Document 8VDa4MYp08OJ124OKgb214Jza
AR226-3384
Reproductive\ and Fluoroalkylethyl Surfactant
S.A. MacKenzie. E. Mvlchreest. S.M. Mmiley, J.C. Stadler. J.F. Hansen, and N.E. Ever DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, DE, USA
Abstract
The objective of these studies was to evaluate the subchronic, reproductive, and deve toxicity of a fluoroalkylethyl ethoxylate surfactant in rats. Test substance was admini
gavage at 0, 25, 100, or 500 mg/kg/day (90-day subchronic toxicity and one-generatio and at 0, 50, 100, or 400 mg/kg/day on gestation days 6-20 (developmental toxicity). subchronic toxicity was 25 mg/kg/day based on clinical signs, reduced body weight a parameters, thyroid follicular hypertrophy, and chronic progressive nephropathy at > Other adverse effects at 500 mg/kg/day were reduced red blood cell mass, increased l activity, and reduced grip strength. Non-adverse liver hypertrophy and splenic effect at >25 and >100 mg/kg/day, respectively. Except for effects on the thyroid at 500 mg kidney at > 100 mg/kg/day, other effects were reversible after a 1- or 3-month recover was determined for reproductive toxicity, based on reduced fertility in P^ rats at >25 m
which was of similar magnitude at all dose levels. Body weights, food efficiency, and
implantation sites were reduced in P^ rats at 500 mg/kg/day, and viability and weights in F^ pups at >100 mg/kg/day. The reduced pup viability was relatively less severe an higher doses than the structurally related perfluorooctane sulfonate. Red blood cell a were similar to those observed with ethylene glycol ethers, but there were no develop testicular effects as seen with glycol ethers. The mechanism for reduced fertility is no developmental toxicity study, the NOELs were 50 and 100 mg/kg/day for maternal an respectively, based on reduced maternal body weight and nutritional parameters, and ^100 mg/kg/day and reduced fetal weight and increased fetal variations at 400 mg/kg malformations were observed.
Introduction
The objective of these studies was to evaluate the subchronic, reproductive, developmental toxicity of a fluoroalkylethyl ethoxylate surfactant in rats. T
substance was administered by gavage, the route selected as the most efficie administer an accurate dose. In a two-month rangefinder study, rats demons weight loss when exposed to 1000 mg/kg/day and male rats demonstrated re weight gain when exposed to 500 mg/kg/day. Blood fluorine levels plateaue
2-3 weeks of dosing. The high dose of 500 mg/kg/day for the 90-day study to produce toxicity without excessive mortality. The low dose of 25 mg/kg/
expected to be the no-observed-effect level, while the 100 mg/kg/day dose w induce minimal or no toxicity. The doses for the developmental toxicity stu 100, and 400 mg/kg/day were selected based on a pilot study in which mater toxicity were observed at ^400 mg/kg/day but no maternal or fetal toxicity w at ^100 mg/kg/day.
Study Design
90-Day and One-Generation Reproduction Study
Sub chronic Toxicity
!i
Pretest g.
90-Day DosingPeriod
10 animals /sex/dose
1-Month Recovery
Pretest g
One-Month Recovely
10 animals / sex in control and high dose
20 animals / sex
3-Month Recovely
90-Day Dosing vnth Blood Collection
90-Day Recovdy with Blood Collection
5 aniiniils / sex
9 Body weights, detailed clinical signs, and food consumption were determined weekly through exposure and 1-month recovery periods.
9 Clinical pathology, gross and microscopic pathology, and biochemical evaluations were perfo the 90-day exposure, 1-month recovery, and 3-month recovery periods.
8 Reproductive evaluations were performed prior to mating and during the mating, gestation, an periods. Developmental landmarks and gross pathology was also assessed on F^ adults.
Developmental Toxicity Study Groups of time-mated rats (22/dose) received oral gavage dose once daily over gestation days
Body weight, food consumption, and clinical observations data were collected. 0 Dams were euthanized on gestation day 21 and examined for gross external and visceral alter 9 Uteri were weighed and dissected and uterine contents were examined. ' Fetuses were removed, weighed, sexed, and examined for external, visceral, head, and skeleta
700
600
500
&JO
^*-'
53
^ ^ 400
o
PQ
u
300
200 ^
100
J:
70 84 98 112 126 140 154 168 182 Days on Test
^^K;:^
Figure 2: Mean Body Weights of Fem
0 14 28 42 56 70 84 98 112 126 140 154 168 182 Days on Test
TABLE 1
CLINICAL PATHOLOGY DATA
Dose (mg/kg/day):
RBC (xlO6/^)
end-of-dosing 1-month recovery 3-month recovery
HGB (g/dL)
end-of-dosing 1-month recovery 3-month recovery
HCT (%)
end-of-dosing 1-month recovery 3-month recovery
SDH (U/L) ALT (U/L)
end-of-dosing 1-month recovery 3-month recovery
end-of-dosing 1-month recovery 3-month recovery
Plasma F (pg/mL) end-of-dosing 1-month recovery 3-month recovery
Urine F(Pg)
end-of-dosing 1-month recovery 3-month recovery
Urine Vol.(mL)
end-of-dosing 1-month recovery 3-month recovery
Males
25
100
500
100%
ND
99%
98%
ND
98%
103% 97% 97%
99%
ND
101%
95% ND
98%
92% 95% 99%
99%
ND
100%
96%
ND
99%
94% 96% 100%
113%
ND
157%
124%
ND
160%
130% 219% 221%
109%
ND
156%
112%
ND
147%
152% 129% 194%
200% ND 100%
200% ND 100%
590% 100% 100%
418% ND 132%
1164% ND 211%
5228% 1214% 459%
134%
ND
77%
85%
ND
78%
368% 123% 111%
]Females
25
100
500
97%
ND
100%
91% ND
99%
89% 98% 101%
97%
ND
101%
90%
ND
94%
85% 97% 97%
98%
ND
104%
97%
ND
96%
96% 97% 100%
89%
ND
86%
68%
ND
120%
75% 112% 131%
76%
ND
83%
61%
ND
94%
73% 55% 109%
100%
ND
100%
200%
ND
100%
500% NC 100%
431%
ND
146%
1080%
ND
168%
5295% 714% 266%
194%
ND
139%
166%
ND
135%
351% 63% 131%
Values are percent of control. Those in green bold italics are statistically significant. ND = No data NC = Not calculable as control value was zero.
CLINICAL PATHOLOGY DATA
Dose (mg/kg/day):
RBC (xlO6/^)
end-of-dosing 1-month recovery 3-month recovery
HGB (g/dL)
end-of-dosing 1-month recovery 3-month recovery
HCT (%)
end-of-dosing 1-month recovery 3-month recovery
SDH (U/L) ALT (U/L)
end-of-dosing 1-month recovery 3-month recovery
end-of-dosing 1-month recovery 3-month recovery
Plasma F (ug/mL) end-of-dosing 1-month recovery 3-month recovery
Urine FGig)
end-of-dosing 1-month recovery 3-month recovery
Urine Vol. (mL)
end-of-dosing 1-month recovery 3-month recovery
Males
25
100
500
100
98
103
ND ND
97
99
98
97
99
95
92
ND ND
95
101
98
99
99
96
94
ND ND
96
100
99
100
113
124
130
ND
ND
219
157
160
221
109
112
152
ND
ND
129
156
147
194
200
200
500
ND
ND
100
100
100
100
418
1164 5228
ND ND 1214
132
211
459
134
85
368
ND
ND
123
77
78
111
Females
25
100
500
97
91
89
ND ND
98
100
99
101
97
90
85
ND ND
97
101
94
97
98
97
96
ND ND
97
104
96
100
89
68
75
ND
ND
112
86
120
131
76
61
73
ND ND
55
83
94
109
100
200
500
ND ND NC
100
100
100
431
1080 5295
ND ND 714
146
168
266
194
166
351
ND ND
63
139
135
131
Values are percent of control. Those in green bold italics are statistically significant. ND = No data NC = Not calculable as control value was zero.
HISTOPATHOLOGY DATA
Dose (mg/kg/day): 0
Malies
25
100
500
End-of-Dosing Thyroid
Hypertrophy, Follicular Alteration, Colloid
Kidney Nephropathy, Chronic Progressive
0/11 6/11
1/10 10/10
9/11 19/11
11/12 11/12
5/11 2/10 5/11 7/12
Liver Hypertrophy, Centrilobular
Spleen Hematopoiesis Pigment
1/11
5/10 19/11 12/12
0/11
1/10
8/11 19/12
0/11 0/10 4/11 6/12
1-Month Recovery Thyroid
Hypertrophy, Follicular Alteration, Colloid
Kidney Nephropathy, Chronic Progressive
0/10 ND
ND
8/19
6/10
ND
ND 19/10
3/10 ND
ND
8/10
Liver Hypertrophy, Centrilobular
Spleen Hematopoiesis Pigment
0/10 ND
ND
7/10
0/10 . ND
ND
8/10
0/10
ND
ND
7/19
3-Month Recovery Thyroid
Hypertrophy, Follicular Alteration, Colloid
0/4
0/5
0/4
4/5
1/4
5/5
4/4
5/5
Kidney Nephropathy, Chronic Progressive
4/4
4/5
3/4
2/5
Liver Hypertrophy, Centrilobular
Spleen Hematopoiesis Pigment
0/4
0/5
0/4
0/5
0/4
1/5
1/4
0/5
0/4
0/5
0/4
0/5
Values in green bold italics are statistically significant.
Pemilies
0
25
100
500
0/10 3/10
0/10 6/10
2/19 19/10
9/19 19/10
2/10 2/10 5/10 5/10
0/10 0/10 9/10 10/19
0/10 1/10
1/10 0/10
6/10 0/10
7/19 19/19
0/10 ND 1/10 ND
ND 6/10 ND 19/10
0/10 ND
ND
8/10
0/10 ND
ND
1/10
0/10
ND
ND
1/10
0/10 ND
ND
0/10
0/4
0/5
0/4
0/4
1/4
3/5
4/4
3/4
2/4
2/5
4/5
4/5
0/4
0/5
0/5
0/5
0/4
0/5
0/5
0/5
0/4
0/5
2/5
2/5
@ Reduced forelimb and hindlimb grip strength in male rats at 500 mg/kg/da to reduced body weight.
No effects considered to represent neurotoxicity.
R___ eproduction E^_^_v^ aluation
9 Reduced body weight parameters at 500 mg/kg/day - P^ males and female 9 Reduced food efficiency at 500 mg/kg/day - P^ females No effect on P^ generation sperm parameters 9 Increased P^ female estrous cycle length at all dose levels Reduced P^ fertility at all dose levels 9 No effect on P^ generation mating or gestation length 9 Reduced P^ uterine implantation sites and litter size at 500 mg/kg/day 9 Reduced F^ lactation pup survival parameters and body weights at 100 an
500 mg/kg/day
Pi GENERATION REPRODUCTIVE PARAMETERS
Dose (mg/kg/day): 0
25
100
500
Percent of Days in Estrus
32
31
34
29
Percent of Days in Diestms
60
59
60
60
Percent of Days in Proestrus
7
10
6
10
Mean Cycle Length (days)
4.3
4.7
4.9
4.8
Mean Precoital Interval (days)
Mating Index (%)
(number copulated/cohoused)
Fertility Index (%)
(number delivered/copulated)
3.4 100.0
85.0
3.3 100.0
55.0
2.8 95.0
3.1 100.0
57.9 65.0
Gestation Length (days)
22.6 22.4 22.5 22.9
Number of Implantation Sites Implantation Efficiency (/of
15.3 90.0
14.1 93.2
14.8 88.6
10.7 86.0
Sperm Motility(% motile)
92.8 91.7 93.4 91.1
Sperm Morphology (% normal)
98.3
98.3
98.9
97.7
Epididymal Sperm (millions) Percauda Per gram Cauda
318.4 1180.2
278.8 979.2
291.1 969.0
279.4 1062.1
Testicular Spermatids (millions) PerTestis Per gram Testis
126.5 83.3
128.8 93.7
128.2 87.4
137.0 83.4
a Number of pups born/number of implantation sites X 100.
Values in green bold italics are statistically significant. Values in blue bold are not statistically significant but considered test substancerelated.
F. GENERATION LITTERS
Dose (mg/kg/day)
0
N
17
25
100
11
11
Mean Number of Pups/Litter
Bom
14.3
Bom Alive Day 4 Preculling Day 4 Postculling
14.3 14.1
8.0
Day 7
8.0
Day 14
8.0
Day 21
8.0
Survival (%) Sex Ratio (males)
Gestation Index'1 Mean % Bom Alive
0-4 Day Viability
Lactation Index13
Litter Survival0
0.48 100.0 100.0
97.6 100.0 100.0
Mean Pup Weights (grams)
DayO
6.9
Day 4 Preculling
11.5
Day 4 Postculling Day 7
11.5 18.4
Day 14
38.0
Day 21
62.6
13.7 13.5 12.8
7.9 7.9 7.9 7.9
0.58 100.0
98.8 98.5 98.2 100.0
6.8 12.2 12.3 18.9 37.7 59.9
13.4 13.1 12.9
8.0 7.7
6J
6.1
0.50 100.0
98.1 98.9 76.1 90.9
6.7 10.8 10.8 15.9 35.7 58.6
500
13
10.3 9.4 7.5 6.3 4.8
1J
0.8
0.46 92.3 86.7 82.8 11.4 25.0
6.2 8.2 8.2 9.9 21.3 35.9
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 green bold italics are statistically significant.
TABLE 5
DEVELOPMENTAL TOXICITY STUDY RESULTS
Dose (mg/kg/day):
0
25
100
Maternal BWG (g) (GD 6-21)
153.1(20.3) 144.5(21.4) 133.3(19.8)
Fetal weight (g)
Supernumerary ribs (litters affected/examined)
5.50(0.28) 6/22
5.49(0.26)
6/21
5.40(0.39) 10/22
Skull, retarded ossification (litters affected/examined)
10/22
9/21
12/22
5
12/21 16/21
Values in green bold italics are statistically significant.
Discussion
9 Exposure to the test substance produced reversible decrements in body weight and parameters and non-specific clinical signs oftoxicity.
8 No evidence ofneurotoxicity was observed. Reductions in grip strength were attri body weight.
8 Liver, thyroid, kidney, and red blood cells were identified as target organs, based o anatomic pathology evaluations. Effects were generally mild and all adverse effec reversibility, except chronic progressive nephropathy in females.
The reduction in fertility index and mean number of implantation sites, in the abse in implantation efficiency (an indicator of in utero post-implantation loss) suggests
implantation embryonic loss. 9 The reduction in pup viability and survival may be due to the postnatal manifestati
developmental effect due to in utero exposure, maternal factors (insufficient milk s or result from postnatal toxicity from lactational transfer to the nursing pups. 9 In the developmental toxicity study, the test substance did not produce fetal malfor was no evidence of increased sensitivity of the fetus to the test substance.
9 NOEL for Subchronic Toxicity: The no-observed-effect level (NOEL) fo
females was 25 mg/kg/day, based on decrements in body weight and nutr
parameters, clinical signs of toxicity, and thyroid and kidney histopatholo
observed at 100 mg/kg/day.
9 NOEL for Reproductive Toxicity: There was no NOEL for the reproduct parameters evaluated under the conditions of this study; this was based on
dose levels on the fertility index in the P^ generation.
9 NOEL for Developmental Toxicity: The maternal NOEL was 50 mg/kg/d reduced maternal body weight and nutritional parameters. The fetal NOE
100 mg/kg/day, based on reduced fetal weight and increased fetal variatio malformations were observed.
j
y
n
-s
9
We wish to thank the following individuals for their technical assista Linda A. Malley for neuropathology evaluations Primary Technician: Nita B. Baker e Poster Preparation: Maryanne M. Wilford
Evaluation of the Subchronic, Reproductive, and Developmental Toxicity of a
S.A. MacKenzie, E. Mylchreest, S.M. Munley, J.C. Stadler, J.F. Hansen, andN.E. Everds The DuPont Company, Haskell Laboratory for Health and Environmental Sciences, Newark, Delaware, USA
Fluoroalky
Dose N
VUs Abstract
The objective ofihese studies was to evaluate the subchtonic. reproductive, and developmental toxicky of a nuoroalkyiethyl eihonylate surfaciant in rats. Test substance was administered by gavage at 0,25, 100, or 500 ing/kg/day (90-day subclironic toxicity and one-generation reproduction), and at 0, 50,100. or 400 mg/kg/day on gestation days 6-20 (developmental toxicity). The NOEL for subchronic toxicity was 25 nig/kg/day based on clinical signs, reduced body weight and nutritional parameters, thyroid follicular hypertrophy, ami chronic progressive nephropatny at >100 nig/kg/day. Other adverse effects at 500 ing/lcg/day were reduced red blood eel! mass, increased liver enzyme activity, and reduced grip strength. Non-adverse liver hypertrophy and splenic effects were observed at >25 and 100 ing/kg/day, respectively. Except for effects on the thyroid at 500
mg/kg/day and kidney at y00 ing/kg/day, other effects were reversible after a i- or
3-month recovery. No NOEL was determined for reproductive toricily, based on reduced fertility in P; rats ai 25 nig/kg/day, which was of similar magnitude at all dose levels. Body weights, food efficiency, and number of implantation sites were reduced in P] rats at 500 nig/kg/day, and viability and weights were reduced in F, pups at SlOO nig/kg/day. The reduced pup viabiiity was relatively less severe and required much higher doses than the structurally related perfluorooctane sulfonate,
Red blood cell and spleen effects were similar to dose observed with ethylene glycol ethers, but there were no developmental or testicular effects as seen with glycol ethers. Tlie mechanism for reduced fertility is not known, Indie developmental toxicity study, the NOELs were 50 and 100 ing/kg/day for maternal and fetal toxicity. respectively, based on reduced maternal body weight and nutritional parameters, and clinical signs at 100 mg/kg/day and reduced fetal weight and increased fetal variations at 400 nig/kg/day. No fetal malformations
were observed,
Table 2 - Histopatfiology Dala
H)pcnniph,Cal IknElBpfflMil
l.MonlbKm -rh)TMi
Bom Bom Day Day Day Day Dny
S^ SCI.R
Gesu MEO 0-4 D UlCi Litte
Day Day Day Day Day Day
Table 5
Maenid BWG
FMBiweighlQi
mi Introduction
The objective of these studies was to evaluate the subchronic. reproductive, and developmental toxicily of a fluoroalkylethylethoxylaleEurfaclant in rats. The test substance was administered by gavage, the route selected as the most efficient way to administer an accurate dose. In a two-month rangefinder study, rats demonstrated body weight loss when exposed to 1000 mg/kg/day and male rais demonstrated reduced body weight gain when exposed to 500 mg/kg/day. Blood fluorine levels platcaucd within 2-3 weeks of dosing. The high dose of 500 ing/kg/day for the 90-day study was expected to produce toxicity without excessive mortality. The low dose of 25 mg/kg/day was expected lobe the no-observedcffcct level, while the 100 mg/kg/day dose was expected to induce minimal or no loxicity. The doses for the developmental loxicity study of 0,50,100, and 400 nig/kg/day were selected based on a pilot study in which maternal and fetal toxicity were observed at 400 mg/kg/day but no maternal or fetal toxicily were observed at SlOOrog/kg/day.
HUStudy Design
90-Day and One-Generation Reproduction Study
.n- -!=:n "TSswMsM^^1"'^----
Body weights, detailed clinical signs, and food consumption were determined weekly throughout the 90-day exposure and 1-month recovery periods.
Clinical pathology, gross and microscopic pathology, and biochemical evaluations were performed at the end of the 90-day exposure, 1-month recovery, and 3-month recovery periods,
Reproductive evaluations were performed prior to mating and during the mating. gestation, and lactation periods. Developmental landmarks and gross pathology were also assessed on F, adults.
Developmental Toxicity Study Groups of time-mated rats (22/dose) received oral gavage dose once daily over
gestation days 6-20. Body weight, food consumption, and clinical observations data were collected. Dams were euthanized on gestation day 21 and examined for gross external and
iglied and dissected and uterine contents were Fetuses were cmoved. weighed, scxcd, and examined for c;
licad. and skeletal alici
Table 1 - Clinical Patiioloey Data
RBC (xlO'/llU
Dose (mEfcr/day);" 2S 100 500
end-oMosinE
9B 103
ND ND 97 99 98 97
HOB (g/tIL)
end-oMosine
99 95 91
ND ND 95
3-nionl!i recovery 101 oa 99
HCTfa.) SDH (UO-) ALT (tj/L)
cml-of-dosine
99 96 94
ND ND 96
3-monlii recovtiy 100 99 100
124 130 ND ND 219 3-monlh recovery 157 160 211
end-oMosine
152
ND
iSd
194
Plasma F(flE<mLJend-of-ilosinE
200 200 500
1-monlli recovery NO ND 100
3-nionui recovery 100 100 100
Urine F W
1-month iccovcly
1164 522S
ND 1214 132 311 459
Urine Vol. (mL)
85 S68 ND ND 123
3-monlii [eTMtc.y
25 100 500
97 91 89 ND 98
100 99 101
9B 85 ND ND 97
101 94 97
9B 97 96 ND ND 97 104 96 100
89 68 75 ND ND 112
B6 120 131
76 61 73 ND S5
G3 94 109
100 20Q 5BS ND ND A'C
100 100 100
431 1080 5295 ND ND 714
168 266
194 I6G 351 ND ND 63
139 133 131
Ncuroloxicologi1 Effects
Reduc^forclimbaadhindliinbgripstrengthin reduced body weigh!.
ai 500 ing/kg/day. attributed ic
Reproduction Evaluation Reduced body weight parariKiers ai 500 ing/kg/day ~ P, males and females
Reduced food efficiency at 500 nig/kg/day - P, Females
NoeffeclonP] generation spennparamcters Increased-Pi female esuous cycle length ai all dose levels Reduced P, fertility at all dose levels No effect on P| generaiion mating or gestation length Reduced Pi uterine implantation sUcs mid liner sitte at 500 nig/kg/day Reduced F[ lactation pup survival parameters and body weights at 100 and
500 rog/fcg/day
Table 3 P, Generation Reproductive Parameters PercenI of Days in Eslnis
Mean Cycle LeiigUi (days)
Mating Index W
100.0
(number coiiulalciUl.-uliouscd)
Fenilily Indent)
i
100.0
95,0 100.0
Gesladon Length (days)
NmiiberonnipltlitBlionSiIcE IlDplniimdon Euicicliry TO' Sperm Motiiily (t, mnlilc) Spenn Moipliology (1 nomial) Hpididymal Sperm <mi II in us)
22.6 15.3 90.0 92.8 98,3
214 22S 22,9
HI 14.S ". '
93.2 ES.6 B6.0 91,7 934 91.1 98.3 98,9 97.7
grani Cauda ut Spcrmalids (n
1180!979.2969.01062 !
m.,n,di.inMi. t.lttiW""'""--
fliilcrs affee
^Discuss
Exposure to tlie lest su nutiitionai parameters
No evidence of neuroi 10 lower body weighi.
Liver, [hyroid. kidney, clinical and anauunic p
The reduction in fertili reduction in implantati
The reiiuciion in pup v developmental effect d neglect), or result from
In the developmental t
^Conclus
NOEL for Suhdironic females was 25 ing/kg parameters, clijiicai sig at 100 ing/kg/day.
NOEL for Reproductiv dose levels on ilie fert
NOELforDevelopinen reduced maternal body 100 BiB/kg/day. based
llf Acknow
WE wish 10 thank Ilie fol Linda A. Mnlley for nc Primary TecliiiiciBti N Posler Preparaiiiin: Ma