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https://ntp.niehs.nih.gov/go/dart08abs

Abstract for DART-08

Modified One-Generation Study of Bisphenol AF Administered in Feed to Sprague Dawley (Hsd:Sprague Dawley SD) Rats with Prenatal, Reproductive Performance, and Subchronic Assessments in F1 Offspring

CASRN: 1478-61-1
Chemical Formula: C15H10F6O2
Molecular Weight: 336.23
Synonyms/Common Names: 4,4’-(Hexafluoroisopropylidene)diphenol; 2,2-bis(4-hydroxyphenyl)hexafluoropropane; 2,2-bis(4-hydroxyphenyl)perfluoropropane; phenol, 4,4’-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]bis-; hexafluorobisphenol a
Report Date: September 2022

FULL REPORT PDF

Abstract

Bisphenol AF (BPAF) is used as a curing or crosslinking agent in the processing of fluorocarbon elastomers, rubber processing, and specialty polymers due to its material characteristics, including thermal stability, chemical resistance, and compression set resistance, which are useful in plastics manufacturing and other fabrication processes.

BPAF was selected for evaluation based on a review of compounds that are potentially endocrine-active after concerns were raised about possible effects of bisphenol A (BPA) on the brain, behavior, and prostate gland of fetuses, infants, and children at current human exposure levels. The review assessed a number of agents that could have endocrine activity and are either persistent in the environment or have high human exposures, including chemicals that are structurally related to BPA. BPAF was selected because of its potential for endocrine activity, lack of adequate toxicity data, and potential environmental persistence due to the presence of fluorine atoms.

The objective of the present study was to characterize the potential for BPAF to adversely affect any phase of rat development, maturation, and ability to reproduce. The potential for BPAF to induce subchronic toxicity in the F1 generation, adversely affect the ability of the F1 generation to reproduce viable F2 offspring, and adversely affect F2 embryo-fetal development was assessed in Sprague Dawley (Hsd:Sprague Dawley SD) rats administered BPAF in 5K96 feed, a diet low in phytoestrogens, using the National Toxicology Program (NTP) modified one‑generation (MOG) study design.

NTP conducted a dose range-finding study with exposure concentrations of 0, 937.5, 1,875, 3,750, 7,500, and 15,000 ppm and based on findings of maternal and pup toxicity (significantly decreased body weights) observed at ≥7,500 ppm, exposure concentrations of 338, 1,125, and 3,750 ppm were selected for the MOG study.

Modified one-generation study

F0 dietary exposure began on gestation day (GD) 6 and continued throughout the study. Biological samples were collected on GD 18 (maternal and fetal), on lactation day (LD) 4 (maternal), and on postnatal day (PND) 4 (pup) to determine maternal transfer. At weaning on PND 28, offspring were randomly assigned to the reproductive performance (1/sex/litter), prenatal (1/sex/litter), subchronic (1/sex/litter from 10 litters), or biological sampling (6/sex for sample collection on PND 28 to determine internal concentrations of BPAF and up to 12 females for sample collection at vaginal opening) cohort. Upon sexual maturity, F1 mating and pregnancy indices were evaluated. In the prenatal cohort, F2 prenatal development (litter size, fetal weight, and morphology) was assessed on GD 21. In the reproductive performance cohort, littering indices and F2 viability and growth were assessed until PND 91. The likelihood of identifying potential BPAF-induced adverse effects and their similarity and magnitude—at any phase of growth or development—was increased by examining related endpoints in multiple pups within a litter throughout life, across cohorts, and across generations.

In this study, dietary consumption of BPAF was associated with lower F0, F1, and F2 mean body weights. The lower F0 female mean body weights and body weight gains during gestation were associated with a significant decrease in PND 1 F1 pup weights (9% and 15% in the 1,125 and 3,750 ppm groups, respectively) that continued through PND 98. Significant decreases in F2 mean body weights were also observed for 1,125 ppm male and female pups (12% on PND 28 for both males and females) through weaning, but only female postweaning mean body weights were significantly decreased through PND 91 for both the 338 and 1,125 ppm groups.

Several biochemical and hematological changes in the F1 generation subchronic cohort were noted. BPAF exposure related changes included significant decreases in serum cholesterol concentrations in both sexes and in serum bile acid concentrations in males, while significant increases in serum triglyceride concentrations were noted in females. Hematological changes were limited to females and included significant decreases in erythrocyte count, hemoglobin concentration, and total white blood cell count.

BPAF-related changes in reproductive performance were observed at all exposure concentrations. For the 3,750 ppm group, a complete absence of pregnant females in the F1 generation resulted in only two concentration groups for evaluation in the F2 generation (338 and 1,125 ppm). The majority (89%) of females in the 3,750 ppm group were not cycling and were in persistent estrus. A slight but significant increase in gestation length for F0 females and a significant decrease in F1 pup survival (PND 1–4) were also attributed to BPAF exposure. Similar findings, although to a lesser extent, were observed at lower concentrations in the prenatal cohort and included a significant decrease in the number of F1 females with live fetuses or live litters, number of corpora lutea, and number of implantation sites in the 1,125 ppm group, which were associated with a significant increase in pre- and postimplantation loss values. Significant decreases in the number of corpora lutea and implantation sites were also noted for the prenatal cohort females in the 338 ppm group. Changes in organ weights were also observed in the F1 generation. In the subchronic cohort, significant increases in the relative weights of the lungs, adrenal glands, and thyroid gland were noted in the 3,750 ppm F1 males. Significant decreases in relative weights for the liver and kidney (left) were also observed at 3,750 ppm for F1 males and microscopic findings were observed in the male kidney (mineral lesions along the junction of the cortex and medulla). In F1 males, lower absolute weights of the dorsolateral prostate, ventral prostate, and seminal vesicles with coagulating glands were observed in the 1,125 and 3,750 ppm groups and of the Cowper’s gland and levator ani/bulbocavernosus muscle (LABC) in the 3,750 ppm group. The organ weight changes in the 3,750 ppm group were more than the magnitude of the reductions in body weight and, along with histopathology observations of hypoplasia, indicated a potential direct BPAF-mediated suppression of maturation of these tissues. F2 males exhibited similar findings in the same reproductive tissues as F1 males in the 338 and 1,125 ppm groups. Changes in reproductive organ weights that appeared secondary to the effect of BPAF on body weight were limited to lower absolute weights of the testes, epididymides, and preputial glands in all three F1 exposed groups. The lower testes weights may also be due to direct (germinal epithelium degeneration and Leydig cell atrophy) effects of BPAF exposure. Changes in reproductive organ weights that appear to be secondary to the effect of BPAF on body weight for the F2 exposed males were limited to the testes and epididymides. Histopathology was not performed on the F2 generation. BPAF-related changes in andrology parameters were noted in both F1 and F2 males.

In F1 females, reproductive toxicity associated with exposure to BPAF included significant decreases in absolute ovarian and uterus/cervix/vagina weights, with gross observations of reduced size and hypoplasia in the 3,750 ppm group. In the subchronic cohort, significant increases in the relative weights of the thyroid gland and liver were noted in the 3,750 ppm F1 females. Significant decreases in absolute ovarian weights were also observed in the 338 and 1,125 ppm F2 females. The magnitude of the reduction in weights of the ovaries in the 1,125 ppm group was more than the magnitude of the reduction in body weight, suggesting a direct BPAF-mediated suppression of maturation of this tissue.

BPAF-related changes consistent with impaired development include lower mean body weights for all generations, including fetal or pup weights and reduced litter sizes, as well as impacts on fetal parameters and select developmental markers. Developmental landmarks impacted by BPAF exposure included time to vaginal opening (VO), testicular descent, and balanopreputial separation (BPS). No impacts on anogenital distance or areolae and nipple retention were observed in this study. The time to VO was significantly accelerated in all BPAF-exposed groups for both the F1 and F2 generations at all exposure concentrations. The mean day of testicular descent was not affected in the F1 generation, although one male in the 1,125 ppm group and 11 males in the 3,750 ppm group did not attain testicular descent by study termination; however, the mean day of testicular descent was significantly delayed by approximately 2 days for the F2 offspring in the 1,125 ppm group. In addition, 10 F1 males in the 3,750 ppm group did not attain BPS. The time to BPS was significantly delayed in both the F1 and F2 offspring in the 1,125 and 3,750 ppm groups for the F1 generation and the 1,125 ppm group for the F2 generation. BPAF exposure resulted in fetal malformations of the penis and vagina in two F1 males and three F1 females in the 3,750 ppm group. Additional findings were limited to an increase in the incidence of dilated and/or misshapen lateral ventricle (brain) in the 1,125 ppm group, which NTP has not recorded in its previous studies, and increases in the incidences of rudimentary and full lumbar I (L1) ribs in the 338 ppm group and rudimentary L1 ribs in the 1,125 ppm group for the prenatal cohort. These last findings were outside the NTP historical control ranges; however, the lack of an exposure-related response impedes a more thorough assessment to determine if they may have been related to BPAF exposure.

As previously reported, average dam daily BPAF intake was estimated on the basis of feed consumption per cage during gestation and lactation for all exposure groups. Average BPAF intake during gestation for F0 females was lower (26–259 mg BPAF/kg body weight/day [mg/kg/day] when assessed at GD 15–18) than for the lactation period (41–770 mg/kg/day, when measured at LD 1–4). Average BPAF intake estimated around weaning, PND 25–28, was higher than during the earlier part of lactation and was 131, 446, and 1,684 mg/kg/day in the 338, 1,125, and 3,750 ppm groups, respectively. During all three periods, average BPAF intake increased proportionally to exposure concentration. Free (parent only) and total (combined parent and conjugated forms) BPAF concentrations were quantified in maternal plasma and fetuses at GD 18 and maternal and pup plasma at LD 4 and LD 28. Free BPAF F1 concentrations were higher than corresponding dam concentrations in both GD 18 fetuses and PND 4 pups, demonstrating considerable transfer of BPAF from mother to offspring, whereas total BPAF concentrations were lower than corresponding concentrations in dams, suggesting either preferential transfer of free BPAF and/or inability of fetuses and pups to conjugate BPAF. Free and total concentrations in PND 28 pups were similar to LD 28 maternal concentrations, demonstrating direct exposure of pups via feed and indicating that conjugating enzymes are developed in PND 28 pups.

Genetic toxicology

BPAF was not mutagenic in tests conducted with three strains of bacteria, with and without induced rat liver S9 mix. BPAF was also evaluated in the in vivo peripheral blood micronucleus assay for its ability to induce chromosomal damage in the form of structural or numerical alterations. No significant increases in the frequencies of micronucleated immature erythrocytes (PCEs) were observed in male or female rats, and no significant changes in % PCE were observed, suggesting that BPAF exposure did not affect erythropoiesis.

Conclusions

Under the conditions of of this modified one-generation (MOG) study, there was clear evidence of reproductive toxicity of bisphenol AF (BPAF) in Hsd:Sprague Dawley SD rats based on the increased disruption of estrous cyclicity, the inability of the F1 generation to reproduce, decreases in F1 pup survival, and a slight increase in gestation length for F0 females at the highest dietary exposure concentration and, at lower concentrations, decreases in the number of implants, corpora lutea, and live fetuses or litters.

Under the conditions of this MOG study, there was clear evidence of developmental toxicity of BPAF in Hsd:Sprague Dawley SD rats based on the presence of fetal malformations and abnormal histopathology of both the male and female reproductive tract in the F1 generation, impacts on developmental markers, including accelerated vaginal opening and delayed balanopreputial separation, and lower F1 and F2 mean body and organ weights.

National Toxicology Program (NTP). 2022. NTP developmental and reproductive toxicity technical report on the modified one-generation study of bisphenol AF (CASRN 1478-61-1) administered in feed to Sprague Dawley (Hsd:Sprague Dawley SD) rats with prenatal, reproductive performance, and subchronic assessments in F1 offspring. Research Triangle Park, NC: National Toxicology Program. DART Report 08. https://doi.org/10.22427/NTP-DART-08

Studies

Summary of Exposure-related Findings in Rats in the Modified One-Generation Study of Bisphenol AF

0 ppm 338 ppm 1,125 ppm 3,750 ppm
F0 generation
Maternal parameters
Number mated 35 35 35 35
Number pregnant (%) 30 (85.7) 32 (91.4) 33 (94.3) 29 (82.9)
Number not pregnant (%) 5 (14.3) 3 (8.6) 2 (5.7) 6 (17.1)
Number littered (%)[a] 25 (92.6) 28 (96.6) 29 (96.7) 25 (96.2)
Gestation length (days) 22.1 ± 0.1** 22.2 ± 0.1 22.1 ± 0.1 22.4 ± 0.1**
Clinical observations None None None None
Mean body weight and feed consumption[b],[c]
Body weight: GD 21 356.8 ± 7.6** 359.7 ± 5.9 333.2 ± 3.8** 309.9 ± 4.4**
Body weight gain: GD 6–21 119.3 ± 7.0** 120.6 ± 4.2 94.8 ± 2.9** 71.9 ± 4.1**
Feed consumption: GD 6–21 20.0 ± 0.3* 20.5 ± 0.4 19.5 ± 0.7 19.2 ± 0.5
Body weight: LD 28 279.9 ± 3.3 273.7 ± 3.1 270.3 ± 3.0 274.4 ± 3.0
Body weight gain: LD 4–28 1.7 ± 2.5** −1.6 ± 2.6 25.4 ± 2.5** 41.6 ± 2.8**
Feed consumption: LD 1–13 49.0 ± 0.7 47.6 ± 1.1 50.7 ± 1.4 54.4 ± 2.3
Necropsy observations None None None None
F1 generation (preweaning)[c]
Clinical observations None None None Yellow fur
Live litter size
PND 0 13.2 ± 0.4 11.9 ± 0.6 12.9 ± 0.4 12.5 ± 0.5
PND 1 13.0 ± 0.4 11.4 ± 0.7 12.6 ± 0.4 11.4 ± 0.7
PND 4 (prestandardization) 13.1 ± 0.4* 11.6 ± 0.6 12.4 ± 0.4 10.6 ± 0.8**
PND 4 (poststandardization) 9.8 ± 0.2 9.5 ± 0.3 10.0 ± 0.0 9.0 ± 0.5
PND 28 9.7 ± 0.2 9.0 ± 0.4 9.2 ± 0.3 8.6 ± 0.4
Male pup mean body weight
Body weight: PND 1 6.82 ± 0.07** 6.58 ± 0.17 6.18 ± 0.12** 5.84 ± 0.19**
Body weight: PND 28 77.54 ± 1.30** 76.34 ± 1.38 68.49 ± 1.31** 53.94 ± 1.48**
Body weight gain: PND 4–28 67.77 ± 1.16** 66.60 ± 1.29 59.79 ± 1.18** 45.82 ± 1.32**
Female pup mean body weight
Body weight: PND 1
6.57 ± 0.07** 6.34 ± 0.12 5.98 ± 0.11** 5.56 ± 0.12**
Body weight: PND 28
71.32 ± 1.34** 69.14 ± 1.13 64.65 ± 1.31** 51.92 ± 1.33**
Body weight gain: PND 4–28 62.05 ± 1.21** 59.93 ± 1.00 56.09 ± 1.18** 44.15 ± 1.18**
F1 generation (postweaning)
Mean body weight and feed consumption[b],[c]
Male body weight: PND 28 76.3 ± 1.4** 75.5 ± 1.5 67.6 ± 1.4** 53.6 ± 1.6**
Male body weight: PND 98 386.3 ± 4.4** 373.7 ± 5.2 334.4 ± 5.0** 238.3 ± 4.6**
Male body weight gain: PND 28–91 299.7 ± 3.0** 286.9 ± 4.4 257.8 ± 4.1** 177.5 ± 4.0**
Male feed consumption: PND 28–98 21.8 ± 0.2** 21.3 ± 0.4 19.8 ± 0.3** 18.5 ± 0.3**
Female body weight: PND 28 69.5 ± 1.4** 69.3 ± 1.3 63.5 ± 1.6** 51.5 ± 1.4**
Female body weight: PND 98 242.9 ± 3.4** 227.2 ± 3.7** 206.0 ± 2.3** 172.6 ± 2.3**
Female body weight gain: PND 28–98 173.3 ± 2.9** 157.8 ± 3.1** 142.6 ± 2.1** 121.2 ± 1.7**
Female feed consumption: PND 28–98 15.6 ± 0.2** 16.3 ± 0.4 15.6 ± 0.4     14.5 ± 0.4*
F1 and F2 generations
Endocrine endpoints, developmental landmarks, and pubertal endpoints[c]
Vaginal opening (F1)
Adjusted mean day of vaginal opening (litter mean)[d] 35.8 ± 0.3** 33.8 ± 0.3** 27.8 ± 0.3** 27.9 ± 0.7**
Body weight at acquisition[b] 103.2 ± 1.7** 90.9 ± 1.6** 63.2 ± 1.4** 60.5 ± 2.6**
Balanopreputial separation (F1)
Adjusted mean day of balanopreputial separation (litter mean)[d] 46.4 ± 0.2** 46 ± 0.3 50.8 ± 0.7** 78.3 ± 2.1**
Body weight at acquisition[b] 200.4 ± 1.8** 188.0 ± 2.1** 195.1 ± 3.5 217.9 ± 3.6**
Number not attaining 0 0 0 10(9)
Testicular descent (F1)
Mean day of testes descent (litter mean) 18.0 ± 0.2 17.9 ± 0.2 17.7 ± 0.3 18.1 ± 0.5
Number not attaining 0 0 1(1) 11(7)
Vaginal opening (F2)
Adjusted mean day of vaginal opening (litter mean)[d] 34.7 ± 0.3** 31.3 ± 0.6** 25.1 ± 0.5** [e]
Body weight at acquisition[b] 113.8 ± 1.8** 94.3 ± 2.5** 65.9 ± 2.2** –  
Balanopreputial separation (F2) –  
Adjusted mean day of balanopreputial separation (litter mean)[d] 46.5 ± 0.7** 45.0 ± 0.4 52.1 ± 1.1** –  
Body weight at acquisition[b] 209.5 ± 4.4 195.1 ± 2.6* 222.1 ± 7.4   –  
Testicular descent (F2) –  
Mean day of testes descent (litter mean) 15.8 ± 0.4** 16.4 ± 0.3 17.7 ± 0.3* –  
Number not attaining 1 (1) 0 0 –  
Prenatal Cohort
Mating and fertility performance
Number of mating pairs 21 21 22 19
Mated females/paired (%) 81.0** 95.2 86.4 0.0**
Pregnant females/mated (%) 100.0 100.0 94.7 –  
Mean body weight and feed consumption[b],[c]
Body weight gain: GD 0–21 169.2 ± 2.7** 143.9 ± 4.4** 90.4 ± 9.1** –  
Feed consumption: GD 0–21 22.7 ± 0.3** 21.6 ± 0.5 19.7 ± 0.5**
Uterine content data[c]
Mean number of corpora lutea/female 15.82 ± 0.44** 14.20 ± 0.47** 11.89 ± 0.52** –  
Implantations/female 15.00 ± 0.37** 13.85 ± 0.39* 8.73 ± 0.69** –  
Live fetuses/litter 14.63 ± 0.34 13.25 ± 0.52 7.29 ± 1.06** –  
Fetal weight/litter 5.09 ± 0.07** 4.98 ± 0.06 3.81 ± 0.35** –  
Fetal findings
External findings None None None –  
Visceral findings None None None –  
Head findings[f]
Dilated lateral ventricle, bilateral – [V]
Fetuses 0 (0.00) 0 (0.00) 4 (8.33) –  
Litters 0 (0.00) 0 (0.00) 4 (30.77) –  
Misshapen lateral ventricle, left – [V]
Fetuses 0 (0.00) 0 (0.00) 1 (2.08) –  
Litters 0 (0.00) 0 (0.00) 1 (7.69) –  
Skeletal findings[f]
Lumbar, 1, rudimentary, total – [V]
Fetuses 11 (4.70) 19 (7.17) 14 (13.73)
Litters 6 (37.50) 10 (50.00) 4 (28.57)
Lumbar, 1, full, total – [M]
Fetuses 0 (0.00) 4 (1.51) 0 (0.00)
Litters 0 (0.00) 3 (15.00) 0 (0.00)
Reproductive performance cohort
Mating and fertility performance
Number of mating pairs 22 23 21 19
Mated females/paired (%) 100.0** 100.0 76.2* 5.3**
Pregnant females/mated (%) 81.8* 95.7 75.0 0.0
Littered females/mated (%) 81.8** 87.0 56.3 0.0
Mean body weight and feed consumption[b],[c]
Body weight gain: GD 0–21 158.0 ± 6.1** 132.8 ± 6.8* 95.3 ± 10.7** –  
Feed consumption: GD 0–21 23.5 ± 0.4** 22.3 ± 0.7 20.1 ± 1.0** –  
Body weight: LD 28 305.4 ± 3.7** 281.1 ± 3.8** 264.6 ± 6.9**
Body weight gain: LD 4–28 −8.5 ± 2.8** −7.7 ± 2.3 16.1 ± 4.8**
Feed consumption: LD 1–13 44.9 ± 1.6 45.8 ± 0.9 37.0 ± 4.0
Live litter size[c]
PND 0 11.2 ± 1.0* 10.6 ± 0.8 6.4 ± 1.4* –  
PND 4 (prestandardization) 10.9 ± 1.0* 11.1 ± 0.6 6.4 ± 1.4* –  
PND 4 (poststandardization) 7.3 ± 0.4 7.9 ± 0.1 5.7 ± 1.1 –  
PND 28 7.2 ± 0.4 7.7 ± 0.1 5.2 ± 1.1 –  
Male pup mean body weight (preweaning)[b],[c]
Body weight: PND 1 7.32 ± 0.15 7.00 ± 0.16 6.80 ± 0.25 –  
Body weight: PND 28 88.86 ± 2.01** 86.96 ± 1.49 77.82 ± 4.07* –  
Body weight gain: PND 4–28 77.63 ± 1.64** 76.12 ± 1.20 67.07 ± 3.63**
Female pup mean body weight (preweaning)[b],[c]
Body weight: PND 1 7.15 ± 0.15** 6.79 ± 0.14 6.28 ± 0.32* –  
Body weight: PND 28 81.62 ± 1.31** 78.23 ± 1.15 71.69 ± 2.52** –  
Body weight gain: PND 4–28 70.82 ± 1.09** 67.83 ± 0.94 62.17 ± 1.98**
Mean body weight and feed consumption (postweaning)[b],[c]
Male body weight: PND 28 87.5 ± 2.5* 85.8 ± 1.6 78.6 ± 4.1 –  
Male body weight: PND 91 387.9 ± 6.7* 372.4 ± 5.3 360.2 ± 9.8 –  
Male body weight gain: PND 28–91 300.4 ± 5.8 286.6 ± 4.5 281.6 ± 7.3 –  
Male feed consumption: PND 28–91 22.0 ± 0.2** 21.5 ± 0.2 20.7 ± 0.4** –  
Female body weight: PND 28 81.1 ± 1.7** 76.8 ± 1.2 73.9 ± 1.9* –  
Female body weight: PND 91 240.3 ± 4.2** 217.6 ± 4.0** 203.9 ± 5.9** –  
Female body weight gain: PND 28–91 159.2 ± 3.6** 140.8 ± 3.9** 130.0 ± 5.9** –  
Female feed consumption: PND 28–91 16.0 ± 0.2* 15.4 ± 0.3 14.8 ± 0.6 –  
Adult necropsies
Clinical pathology (subchronic cohort)
Hematology
Male NA None None None
Female NA None None ↓ Erythrocytes, hemoglobin, total white blood cells
Clinical chemistry
Male NA None ↓ Cholesterol, bile acids ↓ Cholesterol, bile acids
Female NA ↓ Cholesterol ↓ Cholesterol ↓ Cholesterol

↑ Triglycerides
Gross necropsy findings
Prenatal cohort
Male
Cowper’s gland
Missing, left[g] 0 0 1 (1) 0
Missing, bilateral 0* 0 0 2 (2)
Missing, total 0 0 1 (1) 2 (2)
Size, reduced, bilateral 0** 0 0 14 (14)**
LABC
Size, reduced 0** 0 0 18 (18)**
Prostate gland
Size, reduced 0** 0 0 20 (20)**
Seminal vesicles
Size, reduced, left 0 0 1 (1) 0
Size, reduced, right 0 1 (1) 0 0
Size, reduced, bilateral 0** 0 0 20 (20)**
Size, reduced, total 0** 1 (1) 1 (1) 20 (20)**
Female
Ovaries
Size, reduced, left 0* 0 0 2 (2)
Size, reduced, right 0* 0 0 2 (2)
Size, reduced, bilateral 0** 0 1 (1) 17 (17)**
Size, reduced, total 0** 0 1 (1) 19 (19)**
Uterus
Size, reduced, bilateral 0** 0 1 (1) 19 (19)**
Vagina
Misshapen 0 0 0 1 (1)
Reproductive performance cohort
Male
Cowper’s gland
Missing, bilateral 0* 0 0 2 (2)
Size, reduced, left 0 1 (1) 0 0
Size, reduced, bilateral 0** 1 (1) 0 14 (14)**
Size, reduced, total 0** 2 (2) 0 14 (14)**
LABC
Size, reduced 0** 0 0 16 (16)**
Dorsolateral prostate gland
Size, reduced 0** 0 0 18 (18)**
Ventral prostate gland
Size, reduced 0** 0 0 18 (18)**
Seminal vesicles
Size, reduced, bilateral 0** 0 0 18 (18)**
Phallus
Misshapen 0 0 0 1 (1)
Female
Ovaries
Size, reduced, bilateral 0** 0 0 18 (18)**
Vagina
Deformity 0 0 0 1 (1)
Misshapen 0 0 0 1 (1)
Subchronic cohort
Male
Prostate gland
Size, reduced 0** 0 0 10 (10)**
Seminal vesicles
Size, reduced, bilateral 0** 0 0 10 (10)**
Phallus
Deformity 0 0 0 1 (1)
Female
Ovaries
Size, reduced, bilateral 0** 0 0 9 (9)**
Uterus
Size, reduced, bilateral 0** 0 0 9 (9)**
F2 pups
Male
Cowper’s gland
Size, reduced, left 1 (1) 0 0
Size, reduced, bilateral 1 (1) 0 3 (3)
Size, reduced, total 2 (2) 0 3 (3)
LABC
Size, reduced 0 0 2 (2)
Dorsolateral prostate gland
Size, reduced 1 (1) 0 4 (3)
Ventral prostate gland
Size, reduced 1 (1)* 0 5 (3)
Seminal vesicles
Size, reduced, bilateral 0* 0 5 (3)
Organ weights
Prenatal cohort
Male NA None

↓ Absolute dorsolateral prostate gland weight

↓ Absolute and relative ventral prostate gland weights

↓ Absolute seminal vesicles with coagulating gland weight



↓ Absolute and relative dorsolateral prostate gland weights

↓ Absolute and relative ventral prostate gland weights

↓ Absolute and relative seminal vesicles with coagulating gland weights

↓ Absolute and relative LABC weights

↓ Absolute and relative Cowper’s gland weights

Female NA None

↓ Absolute right ovary weight

↓ Absolute left ovary weight

Reproductive performance cohort

Male NA None ↓ Absolute ventral prostate gland weight

↓ Absolute seminal vesicles with coagulating gland weight
↓ Absolute and relative dorsolateral prostate gland weights

↓ Absolute and relative ventral prostate gland weights

↓ Absolute and relative seminal vesicles with coagulating gland weights

↓ Absolute and relative LABC weights

↓ Absolute and relative Cowper’s gland weights
Female NA None

↓ Absolute and relative right ovary weights

↓ Absolute and relative left ovary weights

Subchronic cohort
Male NA None ↓ Absolute and relative ventral prostate gland weights

↑ Relative adrenal glands weight

↑ Relative thyroid gland weight

↑ Relative lung weight

↓ Absolute lung weight

↓ Absolute and relative liver weights

↓ Absolute and relative left kidney weights

↓ Absolute and relative dorsolateral prostate gland weights

↓ Absolute and relative ventral prostate gland weights

↓ Absolute and relative seminal vesicles with coagulating gland weights

Female NA None ↓ Absolute right ovary weight

↓ Absolute and relative left ovary weights

↓ Absolute uterus/cervix/ vaginal weight

↓ Absolute and relative right ovary weights

↓ Absolute and relative left ovary weights

↓ Absolute uterus/cervix/ vaginal weight

↑ Relative thyroid gland weight

↑ Relative liver weight

F2 pups
Male NA ↓ Absolute and relative dorsolateral prostate gland weights

↓ Absolute seminal vesicles with coagulating gland weight

↓ Absolute and relative Cowper’s gland weights

↓ Absolute LABC weight

↓ Absolute and relative dorsolateral prostate gland weights

↓ Absolute and relative ventral prostate gland weights

↓ Absolute and relative seminal vesicles with coagulating gland weights

↓ Absolute and relative Cowper’s gland weights

↓ Absolute and relative LABC weights

Female NA ↓ Absolute right ovary weight

↓ Absolute left ovary weight

↓ Absolute and relative right ovary weights

↓ Absolute and relative left ovary weights

Nonneoplastic lesions
Reproductive performance cohort
Male
Prostate gland
Hypoplasia, dorsolateral[g] 0** 0 0 18 (18)**
Hypoplasia, ventral 0** 0 0 18 (18)**
Seminal vesicles
Hypoplasia, bilateral 0** 0 0 18 (18)**
Coagulating gland
Hypoplasia, bilateral 0** 0 0 18 (18)**
Cowper’s gland
Hypoplasia, bilateral 0** 0 0 15 (15)**
Hypoplasia, unilateral 0 1 (1) 0 0
Hypoplasia, total 0** 1 (1) 0 15 (15)**
LABC
Hypoplasia 0** 0 1 (1) 17 (17)**
Testis
Germinal epithelium, degeneration 0** 0 1 (1) 6 (6)**
Leydig cell, atrophy 0** 0 0 11 (11)**
Seminiferous tubule, retention, Spermatid 0** 0 0 8 (8)**
Epididymis
Duct, atrophy 0** 0 0 10 (10)**
Duct, hypospermia 0** 0 1 (1) 6 (6)**
Duct, exfoliated germ cell 0** 0 1 (1) 5 (5)*
Female
Ovary 0** 1 (1) 0 20 (20)**
Hypoplasia, bilateral 0 2 (2) 0 0
Hypoplasia, unilateral 0** 3 (3) 0 20 (20)**
Hypoplasia, total
Uterus
Hypoplasia 0** 0 0 18 (18)**
Epithelial, metaplasia, squamous 0** 0 0 20 (20)**
Dilation, glandular, cystic 0** 0 0 8 (8)**
Stroma, hyalinization 0** 0 8 (8)** 18 (18)**
Subchronic cohort
Male
Prostate gland
Hypoplasia, dorsolateral 0** 0 0 10 (10)**
Hypoplasia, ventral 0** 0 0 10 (10)**
Seminal vesicles
Hypoplasia, bilateral 0** 0 0 10 (10)**
Female
Ovary
Hypoplasia, bilateral 0** 0 0 10 (10)**
Uterus
Hypoplasia 0** 0 0 10 (10)**
Epithelial, metaplasia, squamous 0** 0 0 10 (10)**
Dilation, glandular, cystic 0** 0 0 6 (6)**
Stroma, hyalinization 0** 0 0 10 (10)**
Andrology[c]
F1 males
Left cauda epididymis weight 0.262 ± 0.004** 0.249 ± 0.004 0.222 ± 0.006** 0.130 ± 0.007**
Left testis weight 2.039 ± 0.026** 1.965 ± 0.028 1.876 ± 0.047** 1.469 ± 0.057**
Sperm (10e+6/g cauda epididymis) 843.4 ± 27.3** 835.2 ± 26.3 796.9 ± 38.3 704.1 ± 27.1 **
Spermatid heads (10e+6/g testis) 120.9 ± 3.9** 128.5 ± 3.5 128.0 ± 3.9 148.8 ± 6.3**
F2 males
Left cauda epididymis weight 0.211 ± 0.005** 0.198 ± 0.003** 0.171 ± 0.005**
Left testis weight 2.014 ± 0.023** 1.855 ± 0.026** 1.851 ± 0.041**
Vaginal Cytology
F1 females NA None ↑ Estrous cycle length

↓ Diestrus stage length

↑ Proestrus stage length

↑ Estrus stage length

F2 females NA

↑ Estrous cycle length

↓ Proestrus stage length

↓ Estrus stage length

↑ Estrous cycle length

Level of evidence of reproductive toxicity: Clear evidence
Level of evidence of developmental toxicity: Clear evidence
Genetic Toxicology
Assay Results
Bacterial mutagenicity:
Salmonella typhimurium strains TA98 and TA100:
Escherichia coli strain WP2 uvrA (pKM101):

Negative
Negative
Peripheral blood micronucleus assay:
Male and female Sprague Dawley rats:

Negative

Statistical significance for an exposed group indicates a significant pairwise test compared to the vehicle control group. 
Statistical significance for the vehicle control group indicates a significant trend test.
* Statistically significant at p ≤ 0.05.
** p ≤ 0.01.
GD = gestation day; LD = lactation day; PND = postnatal day; NA = not applicable; LABC = levator ani/bulbocavernosus muscle.
[V] = variation.
[M] = malformation.
[a] Three F0 dams were removed in each exposure group on GD 18 for biological sample collection.
[b] Body weight results given in grams. Feed consumption results given in grams/animal/day.
[c] Data are presented as mean ± standard error.
[d] Adjusted based on body weight at weaning.
[e] No F1 females were confirmed pregnant for the 3,750 ppm group.
[f] Upper row denotes number of affected fetuses (%) and lower row the number of affected litters (%).
[g] Number of animals (number of litters) with lesion.