Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

BADGE displayed no indications of any adverse effects on reproduction in rats over two generations at dose levels which greatly exceed potential human exposure levels. The NOEL for adult males was considered to be 50 mg/kg/day, NOEL for adult females 540 mg/kg/day, and the NOEL for reproductive effects was 750 mg/kg/day, the highest dose tested.

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-08-30-1995-9-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP as well as Guidelines .
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
other: TSCA section 416
Qualifier:
according to
Guideline:
other: EEC, No. L,383
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Four-week old male and female Sprague-Dawley rats were obtained from a commercial supplier. They were examined for health status by a veterinarian, and acclimated to the laboratory for approximately two weeks before study start. The animals were randomized based on body weights, and assigned to treatment groups. Animals were identified via a uniquely-coded alphanumeric metal ear tag.

The rats were housed singly in wire mesh cages. During gestation and lactation, females were housed in plastic shoe-box type cages with corn cob bedding. Animal rooms were designed and monitored to maintain appropriate temperature, relative humidity, air changes, and photocycles for the species. They were fed a commercial diet and municipal water ad libitum throughout the study. The feed was analyzed by the supplier for contaminants and nutritional content, and water was analyzed by the municipal water authority for contaminants. Copies of the analyses are maintained at Dow Chemical.
Route of administration:
oral: gavage
Vehicle:
other: aqueous solution of 0.5% Methocel A4M (Trademark, Dow Chemical) with 0.1% Tween 80
Details on exposure:
Two consecutive generations of Sprague-Dawley rats were administered BADGE once daily at dose levels of 0, 50, 180, 540 or 750 mg/kg/day in an aqueous solution of 0.5% Methocel A4M (Trademark, Dow Chemical) with 0.1% Tween 80 (dose volume of 10 mL/kg to deliver the appropriate dose). The study groups for both generations consisted of 30 male and 30 female rats per dose level. Gavage doses were administered to the P1 and P2 adult generations for 14 and 12 weeks, respectively, prior to the first mating. Both female adult generations were dosed during the mating, gestation, lactation, and weaning periods until necropsy. Doses for gestating and lactating females were adjusted based on body weight at day 0 and 6 of gestation. After the mating periods, the adult males from both generations received gavage doses until necropsied.
Details on mating procedure:
Breeding Procedure
Three 7-day cohabitation periods of one male and one female of the respective treatment group was allowed. For the P2/F2 mating, cohabitation and mating of male and female littermates was avoided. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal plug was discovered in situ was considered gestation day 0. Sperm-positive females were placed in nesting cages. Females that failed to mate were placed with an alternate male from the same treatment group. Adult females continued to receive the test material on a daily basis throughout the breeding, gestation, and lactation periods until necropsy. Litters were culled to 4 males and 4 females if possible. Litters with 8 or fewer pups were not culled; culling of runts was not performed. Weaning was 3 weeks after delivery.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis to confirm the stability of DGEBPA in the vehicle was conducted during the course of the study. The homogeneity of the dose suspensions was also determined during the first week of the study. Analysis of the dose suspensions to confirm the concentrations was performed at least 3 times per generation.
Duration of treatment / exposure:
238 days total
Frequency of treatment:
once daily
Details on study schedule:
Treatment of the P1 adults began on August 30, 1994 when these animals were approximately 6 weeks of age. After approximately 14 weeks of treatment, the P1 rats were mated (one male to one female of the respective treatment group) to produce the F1a litters. The P1 adults were co-housed to produce the F1a litters beginning on December 6, 1994. Approximately one week following weaning (3 weeks of age) of the last F1a litter, the P1 adults were co-housed a second time (beginning February 13, 1995) to produce the F1b litters (weeks 25-27). Following weaning of the F1b litters, 30 males and 30 females from each treatment group representing the maximum number of litters available within each treatment group were randomly selected and assigned to become the P2 parental generation. Dosing of the P2 generation was initiated after all F1b litters had been weaned and began on April 17, 1995. The P2 adults were treated for approximately 12 weeks and then bred (beginning July 10, 1995) to produce the F2 litters (weeks 46-48). The F2 litters were weaned on day 21 post-partum with 10 F2 pups/sex/dose selected for necropsy. On week 55 the P2 adults were necropsied.
Remarks:
Doses / Concentrations:
0, 50, 180, 540 or 750 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
30/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Two consecutive generations of Sprague-Dawley rats were administered BADGE once daily at dose levels of 0, 50, 180, 540 or 750 mg/kg/day in an aqueous solution of 0.5% Methocel A4M (Trademark, Dow Chemical) with 0.1% Tween 80 (dose volume of 10 mL/kg to deliver the appropriate dose). The study groups for both generations consisted of 30 male and 30 female rats per dose level. Gavage doses were administered to the P1 and P2 adult generations for 14 and 12 weeks, respectively, prior to the first mating. Both female adult generations were dosed during the mating, gestation, lactation, and weaning periods until necropsy. Doses for gestating and lactating females were adjusted based on body weight at day 0 and 6 of gestation. After the mating periods, the adult males from both generations received gavage doses until necropsied.

The dose levels selected for this study represented extremely large multiples of the maximum human exposure, but were selected based on data from the one-generation reproduction study conducted in rats (Smith et al., 1989). Administration of the two top dose levels (540 and 750 mg/kg/day) was expected to result in lower body weights in the PI parental animals when compared to the controls, and also to result in lower pup weights. Based on the expectation that 750 mg/kg/day would result in extreme parental toxicity, and that sufficient toxicity would be observed at 540 mg/kg/day to satisfy regulatory requirements, termination of the 750 mg/kg/day dose level was considered in the original study design. However, a dose of 750 mg/kg/day did not produce evidence of significant toxicity in the P1 adults through the planned 10-week treatment period prior to breeding. This information was conveyed to the EPA, and while this information was under consideration, the P1 adults continued to receive DGEBPA as appropriate for an additional 4 week period. As a result of discussions with the EPA, a decision to terminate the mid-low (180 mg/kg/day) dose group without conducting a breeding phase, and to retain both the 540 and 750 mg/kg/day dose groups for breeding was made.
Positive control:
no positive control group
Parental animals: Observations and examinations:
Observations
Each rat was observed daily for changes in appearance or behavior. Body weights and food consumption was measured weekly during pre-breeding on P1 females, and males weekly throughout the study. Sperm-positive female were weighed on GD 0, 7, 14, and 21, and on LD 1, 4, 7, 14, 21. Food consumption was not measured during breeding. Food consumption of the dam and litter was measured twice weekly during the first 2 weeks of lactation, and at 2-3 day intervals during the last lactation week.
Oestrous cyclicity (parental animals):
During each breeding period, daily vaginal lavage samples were evaluated for the presence of spenn as an indication of mating. The day on which sperm were detected, or a vaginal plug was observed in situ, was considered day 0 of gestation. Sperm positive females were placed in nesting cages. Females which failed to mate during the first 7-day breeding period were placed with an alternate male from the same treatment grocp for the second 7-day period; the same procedure was followed for the third 7-day mating period in each breeding.
Sperm parameters (parental animals):
Sperm were examined as part of the vaginal lavage sample. At necropsy, testes and epididymides were preserved in Bouin's fixative.
Litter observations:
Litters were examined as soon as possible after delivery. Littering date, size, number live/dead, sex and weight on day 1,4,7,14,21, and visible abnormalities or demeanor changes were noted during lactation.
Postmortem examinations (parental animals):
Upon completion of the dosing regimen, the P1 and P2 adults were fasted overnight and necropsied. The terminal body weights were obtained and the animals were anesthetized with methoxyflurane. The tracheas were exposed and clamped and the rats were euthanatized by decapitation. The eyes were examined in situ by gently pressing a glass slide against the cornea and observing the globe and its contents under fluorescent illumination. A complete necropsy was performed by a board certified veterinary pathologist. The weights of the liver and kidneys were recorded. The lungs were distended to their approximately normal inspiratory volume with neutral phosphate-buffered 10% formalin instilled via the trachea. The nasal cavity was flushed with neutral phosphate-buffered 10% formalin via the pharyngeal duct. Representative samples of the organs and tissues listed in Table 1 were saved and preserved in formalin, with the exception of the testes and epididymides, which were preserved in Bouin's fixative. Histopathologic examination was performed on the liver, kidneys, pituitary gland, mammary gland and all reproductive organs of all control and high dose P1 and P2 adults. Histopathologic examination of tissues from the lower dose groups was limited to the kidneys and grossly visible lesions. Tissues were prepared for light microscopic evaluation by standard procedures, sectioned at approximately 6 µm thickness, and stained with hematoxylin and eosin. Rats that died, or were euthanatized due to moribund condition prior to completion of the study were necropsied in a similar manner, except the body and organ weights were not obtained. A complete histopathologic examination of all organs and tissues listed in Table 1 (with the exception of auditory sebaceous gland and joint) was performed on the dead or moribund rats.

Grading of histopathologic findings was done to reflect the severity of specific lesions when considered necessary to evaluate the contribution of a specific lesion to the health status of the animal. Grades of very slight and slight were used for conditions that were present in excess of the normal textbook appearance of an organ/tissue, but were of low severity and involved less than 10% of the parenchyma. This type of change would not be expected to significantly affect the function of the specific organ/tissue involved nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue may have been adversely affected, but not to the point of organ failure. A severe grade was used for conditions that were extensive enough to result in organ failure, and may have been life-threatening.
Postmortem examinations (offspring):
At the time of weaning, 10 pups/sex/dose level from the F1 and F2 litters were randomly selected for a complete necropsy. The necropsies were performed by a board certified veterinary pathologist. The pups were anesthetized with methoxyflurane, their tracheas were exposed and clamped, and euthanasia was accomplished by decapitation. Gross pathologic examinations were performed as described above for adults, except that terminal body and organ weights were not obtained, and the testes and epididymides were preserved in formalin rather than Bouin's fixative. Histopathologic examination was not performed on the weanlings.
Statistics:
Descriptive statistics were reported for feed consumption. Body weights and relative and absolute organ weights were first evaluated by Bartlett's test for equality of variances. Based on the outcome, either a parametric or nonparametric ANOVA was performed. If the ANOVA was significant, a Dunnett's test or the Wilconxon Rank-Sum Test with Bonferroni's correction was performed.

Gestation length, average time to mating, and litter sizes were analyzed using a nonparametric ANOVA. If the ANOVA was significant, a Dunnett's test or the Wilconxon Rank-Sum Test with Bonferroni's correction was performed. Statistical outliers were identified by the method of Grubbs (1969) and were routinely excluded from feed analyses only. Outliers for body weight, gestation length, litter size, and time to mating were excluded from analyses only for sound scientific reasons. Fertility indices were analyzed by the Fischer exact probability test and Bonferroni's correction was used for multiple testing of groups in comparison to a single control. Evaluation of the neonatal sex ratios was performed using a binomial distribution test. Survival indices, and other incidence data among the neonates were analyzed using the litter as the experimental unit by the Wilcoxon test as modified by Haseman and Hoel (1974).
Reproductive indices:
Measures of male or female reproductive performance, time to mating, gestation length were conducted.
Offspring viability indices:
Pup survival (days 1, 4, 7, 14 and 21), litter size, pup weights, and litter observations were conducted.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Test Material
Average test material concentrations ranged from 93%-115% of target. Homogeneity was confirmed, within 5% of target concentration for the solution. Stability was confirmed.

Clinical Observations
No treatment-related observations were noted in P1 animals at any dose level.

Body Weights
P1 adult test day 77- 238 (terminal) male mean body weight at 540 and 750 mg/kg was decreased and considered treatment-related. Female P1 adult bodyweight was lower on test day 168 (prior to breeding for F1b litters) at 750 mg/kg/day. There were no bodyweight effects on F1a at breeding or during lactation. Body weights among female F1b breeding at the 750 mg/kg/day dose group was ~8% lower than controls and was considered treatment-related.

Food Consumption
There were no treatment-related changes in P1 males or females.

Reproductive Indices
There were no treatment-related effects on any of the measures of male or female reproductive performance, time to mating, gestation length, pup survival, litter size, pup weights, or litter observations in any dose group of either generation. There were, however, lower numbers of litters in low and mid dose groups than required by guidelines (though not considered treatment-related based on lack of dose-response), so P1 adults were bred again to produce F1b litters.

Gross Pathology
There were no remarkable treatment-related effects on gross pathology or organ weights. There were slight variations in organ weights in some males and females at the mid and high doses, but were considered secondary to body weight effects or normal variation in the strain.

P1 and P2 Adults - Histopathologic Observations
There were no treatment-related histopathologic observations in any of the rats from the P1 or P2 adults administered up to 750 mg/kg/day. All histopathologic observations from the control and treatment groups were interpreted to be spontaneous alterations, unrelated to oral administration of DGEBPA.

In the P1 adults, there was an increased incidence of mineralization of the pelvic epithelium of the kidneys in males and females administered 540 or 750 mg/kg/day. In addition, males administered 750 mg/kg/day from the P1 adults had an increased incidence of hyperplasia of the pelvic epithelium of the kidneys. In most instances, the hyperplasia was present in association with mineralization of the pelvic epithelium, probably representing a secondary response to the mineral deposits. The hyperplasia and mineralization of the pelvic epithelium were most commonly located near the base of the renal papilla, where the urinary space extends deepest into the medulla. Because of these kidney alterations in the P1 adults, the kidneys from all dose levels in the P1 and P2 adult generations were examined microscopically.

The increased incidence of mineralization and hyperplasia of the pelvic epithelium in P1 adults was interpreted to be not treatment-related, because the incidence of these findings was not increased in the subsequent P2 adult generation. If the mineralization and hyperplasia of the pelvic epithelium were indeed treatment-related, the P2 adults would be expected to have an equal or higher incidence of these alterations, because of their additional exposure to BADGE during the gestation and lactation periods. This, however, did not occur. Instead, the incidence of mineralization and hyperplasia of the pelvic epithelium of P2 adults was much lower than in P1 adults. The incidence of hyperplasia of the pelvic epithelium was actually lower in the treatment groups of the P2 adults as compared to controls, and the incidence of mineralization of the pelvic epithelium in all treatment groups from the P2 adults was comparable to controls.

The cause of the increased incidence of mineralization and hyperplasia of the pelvic epithelium of the kidneys in the P1 adults is unknown. The incidence of these alterations in control and treated animals was higher than recently performed two-generation reproduction studies in Sprague-Dawley rats from the Toxicology Research Laboratory of The Dow Chemical Company in Midland, Michigan. Historic control data show a range of 0 to 2 rats per 30 controls with mineralization or hyperplasia of the pelvic epithelium. Based on this higher incidence relative to historic controls, non-treatment related alterations in factors such as dietary content or hydration status may be considered as possible etiologies for the renal changes observed.

The reproductive organs of males (testes, epididymides, prostate, coagulating glands and seminal vesicles) and females (uterus, ovaries, oviducts, cervix, and vagina), plus the pituitary gland and mammary gland from both sexes, were evaluated microscopically in the high dose and control adult rats from both generations. Observations in these organs were limited to spontaneous alterations commonly encountered in rats of this age and strain. The most common observation in reproductive organs was normal postpartum hemosiderin pigment in the uterine wall, representing previous placental attachment sites. In both adult generations, there were no treatment-related toxicologic alterations of the reproductive organs at any dose level.

Spontaneous deaths occurred in 12 rats from the P1 adult generation, and 7 rats from the P2 adult generation. None of the spontaneous deaths were attributed to toxicity from oral administration of BADGE.

In conclusion, there were no treatment-related histopathologic effects in male and female rats from the P1 and P2 adult generations administered up to 750 mg BADGE/kg/day.

Supplemental Report- Based on the study results there were no additional systemic target tissues identified in rats in this supplementary histologic examination of tissues from the P1 generation of the 2-generation oral gavage reproduction study of DGEBPA, including the glandular and non-glandular stomach at all dose levels. The local effects observed in the nasal tissues of highdose rats are considered to be an artifact of administration.
Dose descriptor:
other: NOEL- adult males
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: 8-11% body weight decrease observed at 540 mg/kg/day.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
other: NOEL- adult females
Effect level:
540 mg/kg bw/day
Sex:
female
Basis for effect level:
other: Decreased body weight noted at 750 mg/kg/day.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
other: NOEL- reproductive effects
Effect level:
750 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Highest dose examined.
Remarks on result:
other: Generation: F1a, F1b and F2 offspring (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
F1a, F1b and F2- There were no treatment-related gross changes noted in these litters.

F1a , F1b and F2 Litters- There were no treatment-related effects on any of the measures of male or female reproductive performance, time to mating, gestation length, pup survival, litter size, pup weights or litter observations in any dose group
Dose descriptor:
NOEL
Generation:
other: F1a, F1b and F2 offspring
Effect level:
750 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no effects at highest dose examined
Reproductive effects observed:
not specified

Administration of DGEBPA to adult rats by oral gavage at dose levels which represent extremely large multiples of potential human exposures resulted in only slight toxicity. Among adult males, body weights were decreased approxinately 11% at dose levels of 540 and 750 mg/kg/day in both the P1 and P2 generations. In females, body weights were also affected in both generations, but only at the highest dose level (750 mg/kg/day). Secondary changes in absolute and/or relative liver and kidney weights were also observed in these dose groups. There were no treatment-related effects on body weights among males given 50 mg/kg/day, or among females given 540 or 50 mg/ kg/day in either generation.

There were no treatment-related histologic changes noted in any dose group. In the P1 adults, there was an increased incidence of mineralization of the pelvic epithelium of the kidneys in males and females administered 540 or 750 mg/kg/day. However, the increased incidence of mineralization and hyperplasia of the pelvic epithelium in P1 adults was interpreted to be not treatment-related, because the incidence of these findings was not increased in the subsequent P2 adult generation. If the mineralization and hyperplasia of the pelvic epithelium were indeed treatment-related, the P2 adults would be expected to have an equal or higher incidence of these alterations, because of their additional exposure to DGEBPA during the gestation and lactation periods. This, however, did not occur. Instead, the incidence of mineralization and hyperplasia of the pelvic epithelium of P2 adults was much lower than in P1 adults. The incidence of hyperplasl: of the pelvic epithelium w& actually lower hi the treatment groups of the P2 adults as compared to controls, and the incidence of mineralization of the pelvic epithelium in all treatment groups from the P2 adults was comparable to controls.

The cause of the increased incidence of mineralization ard hyperplasia of the pelvic epithelium of the kidneys in the P1 adults is unknown. The incidence of these alterations in control and treated animals was higher than recently performed two-generation reproduction studies in Sprague-Dawley rats from the Toxicology Research Laboratory of The Dow Chemical Company in Midland, Michigan. Historic control data show a range of 0 to 2 rats per 30 controls with mineraiization or hyperplasia of the pelvic epithelium. Based on this higher incidence relative to historic controls, non-treatment related alterations in factors such as dietary content or hydration status may be considered as possible etiologies for the renal changes observed.

Despite the extremely high dose levels used in this study, there were no treatment-related effects on reproductive performance in any dose group. Mating and conception indices for both males and females were lower than normal in the P1 generation, but the lowest conception values were found in the control group, where only 50-60% of the females produced litters. The largest numbers of litters were found in the highest dose group, where fertility indices in females ranged from 73% to 93%. Inspection of these parameters across the study revealed that overall fertility tended to increase as the dose level increased. Thus, any differences seen in fertility were considered a function of the population used for this study, and not related to exposure to DGEBPA.

Gross necropsy examination revealed a partial explanation for the lower than normal fertility seen in these animals. A gross lesion in females, characterized as an inpatent cervix, was found in a total of 9 of 120 females (7.5%) from the P1 generation. This lesion consisted of a membranous transverse septum at the proximal end of the vagina acting as a physical barrier to impregnation. Other studies conducted in our laboratory have reported similar incidences of this lesion. A similar lesion has also been reported in Wistar rats (DeSchaepdrijver, et al., 1995). Though this lesion did not account for all unsuccessful matings, its presence did affect the mating success for a number of animals.

Evaluation of the various parameters of neonatal growth and survival over the three sets of litters produced by the two generations of adults revealed no indication of any significant adverse effect of DGEBPA. The only suggestion of any possible effect of DGEBPA on neonatal growth was a lower mean pup weight on lactation day 14 among the F2 high dose litters (750 mg/kg/day). This difference was only transient, however, and there was no significant difference between controls and any dose group by weaning on lactation day 21.

The results obtained in this study were consistent with those seen in the one-generation reproduction study conducted previously (Smith et al., 1989). In that study, male and female rats were given dose levels of 20 - 540 mg/kg/day. a Smith reported decreased body weights in males at 540 mg/kg/day of approximately 8%, similar to the effects noted in this study at the same dose level. A lower pup weight on lactation day 12 and 21 at the top dose level was considered a function of the slightly larger litter size in the top dose group, and there were no indications of any effects on reproductive performance.

DeSchapdrijver, L.M., Fransen, J.L., Van der Eycken,E.S., and Coussement, W.C. (1995). Transverse Vaginal Septum in the Specific-Pathogen-Free

Wistar Rat. Lab. Anim. Sci. 181-183.

Smith, J.A., Bryson, A., Offer, J.M., Parker, C.A. and Anderson, A. (1989). A Study of the Effect of TK 10490 on the Reproductive Function of One

Generation in the Rat. Report to Ciba-Geigy Limited, Huntingdon Research Centre, LTD.

Conclusions:
BADGE displayed no indications of any adverse effects on reproduction in rats over two generations at dose levels which greatly exceed potential human exposure levels. The NOEL for adult males was considered to be 50 mg/kg/day, NOEL for adult females 540 mg/kg/day, and the NOEL for reproductive effects was 750 mg/kg/day.

Based on the study results there were no additional systemic target tissues identified in rats in the supplementary histologic examination of tissues from the P1 generation of the 2-generation oral gavage reproduction study of DGEBPA. The local effects observed in the nasal tissues of highdose
rats are considered to be an artifact of administration.
Executive summary:

A reproduction study was conducted with diglycidyl ether of bisphenol A (DGEBPA) in which groups of rats were administered the test material daily over two generations. Groups of 30 male and 30 female Sprague-Dawley rats were administered dose levels of 0, 50, 180, 540 and 750 mg/kg/day by oral gavage for approximately 14 weeks prior to breeding. Immediately prior to breeding, a decision to terminate the 180 mg/kg/ day dose group was made. This decision was based on the fact that only three treatment groups are required by EPA guidelines, and the animals clearly tolerated the 750 mg/kg/day dose level without significant toxicity. The remaining P1 adults were bred twice to produce Fla and Flb sets of litters. Groups of 30 males and 30 females per dose group were then randomly selected from the Flb litters to become the P2 generation and were administered DGEBPA by gavage at the dose level administered to their parents for approximately 12 weeks prior to breeding to produce the F2 litters. Parameters evaluated over the course of the study included body weights, feed consumption, clinical observations, mating performance, and gross pathology and histopathology of the adults, as well as neonatal growth and survival of the offspring.

Administration of DGEBPA to adult rats by oral gavage at dose !evels which represent extremely large multiples of potential human exposures resulted in only slight toxicity. Among adult males, body weights were decreased approximately 8 -1l% at dose levels of 540 and 750 mg/ kg/day in both the P1 and P2 generations, although there was not a statistically significant decrease in the body weights of the P1 males treated with 750 mg/kg/day until test day 238 {i.e., termination). In females, body weights were also affected in both generations. but only at the highest dose level (750 mg/ kg/day). Secondary changes in absolute and/or relative liver and kidney weights were also observed in these dose groups. There were no treatment-related effects on body weights among males given 50 mg/kg/day, or among females given 540 or 50 mg/kg/day in either generation. There were no treatment-related histologic changes noted in any dose group.

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18 January 1988 - 10 June 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
Remarks:
Not specified in report
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CrL:CD(SD) BR VAF/Plus
Sex:
male/female
Details on test animals and environmental conditions:
Four-week old males and females were obtained from a commercial supplier. They were weighed and examined upon arrival to the testing facility, and allowed to acclimate to the laboratory for 3 days. They were then randomized by a computer program by body weight, earmarked for identification, and treatment commenced when the males and females were approximately 6 weeks old.

Animal room conditions (temperature, relative humidity, air changes, and photocycle) were maintained appropriately for the species. During pre-mating, they were housed 4 per cage by sex in metal mesh cages. During mating, they were housed one male and one female per plastic breeding cage. Females were provided nesting material after the breeding period, and males were returned to steel caging. Animals were given water and food ad libitum.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethylcellulose and 0.1% (v/v) Tween 80
Details on exposure:
Suspensions of test material were mixed daily and dosed within 2 hours of preparation.
Details on mating procedure:
During the mating period the rats were housed on the basis of one male to one female in plastic breeding cages. Due to there being two male mortalities at 180 mg/kg/day, this necessitated a one male to two female pairing in two instances.

At the end of the mating period, the males were returned to clean metal cages and the females provided with suitable nesting material prior to the birth and rearing of young.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses for concentration were performed throughout the study.
Duration of treatment / exposure:
Males were dosed 10 weeks prior to mating, through mating, gestation, and lactation up until sacrifice of most females on day 21 post-partum. Females were treated 2 weeks prior to mating and continued until sacrifice on day 21 post-partum.
Frequency of treatment:
daily
Details on study schedule:
Male animals were approximately 6 weeks of age at the commencement of treatment. They were treated each day for at least 70 days prior to mating, i.e., until they were approximately 16 weeks of age. Sexually mature females were treated for 2 weeks prior to mating.
Remarks:
Doses / Concentrations:
0, 20, 30, 180, 540 mg/kg/day
Basis:
nominal conc.
Analytical analysis of the prepared dosing solutions revealed ranges of 13.1-17.4 mg/kg/day, 50.6-55.0 mg/kg/day, 154.8-166.3 mg/kg/day and 464.4-499.0 mg/kg/day for nominal doses of 20, 60, 180 and 540 mg/kg/day, respectively. Thus values were 8-35% bel
No. of animals per sex per dose:
24 males and 24 females/dose
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
In-life observations, mortality, food consumption, and water consumption were recorded.
Litter observations:
Pre-weaning, pups were examined for surface righting reflex, startle reflex, air righting reflex, and pupil reflex. On post-partum day 22, they were sacrificed and examined internally and externally for abnormalities.
Postmortem examinations (parental animals):
Male and female parents were sacrificed, and females examined for number of implantation sites. Organs were weighed and examined. Reproductive tract and associated tissues were examined histologically. The alimentary tract from control and high dose males and females were examined histologically also.
Postmortem examinations (offspring):
Pups were sacrificed and examined macroscopically on day 22 post-partum.
Statistics:
ANOVA (Williams Test) were used for intergroup differences in mean water, food consumption and bodyweights. Means were determined for pre-birth loss, litter size, pup loss, litter and mean pup weight, sex ration, and pre-weaning development. Kruskal-Wallis and Jonckheere tests were generally applied. Organ weights were analyzed by ANOVA adjusting for bodyweight. Treatment means were compared to controls by Williams test
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
540 mg/kg/day
Post-dosing salivation affecting all animals throughout. Slightly reduced food consumption for females during the first week of treatment.
Reduced mean bodyweight for males through to termination.

180 mg/kg/day
Post-dosing salivation affecting all males from the third week, and females from the sixth week.
Slightly reduced mean food consumption for females during the first week of treatment.

60 mg/kg/day
Post-dosing salivation in 7/24 males following six weeks of treatment.

20 mg/kg/day
No adverse effects of treatment were observed.

Treatment of the F0 generation did not significantly affect mating performance, the duration of gestation, mean pre-birth or total litter loss.
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
60 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: Post-dosing salivation in 7/24 males following six weeks of treatment.
Dose descriptor:
LOAEL
Effect level:
180 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: (a) Post-dosing salivation affecting all males from the third week, and all females from the sixth week of treatment. (b) Slightly reduced mean food consumption for females during the first week of treatment.
Clinical signs:
effects observed, treatment-related
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
540 mg/kg/day
Reduced mean pup weight on Days 12 and 21 post-partum, influenced by higher litter size.
Pre-weaning development as assessed by air righting reflex was slightly delayed, probably related to reduced growth rate.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
180 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No effects observed in offspring.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
540 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: (a) Reduced mean pup weight on Days 12 and 21 post partum, this would be influenced by the higher litter size. (b) Pre-weaning development as assessed by air righting reflex was slightly delayed, this is probably related to the reduced growth rate.
Reproductive effects observed:
not specified

The only treatment-related clinical sign observed was post-dosing salivation which was occasionally brown-stained. This occurred in all animals treated at 540 mg/kg/day from the first week of dosing. At 180 mg/kg/day, all animals were also affected, males by the third week of dosing, but the onset in females was slower, all not being affected until the sixth week of dosing. At 60 mg/kg/day only 7/24 males were affected during one to three weeks, generally following at least six weeks of treatment.

There were two mortalities among male animals treated at 180 mg/kg/day during Weeks 9 and 10, respectively. At autopsy animals were found to have congested lungs. Post-dosing salivation was the only dosage-related sign observed prior to death.

Water and Food Consumption:

There were no obvious treatment-related effects on water consumption of males and females during the limited period of assessment; differences from controls were not statistically significant (P>0.05 Anovar).

Weekly food consumption among males was essentially similar in both control and treated groups throughout the pre-mating period. Among the females, treatment at 540 and 180 mg/kg/day was associated with a marginal reduction in food consumption during the first week of treatment, differences from controls attaining statistical significance (P<0.01 Anovar).

Mean food consumption of dams rearing young recorded between Day 1 and 13 post partum was slightly reduced among treated animals compared with thé controls. Differences from controls did not attain statistical significance (P>0.05 Anovar).

Body Weight:

Treatment of males at 540 mg/kg/day was associated with slightly lower bodyweight following one week of treatment, this divergence continued for the first six weeks of treatment and attained statistical significance from Week 4 to 18 inclusive. Thereafter, mean weight gain tended to parallel that of the control group although absolute mean bodyweights remained about 6% below that of controls. At lower dosages mean bodyweights were generally similar, or, following pairing, slightly superior when compared with the control group.

Among females there was no indication of an adverse dosage-related effect on mean weekly bodyweights. Bodyweight change of females during gestation and lactation were essentially similar in all groups.

Mating Performance, Pregnancy Rate and Gestation Interval:

Mating performance, as assessed by the type of vaginal smear observed on the day of mating and by the median pre-coital time, was unaffected by. treatment with TK 10490.

Pregnancy rate was high, varying from 91.7% at 60 and 180 mg/kg/day to 100% at 20 and 540 mg/kg/day.

The mean duration of gestation was essentially similar in both treated and control groups.

Terminal autopsy, Organ Weight Analysis and Microscopic Pathology

At terminal autopsy, neither the incidence nor type of occasional macroscopic abnormality detected suggested an association with treatment with TK 10490.

A decrease in liver weight, when adjusted for bodyweight as covariate, was observed at 540, 180 and 60 mg/kg/day among male animals. This was not dosage-related but differences from control values attained statistical significance (P<0.05). At 540 mg/kg/day the statistically significant (P<0.05) lower brain weight compared with the controls was considered coincidental and not of biological significance but reflected the lack of

variation in the data. Other differences from control values for mean organ weights in both males and females were marginal and did not attain statistical significance (P>0.05).

Histopathological examination of reproductive tract and associated tissues was performed on all FO adult animals. Additionally the alimentary tract from controls, and animals treated at 540 mg/kg/day were also examined. No treatment-related findings were detected in any of the tissues examined. All changes observed were considered to be spontaneous in origin and they were not considered to be of toxicological importance. At terminal autopsy, neither the incidence nor type of occasional macroscopic abnormality detected suggested an association with treatment with TK 10490.

Total Litter Loss, Pre-Birth Loss, Litter Size and Pup Mortality, and Litter and Mean Pup Weight:

Only two total litter losses occurred, one each at 180 and 60 mg/kg/day. Both occurred in the early post natal period prior to Day 4. Additionally one female at 180 mg/kg/day showed a single implantation site by the Salewski test but was not observed giving birth to a live pup. This incidence did not suggest an association with treatment and the following assessment is based on dams rearing young to weaning.

The mean number of implantation sites was essentially similar in both treated and control groups. Differences in the mean incidence of pre-birth loss was not strictly dosage-related and did not attain statistical significance (P>0.05).

Mean litter size at birth was essentially similar in both the control and treated groups and pup mortality was low. Prior to Day 4 cull the % cumulative loss was slightly increased at 540 and 180 mg/kg/day compared with the controls. Culling of individual litters reduced the mean litter size in all groups to just less than 8 and subsequent pup mortality was low.

Litter weight was essentially similar at birth through to weaning in both control and treated groups. At 540 mg/kg/day mean pup weight was slightly lower than the controls at Days 12 and 21, the latter attaining statistical significance P<0.05; this difference in growth rate compared with the controls will be influenced by the higher mean litter size.

Sex Ratio, Pre-Weaning Development, Terminal Autopsy

Sex ratio, as assessed by the percentage of males per litter at birth was unaffected by treatment.

Following the cull on Day 4 post partum there was no evidence of any selective adverse effect on males or females through to weaning.

Pre-weaning development as assessed by the mean day post coitum offspring showed surface righting reflex, startle response and

pupil reflex were essentially similar in both control and treated groups. At 540 mg/kg/day mean age post coitum for air righting reflex was later than for controls, the intergroup difference attaining statistical significance (P<0.05). The response was probably related to the reduced growth rate.

Three pups, two at 20 mg/kg/day and one at 540 mg/kg/day, showed gross structural changes. At 20 mg/kg/day these included one

with thread-like tail and another with unilateral anophthalmia. At 540 mg/kg/day one pup showed multiple changes including harelip and associated cleft palate, cranioschisis and anophthalmia. The isolated nature of these findings did not suggest any association with treatment.

Conclusions:
The NOAEL (parental) was 20 mg/kg bw/day.
Executive summary:

In this assessment of the effect of TK 10490 on reproductive function of one generation in the rat, daily dosages of 0. (Control), 20, 60, 180 and 540 mg/kg/day were administered by oral gavage. Males were treated for ten weeks and females for two weeks, prior to and through mating to termination.

All females were allowed to give birth and rear their offspring to weaning. F0 parents were sacrificed and subjected to organ weight

analysis. Subsequently the reproductive tract of all animals, and alimentary tract of control and high dose animals, were examined

histologically.

Treatment with TK 10490 at 540 mg/kg/day was associated with:

(a) Post-dosing salivation affecting all animals throughout.

(b) Slightly reduced food consumption for females during the first week of treatment.

(c) Reduced mean bodyweight for males through to termination.

(d) Reduced mean pup weight on Days 12 and 21 post partum, this would be influenced by the higher litter size.

(e) Pre-weaning development as assessed by air righting reflex was slightly delayed, this is probably related to the reduced growth rate.

Treatment with TK 10490 at 180 mg/kg/day was associated with:

(a) Post-dosing salivation affecting all males from the third week, and all females from the sixth week of treatment.

(b) Slightly reduced mean food consumption for females during the first week of treatment.

Treatment with TK 10490 at 60 mg/kg/day was associated with:

(a) Post-dosing salivation in 7/24 males following six weeks of treatment.

No adverse effects of treatment were observed following treatment with TK 10490 at 20 mg/kg/day.

Treatment of the F0 generation did not significantly affect mating performance, the duration of gestation, mean pre-birth or total litter

loss.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Administration of DGEBPA to adult rats by oral gavage at dose levels which represent extremely large multiples of potential human exposures resulted in only slight toxicity. Among adult males, body weights were decreased approximately 8 -11% at dose levels of 540 and 750 mg/ kg/day in both the P1 and P2 generations, although there was not a statistically significant decrease in the body weights of the P1 males treated with 750 mg/kg/day until test day 238 {i.e., termination). In females, body weights were also affected in both generations. but only at the highest dose level (750 mg/ kg/day). Secondary changes in absolute and/or relative liver and kidney weights were also observed in these dose groups. There were no treatment-related effects on body weights among males given 50 mg/kg/day, or among females given 540 or 50 mg/kg/day in either generation. There were no treatment-related histologic changes noted in any dose group.



Effects on developmental toxicity

Description of key information
There was no evidence of developmental toxicity at doses levels resulting in maternal toxicity in rats and rabbits following oral administration or rabbits following dermal administration of BADGE.
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP as well as the EPA: TSCA test Guidelines( EPA, 1985).
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: The EPA: TSCA test guidelines ( EPA, 1985)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Female rabbits were received from a commercial supplier, evaluated for general health by a laboratory veterinarian, and were housed singly (suspended stainless steel cages with feed crock and pressure-activated nipple watering system) in rooms designed to maintain adequate environmental conditions and photocycle for rabbits.

Animals were provided food (4oz per day prior to study start, 8oz per day during gestation) and water ad libitum throughout the study. Food was analyzed for nutritional content and contaminants by the supplier. The water was obtained from a municipal water source, analyzed periodically for chemical parameters and biological contaminants by the municipal water department.

Animals were randomized to dose group based on body weights using a computer randomization program. They were identified via a unique animal identification number on a metal ear tag.
Route of administration:
other: dermal
Vehicle:
other: PEG 400 (polyethylene glycol)
Details on exposure:
Groups of 26 inseminated rabbits were treated dermally to varying dose levels of BADGE. A section on the back was clipped free of hair, and test material spread uniformly over the area. The test site was covered and the held in place a minimum of 6 hours. Following the occlusion period, the bandage was removed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose solutions were analyzed by liquid chromatography.

The stability of the test material in the vehicle PEG 400 (polyethylene glycol) was determined to be a minimum of 41 days.
Details on mating procedure:
Females were injected IV with 50 USP units of chorionic gonadotropin to stimulate ovulation following artificial insemination. The day of insemination was considered GD 0.
Duration of treatment / exposure:
minimum of 6 hours/day, GD6-18. Test material was uniformly spread over the clipped area (approximately 10 x 15 cm) using a syringe and blunt-tipped needle. An occlusive bandage of absorbent gauze and non-absorbent cotton was then placed over the dosing area on the back. The bandage was held in place for a minimum of 6 hours using a lycra/spandex jacket. Following the occlusion period, the bandage and jacket were removed.
Frequency of treatment:
daily
Duration of test:
28 days
No. of animals per sex per dose:
26 inseminated rabbits/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 26 inseminated rabbits were treated dermally to varying dose levels of BADGE. A section on the back was clipped free of hair, and test material spread uniformly over the area. The test site was covered and then held in place a minimum of 6 hours. Following the occlusion period, the bandage was removed.
Maternal examinations:
All animals were observed daily for indications of toxicity. The test site was assessed daily for irritation using the OECD (1983) rating scale. In addition, it was ranked for exfoliation and fissuring according to a set scale. Body weights were recorded on GD 0, 6, 9, 12, 15, 19, 28. Maternal liver weights and gravid uterine weights were recorded at time of sacrifice. Liver and skin (test site and virgin site) were preserved.
Ovaries and uterine content:
Fetal examinations included the following: position and number in utero, number of live and dead, number and position of resorption sites, number of corpora lutea, sex, alterations, and body weight. Uteri of non-pregnant animals were stained and examined.
Fetal examinations:
Fetal examinations also included the following: sex, alterations, and body weight. Tissues from half of all fetuses were examined microscopically in addition
Statistics:
Descriptive statistics (mean and SD) were evaluated for skin lesions. Maternal and fetal body weights, gravid uterine weight, absolute and relative organ weights were evaluated by a Bartlett's test for equality of variances. Based on the outcome, a parametric or nonparametric ANOVA was performed. If the ANOVA was significant, analysis by Dunetts test or Wilcoxon Rank-Sum test with Bonferroni's correction was performed.

Statistical frequencies of pre-implantation loss, resorptions, fetal alterations, number of corpora lutea and implantations, and litter size were made by a censored Wilcoxon test with Bonferroni correction. Pregnancy rate was analyzed by the Fisher exact probability test. Statistical outliers were identified by a sequential outlier test, but were not excluded from analyses.
Indices:
1) number and position of fetuses in utero, 2) number of live and dead fetuses, 3) number and position of resorption sites, 4) the number of corpora lutea, and 5) the sex and body weight of each fetus.

Pre- and post-implantation loss
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The dose solutions were analyzed by liquid chromatography and were found to be 105-114% of the targeted concentrations and were homogeneous throughout the study.

Maternal Observations
Dose-related increases in erythema, exfoliation, fissuring, hemmorhage, and edema at the test site were noted at 100 and 300 mg/kg/day. There were no remarkable observations at 30 mg/kg/day for skin changes, body weights, or liver weights. The pregnancy rate of rabbits at 30 mg/kg/day was decreased, and the distribution of male/female fetuses was different than a binomial distribution. A lack of dose-response makes the finding spurious.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal Observations
There was no statistically significant increase in malformations or variations in any dose group as compared to the control.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Dermal application of DGEBPA to pregnant rabbits produced maternal toxicity as exhibited by a dose-related increase in erythema, fissuring, hemorrhage and edema at the site of application. Pregnant rabbits in the 300 mg/kg/day treatment group exhibited moderate to severe erythema, fissures, hemorrhage and slight edema resulting in a maximum mean skin irritation/damage score of 2.7 for erythema/eschar, 2.6 or greater for erythema and/or edema is considered to represent significant maternal toxicity in the rabbit by the Test Rules Development Branch, U.S.E.P.A. (Locke, 1985). The degree of erythema, fissuring and hemorrhage observed in high dose rabbits clearly meets and exceeds the standard for adequate maternal toxicity communicated by the Test Rules Development Branch, U.S.E.P.A. and represents a type of damage to the integument which dramatically altered the integrity of the skin. In general, the skin lesions observed in rabbits in the 100 mg/kg/day dose group were less severe than the lesions observed in rabbits in the high dose group and did not develop to the extent which would result in a mean skin irritation/damage score of 2.0 or greater. However, the individual skin response in rabbits in the 100 mg/kg/day dose group showed considerable variation with some rabbits in this group developing lesions of similar severity to that observed in rabbits from the 300 mg/kg/day dose group. The average dermal response of rabbits in the 30 mg/kg/day dose group was characterized as barely perceptible erythema and was indistinguishable from erythema caused by the occlusive bandages/jacket used to hold the test material in place. Since most pregnant control rabbits showed similar low levels of skin erythema, the skin response observed in rabbits from the 30 mg/kg/day dose group was not considered toxicologically significant. No other treatment-related clinical signs or systemic toxicity were observed in this study.

Although dermal exposure of pregnant rabbits to 300 mg DGEBPA/kg body weight/day produced maternal toxicity, no indication of embryo/fetal toxicity or teratogenicity was observed at any dose level tested. The statistically significant decrease in pregnancy rate and deviation from a binomial distribution of the fetal sex ratio observed in the 30 mg/kg/day dose group were considered random events in view of the lack of a dose-response for these parameters. A small number of fetuses scattered throughout the dose levels exhibited malformations. Although the majority of these malformations occurred in the DGE BPA exposure groups, the frequency of specific malformations were low and did not show a dose-response. In addition, most of the malformations observed also occurred at low frequencies in historical control New Zealand White rabbit data generated in this laboratory and thus were not considered due to exposure to DGEBPA.

Conclusions:
The no observed-adverse-effect level for maternal toxicity is 30 mg/kg bw. The embryo/ fetal no observed-effect level was >300 mg/kg body weight.
Executive summary:

Diglycidyl ether of bisphenol A (DGEBPA) was tested for its embryo/fetal toxicity and teratogenicity in pregnant rabbits. DGEBPA was applied daily to the backs (clipped free of hair) of New Zealand White rabbits at dose levels of 0 (polyethylene glycol, vehicle control), 30, 100 or 300 mg/kg body weight/day at a dose volume of 1 ml/kg body weight/day on days 6 through 18 of gestation. Twenty six inseminated rabbits were used per dose group resulting in a minimum of 20 pregnant rabbits per exposure level. An occlusive bandage of absorbent gauze and non-absorbent cotton was placed over the dosing area on the back of each rabbit. The bandage was held in place for a minimum of 6 hours/day using a lycra/spandex jacket. Following the occlusion period the bandage and jacket were removed.

Maternal toxicity was observed among pregnant rabbits in the 300 mg/kg dose group as evidenced by moderate to severe erythema, fissures, hemorrhage and slight edema at the exposure site. Similar, but less severe skin lesions were observed in pregnant rabbits in the 100 mg/kg/day exposure group. Skin effects (slight erythema) observed in pregnant rabbits in the 30 mg/kg/day dose group were not considered toxicicologically significant. No evidence of embryo/fetal toxicity or teratogenicity was observed at any dose level resulting in a embryo/fetal no-observed-effect level of 300 mg/kg body weight/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987-06-08-1987-07-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
Not specified in report
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Seventy-two time-mated females were obtained from a commercial supplier. They were weighed and examined upon arrival to the testing facility. They were uniquely identified, and treatment commenced when the animals were approximately 12-24 weeks old.

Animal room conditions (temperature, relative humidity, air changes, and photocycle) were maintained appropriately for the species. They were housed singly in metal mesh cages. Animals were given water and food ad libitum.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethylcellulose / 0.1% v/v Tween 80 solution in water
Details on exposure:
Each concentration of suspension was obtained by mixing an appropriate weight of TK 10490 into 0.5% carboxymethylcellulose and 0.1% v/v Tween 80 in water. Uniformity of suspensions was achieved using a Silverson homogeniser. Suspensions were formulated daily and dosed within 2 hours of preparation.

Groups of 18 female rabbits were dosed with 0, 20, 60 or 180 mg/kg/day on gestation days 7-19
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suspensions were formulated daily and used within 2 hours. Concentration was verified analytically on three occasions.
Details on mating procedure:
time-mated animals
Duration of treatment / exposure:
GD7-19
Frequency of treatment:
daily
Duration of test:
GD29
No. of animals per sex per dose:
18 female rabbits/dose
Control animals:
yes, concurrent vehicle
Details on study design:
see below
Maternal examinations:
In-life observations, mortality, food consumption, and body weights were measured.

On GD 29, animls were sacrificed and examined for changes and abnormalities in maternal organs.
Ovaries and uterine content:
Ovaries and uteri were examined for number of corpora lutea, distribution of live young, distribution of embryo fetal death, individual fetal weight, fetal abnormalities.
Fetal examinations:
Live young were examined externally, weighed, sexed and examined for visceral abnormalities. After fixation in 74 OP industrial methylated spirit, the brain was examined for visible abnormalities prior to skeletal examination.
Statistics:
Pre- and post-implantation losses were calculated, means were calculated from individual litter values. Mean values for litter size pre-and post-implantation loss, litter weights, young weight, incidence of offspring with anomalies were analyzed by Kruskal-Wallis test. Intergroup comparisons were made with nonparametric equivalient t-tests with the Jonckheere test or by the nonparametric equivalent of the Williams test. ANOVA was used, followed by Williams test, to analyze food consumption and body weights.
Indices:
Pre- and post-implantation loss was calculated
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
PARENT ANIMALS
Treatment at 180 mg/kg was associated with an increased incidence of animals with general signs of anorexia, and an increased incidence of animals with cold ears during 4 or more days of the dosing period. There was one non-treatment related death in the 180 mg/kg group, and one death associated with a dosing error. Food consumption at the high dose was also significantly lower than the controls, as well as a concomitant decrease in bodyweights. There was no statistically-significant difference in animals dosed at 20 or 60 mg/kg when compared to the control. Pregnancy rates were high in all dose groups (88.2-100%). There were no macroscopic findings in maternal organs noted at necropsy and attributed to treatment

LITTER DATA
There were only two instances of total litter loss; a control animal resorbed the entire litter, and a dam at 20 mg/kg aborted her litter.

Mean corpora lutea rates were similar among all groups. There was an increase in pre-implantation loss at the high dose level; post-implantation loss was elevated in treated animals, and litter size was decreased, though no dose-response was evident.
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean fetal weights, sex ratios, malformations, anomalies, and skeletal variants were unaffected by treatment.
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (nominal)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Results indicate that the dosages (mg/kg/day) were generally 4 -23% below that intended. These data were consistent with previous experience of analysing the material in a biologically acceptable medium within the constraints of an acceptable formulation for dosing. Clear separation and a three-fold interval between dosages was still achieved.

Treatment with TK 10490 at 180 mg/kg/day was associated with: a) anorexia and cold ears in approximately half of the animals during the dosing period, and b) reduced food consumption resulting in initial weight loss during the dosing period.

Treatment at 60 and 20 mg/kg/day did not produce any clear or consistent adverse effects on the parent female.

Mean litter parameters at 180, 60 and 20 mg/kg/day were essentially similar to the controls, none of the differences from controls attained statistical significance (P>0.05).

Treatment with TK 10490 showed no obvious adverse effect on embryonic and foetal development as assessed by overall incidences and types of malformations, visceral and skeletal anomalies.

Conclusions:
The no observed-adverse-effect level for maternal toxicity is 60 mg/kg/day. The no observed-adverse-effect level for teratogenicity is >180 mg/kg/day.
Executive summary:

In this assessment of the effect of TK 10490 on the pregnancy and embryofoetal development of the rabbit, daily dosages of 0 (Control), 20, 60 and 180 mg/kg/day were administered by oral gavage to the parent females from Day 7 throught to Day 19 of pregnancy inclusive. On Day 29 of pregnancy the females were killed and subjected to post mortem examination, litter parameters determined and foetuses were examined for visceral, and subsequently, for skeletal anomalies.

Treatment with TK 10490 at 180 mg/kg/day was associated with: a) anorexia and cold ears in approximately half of the animals during the dosing period, and b) reduced food consumption resulting in initial weight loss during the dosing period.

Treatment at 60 and 20 mg/kg/day did not produce any clear or consistent adverse effects on the parent female.

Within the context of this study, oral administration of TK 10490 during days 7 to 19 of gestation produced evidence of maternal toxicity at 180 mg/kg/day in terms of anorexia and an initial weight loss. Treatment at 60 and 20 mg/kg/day was not associated with any maternal toxicity. There was no evidence that treatment of the dam with TK 10490 adversely affected mean litter parameters. Embryofoetal development and morphology was unaffected at all the dosages investigated.

Mean litter parameters at 180, 60 and 20 mg/kg/day were essentially similar to the controls, none of the differences from controls attained statistical significance (P>0.05).

Treatment with TK 10490 showed no obvious adverse effect on embryonic and foetal development as assessed by overall incidences and types of malformations, visceral and skeletal anomalies.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987-07-13-1987-07-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
Not specified in report
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CrL:COBS CD (SD) BR
Details on test animals and environmental conditions:
Eight to ten week-old time-mated females were obtained from a commercial supplier.

Animal room conditions (temperature, relative humidity, air changes, and photocycle) were maintained appropriately for the species. They were housed 5 per cage by sex in metal mesh cages. Animals were given water and food ad libitum.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethylcellulose / 0.1% v/v Tween 80 solution
Details on exposure:
dose volume= 0.6 ml/100g bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suspension of TK 10490 was prepared in a 0.5% carboxymethylcellulose / 0.1% v/v Tween 80 solution. The suspensions were formulated daily and used within 2 hours. Concentration was verified analytically.
Details on mating procedure:
purchased time-mated females
Duration of treatment / exposure:
GD6-15
Frequency of treatment:
daily
Duration of test:
GD20
No. of animals per sex per dose:
25 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Daily dosages of 0 (control), 60, 180 and 540 mg/kg/day were administered by oral gavage to the parent females from Day 6 through Day 15 of gestation. On Day 20 of pregnanacy the females were killed and subjected to post mortem examination, litter parameters determined and foetuses fixed prior to examination for skeletal or visceral anomalies.
Maternal examinations:
In-life observations, water and food consumption, and body weights were measured.

On GD 20, animals were sacrificed and examined for changes and abnormalities in maternal organs.
Ovaries and uterine content:
Ovaries and uteri were examined for number of corpora lutea, distribution of live young, distribution of embryo fetal death.
Fetal examinations:
Fetuses were weighed, sexed, uniquely identified by litter and examined for fetal abnormalities.
Statistics:
Pre- and post-implantation losses were calculated, means were calculated from individual litter values. Mean values for litter size pre-and post-implantation loss, litter weights, pup weight, incidence of offspring with anomalies were analyzed by Kruskal-Wallis test. Intergroup comparisons were made with nonparametric equivalient t-tests with the Jonckheere test or by the nonparametric equivalent of the Williams test. ANOVA was used, followed by Williams test, to analyze food and water consumption and body weights.
Indices:
pre- and post-implantation loss
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
PARENT ANIMALS
At 540 mg/kg, all animals showed salivation post-dosing, and in some cases brown staining lasting 3-7 days. Incidences of salivation and staining decreased with decreasing dose. There was no effect of treatment on food or water consumption. Decreased mean body weight gains were statistically-significant at the high dose, and marginally affected at 180 and 60 mg/kg. Pregnancy rate was 88-96%. Preimplantation loss was increased in treated animals, though not dose-related or statistically significant. Sporadic findings at necropsy were not considered related to treatment.
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
LITTER DATA
Three malformations were noted, one in each of the treated groups. One animal had a reduced/absent eye, one had a reduced cranium, arrinencephaly, and microencephaly, and one was noted with a shortened trunk, anury, and umbilical hernia. The incidence of anomalies and malformations was not dose-related, and occurred at a low rate. Sex ratios and litter size were similar to controls in all groups. Post-implantation loss was decreased in treated animals when compared to the controls. Mean litter and fetal weights were comparable to controls. The incidence of fetuses with thoracolumbar ribs was low in all groups. The mean percentage of fetuses with variant sternbrae was slightly higher in the control group.
Dose descriptor:
NOAEL
Effect level:
> 540 mg/kg bw/day (nominal)
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Results indicate that the dosage (mg/kg/day) were generally 5% to 16% below that intended. These data were consistent with previous experience of analysing the material in a biologically acceptable medium within the constraints of an acceptable formulation for dosing. Clear separation and a three-fold interval between dosages were still achieved despite the variation recorded.

Treatment with TK 10490 at 540 mg/kg/day was associated with: a) post-dosing salivation in all animals generally for five days and b) retarded weight gain during the dosing period. Treatment at 180 mg/kg/day was associated with post-dosing salivation in occasional animals for one to four days. At 60 mg/kg/day no adverse effects were detected in the parent female.

Litter parameters as assessed by mean values for litter size, pre- and post-implantation losses, litter and mean foetal weight were not adversely affected by treatment.

Treatment with TK 10490 had no adverse effect on the incidence of malformations, visceral and skeletal anomalies or skeletal variants.

Conclusions:
The maternal no observed-adverse-effect level is 180 mg/kg/day. The fetal no observed-adverse-effect level is >540 mg/kg/day.
Executive summary:

In this assessment of the effect of TK 10490 on the pregnancy and embryofoetal development of the rat, daily dosages of 0 (Control), 60, 180 and 540 mg/kg/day were administered by oral gavage to the parent females from Day 6 through to Day 15 of pregnancy inclusive. On Day 20 of pregnancy the females were killed and subjected to post-mortem examination, litter parameters determined and foetuses fixed prior to examination for skeletal or visceral anomalies.

Treatment with TK 10490 at 540 mg/kg/day was associated with: a) post-dosing salivation in all animals generally for five days and b) retarded weight gain during the dosing period. Treatment at 180 mg/kg/day was associated with post-dosing salivation in occasional animals for one to four days. At 60 mg/kg/day no adverse effects were detected in the parent female.

Litter parameters as assessed by mean values for litter size, pre- and post-implantation losses, litter and mean foetal weight were not adversely affected by treatment.

Treatment with TK 10490 had no adverse effect on the incidence of malformations, visceral and skeletal anomalies or skeletal variants.

Within the context of this study, oral administration of TK 10490 during days 6 to 15 of gestation produced evidence of maternal toxicity at 540 mg/kg/day in terms of slightly retarded mean body weight gain. A physiological change (increase in post-dosing salivation) which is not necessarily indicative of a toxic response per se was recorded at the high and intermediate dosages but there was no evidence that treatment of the dam had any adverse effect on embryofoetal development or morphology at any of the dosages investigated.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
180 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Additional information

Rat and rabbit developmental toxicity studies have been conducted via the oral route. Oral administration of TK 10490 to rabbits during days 7 to 19 of gestation produced evidence of maternal toxicity at 180 mg/kg/day in terms of anorexia and an initial weight loss. Treatment at 60 and 20 mg/kg/day was not associated with any maternal toxicity. There was no evidence that treatment of the dam with TK 10490 adversely affected mean litter parameters. Embryofoetal development and morphology was unaffected at all the dosages investigated. Rats dosed on days 6 to 15 of gestation had evidence of maternal toxicity at 540 mg/kg/day with slightly retarded mean body weight gain. A physiological change (increase in post-dosing salivation) which is not necessarily indicative of a toxic response per se was recorded at the high and intermediate dosages but there was no evidence that treatment of the dam had any adverse effect on embryofoetal development or morphology at any of the dosages investigated.

Dermal exposure of pregnant rabbits to 0, 30, 100 or 300 mg DGEBPA/kg body weight/day on gestation days 6 through 18 produced no evidence of embryo/fetal toxicity or teratogenicity resulting in a embryo/fetal no observed effect level (NOEL) of 300 mg/kg body weight/day. Exposure to 300 mg DGEBPA/kg body weight/day produced maternal toxicity as evidenced by moderate to severe erythema, fissures, hemorrhages and slight edema at the site of exposure. Similar, but less severe skin lesions were observed in pregnant rabbits in the 100 mg/kg/day exposure group. Exposure to 30 mg DGEBPA/kg body weight/day produced slight erythema (barely perceptible) which was not considered toxicologically significant.

Toxicity to reproduction: other studies

Additional information

No additional data.

Justification for classification or non-classification

BADGE displayed no indications of any adverse effects on reproduction in rats over two generations at dose levels which greatly exceed potential human exposure levels. There was no evidence of developmental toxicity at doses levels resulting in maternal toxicity in rats and rabbits following oral administration or rabbits following dermal administration of BADGE. Based upon these results, no classification is warranted.