.] Lab Request Number E l282. St. Paul, MN.
OTHER
General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
382
000281
TOXICITY TO MICROORGANISMS DATA
Title: Microbics Microtoxi i Toxicity Test TEST SUBSTANCE
Identity: PFOA ammonium salt also referred to as ammonium perfluorooctanoate, PFO, FC-116, FC126, FC-169, FC-143, or as a major component of FX-1003 (octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1), as stated in the submitter's summary.
Remarks: The 3M production lot number was 2327; the test substance was FX-1003. The purity of the test substance was not sufficiently characterized, though current information indicated it was a solution of < 45% ammonium perfluorooctanoate, 50% water, < 3% inert perfluorinated compound, and 1 - 2% C5 and C7 perfluoro-analogue compounds.
METHOD
Method/guideline followed: Microbics Microtox "BASIC" Procedure
Test type: Static
GLP (Y/N): No
Year study performed: 1990
Species/strain/supplier: Photobacterium phosphoreum/3M
Doses (concentrations) tested: 0 (blank control), 125, 250, 500, and 1000 mg/L
Exposure period: 30 minutes
Analytical monitoring: Concentrations of the test substance were not measured during the study.
Test conditions: Two replicates of each of the four test concentrations and blank control were prepared using aliquots of the stock solution, a 2000 mg/L solution prepared in Millipore Milli-Q f I water. 2% NaCl Microbic's Reagent was used as the diluent. The initial pH of 6.8 was adjusted to pH 6.7 with dilute IL SO.,: for osmotic adjustments, 200 mg NaCl was added to 10 ml of sample. The final solutions were clear and colorless. Cuvettes were used as the exposure vessels. Percent of light-loss by the dosed organisms, in reference to the mean light generated by the control organisms, was used to assess the toxicity of the test substance.
Statistical methods: EC|0and EC50values were calculated using a statistical linear-regression program provided for Microtoxi j. Remarks: The test conditions and procedures were not further described.
383
000282
RESULTS Dose of each endpoint (calculated by statistical linear-regression program):
5-min EC50>1000 mg/L 15-min EC50>1000 mg/L 30-min EC5o>1000 mg/L Was control response satisfactory (yes/no/unknown): Unknown Statistical results, as appropriate: Not specified Submitter's remarks: Testing met all criteria for quality testing, but lacked analytical confirmation of test-substance concentrations. Also, there was a lack of characterization of the test sample.
Reviewer's remarks: Unable to determine % mortality of the controls.
CONCLUSIONS The authors conclude the FX-1003 30-minute EC50for Photobacterium phoshphoreum to be >1,000 mg/L (nominal concentration). Submitter's remarks: Klimisch-ranking 2. The study summary applies to the test sample as a mixture of the test substance in water solution with incompletely characterized concentrations of impurities. Data may not accurately relate toxicity of the test sample with that of the test substance. Reviewer's remarks: None REFERENCE 3M Environmental Laboratory. 1990. Microbics Microtox! I Toxicity Test. St. Paul, Minnesota. Lab request number G2882. OTHER
Remarks: This summary was based on a summary report and limited raw data. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
384
000283
TOXICITY TO MICROORGANISMS
Title: Microbics Microtox Toxicity Test of FC-143
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1)
Remarks: The test sample is FC-143. Its purity was not sufficiently characterized, though current information indicates it is a mixture of 96.5 - 100% test substance and 0 - 3.5% Q,, C7, and C9 perfluoro analog compounds. The 3M product lot number was 427.
METHOD
Method/guideline followed: Microbics Microtox "BASIC" Procedure
Type (test type): static
GLP (Y/N): N
Year study performed: 1996
Species/strain: Photobacterium phosphoreum
Supplier: Microbics
Concentrations tested: 0, 125, 250, 500, and 1000 mg/L. The concentrations were nominal. Two replicates of each were tested at each concentration.
Exposure period: 30 minutes
Analytical monitoring: none
Statistical methods: EC50 values were calculated by a statistical linear regression program provided for Microtox.
Test conditions: A primary 2000 mg/L stock solution was prepared in Millipore Milli-QTM water. The pH of the stock solution was then adjusted from 5.8 to 7.6 using 1.0 N NaOH. An osmotic adjustment was made using 200 mg NaCl dissolved in 10 mL stock solution. The water hardness was not reported. Appearance of test solutions was noted as "clear and colorless." All test solutions were made by proportional dilutions. The test temperature was not noted. Cuvettes (3 mL) were used as the exposure vessels. Two replicates at each of four concentrations were tested, along with two controls.
R em arks: The number of microbes per replicate and the growth phase of the microbes were not indicated.
RESULTS
385
000284
D ose o f each en d p o in t (as rag/L): 5 minute EC50 > 1000 mg/L 15 minute EC50 = 800 (790 - 820) mg/L 30 minute EC50 = 730 (630 - 850) mg/L
Was control response satisfactory (yes/no/unknown): unknown Statistical results, as appropriate: none Remarks: none CONCLUSIONS The FC-143 30 minute EC50 for Photobacterium phosphoreum was determined to be 730 mg/L with a 95% confidence interval of 630 to 850 mg/L. Submitters' remarks: For data reliability, the study was assigned a Klimisch ranking of 2. Testing meets all criteria for quality testing, but lacks analytical confirmation of test substance concentrations. There is also a lack of characterization of the test sample. Reviewers' remarks: none REFERENCE 3M Environmental Laboratory. 1996. Microbics Microtox Toxicity Test of FC-143. Lab Request number P1626. St. Paul, Minnesota. OTHER General Remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
386
0002S5
TOXICITY TO MICROORGANISMS
Title: Microbics Microtox Toxicity Test of FC-118
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143, or as the major component of FC-118. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1)
Remarks: The test sample is FC-118. Its purity was not sufficiently characterized, though current information indicates it is 20% FC-143 in 80% water. The 3M product lot number was not noted. Data may not accurately relate toxicity of the test sample with that of the test substance.
METHOD
Method/guideline followed: Microbics Microtox "BASIC" Procedure
Type (test type): static
GLP (Y/N): N
Year study performed: 1996
Species/strain: Photobacterium phosphoreum
Supplier: Microbics
Doses (concentrations) used: 0, 625, 1250, 2500, and 5000 mg/L. The concentrations were nominal. Two replicates were tested at each concentration.
Exposure period: 30 minutes
Analytical monitoring: none
Statistical methods: EC50 values calculated by statistical linear regression program provided for Microtox.
Test Conditions: A primary 10 g/L stock solution was prepared in Millipore Milli-QTM water. The pH of the stock solution was 6.0 (no adjustment) and an osmotic adjustment was made using 200 mg NaCl dissolved in 10 mL stock solution. Appearance of test solutions was noted as "clear and colorless." All test solutions were made by proportional dilutions. The test temperature was not noted. Cuvettes (3 mL) were used as the exposure vessels. Two replicates were tested at each concentration, as well as the control.
R em arks: The number of microbes per replicate and the growth phase of the microbes were not indicated.
RESULTS
387
000256
D ose o f each en d p oin t (as m g/L ): 5 minute EC50 = 4460 (4020 - 4950) mg/L 15 minute EC50 = 3360 (3090 - 3641) mg/L 30 minute EC50 = 3150 (2910 - 3420) mg/L
Was control response satisfactory (yes/no/unknown): unknown Statistical results, as appropriate: none Remarks: none CONCLUSIONS The FC-118 30 minute EC50 for Photobacterium phosphoreum was determined to be 3150 mg/L with a 95% confidence interval of 2910-3420 mg/L. Submitters' remarks: For data reliability, the study was assigned a Klimisch rating of 2. Testing meets all criteria for quality testing, but lacks analytical confirmation of test substance concentrations. There is also a lack of characterization of the test sample. Reviewers' remarks: none REFERENCE 3M Environmental Laboratory. 1996. Microbics Microtox Toxicity Test of FC-118. Lab Request number P I626. St. Paul, Minnesota. OTHER General Remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
388
000287
TOXICITY TO MICROORGANISMS
Title: Microbics Microtox Toxicity Test of FC-1015-X
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143, or as the major component of FC-1015 orFC-1015-X. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1)
Remarks: The test sample is FC-1015-X. Its purity was not sufficiently characterized, though current information indicates it is a 30% straight carbon chain version of FC-143 in 80% water. The 3M product lot number was not noted. Data may not accurately relate toxicity of the test sample with that of the test substance. Data were used to compare toxicity of the branched/ straight chain ammonium perfulorooctanoate homolog mixture in FC-143 vs. FC-1015-X.
METHOD
Method/guideline followed: Microbics Microtox "BASIC" Procedure
Type (test type): static
GLP (Y/N): N
Year study performed: 1996
Species/strain: Photobacterium phosphoreum
Supplier: Microbics
Doses (concentrations) used: 0, 416, 832, 1665, and 3330 mg/L. The concentrations were nominal. Two replicates were tested at each concentration.
Exposure period: 30 minutes
Analytical monitoring: none
Statistical methods: EC50 values calculated by statistical linear regression program provided for Microtox.
Test Conditions: A primary 6.660 g/L stock solution was prepared in Millipore Milli-QTM water. The pH of the stock solution was 6.9 and an osmotic adjustment was made using 200 mg NaCl dissolved in 10 mL stock solution. Appearance of test solutions was noted as "clear and colorless." All test solutions were made by proportional dilutions. The test temperature was not noted. Cuvettes (3 mL) were used as the exposure vessels. Two replicates were tested at each concentration, as well as two controls.
R em arks: The number of microbes per replicate and the growth phase of the microbes were not indicated.
RESULTS
389
000288
D oses o f each en d p oin t (as m g/L ): 5 minute EC50 = 2300 (2070 - 2560) mg/L 15 minute EC50 = 1960 (1730 - 2210) mg/L 30 minute EC50 = 1950 (1760 - 2160) mg/L
Was control response satisfactory (yes/no/unknown): unknown Statistical results, as appropriate: none Remarks: none CONCLUSIONS The FC-1015-X 30 minute EC50 for Photobacterium phosphoreum was determined to be 1950 mg/L with a 95% confidence interval of 1760-2160 mg/L. Submitters' Remarks: For data reliability, the study was assigned a Klimisch rating of 2. Testing meets all criteria for quality testing, but lacks analytical confirmation of test substance concentrations. There is also a lack of characterization of the test sample. Reviewers' Remarks: none REFERENCE 3M Environmental Laboratory. 1996. Microbics Microtox Toxicity Test of FC-1015-X. Lab Request number P 1626. St. Paul, Minnesota. OTHER General Remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of this study, are limited.
390
000289
TOXICITY TO BACTERIA Title: Activated Sludge Respiration Inhibition
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1) Remarks: The 3M production lot number was 37. The test sample was FC-143. The purity of the substance was not sufficiently characterized, though current information indicates it was a mixture of 96.5 - 100% test substance and 0 - 3.5% C6, C7, and C9 perfluoro analogue compounds. METHODS
Method/guideline followed: Protocol reference EAR, 11/8/79
Test type: Not noted GLP (Y/N): No
Year study performed: 1980 Test organism: Activated sludge mixed liquor Source: Metro Wastewater Treatment Plant, St. Paul, MN. Concentrations tested: Blank control, 1000 mg/L Exposure period: Seven minutes Analytical monitoring: None Statistical methods: Graphed dissolved oxygen versus time in minutes. Test conditions: Not noted other than the stock solution was prepared with 0.6 g FC-143 diluted to 100 mL with D.I. water. Remarks: No further details on testing methods were provided by the submitter.
RESULTS
391
000290
Dose of each endpoint (as mg/L): No acute inhibitory effect on activated sludge respiration rate at 1000 mg/L with a contact time of 7 minutes. Remarks: Results based on nominal concentrations. Was control response satisfactory (yes/no/unknown): unknown Statistical results, as appropriate: Not noted CONCLUSIONS Limited contact time estimates that ammonium perfluorooctanoate is not expected to inhibit the activity of activated sludge. Submitters' remarks: Klimisch ranking 3. Testing lacks record of methodology used. The method was not an agency approved method. There is lack of characterization of the test sample and test solutions not analyzed for test substance concentrations. Reviewers' remarks: No EC50 or other effect level was given. REFERENCE
3M Company. [No title given]. Lab request number 5625S. St. Paul, MN. OTHER
General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
392
000291
TOXICITY TO BACTERIA Title: Activated Sludge Respiration Inhibition Test
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; also referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, FC-116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS# 3825-26-1.)
Remarks: The 3M production lot number is 390. The test sample was FC-126, a white powdery solid. The purity was not completely characterized, although information suggested it was a mixture of 78-93 percent test substance and 7-22 percent C5, C6, and C7 perfluoro analogue compounds.
METHODS
Method/guideline followed: OECD 209
Test type: Static
GLP (Y/N): No
Year study performed: 1987
Test Organism: Activated sludge mixed liquor was used the same day it was collected. Source: Metro Wastewater Treatment Plant, St. Paul, MN.
Concentrations tested: 0, 100, 180, 320, 560, and 10000 mg/L test material solution. (Two blank controls - 0 mg/L - were used, as well as a reference substance.)
Exposure period: 30 minutes; 3 hours
Analytical monitoring: It was not stated whether the concentrations were monitored throughout the test.
Statistical methods: Not stated. Test conditions:
- Dilution water was aerated, distilled, deionized water with a pH of 6.7 - Synthetic sewage was prepared according to OECD Guideline # 209 - Suspended solids were 2.7 g/L
- Test solutions were prepared by adding individual weights to solutions containing 284 mL distilled water, 16 mL synthetic sewage feed, and 200 mL inoculum. The pH of the solutions was adjusted to 7.0 using 1 N NaOH. The reference solutions were created using 500 mg/L stock solution of 3,5-diochlorophenol.
393
000292
- pH of the activated sludge mixed liquor was 7.8 (initial) and 8.0 (final); pH of the 1000 mg/L test concentration was 7.4 (initial) and 8.1 (final). - Temperature of the test was 20-21 C (for both 30 min and 3 hr).
- Exposure vessel type was not described. - Number of replicates not stated, except that two blank controls were used. - Respiration inhibition was determined by measuring dissolved oxygen consumption.
Remarks: Hardness of the dilution water was not presented.
RESULTS
Dose of each endpoint (as mg/L): At 30 min: EC50 (for respiration inhibition) = > 1000 mg/L
At 3 hrs: EC50 (for respiration inhibition) = > 1000 mg/L
Remarks: - A reference substance was used, but no results were presented.
- None of the doses resulted in 100 percent inhibition.
Was control response satisfactory (yes/no/unknown): Control response appeared adequate; less than 1 percent respiration rate inhibition occurred. Also, the difference between the two controls was 2.2 percent at 30 minutes and 0.8 percent at 3 hours; this meets the guideline criterion of being within 15 percent of each other.
Statistical results, as appropriate: none
CONCLUSIONS
The authors sate that the test material induced 38 percent inhibition in respiration rate at 1000 mg/L after 3 hours of exposure.
Submitters' remarks: The authors state that the Klimisch data quality ranking was 2, and that the study meets the criteria for quality testing. However, the study lacked characterization of the test substance purity and analytical confirmation of concentrations. Also, no results were presented for the reference compound.
Reviewers' remarks:
REFERENCE
3M Company. Activated Sludge Respiration Inhibition Test. Environmental Laboratory; Lab Request Number E1282. St. Paul, MN.
OTHER
General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
394
000293
TOXICITY TO BACTERIA
Title: Activated Sludge Respiration Inhibition
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC-116, FC-126, FC-169, FC-143, or as a major component of FX1003. (Octanoic acid, pentadecafluoro-, ammonium salt, CASRN 3825-26-1)
Remarks: The 3M production lot number was 2327. The test sample was FX-1003. The purity of the test substance was not sufficiently characterized, though available information indicated it was a solution of <45% ammonium perfluorooctanoate, 50% water, <3% inert perfluorinated compound and 1-2% C5and C7perfluoro- analogue compounds. The test substance was a clear liquid. The reference substance used for this study, 3,5-dichlorophenol, was Aldrich red label, lot number D7-060-0.
METHODS
Method/guideline followed: OECD 209
Test type: Static
GLP (Y/N): No
Year study performed: 1990
Test organism: Mixed liquor activated sludge was collected from Metro Wastewater Treatment Plant. The test organisms were used on the day obtained. The condition of the organisms was not specified.
Supplier: Metro Wastewater Treatment Plant
Concentrations tested: 0 (blank control) and 1000 mg/L FX-1003 were used for each exposure period (30 minutes and 3 hours). Two replicates of the blank control and one replicate of 1000 mg/L FX-1003 were used for each exposure period. A reference control, 3,5-dichlorophenol, was included at a concentration of 10 mg/L. One replicate of the reference control was used for each exposure period. Nominal concentrations of the test substance and reference control were used during this study.
Exposure period: 30-minutes and 3-hours
Analytical monitoring: Dissolved oxygen concentrations were monitored in order to determine respiration inhibition, as determined by oxygen consumption.
Statistical methods: Not specified
Test conditions: The inoculum contained 3.2 g/L of mixed liquor suspended solids. The test substance was created by a mass addition to a solution containing 284 mL Millipore Milli-Q water, 16 mL
395
000294
synthetic sewage feed, and 200 mL inoculum. The test substance was apparently miscible with water. A reference solution was created using a 500 mg/L stock solution of 3,5-dichlorophenol. The initial and final pH of the activated sludge mixed liquor was 6.7 and 7.1, respectively. The initial pH of the 1000 mg/L test solution was 7.3. The initial pH of the reference solution, 10 mg/L 3,5-dichlorophenol, was 7.5. The temperature range was 20 - 21C during the study. Two replicates of the blank control, and one replicate of each concentration of FX-1003 (1000 mg/L) and 3,5-dichlorophenol (10 mg/L), were used for each exposure period (30 minutes and 3 hours).
Remarks: No additional comments
RESULTS Dose of each endpoint (as mg/L): 30-minute EC50 >1000 mg/L
3-hour EC50 >1000 mg/L
Remarks: Testing was conducted on the mixture of the described test substance. The values reported apply to that mixture and not the test substance.
Was control response satisfactory (yes/no/unknown): Yes
Statistical results, as appropriate: No additional comments
CONCLUSIONS The FX-1003 3-hour EC50 for activated sludge respiration inhibition was determined to be >1000 mg/L.
Submitters' remarks: Klimisch ranking 2. Testing meets all criteria for quality testing, but lacks analytical confirmation of test substance concentrations. There is a lack of characterization of the test sample. The testing procedure was not fully documented.
Reviewers' remarks: The conclusions appear to be supported by the data, however, limited data were available to adequately assess the study.
REFERENCE 3M Environmental Laboratory. 1990. Activated Sludge Respiration Inhibition. St. Paul, Minnesota. Lab Request number G2882.
OTHER
General remarks: This summary was based on a summary report and limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
396
000295
TOXICITY TO BACTERIA
T itle: Activated Sludge Respiration Inhibition Test
TEST SUBSTANCE
Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA tetrabutylammonium salt, Ammonium perfluorooctanoate PFO, FC-116, FC-126, FC-169, FC-143, or as a major component of GC-1015 or FC-1015-X. (Octanoic acid, pentadecafluoro-, ammonium salt, CAS # 3825-26-1)
Remarks: The test sample was FC-1015-X. It's purity was not sufficiently characterized, though current information indicates it is a 30% straight chain version of FC-143 in 80% water. The 3M product lot number was "HOGE 205." Data were used to compare the toxicity of the branched/ straight chain ammonium perfluorooctanoate homologue mixture in FC-143 with what is supposed to be the 100% straight carbon chain ammonium perfluorooctanoate in FC-1015-X.
METHODS
Method/guideline followed: OECD Test #209
Test type: static
GLP (Y/N): no
Year study performed: 1996
Test Organism: activated sludge
Supplier: The activated sludge mixed liquor was collected from the Metro Wastewater Treatment Plant in St. Paul, MN.
Concentrations tested: 420, 840, 1660, and 3320 mg/L. Two blank controls and a reference substance were also tested. The concentrations were nominal.
Exposure period: 30 minutes and 3 hours
Analytical monitoring: none
Statistical methods: none
Test conditions: The dilution water used was Millipore Milli-QTM water. A stock solution of the reference substance, 3,5-dichlorophenol, was prepared by dissolving 500 mg in 10 mL IN NaOH, diluted to 30 mL, then brought to the point of incipient precipitation with IN H2S 0 4 and diluted to 1L. The pH of the reference solution was measured to be 7.2. The test substance was added directly to the test vessels.
Synthetic sewage per OECD guidelines (Test #209) was used.
397
000296
The temperature ranged from 19.1 to 22.1 C. Initially, total suspended solids were measured at 3.22 g/L. At the end of the test, TSS = 1.3 g/L. The initial pH = 7.8, while the final pH = 7.9. The water hardness was not indicated. The element basis was respiration inhibition as determined by oxygen consumption. Remarks: Authors reported that values were corrected to 20C for calculations. RESULTS Dose of each endpoint (as mg/L): 30 min EC50 > 3320 mg/L
3 h EC50 > 3320 mg/L Remarks: Testing was conducted on the mixture of the test substance as described in the test substance remarks field. The values reported apply to that mixture and not the test substance. A reference substance of 3,5-dichlorophenol was used, but EC50 values were not reported. Was control response satisfactory (yes/no/unknown): unknown Statistical results, as appropriate: none CONCLUSIONS
The FC-1015-X 3 hour EC50 for activated sludge respiration inhibition was determined to be greater than 3320 mg/L. Submitters' remarks: The study was assigned a Klimisch ranking of 2. Testing meets all criteria for quality testing, but lacks analytical confirmation of test substance concentrations. There is a lack of characterization of the test sample. Reviewers' remarks: none REFERENCE 3M Company Environmental Laboratory. 1996. Lab Request N 2169. St. Paul, MN. OTHER General remarks: This summary was based on a summary report and only limited data tables. No detailed report was available. Therefore, the contents of this summary, in reference to the protocols and results of the study, are limited.
398
000297
MICROBIOS' MICROTOX TOXICITY TEST TEST SUBSTANCE Identity: Perfluorooctanoic acid, tetrabutylammonium salt; may also be referred to as PFOA tetrabutylammonium salt, tetrabutylammonium perfluorooctanoate, N2803-2, or as a major component of L-13492. (Octanoic acid, pentadecafluoro-, tetrabutylammonium salt, CAS # 95658-53-0) Remarks: The 3M production lot number was 2 . The test sample is referred to by the testing laboratory as L-13492. The purity of the sample was not sufficiently characterized, although current information indicates it is a solution of 44.9% tetrabutylammonium perfluorooctanoate, 27.9% water, and 27.2% isopropanol. Thefollowing summary applies to the test sample as a mixture of the test substance in an isopropanol/water solution with incompletely characterized concentrations o f impurities. Data may not accurately relate toxicity o f the test sample with that o f the test substance. METHOD: Method: Microbios' Microtox "BASIC" Procedure GLP: No Year Completed: 1995 Species: Photobacterium phosphoreum Analytical monitoring: pH, light output Replicates: 2 Statistical methods: EC50 values calculated by statistical linear regression program provided for Microtox Test organism source: Microbics Corporation, Carlsbad, CA Test Conditions: Dilution water: Millipore Milli-Q TM water. Stock and test solution preparation: A primary 2000 mg/L stock solution was prepared in Millipore Milli-QTM water. The pH of the stock solution was then adjusted from 4.7 to 6.7 using 0.1 N NaOH and an osmotic adjustment was made using 200 NaCl dissolved in 10 mL stock solution. Appearance of test solutions was noted as "clear and colorless". A 11test solutions were made by proportional dilutions. Exposure vessels: 4 mL glass cuvettes. Number of replicates: Two Number of concentrations: Four plus blank control. Element Basis: Percent light loss. RESULTS Nominal concentrations: Blank control, 125, 250, 500, 1000 mg/L Element value and 95% confidence interval: 5-minute EC50 = 630 (590 - 665) mg/L 15-minute EC50 = 300 (270 - 330) mg/L 30-minute EC50 = 260 (220 - 300) mg/L Element values based on nominal concentrations. Remarks: Testing was conducted on a mixture as described in the Test Substance Remarks field. The values reported apply to that mixture and not the fluorochemical component alone. CONCLUSIONS
399
The test sample 30-minute EC50 for Photobacterium phosphoreum was determined to be 260 mg/L with a 95% Confidence Interval of 220-300 mg/L. Subm itter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 2. Testing meets all criteria for quality testing, but lacks analytical confirmation of test substance concentrations in the test solutions and sample purity is not sufficiently characterized. REFERENCES This study was conducted by the 3M Company, Environmental Laboratory, St Paul, MN, Lab Request number N 2169, 1995 OTHER
400
000299
Title: Bioconcentration test of Perfluoroalkylcarboxylic acid (C=7-13) [This test is performed using Perfluorooctanoic acid (Test substance number k-1519)] in carp
TEST SUBSTANCE Identity: K-1519. Perfluorooctane sulfonic acid, potassium salt. Lot number A37626B Remarks: Test substance number K-1519. The test substance is a white powder. Purity determined to be 98%. The test substance was treated as 100% purity.
METHOD: M ethod/guideline followed: Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body" stipulated in the "Test Method for New Chemical Substance" July 13, 1974, Revised October 8, 1998, Kanpogyo No. 5, Planning and Coordination Bureau, Environmental Agency; No.615, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare: and No. 392, Basic Industries Bureau, Ministry of International Trade and Industry, Japan), and Bioconcentration : Flow-through Fish test ( Guideline 305, June 14, 1996)" in the OECD Guidelines for Testing of Chemicals. Type: Flow-through system GLP (Y/N): Yes Year: December 18, 2000 Species: Carp ( C yprinus carpio) Supplier: Fuluokaken yabeqawa fishermen's cooperative association Length and weight at test termination:
Mean length = 6.8-8.6 Mean weight = No weight recorded Loading: 2 and 20 ug/L respectively Fish Age: Yearling fish Analytical monitoring: High-performance liquid chromatography-Mass spectrometry Pretreatment: Test water: Aliquot of test water for level 1 and level 2 were one and two ml respectively for HPLC-mass spectrometry. Test Fish: Fish were taken from each test tank and pretreated for HPLC-Mass Spectrometry. Measurement of body weight and length, chopped into pieces, making sample fine, taking out 1 -5 grams. Treated with acetonitrile (20 mL), homogenized, washed, and centrifuged. Residue Supernatant- Filtration and sampled for LC-MS analysis
Number o f concentrations: Two plus a negative control Test concentration: (mean measured): Negative control, 5 and 50 ug/L Uptake period: 28 days Depuration period: Not stated. Test conditions:
Dilution water: Groundwater from the premises of Kurume Laboratory Dilution water chemistry:
Specific conductance: Not recorded Hardness: 111 mg/L Alkalinity: 96.1
401
000300
pH: 7.8
Dissolved Oxygen: <6 Tem perature: 24.3 to 25.6C Stock and test solution preparation: Based on preliminary test results for the 96 hour LC50 value and analytical detection limits, test concentrations of the test substance were decided as follows. The control was set as a blank test. Level 1 was 5 ug/L and Level 2 was 50 ug/L. The test substance was dissolved with ion-exchanged water to prepare 500 mg/L stock solution. Diluter flow rate: 2 mL/min for stock solution and 800 mL/min for dilution water; 1155 liters/day for test water were supplied. Exposure Vessels: 100 liter glass tank Num ber o f replicates: None Number of fish per vessel: Level 1 and 2: 28 Contol: 8 Diet: Nippon Formula Feed Mfg. Co., Ltd.
W ater chemistry ranges during the study:
Neg. Control
50 ug/L
Dissolved Oxygen: 80.0-8,1 mg/L
7.9 - 8.1 mg/L
Temperature C:
25.2 - 25.8C
2 4 .9 -2 5 .4 'C
OO
r-'
1 kO
_Hi_________________ 7 .6 -7 .8
5 ug/L 7.9 - 8.1 mg/L 24.3 - 25.6C 7.6-7.8
Photoperiod: Artificial light of white fluorescent lamp (14 hrs./day) Light intensity: Artificial light of white fluorescent lamp
Collection o f tissue samples: Analysis of test fish was performed six times at each level in duration of exposure. Four fish were taken out at each sampling time and divided into two groups, and then both were analyzed individually. Analysis of control fish was performed before the experimental starting and after the experimental completion. Four fish were taken out at each sampling time and divided into two groups, and then both were analyzed individually. The fish were sampled for there lipid content by fixing theme in chloroform-methanol extraction with gravimetric analysis. Statistical methods: Calculation of lipid content was as follows: Lipid content (%) = (T-To)/S x 100 Where To = Weight of vessel (g) T = Weight of sample of gravimetric analysis (containing vessel) (g) S = Weight of fine sample taken out for analysis of lipid content. Two fish were employed to measure the lipid content because of insufficient size.
Confirmation of the steady-state was reached. It was evaluated that a steady-state had been reached when there successive analysis of BCFs made on samples taken at intervals of at least 48 hours were within 20% of each other. When BCFs were aless than 100, it was evaluated that a steady-state had been reached after 28 days even if BCF were over 20% of each other.
402
000301
RESULTS
Nom inal concentrations: Negative control, M ean measured concentrations: 5 and 50 ug/L Bioconcentration factors (BCF): Level 1 (5 ug/L) apparent steady-state BCF Time to reach 50% clearance:
PFOAConcentration in test water:
Cone.
After 1 After 3 After 10
Day Days Days
5 ug/L 50.3
47.6 46.9
50 ug/L 4.83
4.69 4.61
After 16 Days 48.1 4.66
After 23 Days 46.7 4.83
After 28 Days 45.8 4.61
Average (STD) 47.6+ 1.52 4.71 +0.101
PFOA Concentration in Tissue of Carp Exposed to 5 and 50 ug/L:
Cone.
After 3 After 10 After 16 After 23 After 28
Days
Days
Days
Days
Days
5 ug/L 2.9 2.4 3.0 3.0 4.2
2.1 2.5 3.0 2.0 3.3
50 ug/L 6.5 <5.1 7.7 6.1 6.8
<5.1 <5.1 9.4 5.1 <5.1
Test organism mortality: Negative control: None documented. Level 1 (5 ug/L): None documented. Level 2 (50 ug/L): None documented.
Analytical methodology: Analysis of PFOA in the test water and carp was performed using high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. The test water of each level was analyzed once before first analysis of test fish and at the same time as the analysis of the test fish. Steady state was reached when three successive analyses of BCFs made on samples taken at intervals of at least 48 hours were within + 20% of each other. When BCFs were less that 100, it was evaluated that a steady-state had been reached after 28 days.
Concentration of test substance in test water at a steady-state The mean concentration of the test substance in test water at a steady state were 94% of nominated concentrations. The measured lipid contents in the test fish were 3.10% before initiation of exposure and 2.82% after termination of exposure.
CONCLUSIONS In this study, PFOA BCF ranged from 31 for level 1 and 5.1-9.1 for level 2 in the tissues of carp. Test concentrations of 5 and 50 ug/L were used. The fish were exposed for 28 days.
REFERENCES Kurume Laboratory (2001). Chemicals Evaluation and Research Institute, Japan. Test number:
000302
