Reaction mass of 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane and Orthophosphoric acid, oligomeric reaction products with 4,4'-isopropylidenediphenol -1-chloro-2,3-epoxypropane co-oligomer

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane

EC Number:

500-033-5

EC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane

Cas Number:

25068-38-6

Molecular formula:

(C15 H16 O2 . C3 H5 Cl O)x

IUPAC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane

Constituent 2

Reference substance name:

2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

216-823-5

EC Name:

2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Cas Number:

1675-54-3

Molecular formula:

C21H24O4

IUPAC Name:

2,2'-[propane-2,2-diylbis(4,1-phenyleneoxymethylene)]dioxirane

Details on test material:

The identity of the test material was confirmed through the use of gas / chromatography / mass spectroscopy using the electron impact mode. The purity of BADGE was found to be 99.65% by gas chromatography using area percent quantitation. A subsequent analysis confirmed the stability of the test material.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system 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.

Administration / exposure

Route of administration:

oral: gavage

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.

Doses / concentrations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

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

2

Dose descriptor:

NOEL

Remarks:

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

3

Dose descriptor:

NOEL

Remarks:

reproductive effects

Effect level:

750 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: highest dose examined

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.