Document ExxJzbao8GjrV8KDbKGRB76Nj

AR226-3385 Subchronic, Developmental, and Reproductive Toxicity o f a Fluoroalkylethyl Phosphate Surfactant J.C. Stadler. D.A. Delker. G.T. Makovec, J.F. Hansen, S.M. Munley, and E. Mylchrgest. DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, Delaware, U SA Abstract A fluoroalkylethyl phosphate surfactant was evaluated in rats in 90-day, one-generation reproduction, and developmental toxicity studies. In the 90-day study, the test substance was given as a suspension in isopropanol (IPA) at oral doses of 0, 10, 60 or 300 mg/kg/day (35% a.i.) and 60 mg/kg/day IPA. There were no compound effects on mortality, clinical signs or neurobehavior. Adverse effects on body weights, food parameters, red cell mass, and hepatic enzymes were seen at the highest two doses, and increases in thyroid weight and cellular hypertrophy were observed at the high dose. Adverse microscopic changes in the liver (focal hepatocellular necrosis) occurred at all dose levels in males and at only the high dose in females. These effects were not seen with IPA dosed alone. Other non-adverse physiological changes included increased liver and kidney weights, hepatocellular hypertrophy, and an increased rate of peroxisome proliferation. The NOEL for female rats in this study was 10 mg/kg/day. In the reproductive study, where surfactant doses were 0, 75, 500 and 3500 mg/kg/day (20% a.i. in water, no IPA), parental body weights, weight gains and food parameters were reduced at the high dose, with males more affected than females. There were no effects on reproduction parameters at any dose level, except for Fj pup weights, which were progressively reduced from birth to weaning (92% and 76% of control, respectively) at the high dose level. In these rats dosed with the active in water, thyroid hypertrophy and liver necrosis were not observed. The NOEL for reproductive parameters was 500 mg/kg/day. In the developmental study, doses were 625, 1250 or 2500 mg/kg/day (20% a.i. in water). At 2500 mg/kg/day, effects on maternal weigfit, food consumption, and fetal weight occurred. At 1250 mg/kg/day, maternal body weight reductions occurred. No effects were seen at 625 mg/kg/day. Overall, the most sensitive target of toxicity was the liver, but the test substance was not considered a reproductive or developmental toxin. Introduction The objective of these studies was to evaluate the potential subchronic, developmental, and reproductive toxicity of a fluorotelomer-based phosphate surfactant in male and female rats. The surfactant is generally sold as a dispersion in isopropanol (IPA) and is used in numerous surface coating applications. In the subchronic study, the telomer product was administered as formulated product that contained approximately 35% active ingredient in IPA. In the developmental and reproductive toxicity studies, the telomer surfactant was suspended in water, with a 20% concentration of active ingredient. The test material was administered by gavage, the route selected as the most efficient way to deliver an accurate dosage. Separate range-finding studies were conducted to select appropriate dosages for each study. Study Designs AH studies: Crl:CD(SD)IGS BR rats 90-Day Subchronic Toxicity Dosages: 0, 10, 60 or 300 mg/kg/day (35% a.i. in IPA), 60 mg/kg/day IPA No. of animals: 10/sex/group --main study, 10/sex/group intermediate, low --1-month recovery, 5/sex/group - 3-month recovery Dosing Regimen: 90 days all groups, 1-month and 3-month recovery Parameters: U.S. EPA Health Effects Test Guideline OPPTS 870.3100(1998) Developmental Toxicity Dosages: 0, 625, 1250, or 2500 mg/kg/day (20% a.i. in water) No. of animals: 22 pregnant females/group Dosing regimen: Gestation Days 6-20 Parameters: U.S. EPA Health Effects Test Guidelines OPPTS 870.3700 Prenatal Developmental Toxicity Study Study Designs (Continued) One-Generation Reproduction Dosages: 0, 75, 500, and 3500 mg/kg/day (20% a.i. in water) No. of animals: 20/sex/group Dosing Regimen: 70 days prior to mating and throughout mating -- males and females During gestation and lactation - dams only Parameters: Special Design (Figure 1) Pretest Reproductive Evaluation^ Figure 1 - Study Design Pj Adult Dosing Period o\ Vaginal or- Smears CO Q t fating /Cjc.\talion/ Lactation Fj Adult Evaluations & JLJttfttsGFfts IJJj Subchronic Study Results In-Life Parameters No mortality, clinical signs of toxicity, or behavioral abnormalities ' Significant lower body weight at 300 mg/kg/day - males only (Figure 2) Low food consumption and food efficiency at 300 mg/kg/day - males only Figure 2 - M ale Body W eights Days on Test m Subchronic Study Results Liver Effects Effects commonly observed in rats with many organofluoride materials, such as peroxisome proliferation, hepatomegaly, and increased liver weights were observed in this study Increased hepatic B-oxidation activity at 60 and 300 mg/kg/day - males and females (Figure 3) Elevated liver weights and hypertrophy at 60 and 300 mg/kg/day in males and at 300 mg/kg/day in females (Figure 3) Elevated liver enzymes at all doses - males; at 300 mg/kg/day --females Figure 3 - Liver Effects Beta Oxidation --Males Beta Oxidation -- Females ES0 mg/kg/day E310 mgfc^tlay S 0 ntg/kg/day B 300 ntgft^day nmol/min/mg protein 5.00Y Liver Weight Effects in Male Rats S 0 ntg/kg'day 10 mg/kg^dny S360 ntg/kg/day B300 nig'k^day Liver Weight Effects in Female Rats liver weight as % body weigh Subchronic Study Results Clinical Pathology Red cell mass parameters were affected at 60 and 300 mg/kg/day --male rats only (Table 1). These parameters were not affected in the reproduction study. TABLE 1 PARAMETERS OF RED CELL MASS Dosage (mg/kg/day): 10 60 300 Red Blood Cells Day 38 Day 92 1-month Recovery 3-month Recovery 101% 99% 96% 99% 97% 94% ND ND 90% 97% 94% 97% Hemoglobin Day 38 Day 92 1-month Recovery 3-month Recovery 99% 97% 93% 95% 95% 91% ND ND 94% 97% 94% 92% Hematocrit Day 38 Day 92 1-month Recovery 3-month Recovery 0% 98% 94% 96% 95% 91% ND ND 93% 98% 94% 93% Values are percent o f control. Those in bold italics are statistically significant. N D = N o data I m Subchronic and Reproductive Study Results Histopathology In the subchronic study with active suspended in alcohol there were adverse liver lesions (focal necrosis) that did not occur in the study with active ingredient suspended in water. Similarly, chronic progressive nephropathy was evident in the kidneys of animals dosed with active in water (reproductive study), but not in the subchronic study. Other histopathological lesions were similar. Table 2 - Histopathology Effects Dosage Suhchronic Adult Subchronic Adult PI Adult FI Adults (mg/kg/day)____________Males_________ Females_____________ Males___________ (Male and Female) 3.5 (active in IPA) Liver: focal necrosis None 15 (active in water) Thyroid: hypertrophy Thyroid: hypertrophy(M) 20 (active in EPA) Liver: hypertrophy, None focal necrosis Thyroid: hypertrophy 100 (active in water or Liver: hypertrophy, Liver: hypertrophy in IPA) focal necrosis Thyroid: hypertrophy Thyroid: hypertrophy Thyroid: hypertrophy Thyroid: hypertrophy(M) Kidney: tubular Kidney: chronic hypertrophy progressive nephropathy 700 (active in water) ? Liver: hypertrophy, Thyroid: hypertrophy Thyroid: hypertrophy(M&F) Kidney: chronic progressive nephropathy Developmental Toxicity Results Maternal Toxicity Reductions in body weight parameters at 1250 and 2500 mg/kg/day. (Table 3) Effects on food consumption at 2500 mg/kg/day. Fetal Toxicity Reduced body weight parameters at 2500 mg/kg/day No malformations and no-compound-related variations at any dose level TABLE 3 DEVELOPMENTAL EFFECTS 2500 mg/kg/day 1250 mg/kg/day 625 mg/kg/day Maternal Effects Mean body weight (47%) Mean body weight (45%) No effects Adjusted body weight (426%) Adjusted body weight (422%) Food consumption (48%) Fetal Effects Mean body weight (44%) No effects No effects Reproductive Toxicity Results Reduced body weight parameters at 3500 mg/kg/day --Pj and Fj males and females Reduced food consumption at 3500 mg/kg/day - and Fj males and P females Reduced body weight parameters and food consumption at 500 mg/kg/day - Pj males No effect on Pj generation sperm or estrous cycle parameters No effect on Pt generation mating, fertility, gestation length or implantation No effect on x litter size, sex ratio or lactation pup survival indices Reduced Fj pup birth and lactation weights at 3500 mg/kg/day TABLE 4 F, GENERATION LITTERS Dose (mg/kg/day) N 0 18 75 500 19 19 3500 15 Mean Number of Pups/Litter Bom 12.9 11.4 12.4 13.0 Bom Alive 12.8 11.3 12.3 12.5 Day 4 Preculling 12.7 11.3 12.3 12.4 Day 4 Postculling 7.8 7.3 8.0 7.9 Day 21 7.8 7.3 8.0 7.9 Mean Pup Weights (grams) Day 0 6.9 7.1 6.8 6.3 Day 4 Preculling 11.1 11.6 11.5 10.1 Day 4 Postculling 11.0 11.6 11.4 10.1 Day 7 17.4 17.9 18.1 15.0 Day 14 36.9 35.3 35.7 28.4 Day 21 58.8 58.4 57.2 44.6 a Percent litters delivered having at least one live pup. b Mean percent survival from Day 4 Postculling to Day 21. c Percent litters bom with at least one pup alive on Day 21. Values in bold italics are statistically significant. Summary Subchronic Study Based on hepatocellular necrosis at all dose levels, no NOEL was seen in male rats Based on elevated liver enzymes and thyroid gland hypertrophy observed at 300 mg/kg/day, a NOEL of 60 mg/kg/day was established for females Developmental Toxicity Study Based on decreases in body weight parameters at 1250 mg/kg/day, the NOEL for maternal effects was 625 mg/kg/day Based on decreases in fetal body weights at 2500 mg/kg/day, the fetal NOEL was 1250 mg/kg/day Not a selective developmental toxin One-Generation Reproductive Study Based on reductions in mean pup weights throughout the entire lactation period for Fj litters at 3500 mg/kg/day, the NOEL for reproductive effects was 500 mg/kg/day. However, based on thyroid follicular hypertrophy in Pj and Fj adult males at >75 mg/kg/day, a NOEL was not determined for this study. Conclusions The target organs of toxicity in the subchronic study were the liver, thyroid and red blood cells The phosphate surfactant is not a selective developmental toxin The reproductive toxicity of this material was limited to in utero and lactational offspring growth impairment Acknowledgments We wish to thank the following individuals for their technical assistance: Nancy E. Everds for clinical pathology evaluations Linda A. Malley for neuropathology evaluations John C. O'Connor for Biochemical Evaluations Primary Technicians: Robert E. Walker, Jr., Kellie Mcllhatton, and Gregory L. Poindexter Poster Preparation: Maryanne M. Wilford Subchronic, Developmental, and Reproductive Toxicity of a Fluoroalkylethyl Phosphate Surfactant J.C. Stadler, D.A Delker, G.T. Makovec, J.F. Hansen, S.M. Munley, andE. Mylchreest The DuPont Company, Haskell Laboratory fo r Health and Environmental Sciences, Newark, Delaware, USA IBSAbstract A fluoroalkylethyl phosphate surfactant was evaluated in rats in 90-day, onegeneration reproduction, and developmental toxicity studies. !n the 90-day study, the lest substance was given as a suspension in isopropanol (IPA) a t oral doses ofO, 10,60 o r 300 mg/kg/day (35% a.i.) and 6 0 mg/kg/day IPA. H iere were no compound effects on mortality, clinical signs or neurobehavior. Adverse effects on body weights, food parameters, red cell mass, and hepatic enzymes were seen at the highest tw o doses, and increases in thyroid weight and cellular hypertrophy were observed at the high dose. Adverse microscopic changes in the liver (focal hepatocellular necrosis) occurred at all dose levels in males and at only the high dose in females. These effects were not seen with IPA dosed alone. Other non adverse physiological etianges included increased liver and kidney weights, hepatocellular hypertrophy, and an increased rate of peroxisome proliferation. The NOEL for female rats in this study was 10 mg/kg/day. In the reproductive study, where surfactant doses were 0 ,7 5 ,5 0 0 and 3500 mg/kg/day (20% a.i. in water, no IPA). parental body weights, weight gains and food parameters were reduced at the high dose, with males more affected than females. There were no effects on reproduction parameters at any dose level, except for F, pup weights, which were progressively reduced from birth to weaning (92% and 76% of control, respectively) at the high dose level. In these rats dosed with die active in water, thyroid hypertrophy and liver necrosis were not observed. The NOEL for reproductive parameters was 500 mg/kg/day. In the developmental study, doses were 625.1250 o r 2500 mg/kg/day (20% a.i. in water). At 2500 mg/kg/day, effects on maternal weight, food consumption, and fetal weight occurred. At 1250 mg/kg/day. maternal body weight reductions occurred. N o effects were seen at 625 mg/kg/day. Overall, the most sensitive target o f toxicity was the liver, but the test substance was not considered a reproductive o r developmental toxin. ! Introduction The objective of these studies was to evaluate the potential subchronic, developmental, and reproductive toxicity of a fluorotelomer-bascd phosphate surfactant in male and female rats. The surfactant is generally sold as a dispersion in isopropanol (IPA) and is used in numerous surface coating applications. In the subchronic study, the telomer product was administered as formulated product that contained approximately 35% active ingredient in IPA. In the developmental and reproductive toxicity studies, the telomer surfactant was suspended in water, with a 20% concentration of active ingredient. The test material was administered by gavage, the route selected as the most efficient way to deliver an accurate dosage. Separate range-finding studies were conducted to select appropriate dosages for each study. M Study Designs A ll studies: Crl:CD(SD)IGS BR rats 90-Day Subchronic Toxicity Dosages: 0, 10,60 or 300 mg/kg/day (35% a.i. in IPA), 60 mg/kg/day IPA No. o f animals: 10/sex/group - main study, IO/sex/group intermediate, low - 1-month recovery, 5/sex/group --3-month recovery Dosing Regimen: 90 days ail groups, 1-month and 3-month recovery Parameters: U.S. EPA Health Effects Test Guideline OPPTS 870.3100 (1998) Developm ental Toxicity Dosages: 0 ,6 2 5 , 1250, or 2500 mg/kg/day (20% a.i. in water) No. o f animals: 22 pregnant feinales/group Dosing regimen: Gestation Days 6-20 Parameters: U.S. EPA Health Effects Test Guidelines OPPTS 870.3700 Prenatal Developmental Toxicity Study One-Generation Reproduction Dosages: 0, 75, 500, and 3500 mg/kg/day (2 0 % a.i. in water) No. o f animals: 20/sex/group Dosing Regimen: 70 days prior to mating and throughout mating - males and females During gestation and lactation - dams only Parameters: Special Design (Figure 1) Figure 1 - Study Design ~HiSISil I Subchronic Study Results In-Life Param eters . No mortality, clinical signs o f toxicity, or behavioral abnormalities Significant lower body weight at 300 mg/kg/day - males only (Figure 2) Low food consumption and food efficiency at 300 mg/kg/day - males only Figure 2 Male Body Weights L iver Effects Effects commonly observed in rats with many organofluoride materials, such as peroxisome proliferation, hepatomegaly, and increased liver weights were observed in this study Increased hepatic B-oxidation activity at 60 and 300 mg/kg/day - males and females (Figure 3) Elevated liver weights and hypertrophy at 60 and 300 mg/kg/day in males and at 300 mg/kg/day in females (Figure 3) Elevated liver enzymes at ail doses - males; at 300 mg/kg/day - females Figure 3 Liver Effects Clinical Pathology Red cell mass parameters were affected at 60 and 300 mg/kg/day - male rats only (Table 1). These parameters were not affected in die reproduction study. Tabic 1 - Parameters of Red Cell Mass Dosane (ma/ke/dav): 10 60 300 Red Blood Cells Day 38 Day 92 l-month Recovery 3-momJj Recovery 101% 99% 96% 99% 97% 94% ND ND 90% 97% 94% 97% Hemoglobin Day 38 Day 92 l -month Recovery 3-inonth Recovery 99% 97% 93% 95% 95% 91% ND ND 94% 97% 94% 92% Hematocrit Day 38 Day 92 1-monili Recovery 3-month Recovery 0% 98% 94% 96% 95% 91% ND ND 93% 98% 94% 93% Subchronic and Reproductive Study Results Histopathology In the subchronic study with active suspended in alcohol there were adverse liver lesions (focal necrosis) that did not occur in die study with active ingredient suspended in water. Similarly, chronic progressive nephropathy was evident in the kidneys of animals dosed with active in water (reproductive study), but not in the subchronic study. Other histopathoiogica) lesions were similar. Table 2 - Histopathology Effects Subdirwdc Dosage Subchronic Aduli Adult PI Adult FI Adults loie/keMitc)_________ Muja________ Penala_________ Mate________(Maleand Penale) 3.5 (activein IPA) 15(active in water) Liver focal mentii None Thyroid. Mprrtrcytb.v Thyroid. hypenrophytMi 700(active in hyptnrophytM&Fi H f Reproductive Toxicity Results Reduced body weight parameters at 3500 mg/kg/day - P, and F, males and females Reduced food consumption at 3500 mg/kg/day - Pj and F| males and P ( females Reduced body weight parameters and food consumption at 500 mg/kg/day - P| males N o effect on P, generation sperm o r estrous cycle parameters No effect on P, generation mating, fertility, gestation length or implantation No effect on F, litter size, sex ratio or lactation pup survival indices Reduced F, pup birth and lactation weights at 3500 mg/kg/day Table 4 - F | Generation Utters Dose (mg/kg/day) 0 75 500 3503 N 18 19 19 15 Mean Number of Pups/Liner Bnm Day 4 Pieculling 12.9 11.4 12.4 13.0 12.8 11.3 12.3 12.5 12.7 11.3 12J 12.4 Day 4 Pos(culling 7.8 Day 21 7.8 7.3 8.0 Mean Pup Weights (grams)_______________ 6.9 7.1 6.8 63 Day 4 Preculling 11.1 119 11.5 Day 4 Postculliug 119 11.6 17.4 17.9 8.1 15.0 Day 14 36.9 35.3 35.7 Day 21 58.8 584 57.2 I Developmental Toxicity Results M aternal Toxicity Reductions in body weight parameters at 1250and 2500 mg/kg/day. (Table 3) * Effects on food consumption at 2500 mg/kg/day. Fetal Toxicity Reduced body weight parameters at 2500 mg/kg/day No malformations and no-compound-related variations at any dose level Materna) Effe Pelai Effects Table 3 - Developmental Effects 1500 nig/kg/day___________ I250mc/kg/dav 625 mg/kg/day Mean body weight (A' 1*) Mean body cigli! <A5%) Notffecls Adjusted Iwdy weight(A26%) Adjusted body weight(A22%) Food consumption (AS1*) Mean body weight (A4%) Noeffects No effects ||[ f ) Summary Subchronic Study Based on hepatocellular necrosis at all dose levels, no NOEL was seen in male rats Based on elevated liver enzymes and thyroid gland hypertrophy observed at 300 mg/kg/day. a N OEL o f 60 mg/kg/day was established for females Developmental Toxicity Study Based on decreases in body weight parameters at 1 250 mg/kg/day, die NOEL for maternal effects was 625 mg/kg/day Based on decreases in fetal body weights at 2500 mg/kg/day, the fetal NOEL was 1250 mg/kg/day Not a selective developmental toxin One-Generation Reproductive Study Based on reductions in mean pup weights throughout the entire lactation period for F, liners at 3500 mg/kg/day, the NOEL for reproductive effects was 500 mg/kg/day. However, based on thyroid follicular hypertrophy in P, and F[ adult males at >75 mg/kg/day, a NOEL was not determined for this I Conclusions T he target organs o f toxicity in the subchronic study were the liver, thyroid and red blood cells T he phosphate surfactant is not a selective developmental toxin The reproductive toxicity of this material was limited to in utero and lactational offspring growth impairment I Acknowledgements We wish to thank the following individuals for tlteir technical assistance: Nancy E. Everds for clinical pathology evaluations Linda A. Malley for neuropathology evaluations John C. O 'Connor for Biochemical Evaluations Primary Technicians: Robert E. Walker. Jr , Kellie Mclihatiou, and Gregory L. Poindexter Poster Preparation: Maryanne M. Wilford