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https://ntp.niehs.nih.gov/go/dart06abs

Abstract for DART-06

Modified One-Generation Study of 2-Ethylhexyl p-Methoxycinnamate Administered in Feed to Sprague Dawley (Hsd:Sprague Dawley SD) Rats with Prenatal, Reproductive Performance, and Subchronic Assessments in F1 Offspring

CASRN: 5466-77-3
Chemical Formula: C18H26O3
Molecular Weight: 290.40
Synonyms/Common Names: Octinoxate; ethylhexyl methoxycinnamate; octyl methoxycinnamate; 2-propenoic acid, 3-(4-methoxyphenyl)-, 2-ethylhexyl ester; 2-ethylhexyl 3-(4-methoxyphenyl)prop-2-enoate
Report Date: June 2022

FULL REPORT PDF

Abstract

2-Ethylhexyl p-methoxycinnamate (EHMC), also known as octinoxate and octyl methoxycinnamate, is a common component of sunscreens, cosmetics, and personal care products. Mechanistic screening studies have purported that EHMC, and its metabolites, are capable of activating the estrogen receptor to varying degrees. The objective of this study was to characterize the potential for EHMC to adversely affect any phase of rat development, maturation, and ability to reproduce. The potential for EHMC to induce subchronic toxicity in the F1 generation, to adversely affect the ability of the F1 generation to reproduce viable F2 offspring, and to adversely affect the F2 embryo-fetal development was assessed in Sprague Dawley (Hsd:Sprague Dawley SD) rats administered EHMC in 5K96 feed, a diet low in phytoestrogens, using the National Toxicology Program modified one-generation (MOG) study design. The dietary route of administration was selected to approximate continual exposure in group-housed animals. EHMC exposure via the diet, rather than topical application, was selected for this study to sustain internal exposure; if applied topically, the internal dose would have been influenced by intra- and interanimal grooming behavior.

Exposure concentration selection for the MOG study was based on a dose range-finding study in which time-mated rats were exposed to 0, 2,250, 5,000, 10,000, or 20,000 ppm EHMC in the diet from gestation day (GD) 6 through lactation day (LD) 28. Dams exposed to 20,000 ppm displayed significantly decreased mean body weights on GD 21 and body weight gain from GD 6 through GD 21. Dams exposed to 20,000 ppm displayed lower live litter size, and pups in this group displayed significantly decreased PND 1 weights and lower postnatal viability resulting in the group being removed from study on postnatal day (PND) 14. Pup body weights of the 10,000 ppm group were also lower than those in the control group. Therefore, exposure concentrations of 0, 1,000, 3,000, and 6,000 ppm were selected for the subsequent MOG study. Test article consumption was exposure concentration-proportional. EHMC intake for F0 females in the 2,250, 5,000, 10,000, and 20,000 ppm groups, based on feed consumption and dietary concentrations for GD 6 through GD 21, was approximately 161, 365, 714, and 1,841 mg EHMC/kg body weight/day (mg/kg/day), respectively; from LD 1 through LD 14, EHMC intake was approximately 410, 925, and 1,615 mg/kg/day for the 2,250, 5,000, and 10,000 ppm groups, respectively.

Modified one-generation study

F0 exposure began on GD 6 and was continual. At weaning on PND 28, F1 offspring were assigned to the reproductive performance (up to 2/sex/litter, when available), prenatal (1/sex/litter), or subchronic cohort (1/sex from 10 litters). 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, F2 viability, and growth were assessed until PND 28. The likelihood of identifying potential EHMC-induced adverse effects (similarity and magnitude thereof) at any phase of growth or development was increased by examining related endpoints and multiple pups within a litter throughout life, across cohorts, and across generations.

EHMC did not induce overt F0 or F1 maternal toxicity or affect mating or pregnancy indices. Dam feed consumption and body weights were slightly lower during lactation in the 6,000 ppm group. EHMC exposure at 6,000 ppm was associated with significantly decreased F1 and F2 preweaning mean body weights, with an onset at approximately PND 13, consistent with the beginning of pup feed consumption. Significantly decreased F1 preweaning mean body weights were observed in males and females exposed to 3,000 or 6,000 ppm, whereas only F2 male and female preweaning mean body weights of the 6,000 ppm group were significantly decreased relative to their respective control groups. Although mean body weight gains of males (PND 28–105) and females (PND 28–91) in all EHMC-exposed groups were similar to those of the respective control groups, postweaning F1 male and female mean body weights of the 6,000 ppm group were significantly decreased by 5%–14% relative to the respective control animals. Both male and female mean body weights of the 3,000 ppm groups were significantly decreased by approximately 5% on PND 28, but by PND 56, their mean body weights were comparable to those of the control groups. Lower F1 postweaning body weights were not associated with concurrent lower feed consumption. EHMC intake by F0 females in the 1,000, 3,000, and 6,000 ppm EHMC groups, based on feed consumption and dietary concentrations from GD 6 through GD 21, was approximately 70, 207, and 419 mg/kg/day, respectively; from LD 1 through LD 13, EHMC intake was approximately 161, 475, and 920 mg/kg/day, respectively. EHMC intake by the F1 generation postweaning (PND 28 through PND 91) in the 1,000, 3,000, and 6,000 ppm groups was approximately 80, 242, and 491 mg/kg/day (males) and 87, 263, and 528 mg/kg/day (females), respectively. EHMC intake by the adult F1 females in the 1,000, 3,000, and 6,000 ppm groups was approximately 73, 220, and 435 mg/kg/day (GD 0 through GD 21) and 139, 418, and 842 mg/kg/day (LD 1 through LD 13), respectively.

EHMC exposure did not alter anogenital distance or areola/nipple retention. The timing of weaning weight-adjusted vaginal opening (VO) and balanopreputial separation (BPS) was significantly delayed by approximately 2.1 days and 2.2 days, respectively, in the 6,000 ppm group. F1 rats exposed to 6,000 ppm EHMC displayed slightly more time in estrus.

Reproductive performance (fertility and fecundity) was not affected by EHMC exposure. The numbers of live fetuses and pups were not affected. EHMC exposure was not associated with any effects on fetal weight or the incidences of external, visceral, or skeletal malformations. The 6,000 ppm group did exhibit a higher combined fetal incidence of lumbar 1 rudimentary rib variants (approximately 10% versus 4% in the control group).

In the subchronic cohort, no gross findings, changes in organ weights, or histopathological findings were attributed to EHMC exposure.

Conclusions

Under the conditions of this modified one-generation (MOG) study, there was no evidence of reproductive toxicity of 2-ethylhexyl p-methoxycinnamate (EHMC) in Hsd:Sprague Dawley SD rats at exposure concentrations of 1,000, 3,000, or 6,000 ppm. Mating and littering were not affected significantly by EHMC exposure.

Under the conditions of this MOG study, there was equivocal evidence of developmental toxicity of EHMC in Hsd:Sprague Dawley SD rats based on the observed postnatal effects on body weight that showed some indication of recovery by study end, delays in postnatal day 28-adjusted vaginal opening and balanopreputial separation, which could have been influenced by the apparent transient effects on body weight, and time in estrus was slightly longer in EHMC‑exposed females relative to that of the control group. No other signals consistent with alterations in estrogenic, androgenic, or antiandrogenic action were observed. EHMC exposure did not induce any specific fetal malformations.

National Toxicology Program (NTP). 2022. NTP developmental and reproductive toxicity technical report on the modified one-generation study of 2-ethylhexyl p‑methoxycinnamate (CASRN 5466-77-3) 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 06. https://doi.org/10.22427/NTP-DART-06

Studies

Summary of Exposure-related Findings in Rats in the Modified One-Generation Study of 2-Ethylhexyl p-Methoxycinnamate

0 ppm 1,000 ppm 3,000 ppm 6,000 ppm
F0 generation
Maternal parameters
Number mated 26 26 26 26
Number pregnant (%) 22 (84.6) 24 (92.3) 19 (73.1) 22 (84.6)
Number not pregnant (%) 4 (15.4) 2 (7.7) 7 (26.9) 4 (15.4)
Number littered (%) 22 (100.0) 24 (100.0) 19 (100.0) 22 (100.0)
Clinical observations None None None None
Mean body weight[a],[b]
Body weight: GD 21 359.6 ± 4.4 370 ± 3.9 360.8 ± 4.5 360.2 ± 4.6
Body weight: LD 28 283.1 ± 3.3 283.5 ± 2.9 280.4 ± 3.1 282.7 ± 2.3
Necropsy observations None None None None
F1 generation (preweaning)[b]
Clinical observations None None None None
Live litter size
PND 0 10.8 ± 0.7 13.0 ± 0.4* 11.7 ± 0.4 11.1 ± 0.7
PND 4 (prestandardization) 10.7 ± 0.7 12.9 ± 0.4* 11.5 ± 0.4 10.9 ± 0.7
PND 4 (poststandardization) 8.9 ± 0.4 9.9 ± 0.1 9.8 ± 0.2 9.1 ± 0.4
PND 28 8.9 ± 0.4 9.7 ± 0.1 9.7 ± 0.2 8.9 ± 0.4
Male pup mean body weight
PND 1 6.96 ± 0.08 7.01 ± 0.09 7.05 ± 0.08 7.17 ± 0.11
PND 28 82.66 ± 1.00** 82.13 ± 1.07 78.92 ± 0.94* 71.92 ± 0.90**
Female pup mean body weight
PND 1 6.65 ± 0.07 6.64 ± 0.08 6.63 ± 0.07 6.69 ± 0.09
PND 28 75.37 ± 1.11** 73.63 ± 1.03 69.81 ± 1.03** 64.17 ± 0.87**
F1 generation (postweaning)
Mean body weight[a],[b]
Male body weight: PND 28 82.0 ± 1.5** 78.8 ± 1.2 76.3 ± 0.9* 71.9 ± 1.5**
Male body weight: PND 91 396.6 ± 6.6** 392.0 ± 4.2 387.1 ± 3.9 376.3 ± 4.0**
Female body weight: PND 28 75.4 ± 1.8** 70.8 ± 1.1 67.4 ± 1.0** 64.5 ± 1.5**
Female body weight: PND 91 253.0 ± 4.2** 244.5 ± 3.7 241.3 ± 3.0 236.4 ± 2.9**
F1 and F2 generations
Endocrine endpoints, developmental landmarks, and pubertal endpoints[b]
Vaginal opening (F1)
Adjusted mean day of vaginal opening (litter mean)[c] 34.4 ± 0.3** 35.1 ± 0.2 35.7 ± 0.3* 36.5 ± 0.3**
Body weight at acquisition[a] 106.7 ± 2.0 107.3 ± 1.3 107.1 ± 1.4 107.7 ± 2.4
Balanopreputial separation (F1)
Adjusted mean day of balanopreputial separation (litter mean)[c] 45.6 ± 0.3** 45.6 ± 0.6 45.2 ± 0.3 47.8 ± 0.5**
Body weight at acquisition[a] 207.9 ± 3.5 203.5 ± 4.0 199.2 ± 1.9 214.1 ± 3.4
Prenatal cohort
Mating and fertility performance
Number of mating pairs 21 23 19 22
Mated females/paired (%) 90.5 91.3 94.7 90.9
Pregnant females/mated (%) 100.0 85.7 83.3 80.0
Mean body weight[a],[b]
Body weight gain: GD 0–21 168.0 ± 3.5 147.8 ± 8.4* 170.9 ± 3.0 151.9 ± 5.5
Uterine content data[b]
Mean number of corpora lutea/female 17.74 ± 0.73 16.22 ± 0.55 18.71 ± 0.61 17.50 ± 0.74
Implantations/female 15.21 ± 0.68 13.11 ± 1.19 15.75 ± 0.51 14.19 ± 0.88
Live fetuses/litter 14.89 ± 0.65 13.47 ± 1.11 15.25 ± 0.54 13.63 ± 0.93
Fetal findings
External findings None None None None
Visceral findings None None None None
Skeletal findings[d]
Lumbar, 1, unilateral or bilateral, rudimentary – [V]
Fetuses 12 (4.24) 8 (3.79) 7 (3.83) 22 (10.09)
Litters 5 (26.32) 5 (29.41) 2 (16.67) 7 (43.75)
Lumbar, 1, bilateral, rudimentary – [V]
Fetuses 4 (1.41) 4 (1.90) 4 (2.19) 8 (3.67)
Litters 2 (10.53) 3 (17.65) 2 (16.67) 5 (31.25)
Lumbar, 1, left, rudimentary – [V]
Fetuses 0 (0.00)# 4 (1.90) 0 (0.00) 8 (3.67)
Litters 0 (0.00) 4 (23.53) 0 (0.00) 4 (25.00)
Lumbar, 1, right, rudimentary – [V]
Fetuses 8 (2.83) 0 (0.00) 3 (1.64) 6 (2.75)
Litters 5 (26.32) 0 (0.00) 1 (8.33) 4 (25.00)
Reproductive performance cohort
Mating and fertility performance
Number of mating pairs 36 46 35 37
Mated females/paired (%) 94.4 89.1 91.4 91.9
Littered females/mated (%) 76.5 82.9 77.4 76.5
Mean body weight[a],[b]
Body weight: GD 21 431.0 ± 10.2 416.5 ± 6.2 419.2 ± 7.9 402.2 ± 6.9
Body weight: LD 28 318.7 ± 5.8* 311.4 ± 4.5 304.2 ± 5.3 302.6 ± 3.4
Live litter size[b]
PND 0 14.1 ± 0.8 13.0 ± 0.7 15.0 ± 0.6 13.1 ± 0.8
PND 4 (prestandardization) 13.5 ± 0.9 13.1 ± 0.7 14.0 ± 0.8 12.5 ± 0.8
PND 4 (poststandardization) 9.4 ± 0.5 9.4 ± 0.3 9.6 ± 0.4 9.3 ± 0.4
PND 28 7.4 ± 0.7 8.2 ± 0.5 8.0 ± 0.6 8.4 ± 0.6
Male pup mean body weight[b]
PND 1 6.88 ± 0.09 6.78 ± 0.14 6.63 ± 0.09 6.68 ± 0.08
PND 28 78.45 ± 2.28** 78.20 ± 1.68 73.29 ± 2.05 67.29 ± 1.32**
Female pup mean body weight[b]
PND 1 6.50 ± 0.14 6.43 ± 0.10 6.33 ± 0.08 6.43 ± 0.10
PND 28 71.21 ± 2.07** 71.79 ± 1.65 67.82 ± 1.84 63.62 ± 1.31**
Adult necropsies
Clinical pathology
Subchronic cohort None None None None
Gross necropsy findings
All cohorts None None None None
Organ weights
All cohorts None None None None
Histopathological findings
All cohorts None None None None
Andrology None None None None
Vaginal cytology None ↑ Estrus stage length ↑ Estrus stage length ↑ Estrus stage length
Level of evidence of reproductive toxicity: No evidence
Level of evidence of developmental toxicity: Equivocal evidence

Statistical significance for an exposure 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.
# Statistically significant at p ≤ 0.05 in litter-based analysis of fetuses.
GD = gestation day; LD = lactation day; PND = postnatal day.
[V] = variation.
[a] Body weight results given in grams.
[b] Data are presented as mean ± standard error.
[c] Adjusted based on body weight at weaning.
[d] Upper row denotes number of affected fetuses (%) and lower row the number of affected litters (%).