Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is one Key, 2 -gen study available in which reduced viability was observed but this coincided with parental toxicity. The NOAEL parental = 5 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study according to international guideline.
Qualifier:
according to
Guideline:
other: EPA TSCA 40 CFR Part 798
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Sprague-Dawley CRL:CD (SD)BR
- Source: Charles River Laboratories, Inc., Kingston, New York
- Age at study initiation: (P) 45 days ; (F1) 3-6 weeks
- Weight at study initiation: (P) Males: 199.2-238.9 g; Females: 124.1-156.4 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no data
- Housing during growth phase: individually housed in hanging-wire stainless steel cages with automatic watering system
- Housing during mating: one male was housed with one female until mating was confirmed
- Housing during parturition and lactation: on day 20 of presumed gestation, females were transfered to individual nesting boxes with water bottles.
- Diet (e.g. ad libitum): Purina Certified Rodent Chow 5002, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 16 days, during which all rats were examined by an HLA staff veterinarian for general health

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled, but no data on actual value
- Humidity (%): controlled, but no data on actual value
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): controlled, but no data on actual photoperiod cycle
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The required amount of test substance was weighed in a beaker on an Arbor electronic balance, added to approximately 200 grams of basal feed and mixed in a Waring blender for approximately 2 minutes or until a homogeneous mix was achieved. Each premix was then added to the required amount of feed and mixed in a Hobart mixer at a rate of 1 min/kg of feed for a minimum of 5 minutes.
Due to the increased volume of feed required for the F0F1 lactation phase (weeks 15-21) and the F1F2 lactation phase (weeks 17-23), the female feed was mixed in a twin-shell mixer during these several weeks.

Fresh diets were prepared weekly and stored at room temperature until presentation. Dietary mixes were adjusted weekly based on body weights and food consumption measured in the previous week. Feed concentrations for weeks 1 and 2 of each generation growht period were prepared from body weights and food consumption values from historical data. Female diets were available to both sexes in the breeding cages. Diets for female groups were prepared during mating, gestation and lactation periods from body weights and food consumptions measured during week 10.
Details on mating procedure:
- M/F ratio per cage:
- Length of cohabitation:
- Proof of pregnancy: vaginal plug or sperm in vaginal smear is referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility. (F0 mating)
- After 10 days of unsuccessul paring replacement of first male by another randomly selected male, whose fertility was not necessarily proven. (F1 mating)
- No further matings after two unsuccessful attempts.

After 10 weeks of dietary treatment, and following 7 days of acclimation of male rats to the breeding cages, each male was cohabited with one female from the same group. In mating for the F1a and F2 offspring, the parental rats were paired sequentially by number within each group, first to last, until all available animals had been paired.

Due to poor survival of F1a high-dose pups observed in the lactation phase, a second breeding of the F0 control and high-dose parental ratw sith cross-fostering was conducted following the weaning of the F1a pups.
Following delivery, a number of F1b litters were cross-fostered to dams of a different dietary exposure group for lactation. Some dams remained in their natural litters.
In mating for the F1b offspring, the reverse numerical sequence of males was used to avoid remating of the same pairs used in the F1a breeding.

Offspring of the first breeding of the F0 parental rats, the F1a generation, were selected to become the F1 parental generation. There were 705 weanling pups available for selection of the F1 parental animals, 343 males and 362 females. Weanlings remained in nesting boxes by litter until selection. Weanlings were selected and assigned to the same dosage group as their parents. The 120 F1 male rats and 120 F1 female rats were 3 to 6 weeks old at initiation of the F1 growth pase.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to study inititation, samples of the basal diet and all treated diets were analyzed for stability and homogeneity. During the study, an additional analysis for homogeneity of the low- and high-dose mixtures prepared in the twin-shell blender was performed during week 20. Samples reserved from each freshly mixed batch of treated diets and from the basal diet at weeks 1, 2, 3, 4, 8, 12, 16, 20, 24, 28, 32, 36 and 40 were analyzed for concentration. All chemical analyses were performed by Hazleton Biotechnologies Corporation, Vienna, Virginia.
Duration of treatment / exposure:
During quarantine of the F0 parental animals, all rats received the basal diet.
Following acclimation of the F0 rats and from the day of birth of the F1a and F2 offspring, appropriate control or test diets were available ad libitum. F1b pups from that were cross-fostered to dams of a different dietary group received minimal exposure to the diets or milk of the natural dams on the day of their birth. Diets of the foster dams were available to those F1b pups from the time of fostering. Control or test diets were presented throughout the growth, mating, gestation, and lactation phases and until terminal sacrifice of each generation.
Frequency of treatment:
Continuous via the feed. For periods of exposure, see previous section.
Details on study schedule:
The study was initiated on December 21, 1984, with the feeding of F0 rats. The second generation phase was defined as starting on May 10, 1985, and the weaning of the F2 pups was completed on September 19, 1985.

Remarks:
Doses / Concentrations:
5 mg/kg/d
Basis:
nominal in diet
targeted actual dose
Remarks:
Doses / Concentrations:
50 mg/kg/d
Basis:
nominal in diet
targeted actual dose
Remarks:
Doses / Concentrations:
250 mg/kg/d
Basis:
nominal in diet
targeted actual dose
No. of animals per sex per dose:
30 rats per sex per dose
Control animals:
yes, plain diet
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations: mortality, moribundity, obvious indications of toxic effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly in general / twice in the first week post partum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: weekly, except during mating phase (both sexes) or gestation and lactation (females)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Litter observations:
Pregnant females were allowed to deliver their pups. On days 0, 4, 7, 14 and 21 after birth, the number of life and dead pups of each sex per litter, individual pup body weights and clinical observations were recorded. On day 4 of lactation, all litters with more than eight pups were reduced to eight. Selection of pups for culling was by random card draw with equal numbers of males and females remaining in each litter when possible.
Daily cage side obersvations and pup counts on each litter was conducted, and the number of pups missing or found dead was recorded.

For the cross-fostering of F0F1b, high-dose litters were removed from their natural dams and placed with control dams which had given birth on the same day. The control litters were simultaneously moved from the control dams to the high-dose dams. The litters were matched based on order of birth on the first day and each successive day until 13 high-dose litters had been exchanged with 13 control litters. Any control or high-dose litters for which there was no correspondent litter on a given day of delivery remained with the natural dam. On day 4 of lactation al litters were reduced to 8 as described above.

During lactation, all dams and pups were checked once daily for mortality and moribundity. On days 0, 4, 7, 14 and 21 postpartum, the total number of pups (live and dead) per litter, the number of each sex, individual pup body weights and individual signs of external abnormalities were recorded. In addition, on days 0, 4 and 7, pups were examined for the presence of milk in their stomachs as indicated by the presence of a withish distention of the abdomen observed through the thin, hairless skin of the young pups.
Postmortem examinations (parental animals):
After weaning of the F1a pups, all surviving low- and mid-dose F0 males and females were sacrificed by exsanguination under sodium pentobarbital anesthesia. Rats from the F0 control and high-dose groups were retained until F1b pups were weaned and then sacrificed in a like manner. Males from the F1 parental generation were sacrificed using the same procedures as for F0 rats, following completion of breeding for the F2 generation. F1 females were sacrificed following weaning of the F2 offspring.

A complete gross necropsy, in the presence of a pathologist was performed on all parental animals. A complete gross necropsy is defined as examination of external surfaces, all orifices, cranial cavity and cervical, thoracic and abdominal viscera. The following tissues were preserved: vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, urinary bladder (collected with reproductive organs), unusual lesions. The same examinations were carried out on partental animals that died during the treatment periods.
Postmortem examinations (offspring):
Culled pups were sacrificed by intraperitoneal injection of sodium pentobarbital solution, examined for abnormalities of the cervical, thoracic or abdominal viscera, and discarded.

Pups found dead during daily cage side obervations were examined grossly for cervical, thoracic or abdominal viscera abnormalities and an apparent cause of death if discernible. No tissues or carcasses were saved.

All surviving F1b pups were weaned on day 21 of lactation. As soon as possible after weaning, the pups were sacrificed by carbon dioxide asphyxiation, subjected to gross examination of cervical, thoracic and abdominal viscera for microscopic abnormalities, and then discarded. Gross observations were recorded.

Each litter in the F1a generations was weaned on day 21 of lactation. After selection of the F1 parental generation, the remainder of the F1a pups was sacrificed and examinated as described above.

The F2 pups were also sacrificed and necropsied shortly after weaning, as described above.
Statistics:
The control group values representing parental body weights and food consumption during the growth phases, maternal body weights during gestation and lactation, body weight changes during gestation and day 4 postcull pup weights were compared statistically to the data of the compound-treated groups.
Maternal body weights during F1b lactation were analyzed by ANOVA or 2-tailed student's t-test.
Parental fertility and offspring viability indices were analyzed using Fisher's "exact" test.
Statistical analysis of days 0, 4 (precull), 7, 14 and 21 pup body weights was performed using the number of pups in each litter as the covariate.
Control vs. compound-treated group mean comparisons of these data were evaluated at the 5.0% two-tailed probability level.
Reproductive indices:
Duration of gestation
Number of males and females confirmed to have mated.
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:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0 Parental data: There were no apparent compound-related clinical signs in any parental rats during the F0 phases of the study. There was a slightly higher incidence of alopecia in the high-dose females than controls during gestation and lactation, but the occurrence was not clearly dose related.
F1 Parental data: One high-dose male was found dead during week 2 of the F1 growth phase. Death was not attributed to compound exposure. There were no apparent compound-related clinical signs in any F1 parental rats.

BODY WEIGHT (PARENTAL ANIMALS)
F0 Parental data: Mean high-dose body weights were significantly lower than control beginning at week 4 for females and week 5 for males. Mean high-dose body weights continued to be significantly lower than the control for the males at weeks 7, 8 and 9 and at weeks 5 through 10 for the females. Body weight gains from weeks 0-10 for the high-dose males and females were significantly lower than control.

F1 Non-maternal data: Mean high-dose male body weights throughout the premating growth phase (weeks 0-10) were significantly lower than control. Terpstra-Jonckheere test revealed a significant negative trend for the male body weights at weeks 2 through 8. Mean high-dose female body weights were significantly lower than the control at week 0 through 10. Body weight gains from weeks 0-10 for the high-dose males and females were significantly lower than control.

F0F1a Maternal data: Mean high-dose maternal body weights during gestation were significantly lower than control at each weighing interval (days 0, 7, 14 and 20). No significant differences were noted for mean maternal body weight changes between the control and treated groups.
The mean maternal high-dose body weights were significantly lower than control at each weighing interval (days 0, 4, 7, 14 and 21) during lactation. Body weight changes during lactation were comparable among control, low-dose and mid-dose females. However, the high-dose females gained more than twice as much weight during lactation (days 0-21) than the other treated or control lactating females.

F0F1b Maternal data: Mean high-dose maternal body weights during gestation were significantly lower than control at each weighing interval (days 0, 7, 14 and 20). No significant differences were noted for mean maternal body weight changes between the control and high-dose groups except during days 0-7 of gestation. The mean maternal body weights from cross-fostered high-dose dams (HC) were significantly lower than the non-fostered control dams throughout lactation. The mean maternal body weights for non-fostered high-dose dams were significantly lower than the non-fostered controls (CH) at days 0, 4, 7 and 14 of lactation. The treated dams had a mean body weight gain, while the untreated dams had a mean body weight loss for the entire lactation period (days 0-21). Mean maternal body weights of both groups of control dams were comparable during lactation.

FOOD CONSUMPTION (PARENTAL ANIMALS)
F0 Parental data: Mean body weights and mean food consumption values for both sexes in all groups were slightly decreased at week 5. This appeared to have been due to individual animals having difficulty with the watering system or to decreased individual water consumption rather than a treatment effect. All mean body weights and mean fodd consumption values were higher in the weeks that followed than the isolated depressed values seen at that interval.
F1 Non-maternal data: Weekly food consumption values were significantly lower than the controls at week 1-8 and week 10 for the high-dose males and at week 2 and 3 for all treated female groups, at weeks 4 and 5 for the mid-dose females, and at weeks 4-10 for the high-dose females. Mean total food consumption values for weeks 1-10 for high-dose males and mid- and high-dose females were significantly lower than the corresponding control value. With the exception of the first several weeks of the F1 growth period, the compound consumption was 80% or greater of the target level in all groups.
F1 Maternal data: Mean maternal body weights during gestation for the high-dose females at days 0, 7, 14 and 20 were significantly lower than control. Significant differences between the control and the high-dose groups were noted for mean maternal body weight changes at days 0-7, 7-14 and 0-20. The mean maternal high-dose body weights were significantly lower than control at each weighing interval (days 0, 4, 7, 14 and 21) during lactation. Mean maternal mid-dose body weights were also significantly lower at days 4, 7 and 14. Terpstra-Jonckheere test revealed a significant negative trend for body weights at day 0 of lactation after rank transformation of the data. Body weight changes during lactation were similar among the control and treated females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0-F1a reproduction data: The mean gestation index for all groups was 100%. The number of males confirmed to have mated was lowest in the low-dose group. The numbers of mid- and high-dose males confirmed to have mated was comparable to the controls. The number of females compared to have mated was similar for all groups. Fertility of the low-dose animals was lower than control. However, there were no significant trends in the fertility indices.
F0-F1b reproduction data: The mean precoital interval was longer for the high-dose females than the controls. The mean duration of gestation was comparable between control and treated females. The number of high-dose males confirmed to have mated was similar to the controls. The number of females confirmed to have mated was also similar for both groups. Fertility of the high-dose animals was comparable to controls. The mean gestation index was 100% in the control group and slightly but not significantly lower in the high dose group (95%).
One high-dose dam died following the delivery of her F1b litter. The death of this dam was probably due to complications of delivery.

GROSS PATHOLOGY (PARENTAL ANIMALS)
F0 Parental data: One high-dosed female was found dead during the first week. Gross necropsy revealed a small spleen and multiple, small, black pitted areas in the glandular mucosa of the stomach. These findings are common to rats that have been food fasted and/or dehydrated. The low terminal body weight (82.6 g compared to the group mean of 175.9) would indicate anorexia.
A second high-dose female was found dead during week 27, three days after the delivery of her F1 litter. The entire litter was born dead or was partially cannibalized after delivery. Prior to necropsy, this animal was observed to have a vaginal red discharge and a placenta in the vagina. The death of this animal was probably the result of a difficult delivery. Neither death is considered to be compound related. There were no remarkable gross pathology findings among animals sacrificed at termination of the F0 generation.
F1 Parental data: Gross pathology findings for one mid-dose male included small seminal vesicles, enlarged mediastinal lymph nodes, enlarged atria of the heart, dark viscera and fluid-filled peritoneal and thoracic cavities. These findings are not considered to be compound related. Gross pathology findings for the parental rats sacrificed at termination of the F1 generation were essentially unremarkable. Dilated pelvis(es) were noted in the kidney(s) of one, two and two males in the low-, mid- and high-dose groups, respectively. The pelves of the control animals were normal. Alopecia was noted in one, two and two females of the low-, mid- and high-dose groups, respectively.

HISTOPATHOLOGY (PARENTAL ANIMALS)
F0 Parental data: Compound-related lesions seen in the urinary bladder of treated rats were mononuclear cell infiltrate and transitional epithelial hyperplasia. The incidence and/or severity of these lesions increased in mid- and high-dose male rats and in high-dose female rats. Mononuclear cell infiltrate is characterized by the presence of mononuclear cells in the mucosal, submucosal and perivascular areas.
A variety of spontaneous and incidental findings were noted in control and treated animals of both sexes. Frequently observed lesions were interstitial edema of the testes, chronic active inflammation of the prostate and uterus, pigmentation of the ovary and uterus and dilatation of ther uterine lumen.
F1 Parental data: Compound-related histomorphic alterations were noted in the urinary bladders of the high-dose males and females. Compound-related findings in the high-dose urinary bladders were mononuclear cell infiltrate and transitional epithelial hyperplasia. Proteinaceous material was also noted in the urinary bladders of the high-dose males. No compound-related histomorphologic alterations of the reproductive organs were noted.
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Generation: all (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
decreased viability index at day 4 for mid- and high-dose groups
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
mean high-dose pup body weights were significantly lower than control
Gross pathological findings:
no effects observed
VIABILITY (OFFSPRING)
F1a Litter data: Both the viability index at day 4 and the weaning index of the mid- and high-dose offsrping were significantly lower than control. Cochran-Armitage analyses revealed a significant negative trend for F1a offspring survival at days 4 and 21.
F1b Litter data: Pup survival indices were lower in both cross-fostered groups than in the untreated control group. Weaning survival of the control pups fostered to high-dose dams was signficantly lower than the untreated control pups.
F2 Litter data: The viability index at day 4 was significantly lower than control for the low and mid-dose group, but was similar between the high dose group and the control. A significant negative trend for F2 offspring survival was noted at day 21. The weaning index of the high-dose F2 offspring was significantly lower than the control index.

CLINICAL SIGNS (OFFSPRING)
F1a Pups: The majority of clinical signs observed were noted for high-dose pups and were related to size and observations of canibalization. One pup in the mid-dose group had a raised dark area on the head. It was found dead on day 3.
F1b Pups: Two cross-fostered litters and one high-dose litter each contained at least one pup with no milk in the stomach during lactation. One high-dose fostered pup (CH) was pale and cold at day 0 and one fostered control pup (HC) was noted to be small at day 7. Two cross-fostered litters each contained at least one pup with a small raised area on the abdomen at day 14. All F1b pups observed at day 21 appeared normal.
F2 The majority of clinical signs observed were noted for high-dose pups in two litters at days 14 and 21 and were related to small size.

BODY WEIGHT (OFFSPRING)
F1a Litter data: Mean male and female high-dose pup body weights were significantly lower than control at each weighing interval when adjusted for litter size (analysis of covariance). Mean mid-dose body weights were significantly lower than control at days 4, 7 and 21 for the male pups and at days 4, 7, 14 and 21 for female pups.
F1b Litter data: Comparison of mean pup weights at day 0 confirmed the suppression of body weights seen at the high-dose level in the previous breeding of this generation (F1a). The artificial separation of the high-dose group into the fostered and non-fostered subgroup at day 0 resulted in significant differences in body weight between untreated male pups (but not female pups) and high-dose pups of the same sex. The mean weight of high-dose females was lower but not at a statistically significant level. Significantly lower high-dose female pups weights were noted at days 14 and 21 both in the fostered and non-fostered litters compared with untreated controls. Although there was some apparent body weight depression, cross-fostering of the high-dose litters to control dams did not result in the significant differences in body weights that were seen in the non-fostered high-dose group or fostered control pups with high-dose dams.
F2 Litter data: Mean male and female high-dose pup body weights were significantly lower than control at days 4 precull, 7, 14 and 21 when adjusted for litter size (analysis of covariance). Mean male and female high-dose pup body weights were also significantly lower than control at day 4 postcull. Mean mid-dose body weights were significantly lower than control at days 14 and 21 for the male pups and at day 21 for the female pups.

GROSS PATHOLOGY (OFFSPRING)
F1a Pups: Gross pathology of F1a pups found dead during lactation included findings of tail cannibalization, an irregularly shaped skull with a small opening and a pale spleen. Fourteen observations of dilated kidney pelves were noted at the weanling sacrifice. However, nine of the findings occurred in the controls, 3 in the low dose and one each in the mid- and high-dose groups. The remaining gross pathology observations for the F1a weanling sacrifice were not remarkable.
F1b Pups: Gross pathology of pups found dead during lactation included findings of no milk present in stomach, dark red material in thoracic cavity, and pup bodies not cleaned postpartum. Observations of dilated kidney pelves, clear pinpoint cysts in the kidney(s), clear fluid in the uterus and an ovarian cyst were noted at the weanling sacrifice. These findings were not considered treatment-related. The remaining gross pathology observations for the weanling sacrifice were not remarkable.
F2 Pups: Gross pathology of pups found dead during lactation included findings of cannibalization, small size, and no milk present in stomach. Nineteen observations of dilated kidney pelves were noted at the weanling sacrifice. However, 6 of the findings occurred in controls, 2 in the low-dose, 5 in the mid-dose and 6 in the high-dose pups. Uterine horns were distended in two mid-dose pups and one high-dose pup. The remaining gross pathology observations for the weanling sacrifice were not remarkable.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: histomorphologic changes in the bladders of male rats
Dose descriptor:
LOAEL
Generation:
F2
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slight body weight depression and slightly reduced survival of F2 pups.
Reproductive effects observed:
not specified

Nurturing or maternal behaviour did not appear to be the salient factor in pup growth and survival. During the cross-fostering phase, careful examination of the pups was made for the presence or absence of milk in the stomach of the pups during the early days of lactation. The majority of pups appeared to be nursing. The dams did not display abnormal or unusual maternal behavior towards either the F1a or F1b litters such as rejecting or ignoring the pups. The F1b and F2 pups that were found dead did have a common finding of no milk present in the stomach. This was not noted in the F1a offspring; however, it was not specified in the protocol to examine for the presence of milk in the F1a breeding. There were no other gross necropsy findings in the offspring that died which could be indicative of the cause of death.

The reason for the improved viability of F2 offspring is not clear. The dose response of the viability indices in the F1a offspring is linear and therefore appears to be a compound effect.

Conclusions:
There were no effects of compound exposure on the reproductive performance of any group. No treatment effects were ween in rats exposed to the 5 mg/kg dietary level of the test substance.
Executive summary:

To evaluate the effects of the test substance on gonadal function, mating behavior and fertility as well as growth and development of offspring, Sprague-Dawley rats were fed diets containing target dose levels of 5, 50 and 250 mg/kg of body weight through 2 generations. A control group received the basal diet only. A second breeding of the first generation control and high-dose parents with cross-fostering of some litters was carried out to assess the route of toxicity to offspring.

In the high-dose group (250 mg/kg) of both parental generations, body weights and food consumption were significantly reduced compared to the concurrent controls. This is considered a treatment effect. Histomorphologic alterations in the urinary bladder of F0 and F1 parental animals were noted as treatment effects in this group as well. Reduced pup viability in the high-dose group, particularly in the F1a generation, was attributed to compound exposure trhough the milk. Cotnrol pups exposed to the high-dose level of test material by cross-fostering to high-dose dams also showed reduced viability.

In the mid-dose group (50 mg/kg level), the body weights of parental rats in the second generation and survival of the first generation of offspring were slightly lower than the concurrent controls. Histomorphologic changes were seen in the bladders of F0 males only.

There were no effects of compound exposure on the reproductive performance of any group. No treatment effects were ween in rats exposed to the 5 mg/kg dietary level of the test substance.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study available is GLP compliant and of good quality (Klimisch score = 1).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
There is an oral 2-gen study available.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
There is an oral 2-gen study available.
Additional information

In the two-generation reproduction study (Lemen, 1987), rats were fed with diets containing dose levels of 5, 50 and 250 mg/kg bw/day.

In the high-dose group (250 mg/kg) of both parental generations, body weights and food consumption were significantly reduced compared to the concurrent controls. Histomorphologic alterations in the urinary bladder of F0and F1parental animals, were noted as treatment effects in this group as well. Reduced pup viability in the high-dose group was observed. Control pups exposed to the high dose by cross-fostering to high dose dams also showed reduced viability.

In the mid-dose group (50 mg/kg bw/day), the body weights of parental rats in the second generation and survival of the first generation of offspring were slightly lower than the concurrent controls. Histomorphological changes were seen in the bladders of F0males only.

There were no effects of compound exposure on the reproductive performance of any group. No treatment effects were seen in rats exposed to the 5 mg/kg bw/day.




Effects on developmental toxicity

Description of key information

A pilot and a main teratology study are available.

The NOAEL developmental = 300 mg/kg bw/day, while the NOAEL parental = 3 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No international guideline mentioned. However, study is described in acceptable detail.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Pregnant Charles River CD rats were used to evaluate the teratogenic potential of DBPP in this study. The compound was administered orally by gavage as a single daily dose from days 6 through 15 of gestation. The test substance was administered at dosage levels of 3, 30 and 300 mg/kg/d at a constant volume of 10 ml/kg/d. Two control groups received the vehicle, corn oil, on a comparable regimen.

During gestation, the females were observed for mortality, body weight changes and clinical signs of toxicity. Cesarean sections were performed on day 20 of gestation. The number and location of viable fetuses, early and late resorptions, total implantations and corpora lutea were recorded. The fetuses were individually sexed, weighed and examined for external, soft tissue and skeletal malformations and variations.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Portage, Michigan.
- Age at study initiation: approx. 3 months at time of mating
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually (except during mating), in hanging wire-mesh cages
- Diet (e.g. ad libitum):Purina Laboratory Chow, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 4 weeks prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled (no data on actual temperature)
- Humidity (%): controlled (no data on actual humidityà
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): controlled (no data on actual photocycle)
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Mixing appropriate amounts with corn oil at concentrations to permit the administration of applicable dosage levels.
Individual dosages were based on individual body weights recorded on days 6, 9 and 12 of gestation.

DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Storage temperature of food: no data

VEHICLE
- Concentration in vehicle: depending on individual body weight
- Amount of vehicle (if gavage): 10 mL/kg/d
- Lot/batch no. (if required): "Mazola"
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
One female and one male rat of the same strain were placed together for mating. The day of mating was determined by daily inspection for copulatory plug or by a vaginal smear for sperm. The day that evidence of mating was detected was designated day 0 of gestation and the female was returned to an individual cage.
Duration of treatment / exposure:
From day 6 through day 15 of gestation.
Frequency of treatment:
SIngle daily dose.
Duration of test:
Until day 20 of gestation.
No. of animals per sex per dose:
25 female rats per test group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on pilot study
- Rationale for animal assignment (if not random): no data
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality and clinical signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 6, 9, 12, 16 and 20

POST-MORTEM EXAMINATIONS: No data
Ovaries and uterine content:
The uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all fetuses
- Soft tissue examinations: Yes: approx. 1/3 of the fetuses
- Skeletal examinations: Yes: apporx. 2/3 of the fetuses
- Head examinations: Yes: all fetuses
Statistics:
All statistical analyses compared the treatment groups with the control groups, with the level of significance at p<0.05.

Male to female fetal sex ratio and the number of litters with anomalies were compared using the Chi-square test criterion with Yates correction for 2 x 2 contingency tables and/or Fisher's exact probability test as described by Siegel to judge significance of differences.

The proportion of early resorptions, late resorptions and post-implantation losses were compared by the Mann-Whitney U-test as described by Siegel and Weil to judge significance of differences.

The mean number of corpora lutea, implantation sites and live fetuses were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance of differences.

Fetal body weights were compared by analysis of variance (hierarchal classification) and t-test as described by Stell and Torrie using Dunnett's multiple comparison tables to judge significance of differences.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no biologically meaningful differences in appearance or behaviour attributable to treatment with the test substance between any of the treated groups and the control groups.
Hairloss and matting of the haircoat were observed in all of the control and treatment groups. One rat in the second control group delivered prior to its scheduled sacrifice date due to an inaccurate determination of copulation. One rat in the 300 mg/kg/d dosage group died on gestation day 11. A sligh amount of dried red matter round the nose, accentuated lobulation of the liver, slight lung congestion and blood in the thoracic cavity were noted at necropsy. The cause of death was not determined. Survival was 100% in all other groups.
Maternal body weight gains in the 3 and 30 mg/kg/d dosage groups were comparable to the control groups. A very slight reduction in maternal body weight gains over the gestation period was noted in the 300 mg/kg/d dosage group when compared to the control groups.
Remarks on result:
not determinable due to absence of adverse toxic effects
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
CESARIAN SECTION OBSERVATIONS:
There were no statistically significant or biologically meaningful differences in the mean number of viable fetuses, late resorptions, implantations or corpora lutea between any of the treated groups and the control groups. A slight, though not statistically significant increase in early resorptions and post-implantation losses was noted in all of the treatment groups when compared to control groups. However, this increase did not occur in a dose-related pattern.
There were no biologically meaningful differences in mean fetal body weights or in the male to female fetal sex ratios between any of the treated groups and the control groups. A statistically significant increase in mean fetal body weight was noted in the
300 mg/kg/d dosage group when compared to control group I. This difference was not statistically significant to control group II, and therefore is not considered to be biologically meaningful. A statistically significant difference in the male to female fetal sex ratio was noted in the 3 mg/kg/d dosage group when compared to control group II. However, this is not considered to be a compound-related effect as the fetal sex ratios in the 30 and 300 mg/kg/d dosage groups were comparable to the control groups, and this difference was not statistically significant when compared to control group I.

FETAL MORPHOLOGICAL OBSERVATIONS:
There were no statistically significant or biologically meaningful differences in the number of litters with malformations between any of the treated gropus and the control groups. An increase in the number of litters and fetuses with malformations was noted in the 30 mg/kg/d dosage group when compared to the control groups. However, these malformations did not occur in a syndrome-type pattern and no malformations were observed in the 300 mg/kg/d dosage group. Variations were comparable for all groups.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The test substance did not produce a teratogenic effect when administered to rats orally at dosage levels of 300 mg/kg/d or less.
Executive summary:

Pregnant Charles River CD rats were used to evaluate the teratogenic potential of DBPP in this study. The compound was administered orally by gavage as a single daily dose from days 6 through 15 of gestation. The test substance was administered at dosage levels of 3, 30 and 300 mg/kg/d at a constant volume of 10 ml/kg/d. Two control groups received the vehicle, corn oil, on a comparable regimen.

During gestation, the females were observed for mortality, body weight changes and clinical signs of toxicity. Cesarean sections were performed on day 20 of gestation. The number and location of viable fetuses, early and late resorptions, total implantations and corpora lutea were recorded. The fetuses were individually sexed, weighed and examined for external, soft tissue and skeletal malformations and variations.

There were no biologically meaningful differences in appearance or behavior between any of the treated groups and the control groups. Mean maternal body weight gains in the 3 and 30 mg/kg/d dosage groups were comparable to the control groups. A very slight reduction in mean maternal body weight gains was noted in the 300 mg/kg/d dosage group when compared to the control groups.

There were no biologically meaningful differences in the mean number of viable fetuses, implantations, corpora lutea, mean fatal body weights or in the male or female fetal sex ratios between any of the treated groups and the control groups. A slight increase of post-implantation losses was noted in all of the treatment groups when compared to the control groups. However, this increase did not occur in a dose-related pattern.

There were no biologically meaningful differences in the number of litters with malformations and variations in the DBPP treated groups when compared to the control groups.

The test substance did not produce a teratogenic effect when administered to rats orally at dosage levels of 300 mg/kg/d or less.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Two teratology studies, a pilot and a main study, are available. Both are scored with a Klimisch score = 2.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
Only oral developmental studies are available.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
Only oral developmental studies are available.
Additional information

In the pilot teratology study (Goldenthal, 1978), DBPP was administered by gavage at 10, 30, 100, 300 and 1000 mg/kg bw/day to pregnant rats from gestation day 6 through day 15. A control group received the vehicle, corn oil, at 25 mL/kg bw/day. During gestation, the females were observed for clinical signs of effect, mortality and body weight gains. The rats were sacrificed on gestation day 20 and the uterine contents examined for viable and non-viable fetuses, early and late resorptions, the number of corpora lutea and total implantations.

All rats given 1000 mg/kg/day of test substance died by gestation day 14. Survival was 100% for all rats at the lower dosage levels. Abdominal urinary staining and/or lack of grooming was observed in one female in the 10 mg/kg/day group and in all rats at the higher dosage levels. A red nasal discharge was observed for one female given 30 mg/kg/day and for all rats at the higher dosage levels.

There were no compound-related differences for the number of viable or nonviable fetuses, resorptions, implantations or corpora lutea for females given 10 mg/kg bw/day or 300 mg/kg bw/day when compared to the controls. At 100 mg/kg bw/day an increased number of post implantation losses occurred which were not considered to be treatment related.

 

In the key teratology study (Jessup, 1979), the test compound was given orally by gavage to pregnant rats at dosage levels of 3, 30 and 300 mg/kg bw/day. The compound was administered as a single daily dose from day 6 through 15 of gestation. During gestation, the females were observed for mortality, body weight changes and clinical signs of toxicity. Caesarean sections were performed on day 20 of gestation. The number and location of viable fetuses, early and late resorptions, total implantations and corpora lutea were recorded. The fetuses were individually sexed, weighed and examined for external, soft tissue and skeletal malformations and variations.

There were no biologically meaningful differences in the mean number of viable fetuses, implantations, corpora lutea, mean fetal body weights, mean number of litters with malformations or in the male to female fetal sex ratios between any of the DBPP treated groups and the control groups. Variations were comparable for all groups. DBPP did not produce a teratogenic effect when administered to rats orally at dosage levels of 300 mg/kg bw/day or less.

Justification for classification or non-classification

The data of the 2-generation reproduction study and the two teratology studies do not show any treatment-related adverse effects on sexual function and fertility. Only in the 2-generation reproduction study a toxic effect on reproduction was observed. Reduced viability was observed at the high dose (250 mg/kg bw/day)At a dose level of 50 mg/kg bw/day, the survival of the first generation of offspring was slightly lower than the concurrent controls. But also toxic effects for the parental animals were reported at these dose levels. In the high-dose group (250 mg/kg) of both parental generations, body weights and food consumption were significantly reduced compared to the concurrent controls. In the mid-dose group (50 mg/kg bw/day), the body weights of parental rats in the second generation were slightly lower than the concurrent controls and histomorphological changes were seen in the bladders of F0males only.

 

These results provide no evidence of an adverse effect on development in absence of other toxic effects. It is likely that the adverse effect on reproduction is a secondary non-specific consequence of the other effects. Consequently, the test substance should not be classified according to the CLP Regulation as a reproductive toxicant.

In the 2-generation reproduction study, reduced pup viability was observed at the high dose (250 mg/kg bw/day). Control pups exposed to the high dose by cross-fostering to high dose dams also showed reduced viability. At a dose level of 50 mg/kg bw/day, the survival of the first generation of off-spring was slightly lower than the concurrent controls but the survival of the second generation was 99%.

Nurturing or maternal behavior did not appear to be the salient factor in pup growth and survival.

Only in the high dose (250 mg/kg bw) there was a statistically significant effect in reduced pup viability in both generations. In the high dose group, body weight and food consumption of parental animals were significantly reduced and other signs of parental toxicity were observed. In the mid-dose group (50 mg/kg bw) slighter symptoms of toxicity in the parental animals were still observed.

The reduced viability of the pups at the high dose is most probably a non-specific secondary effect. Maternal toxicity is clearly observed at this high dose.

Due to the high dose (250 mg/kg bw) at which this effect is observed and due to the clearly observed maternal toxicity, the test substance should not be classified for effects on or via lactation (Guidance on the Application of the CLP Criteria: 3.7.2.2.2 Substances causing effects on or via lactation).