Viability and Clinical Observations

One F₀ rat in the 3,000 ppm group was euthanized on study day 5 before the start of dosed feed administration due to the presence of excessive red eye discharge (too early to determine pregnancy status); this finding was not attributed to 2H4MBP exposure (Appendix E). No clinical observations were attributed to 2-hydroxy-4-methoxybenzophenone (2H4MBP) exposure in any group during gestation or lactation (Appendix E).

Body Weights and Feed Consumption

F₀ females exposed to 50,000 ppm 2H4MBP displayed lower body weights than the control group (Table 4; Figure 5). The mean body weight of dams in the 50,000 ppm group on gestation day (GD) 21 was significantly decreased by 11% compared to the control group, and the mean body weight gain of dams in the 50,000 ppm group over gestation (GD 6–21) was significantly decreased by 35%. This difference was attributed to a transient body weight loss over the GD 6–9 interval and lower body weight gains over most of the subsequent intervals and not attributed to smaller litters or lower fetal weights (Appendix E). F₀ females exposed to 10,000 or 25,000 ppm 2H4MBP displayed similar 20% significant decreases in body weight gain over the GD 6–21 interval, which were attributed to lower body weights during the early gestation period (Table 4).

Table 4

Summary of Mean Body Weights and Body Weight Gains of F₀ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Gestation and Lactation (Dose Range-finding Study).

Figure 5

Growth Curves for F₀ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Gestation and Lactation (Dose Range-finding Study)

Lactation mean body weights were significantly decreased (16%) in dams exposed to 50,000 ppm 2H4MBP relative to the control group (Table 4; Figure 5). This decrease was similar in magnitude to that observed at the end of gestation and likely related to the significantly decreased body weights observed during gestation.

In general, feed consumption during gestation in the 2H4MBP-exposed groups was higher than in the control group (Table 5). Feed consumption was significantly increased at several time intervals in the 25,000 and 50,000 ppm groups and likely signifies poor palatability and subsequent powdered feed wastage (when the animals were trying to find more palatable feed in the jar). This was also supported by the observation of apparent feed in the animals’ bedding. 2H4MBP intake for F₀ females in the 3,000, 10,000, 25,000, and 50,000 ppm 2H4MBP groups, based on measured feed consumption and dietary concentrations for GD 6–21 interval, was approximately 215, 695, 2,086, and 6,426 mg 2H4MBP/kg body weight/day (mg/kg/day), respectively (Table 5).

Table 5

Summary of Feed and Test Article Consumption of F₀ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Gestation and Lactation (Dose Range-finding Study).

2H4MBP exposure was not associated with lower feed consumption during lactation (Table 5). 2H4MBP intake for F₀ females in the 3,000, 10,000, 25,000, and 50,000 ppm 2H4MBP groups, based on feed consumption and dietary concentrations for lactation days (LDs) 1–14, was approximately 577, 1,858, 4,460, and 12,029 mg/kg/day, respectively (Table 5).

Maternal Reproductive Performance

Across all exposure groups, 13 out of 57 time-mated F₀ females were not pregnant: four in the control group; two each in the 3,000, 10,000, and 50,000 ppm groups; and three in the 25,000 ppm group (Table 6). There were no toxicologically relevant effects of 2H4MBP exposure on the proportion of dams that produced viable litters or on gestation length. There was no effect of 2H4MBP exposure on initial mean litter size or sex ratio.

Table 6

Summary of the Reproductive Performance of F₀ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Gestation (Dose Range-finding Study).

Pup Viability and Body Weights

2H4MBP exposure was associated with a reduction in the mean number of live pups per litter in the 25,000 and 50,000 ppm groups (approximately 2–3 pups/litter from PND 0 through PND 28) (Table 7; Appendix E). Over the lactation period, there were 20 dead pups (from five litters) in the 25,000 ppm group and 16 dead pups (from five litters) in the 50,000 ppm group, compared to 3 dead pups (from two litters) in the control group. In the 25,000 ppm group, 12 of the 20 dead pups were from a single litter. In the 50,000 ppm group, 10 of the 16 dead pups were from a single litter (Appendix E). Male and female pup mean body weights of these exposed groups were significantly decreased (25%–50%) compared to those of control pups (Table 8; Figure 6, Figure 7). Adverse F₁ pup clinical observations in the 25,000 and 50,000 ppm groups were consistent with the effects of 2H4MBP exposure on pup survival (Appendix E). Findings included observations of pups found dead, cannibalized, missing, no milk band, bruised, stained fur, cold to touch, or emaciated. There were no notable gross findings in the limited number of F₁ offspring that received a necropsy. Necropsy findings for pups found dead on or after PND 1 were limited to the absence of milk/food in the stomach (Appendix E). Pups in the 10,000 ppm group displayed mean body weights that were lower (4%–16%) than those of the control group.

Table 7

Summary of F₁ Litter Size and Pup Survival Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone (Dose Range-finding Study).

Table 8

Summary of F₁ Male and Female Pup Mean Body Weights Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone (Dose Range-finding Study)^a,b.

Figure 6

Lactation Growth Curves for F₁ Male Pups Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone (Dose Range-finding Study)

Figure 7

Lactation Growth Curves for F₁ Female Pups Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone (Dose Range-finding Study)

F₀ Viability and Clinical Observations

2H4MBP exposure did not affect viability of the F₀ females (Appendix E). One female in the EE group was removed on GD 11 and was subsequently diagnosed with lymphoma. Given the singular incidence and early onset, this occurrence was not considered related to EE exposure. No clinical observations were attributed to 2H4MBP exposure (Appendix E).

F₀ Gestation Body Weights and Feed Consumption

F₀ females exposed to 10,000 or 30,000 ppm 2H4MBP displayed lower gestation mean body weights and body weight gains (Table 9; Figure 9). On GD 21, female mean body weights were significantly decreased by 5% and 10% compared to those of control animals in the 10,000 and 30,000 ppm 2H4MBP groups, respectively. Body weight gains between GD 6 and GD 21 were significantly decreased by 11%, 25%, and 35% compared to those of the control group in the 10,000 and 30,000 ppm 2H4MBP and 0.05 ppm EE groups, respectively (Table 9). There was a transient loss in mean body weight (−1.0 g) between GD 6 and GD 9 in the 30,000 ppm 2H4MBP group compared to a gain of 13.7 g in the control group. This interval corresponds to the first interval the females were administered dosed feed and likely reflects lower palatability (feed wastage) of the dosed feed; this is also consistent with what was observed in the dose range-finding study. Females in the 30,000 ppm groups also exhibited significantly decreased (approximately 12%–20%) body weight gains in the GD 15–18 and GD 18–21 intervals (Table 9). Gestational mean body weights and weight gains in the EE group were less than those in the control group. Body weight gain in the EE group over the GD 6–21 interval was significantly decreased by approximately 35% compared to the control group (Table 9). There was no effect of 2H4MBP exposure on F₀ female mean body weights during gestation in the 3,000 ppm group. There was no reduction in litter size on PND 0 or pup mean body weight on PND 1 in the 2H4MBP-exposed groups (Appendix E), suggesting the lower relative maternal body weights were due to a maternal body weight effect of 2H4MBP rather than an effect on the collective weight of the uterine contents. Pup body weight on PND 1, but not litter size, was significantly decreased (12%) in the 0.05 ppm EE group (Appendix E) and likely contributed to the lower maternal body weight gain of that group compared to the control group.

Table 9

Summary of Mean Body Weights and Body Weight Gains of F₀ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Gestation.

Figure 9

Growth Curves for F₀ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Gestation

Despite sporadic differences, neither 2H4MBP nor EE exposure adversely affected feed consumption during gestation (Table 10). Observed higher feed consumption in the 30,000 ppm group likely represented feed wastage. 2H4MBP intake for F₀ females in the 3,000, 10,000, and 30,000 ppm groups, based on feed consumption and dietary concentrations over the GD 6–21 interval, was approximately 205, 697, and 2,644 mg/kg/day, respectively (Table 10). EE intake during gestation was approximately 0.004 mg/kg/day.

Table 10

Summary of Feed and Test Article Consumption of F₀ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Gestation.

Maternal Reproductive Performance

Across all exposure groups, 20 of 125 time-mated rats were not pregnant: three each in the control and 10,000 ppm groups, four in the 3,000 ppm group, five in the 30,000 ppm group, and five in the EE group (Table 11; Appendix E). There was no effect of 2H4MBP exposure on the proportion of dams that produced viable litters or on gestation length. There was no effect of 2H4MBP exposure on initial mean litter size, PND 1 pup weight, or sex ratio. PND 1 pup weight in the EE group was significantly decreased by 12% compared to the control group (Table 11). Anogenital distance (AGD) measurements are presented in Appendix E.

Table 11

Summary of the Reproductive Performance of F₀ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Gestation.

Lactation Body Weights and Feed Consumption

F₀ females in the 10,000 and 30,000 ppm 2H4MBP and 0.5 ppm EE groups displayed lower mean body weights during lactation compared to the control group (Figure 10; Table 12). The magnitude of response (approximately 5%–15% decrease) in female body weights at LD 1 and LD 28 was similar to that observed at the end of the gestation interval. These observations collectively suggest that the lower lactation body weight was a consequence of exposure to 2H4MBP or EE during gestation and not a direct effect of exposure during lactation.

Figure 10

Growth Curves for F₀ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Lactation

Table 12

Summary of Mean Body Weights, Body Weight Gains, and Feed and Test Article Consumption of F₀ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Lactation^a.

Feed consumption during lactation was similar among the groups. Dam 2H4MBP intake based on feed consumption and dietary concentrations during lactation from LD 1 through LD 13 (until the pups started consuming feed) for the 3,000, 10,000, and 30,000 ppm groups was approximately 484, 1,591, and 5,120 mg/kg/day, respectively (Table 12). EE intake during lactation was approximately 0.008 mg/kg/day.

Collectively, these data indicate that 30,000 ppm 2H4MBP and 0.05 ppm EE challenged the dams (as demonstrated by significantly decreased GD 6–21 body weights), without adversely affecting F₁ litter size.

F₁ Viability and Clinical Observations

Clinical observations were noted in individual pups in all groups, including the control groups, and were typically indicative of a pup not thriving (e.g., cold to the touch, no milk in the stomach) (Appendix E). There was no effect of 2H4MBP on pup survival (Table 13). The mean number of live pups per litter appeared to be reduced in the 0.05 ppm EE group on PND 4 relative to the control group. That reduction reflected three litters that did not survive to PND 4, resulting in a higher number of dead or missing (presumed dead) pups and a lower survival ratio for the PND 1–4 interval relative to the control group. On PND 28, there was a slight, but significant, decrease in mean litter size in the EE group relative to the control group.

Table 13

Summary of F₁ Litter Size and Pup Survival Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone.

F₁ Body Weights

Male Pups

An exposure concentration- and time-related reduction in male pup mean body weight per litter was observed during lactation in the 10,000 and 30,000 ppm 2H4MBP and the 0.05 ppm EE groups, relative to the control group (Table 14; Figure 12). From PND 1 through PND 28, mean body weight differences were significantly increased between the control group and the 30,000 ppm group and, to a lesser extent, the 10,000 ppm group. On PND 28, male pup mean body weights per litter were significantly decreased by 10%, 24%, and 11% compared to those of the control group in the 10,000 and 30,000 ppm 2H4MBP and 0.05 ppm EE groups, respectively.

Table 14

Summary of F₁ Male and Female Pup Mean Body Weights Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone^a,b.

Figure 12

Lactation Growth Curves for F₁ Male Pups Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone

Female Pups

An exposure concentration- and time-related reduction in female pup mean body weight per litter was observed during lactation in the groups exposed to 10,000 or 30,000 ppm 2H4MBP and 0.05 ppm EE, relative to the control group (Table 14; Figure 13). From PND 1 through PND 28, mean body weight differences became greater between the control group and the 30,000 ppm group and, to a lesser extent, the 10,000 ppm group. On PND 28, female pup mean body weights per litter were significantly decreased by 9%, 24%, and 7% compared to those of the control group in the 10,000 and 30,000 ppm 2H4MBP and 0.05 ppm EE groups, respectively.

Figure 13

Lactation Growth Curves for F₁ Female Pups Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone

F₀ Necropsy

F₀ dams were necropsied on PND 28 following pup weaning when the F₀ dams were 21 weeks of age. Gross findings of pale, discolored kidneys (unilateral/bilateral) were recorded for three females in the 30,000 ppm 2H4MBP group (Appendix E). Histopathological examination identified findings of renal tubule lumen dilatation, tubule epithelium regeneration, interstitial inflammation, papilla necrosis, nephropathy, and transitional epithelium hyperplasia. Similar findings were also observed in the F₁ and F₂ generations exposed to 2H4MBP (Appendix E).

F₁ Viability and Clinical Observations

Neither 2H4MBP nor EE exposure altered viability in the F₁ generation postweaning. Clinical observations were noted in all groups, including the control groups, on a sporadic basis (Appendix E). No clinical observations showed an increase in incidence or severity in association with exposure to 2H4MBP or EE.

F₁ Body Weights and Feed Consumption

Males (Postweaning)

Body weights between PND 28 and PND 91 were significantly decreased in males in the 10,000 and 30,000 ppm 2H4MBP and 0.05 ppm EE groups (Table 15; Figure 15). On PND 91, mean body weights of these groups were significantly decreased by 5%, 16%, and 18%, respectively, compared to those of the control group.

Table 15

Summary of Postweaning Mean Body Weights, Body Weight Gains, and Feed and Test Article Consumption of All F₁ Male Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed.

Figure 15

Postweaning Growth Curves for All F₁ Male Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed

Overall, no adverse effects of 2H4MBP exposure on F₁ male feed consumption were found (Table 15). Sporadic small but significant decreases in absolute feed consumption (g/animal/day) were observed in the 30,000 ppm group between PND 28 and PND 84 (Appendix E) but did not affect overall feed consumption during the postweaning period. Relative feed consumption (g/kg/day) was significantly increased in the 10,000 and 30,000 ppm groups relative to the control group during the postweaning period, likely due to the lower body weights of the animals in these groups. A significant decrease in absolute feed consumption was observed in the 0.05 ppm EE group (14% below the control group) during the postweaning period, suggesting a continued effect of EE exposure on growth during the postweaning phase. 2H4MBP intake for F₁ males, based on feed consumption and dietary concentrations for PND 28 through PND 91, was approximately 267, 948, and 3,003 mg/kg/day at 3,000, 10,000, and 30,000 ppm 2H4MBP, respectively (Table 15). EE intake during the postweaning period was approximately 0.005 mg/kg/day.

Females (Postweaning)

PND 28 through PND 91 mean body weights were significantly decreased in females exposed to 30,000 ppm 2H4MBP or 0.05 ppm EE (Table 16; Figure 16). On PND 91, female mean body weights of the 30,000 ppm 2H4MBP and 0.05 ppm EE groups were significantly decreased by 14% and 17%, respectively, compared to those of the control group. The 10,000 ppm group displayed significantly decreased mean body weights (<10%) on PND 28 and PND 35 (Table 16; Appendix E), after which mean body weights were similar to those of the control group.

Table 16

Summary of Postweaning Mean Body Weights, Body Weight Gains, and Feed and Test Article Consumption of All F₁ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed.

Figure 16

Postweaning Growth Curves for All F₁ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed

In general, 2H4MBP-exposed females displayed similar feed consumption values over the postweaning period (Table 16; Appendix E). There were small (approximately 15%), but significant, increases in absolute feed consumption (g/animal/day) recorded over two weekly intervals in the 30,000 ppm 2H4MBP group between PND 42 and PND 91. There was no overall reduction in absolute feed consumption during the postweaning period in the 30,000 ppm 2H4MBP group. Relative feed consumption (g/kg/day) was significantly increased in the 30,000 ppm group relative to the control group during the postweaning period, likely the result of lower body weights of the 2H4MBP-exposed animals. Absolute feed consumption by the EE group was similar to the control group; however, as these animals weighed less, their relative feed consumption was significantly increased compared to that of the control animals. 2H4MBP intake for F₁ females, based on feed consumption and dietary concentrations for PND 28 through PND 91, was approximately 287, 983, and 3,493 mg/kg/day at 3,000, 10,000, and 30,000 ppm 2H4MBP exposures, respectively. EE intake during the postweaning period was approximately 0.005 mg/kg/day.

Anogenital Distance

F₁ and F₂ male and female offspring exposed to 2H4MBP or EE in feed did not display any alterations in mean PND 1 body-weight-adjusted AGD (Appendix E).

Areolae/Nipple Retention

F₁ and F₂ male offspring exposed to 2H4MBP or EE in feed did not display any signs of areolae/nipple retention (Appendix E).

Testicular Descent

F₁ males in the 30,000 ppm 2H4MBP group displayed a significant 1-day acceleration in the mean day of testicular descent (18.0 ± 0.2) compared to the control group (19.1 ± 0.2) (Appendix E). There was no difference in the mean day of testicular descent in the F₂ generation (Appendix E). The cumulative litter responses for the 30,000 ppm 2H4MBP group (F₁ generation) showed an earlier age at acquisition, whereas the F₂ generation did not display this response. The mean day of achieving testicular descent in control Sprague Dawley (Hsd:Sprague Dawley^® SD^®) rats in two other MOG studies conducted in the testing laboratory was PND 18.2 ± 0.2 and PND 18.0 ± 0.2. For NTP Reproductive Assessment by Continuous Breeding (RACB) studies, the mean day of testicular descent ranged from PND 15.3 ± 0.2 to PND 17.4 ± 0.5 over four studies.^98,99

Vaginal Opening

Females exposed to 30,000 ppm 2H4MBP exhibited a significant delay in litter mean day of VO, relative to the control group (Table 17); however, when adjusted for body weight at weaning, this delay was somewhat mitigated, with the 30,000 ppm group displaying a 1-day delay. Figure 17 shows litter and adjusted litter cumulative response (%), or cumulative probability of attainment, plotted against PND for each exposure group. Exposure increases were associated with higher cumulative probabilities of attainment delays, particularly for the 30,000 ppm group, as seen in the exposure-related rightward shift of curves toward higher attainment days (Figure 17A). These shifts were less pronounced after adjustment for body weight at weaning (Figure 17B). The delay was associated with lower body weight, and these females also exhibited significantly decreased mean body weights during lactation and postweaning (Table 16; Figure 16). As expected, litter mean day of VO in the EE group was greatly accelerated (by approximately 11 days) compared to the control group (Table 17; Figure 17).

Table 17

Summary of Vaginal Opening of F₁ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed.

Figure 17

Time to Vaginal Opening of F₁ Female Offspring Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed

Balanopreputial Separation

Male rats in the 10,000 and 30,000 ppm 2H4MBP and 0.05 ppm EE groups displayed a significant delay in the mean day of attaining BPS (Table 18). Figure 18 shows litter and adjusted litter cumulative response (%), or cumulative probability of attainment, plotted against PND for each exposure group. An exposure-dependent rightward shift is seen for the 10,000 and 30,000 ppm 2H4MBP and 0.05 ppm EE groups, indicating higher cumulative probabilities of attainment at later PNDs (Figure 18). When litter mean day of attainment was adjusted for body weight on day of weaning, these delays were no longer significantly different from control males (Table 18; Figure 18). The observed delay in BPS in 2H4MBP- or EE-exposed animals is likely the consequence of growth retardation as evidenced by lower mean body weights and body weight gains (Table 15; Figure 15). Three males in the 30,000 ppm 2H4MBP group had not achieved BPS as of PND 59, when checks for this marker stopped. These males were from the same litter (dam 202). Two were assigned to the reproductive performance cohort (animals 1901 and 1907) and the other (animal 1903) was assigned to the prenatal cohort. None of them demonstrated evidence of mating or resultant evidence of pregnancy. At scheduled necropsy, two of the males had achieved BPS and the other (animal 1903) had hypospadias.

Table 18

Summary of Balanopreputial Separation of F₁ Male Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed.

Figure 18

Time to Balanopreputial Separation of F₁ Male Offspring Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed

Viability and Clinical Observations

There were no exposure-related deaths or clinical observations in F₁ male and female rats following exposure to 2H4MBP or EE in feed (Appendix E).

Selection and Mating

A male and a female, or two males and two females (1:1), from each litter were allocated to the prenatal and reproductive performance cohorts, respectively, avoiding sibling mating (Figure 19). Vaginal lavage samples were collected for approximately 2 weeks prior to cohabitation and continued until evidence of mating or until the cohabitation period was completed. Estrous cyclicity data are presented in Appendix E.

Vaginal Cytology

Analysis of estrous cyclicity using the continuous-time Markov model demonstrated a slight but significant increase in estrus stage length in the 10,000 and 30,000 ppm 2H4MBP groups compared to the control group (Appendix E). Rats in these two groups spent more time in estrus compared to the control group (approximately 36% and 37% of the days, respectively, versus approximately 31% for the control group). A slight but significant decrease in the length of proestrus was observed in the 10,000 ppm group compared to the control group. These minimal estimated changes in stage length did not impact reproductive performance and likely represent normal biological variability and are not considered biologically adverse. There were no EE exposure-related changes in estrous stage lengths.

Fertility

The precoital interval and number of females that mated (i.e., those that were sperm-positive, littered, or had implantation sites) were similar among the control, 2H4MBP, and EE groups in both cohorts, indicating that neither 2H4MBP nor EE exposure negatively affected mating behavior (Table 19). The number of pregnant females was also similar among the groups, indicating that F₁ male and female fertility were not affected by 2H4MBP or EE exposure at the concentrations examined. Respective responses observed were consistent between the cohorts.

Table 19

Summary of Mating and Fertility Performance of F₁ Male and Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed.

F₁ Reproductive Performance Cohort Andrology

There were no 2H4MBP- or EE-related effects on motile sperm, progressively motile sperm, or testis spermatid head concentration (Appendix E). Males in the 30,000 ppm 2H4MBP group displayed lower cauda epididymal sperm counts (approximately 14%) and epididymis weight (approximately 6%) relative to control animals. Testis weight was lower in the 10,000 and 30,000 ppm 2H4MBP and 0.05 ppm EE groups (approximately 6%, 6%, and 9%, respectively), relative to control animals. These findings were not associated with histopathological changes (Appendix E) or significant changes in reproductive performance (Appendix E).

Gestation Body Weights

As previously mentioned, F₁ female rats exposed to 10,000 or 30,000 ppm 2H4MBP or 0.05 ppm EE displayed significantly decreased preweaning and postweaning mean body weights compared to the control group. Consequently, F₁ female mean body weights of the 30,000 ppm 2H4MBP and 0.05 ppm EE groups in both the prenatal and reproductive performance cohorts at the time of cohabitation were lower relative to control females. Gestation body weight curves of the exposed groups in both cohorts generally paralleled the control group (Figure 20, Figure 21). Dams in both cohorts exposed to 10,000 or 30,000 ppm 2H4MBP or 0.05 ppm EE, however, displayed significantly decreased GD 0–21 mean body weight gains (approximately 13%–14%, 25%–28%, and 22%–24%, respectively) relative to the respective control group (Table 20). This difference in mean body weight gain during pregnancy might be the result of a slight reduction in litter size of one to two fewer fetuses/pups observed in these groups (Appendix E). Respective responses observed were consistent between the two cohorts.

Figure 20

Gestation Growth Curves for F₁ Female Rats in the Reproductive Performance Cohort Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed

Figure 21

Gestation Growth Curves for F₁ Female Rats in the Prenatal Cohort Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed

Table 20

Summary of Gestation Mean Body Weight Gains for F₁ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed^a,b,c.

Gestation Feed Consumption

2H4MBP groups displayed similar absolute feed consumption (g/animal/day) during gestation as the respective control group. Relative feed consumption (g/kg/day) during gestation in the 3,000 and 10,000 ppm 2H4MBP groups was similar to the respective control group (Table 21; Appendix E). Pregnant females in the 30,000 ppm group of the prenatal cohort displayed a significant increase in relative feed consumption between GD 0 and GD 21 (approximately 21%), but this is likely the result of the substantially lower body weights of this group. In the EE group of the reproductive performance cohort, absolute feed consumption between GD 0 and GD 21 was significantly decreased by approximately 19%, and relative feed consumption was similar to that of the control group. The opposite was true for the EE group in the prenatal cohort, in which relative feed consumption was significantly increased by approximately 25% relative to the control group. 2H4MBP intake of both cohorts during gestation, based on feed consumption and dietary concentrations, was approximately 240, 825, and 2,760 mg/kg/day at exposure concentrations of 3,000, 10,000, and 30,000 ppm 2H4MBP, respectively. EE intake was approximately 0.004 mg/kg/day. The respective dose consumed was similar between the two cohorts.

Table 21

Summary of Gestation Feed and Test Article Consumption for F₁ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed^a,b,c.

Maternal Reproductive Performance and Uterine Data

In the prenatal cohort, females were between 109 and 132 days of age at the time of laparotomy. Pregnant females exposed to 10,000 or 30,000 ppm 2H4MBP displayed lower gravid uterine weights (15% and 17%, respectively), fewer implants, and fewer live fetuses (approximately 2 fewer/litter) than control animals; significant decreases were observed for gravid uterine weight and number of implantations at 30,000 ppm (Table 22). In the 30,000 ppm 2H4MBP group, these findings correlated with significant decreases in the mean number of corpora lutea (approximately 4 fewer/litter) relative to the control group and are consistent with the reduction in live litter size on PND 0 relative to control animals observed in the reproductive performance cohort (Appendix E). Females in the 0.05 ppm EE group exhibited significantly decreased gravid uterine weight (20% lower than the control group), mean number of corpora lutea, implantations, and live fetuses (Table 22). Dams exposed to 2H4MBP or EE did not display any adverse changes in postimplantation loss, mean live fetal weights, or fetal sex ratio.

Table 22

Summary of Uterine Content Data for F₁ Female Rats in the Prenatal Cohort Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed.

Fetal Findings

Visceral

Male and female fetuses (combined) exposed to 30,000 ppm 2H4MBP displayed a higher incidence of enlarged liver, a malformation (Table 23), which had not been observed in NTP historical controls.

Table 23

Summary of Select Visceral Findings in Fetuses Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed.

The 30,000 ppm 2H4MBP group displayed a higher incidence of unilateral or bilateral (combined) hydronephrosis, a malformation, relative to the control group (Table 23). This higher incidence was observed in 2.22% of the fetuses (29.41% of the litters), whereas it was observed in 1.12% and 1.15% of the fetuses (16.67% and 13.33% of the litters) from the control group and EE group, respectively. The NTP historical control range for unilateral or bilateral hydronephrosis is 0.00% to 0.81% for fetuses; (0.00% to 16.67% for litters). The incidence of bilateral distended ureter, a variation, was higher in all 2H4MBP-exposed groups as well as the EE group, relative to the control group. When unilateral and bilateral distended ureters were combined, the fetal incidence was 10.68%, 12.72%, and 8.44% (62.50%, 50.00%, and 35.29% of the litters) in the 3,000, 10,000, and 30,000 ppm groups versus 4.83% and 12.64% (44.44% and 46.67% of the litters) in the control and EE groups, respectively. Historical control incidence for distended ureter in fetuses is 10.90% (4.83% to 15.36%) and for litters is 56.70% (43.75% to 68.18%). Hydroureter of the left kidney was observed in one fetus in the control group and in two fetuses in the 3,000 ppm group, but given the low incidence, these were not considered related to 2H4MBP exposure (Appendix E). The NTP historical control range for hydroureter is up to 2.83% and 21.05% for fetuses and litters, respectively. Hydronephrosis and other abnormalities associated with the kidney and ureter (e.g., dilated renal pelvis, distended ureter, hydroureter) are common findings in this strain of rat; therefore, these collective findings may or may not be related to the 2H4MBP-associated microscopic findings observed in the kidney of adult F₁ males and females exposed to 30,000 ppm 2H4MBP (Appendix E).

Other malformations observed in 2H4MBP-exposed fetuses include ventricular septal defects in two fetuses in the 10,000 ppm group and in one fetus in the 30,000 ppm group (Table 23). This finding was not considered related to 2H4MBP due to the low incidence and lack of a clear exposure concentration-response and because it had been observed in a control fetus in a previous study (1/1,385). A single fetus (dam 1950, fetus 01) in the 30,000 ppm 2H4MBP group displayed adrenal gland agenesis, malpositioned kidneys, distended stomach, and agenesis of the gonads (Appendix E). This fetus also had external and skeletal malformations. None of the visceral findings associated with this fetus was considered 2H4MBP-related due to their singular occurrence. One fetus in the 10,000 ppm group displayed small, round kidneys, which were not considered 2H4MBP-related due to the singular occurrence.

There were no additional effects of EE exposure on the incidence of fetal visceral variations.

Reproductive Performance and Littering

Reproductive performance and littering parameters for the reproductive performance cohort are presented in Table 24. Gestation length was similar among the 2H4MBP-exposed groups and the control group. The EE group displayed a significant decrease (approximately 0.4 days) in gestation length compared to the control group.

Table 24

Summary of Reproductive Parameters of F₁ Female Rats in the Reproductive Performance Cohort Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed.

Lactation Body Weights and Feed Consumption

Consistent with their premating and gestation weights, F₁ female mean body weights during lactation were significantly decreased in the 10,000 and 30,000 ppm 2H4MBP and 0.05 ppm EE groups relative to the control group (Table 25; Figure 24). On LDs 1 and 28, female mean body weights of the 10,000 and 30,000 ppm 2H4MBP and 0.05 ppm EE groups were significantly decreased by 5%–7%, 18%–20%, and 19%–21%, respectively, compared to the control group. Body weight gain between LD 1 and LD 28 in the 10,000 and 30,000 ppm 2H4MBP and 0.05 ppm EE groups was higher relative to the control group. In general, feed consumption (g/animal/day) during lactation in the groups exposed to 2H4MBP or EE were similar to the control group (Table 25). 2H4MBP intake during lactation in the 3,000, 10,000, and 30,000 ppm 2H4MBP groups, based on feed consumption and dietary concentrations for the LD 1–13 interval, was approximately 426, 1,621, and 5,944 mg/kg/day, respectively (Table 25). EE intake during the postweaning period was approximately 0.009 mg/kg/day.

Table 25

Summary of Mean Body Weights, Body Weight Gains, and Feed and Test Article Consumption for F₁ Female Rats in the Reproductive Performance Cohort Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed during Lactation.

Figure 24

Lactation Growth Curves for F₁ Female Rats in the Reproductive Performance Cohort Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed

F₂ Viability and Clinical Observations

Clinical observations noted in individual pups in all groups, including the control group, were typically indicative of an individual pup not thriving (e.g., cold to the touch, no milk in the stomach). Exposure-related reductions in mean total and live litter size were observed in the 2H4MBP- and EE-exposed groups. Dams in the 10,000 and 30,000 ppm 2H4MBP groups had lower total and live litter size than the control group on PND 0 (by ~1 pup/litter). PND 0 total and live litter sizes in the EE-exposed group were significantly decreased (by ~2 pups/litter) relative to the control group (Table 26). Although the reductions in mean live litter size in the 2H4MBP-exposed groups did not achieve statistical significance compared to the control group after PND 0, the findings were consistent with the reductions in the mean number of live fetuses/pregnant females that were observed in the prenatal cohort (Table 22).

Table 26

Summary of F₂ Litter Size and Pup Survival Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone.

F₂ Body Weights

Male Pups

Male pups exposed to 30,000 ppm 2H4MBP displayed lower mean body weights (litter means) over time compared to the control group (Table 27; Figure 25; Appendix E). On PND 21, male pup mean body weights per litter of the 30,000 ppm group were lower by approximately 8% and by PND 28 they were significantly decreased 14% relative to the control group. A significant decrease in pup mean body weight was first observed in male offspring on PND 25 (Appendix E). These effects are consistent with what was observed in the F₁ generation, but the magnitude of change with exposure concentration is not as severe. EE exposure had no adverse effect on male pup mean body weights.

Table 27

Summary of F₂ Male and Female Pup Mean Body Weights and Body Weight Gains Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone^a,b.

Figure 25

Lactation Growth Curves for F₂ Male Pups Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone

Female Pups

Female pups exposed to 30,000 ppm 2H4MBP also displayed lower mean body weights (litter means) relative to the control group (Table 27; Figure 26; Appendix E). On PND 21 and PND 28, female pup mean body weights per litter of the 30,000 ppm group were significantly decreased by approximately 12% and 22% relative to the control group, respectively. A significant decrease in pup mean body weight was first observed in female offspring on PND 19 (Appendix E). These effects are consistent with what was observed in the F₁ generation, but the magnitude of reduction with exposure concentration is not as severe. There was no adverse effect of EE exposure on female pup mean body weights.

Figure 26

Lactation Growth Curves for F₂ Female Pups Following Perinatal Exposure to 2-Hydroxy-4-methoxybenzophenone

F₁ Male Necropsies

F₁ males in the reproductive performance cohort were euthanized following the mating period at 153–155 days of age. The F₁ males in the prenatal cohort were euthanized following completion of pairing at 111–113 days of age.

Male rats exposed to 30,000 ppm 2H4MBP displayed a higher incidence of discolored (pale or dark) or enlarged kidneys and discolored (brown) urinary bladders (Table 28). Necropsy mean body weights of rats exposed to 30,000 ppm 2H4MBP or 0.05 ppm EE in both cohorts were significantly decreased by 14% and 15%−20%, respectively, compared to control animals (Table 29). Rats in both cohorts from all 2H4MBP-exposed groups displayed higher left and right absolute and relative kidney weights (Table 29). Absolute kidney weights were 5%−12%, 12%−14%, and 13%−22% higher and relative weights were 7%−10%, 15%−16%, and 30%−42% higher than those of control animals in the 3,000, 10,000, and 30,000 ppm groups, respectively. Gross findings in the kidney and bladder correlated with histopathological changes consistent with a retrograde nephropathy. One male rat in the 30,000 ppm 2H4MBP group in the reproductive performance cohort exhibited a diaphragmatic hernia. These hernias were also observed in F₁ females and in the F₂ generation. One male in the 30,000 ppm 2H4MBP group displayed hypospadias and another displayed bilateral smaller testes (Appendix E).

Table 28

Summary of Gross Necropsy Findings in Adult F₁ Male Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed^a.

Table 29

Summary of Organ Weights of Adult F₁ Male Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed^a,b,c.

Male rats in all 2H4MBP-exposed groups in both cohorts displayed higher absolute and relative liver weights compared to the control animals (Table 29). Absolute liver weights of males exposed to 3,000 ppm 2H4MBP in the reproductive performance and prenatal cohorts were higher by 6% and 11%, respectively, relative to control animals. Absolute liver weights of males in both cohorts exposed to 10,000 and 30,000 ppm were significantly increased 14%−20% relative to control animals. Relative liver weights of the 3,000, 10,000, and 30,000 ppm 2H4MBP groups in both cohorts were significantly increased approximately 7%−9%, 20%−23%, and 32%−34%, respectively, relative to the control group. The reproductive performance and prenatal cohorts displayed generally similar responses.

Rats in both cohorts exposed to 30,000 ppm 2H4MBP displayed slightly lower right and left absolute testis weights (approximately 4%−6%) (Table 29). Rats exposed to 30,000 ppm in the reproductive performance cohort exhibited a slight but significant decrease (5%−6%) in right and left absolute epididymis weights. Absolute ventral prostate gland weights of the 30,000 ppm 2H4MBP groups were lower by 19% and 9% relative to control animals in the reproductive performance and prenatal cohorts, respectively. This difference in cohort response might be due to duration of exposure being longer in the reproductive performance cohort. No 2H4MBP-related histopathological effects in the testis or epididymis were found. No exposure-related changes in sperm motility, sperm concentration, or testicular sperm head concentration were found (Appendix E). Rats in the 30,000 ppm 2H4MBP group in both cohorts displayed significantly decreased absolute levator ani/bulbocavernosus (LABC) muscle weights (10%−12%); however, when adjusted for body weight, this difference was negligible (Table 29). No gross pathological findings in the males exposed to 0.05 ppm EE were considered to be related to exposure. In general, male rats exposed to EE displayed lower absolute weights of the testes, epididymides, prostate gland, kidney, liver, seminal vesicles with coagulating glands, and LABC. These observations are likely the result of exposed animals weighing 15%−20% less than control animals.

F₁ Female Necropsies

F₁ females (and F₂ offspring) in the reproductive performance cohort were euthanized and necropsied on PND 28, when the F₁ females were between 127 and 168 days of age. F₁ females in the prenatal cohort were between 109 and 132 days of age at the time of necropsy and the collection of organ weight data.

There were no gross observations in the prenatal cohort attributed to 2H4MBP exposure. Females in the reproductive performance cohort exposed to 30,000 ppm 2H4MBP displayed a higher individual and litter incidence of abnormal kidney findings (dilation, discoloration) (Table 30). These findings were also observed at a low incidence in the 3,000 ppm group and are consistent with what was observed in the F₁ males. This difference in response between the two cohorts might have been the result of duration of exposure and is consistent with what was observed in the F₁ males.

Table 30

Summary of Gross Necropsy Findings in Adult F₁ Female Rats in the Reproductive Performance Cohort Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed.

The reproductive performance and prenatal cohorts exposed to 10,000 or 30,000 ppm 2H4MBP displayed terminal/adjusted body weights that were significantly decreased (5%−8% and 18%−19%, respectively) compared to the control females (Table 31). Females in all 2H4MBP-exposed groups from both cohorts displayed significantly increased relative liver weights (10%–14%, 17%–32%, and 28%–53% in the 3,000, 10,000, and 30,000 ppm groups, respectively) compared to the control females (Table 31). Rats in the reproductive performance cohort exposed to 3,000, 10,000, and 30,000 ppm 2H4MBP displayed higher (approximately 5%−7%, 11%, and 24%−30%, respectively) relative right and left kidney weights compared to the control group. Absolute kidney weights were significantly decreased (12%−14%) compared to the control group in females in the reproductive performance cohort exposed to 0.05 ppm EE. Relative liver weights were significantly increased in the 0.05 ppm EE groups in both cohorts compared to the control groups, likely because necropsy body weights were lower than those of the control group.

Table 31

Summary of Organ Weights of Adult F₁ Female Rats Exposed to 2-Hydroxy-4-methoxybenzophenone in Feed^a,b,c.

Females exposed to 10,000 or 30,000 ppm 2H4MBP in both cohorts displayed lower absolute right and left ovarian weights (Table 31). Females in the reproductive performance cohort exposed to 30,000 ppm 2H4MBP displayed significantly decreased absolute adrenal gland weight compared to the control group. Both cohorts of the EE groups had lower absolute ovarian and adrenal gland weights. These changes are likely the result of lower body weights of exposed animals relative to control animals.

F₂ Necropsy

Pups were euthanized on PND 28; gross pathology findings are reported in Appendix E. One male each in the 3,000 and 30,000 ppm 2H4MBP groups exhibited bilateral undescended testes. Three males each in the 3,000 and 10,000 ppm 2H4MBP groups exhibited unilateral undescended testes. Several females in the 30,000 ppm 2H4MBP group displayed dilated, discolored, or enlarged kidneys consistent with what was observed in adults. Diaphragmatic hernias were observed in three males in the 30,000 ppm 2H4MBP group and in one male in the EE group (Appendix E). Diaphragmatic hernias were also observed in 2H4MBP- or EE-exposed F₁ rats in the reproductive performance cohort (Appendix E). The collective EE group had two males with diaphragmatic hernias. No hernias were observed in control animals or in the F₀ females (Appendix E). These hernias consist of a small protrusion of the liver through the diaphragm and are sometimes recorded grossly as diaphragmatic hernias and sometimes as hepatodiaphragmatic hernias.