Registration Dossier

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:
publication
Title:
A two-generation reproductive toxicity study of benzophenone in rats
Author:
Hoshino N., Tani E., Wako Y. & Takahashi K.
Year:
2005
Bibliographic source:
J Toxicol Sci 30, 5-20

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzophenone (BZP)
- Analytical purity: 99.98% or higher
- Lot/batch No.: 112D2013

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Astugi breeding Center)
- Age at study initiation: (P) 5 wks; (F1) 3 wks
- Housing: Polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-25.0
- Humidity (%): 35-75
- Air changes (per hr): 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and /or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: non known
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Administration of F0 parental animals started from an age of 5 weeks and continued in males for 10 weeks. For females, administration lasted through 10 weeks or more of the pre-mating, mating, gestational, lactational and during weaning of the F1 offspring (PND 21).
Administration to F1 parental animals was started from the time of weaning (three weeks old); in F1 males it was continued until necropsy trough 10 weeks or more of the pre-mating and mating periods, and in F1 females until necropsy through 10 weeks or more of the pre-mating, mating, gestational, lactational periods, and during weaning of the F2 offspring (PND 21).
Frequency of treatment:
continously via diet
Doses / concentrations
Remarks:
0, 100, 450 or 2000 ppm (nominal in diet)
F0 males: ca. 0, 6.5, 29 or 130 mg/kg bw
F0 females: ca. 0, 8.4, 38.2 or 166.5 mg/kg bw
F1 males: ca. 0, 7.8, 34.6 or 159.4 mg/kg bw
F1 females: ca. 0, 8.8, 40.5 or 179.2 mg/kg bw
No. of animals per sex per dose:
24 males and 24 females
Control animals:
yes, plain diet
Details on study design:
Dosing was based on the results of a preliminary dose-range finding study over 4 weeks with dietary concentrations of 0, 600, 2000, 6000 or 20000 ppm. The highest dose for the definite study was set at 2000 ppm.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (once daily)
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes (time schedule: on gestation days 0, 7, 14 and 20, on lactation days 0, 4, 7, 14 and at necropsy)
FOOD CONSUMPTION AND COMPOUND INTAKE: yes
Oestrous cyclicity (parental animals):
Vaginal smears were collected from female animals everyday in the morning to examine the estrus cycle during the two weeks before mating, starting from 13 weeks of age for the F0 parents and from 11 weeks for the F1 parents.
Sperm parameters (parental animals):
Parameters examined in F0 and F1 male parental generations: In 10 animals of the control and 2000 ppm group, the number of homogenization-resistant spermatids in the testis and number of sperms in the cauda epididymal were counted.
Litter observations:
STANDARDISATION OF LITTERS: Yes (on day 4 postpartum; maximum of 8 pups/litter as nearly as possible, excess pups were killed and discarded)
PARAMETERS EXAMINED: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, reflex tests
GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
SACRIFICE: All surviving male animals as soon as possible after the last litters in each generation were produced. All surviving maternal animals after the last litter of each generation was weaned.

GROSS NECROPSY: no data

HISTOPATHOLOGY / ORGAN WEIGHTS: Organ weights in all F0 and F1 parental animals: brain, pituitary gland, thyroid including parathyroid, liver, kidneys, adrenal glands, spleen, testes, epididymes, prostate, seminal vesicle, ovaries and uterus. Histopathological examinations were conducted for the following organs from all males and females of the control and 2000 ppm groups in the F0 and F1 parents. The brain, pituitary gland, thyroid and parathyroid, liver, kidneys, adrenal glands, spleen, testes, epididymes, seminal vesicles (including coagulating glands), prostate (ventral lobe), ovaries, uterus (including the cervical region), vagina, and mammary glands. Furthermore, the liver and kidneys in the 100 and 450 ppm groups in the F0 and F1 parent animals and any macroscopically abnormal sites were also histopathologically examined. Besides, any animals that died during the course of the study or sacrificed upon becoming moribund were examined to investigate the causes.
Postmortem examinations (offspring):
SACRIFICE: Excluding the pups that died before selection on PND 4 or the pups not selected on PND 4 (when their numbers were adjusted), all the remaining pups were necropsied when they were sacrificed or were found dead.
GROSS NECROPSY: consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Organ weights (at weaning)
ORGAN WEIGTHS: The brain, thymus, and spleen of one F1 and F2 male and female pups each selected from each litter were weighed on PND 21.
Statistics:
Data concerning effects on the offspring until their weaning were based on values calculated per litter as the specimen unit. Using weights of bilateral organs, the sums of the left and right organs were employed for statistical analysis. Metric data were analyzed for homogeneity of variance by Bartlett’s method. When the variance was homogeneous, one-way ANOVA was carried out. When not homogeneous, on the other hand, a Kruskal-Wallis’s test was performed. When a significant inter-group difference was found, Dunnett’s method or a Dunnett type multiple-comparison method were applied. For some examination items, the Kruskal-Wallis test was applied first, and when a significant inter-group difference was found, Dunnett type multiple-comparison method was conducted. Numerical data were analyzed by the Fisher’s exact probability method. The level of statistical significance was basically set at 5%.
Reproductive indices:
No. of days until copulation (days)
Mating index (%) = (Number of males and females showing evidence of copulation / number of males and females used for mating) × 100.
Fertility index (%) = (Number of pregnant females / number of copulated females) × 100.
Gestation length (days) = Number of days from GD 0 till the delivery day.
Gestation index (%) = (Number of females with normal delivery / number of pregnant females) × 100.
Sex ratio = Total number of male pups delivered / total number of the pups delivered.
Offspring viability indices:
Birth index (%) = (Number of live pups delivered / number of implantations) × 100
Viability rate on PND 21 (weaning rate)(%) = (Number of live pups on PND 21 / number of live pups on PND 4) × 100
Viability on PND 4 (the survival rate on day 4)(%) = (Number of live pups on PND 4 / number of live pups on PND 0) × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
dosing with >= 450 ppm inhibited body weight gain in both males and females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
dosing with >= 450 ppm inhibited food consumption in both males and females
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
100 ppm: hypertrophy of centrilobular hepatocytes in males and females
>= 450 ppm: dilation of renal proximal tubules in both males and females. Regeneration of the proximal tubular epithelium was also apparent in all groups of males and females, including the control group. The incidence of the renal change was higher in the males receiving 450 ppm or 2000 ppm and in the females given 2000 ppm as compared with the control, and cases with moderate or marked change were included only in the former groups. Regeneration of tubular epithelium was more remarkable in males than females, and was pathologically prevalent around the dilated tubules
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Serum hormone levels: >= 100 ppm: testosterone levels in males showed a tendency to be elevated. No effects were found with regard to the mating index or the fertility index. Furthermore, when testosterone levels were compared between the F0 and F1 males, the dosing was found to have had no biologically significant effect. The change observed in F0 males was judged to be due to the low value in one control male. With serum levels of FSH, LH and estradiol, no change related to the treatment was found in any dose groups of F0 and F1 animals of either sex. In addition, no abnormalities were found in the hormone levels measured in three F1 control and one 100 ppm F1, all of which were non-copulating or non-pregnant.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

see above

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
< 100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOEL
Remarks:
concerning reproduction and endocrine system
Effect level:
> 2 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects at any dose level

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
dosing with >= 450 ppm inhibited body weight gain in both males and females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
dosing with >= 450 ppm inhibited food consumption in both males and females
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
450 ppm: significant increase in relative liver and kidney weights in both males and females
2000 ppm: significantly elevated values were found for both the absolute and relative weights of the liver and kidneys in males and females
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
100 ppm: hypertrophy of centrilobular hepatocytes in males and females
>= 450 ppm: dilation of renal proximal tubules in both males and females. Regeneration of the proximal tubular epithelium was also apparent in all groups of males and females, including the control group. The incidence of the renal change was higher in the males receiving 450 ppm or 2000 ppm and in the females given 2000 ppm as compared with the control, and cases with moderate or marked change were included only in the former groups. Regeneration of tubular epithelium was more remarkable in males than females, and was pathologically prevalent around the dilated tubules
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Serum hormone levels: >= 100 ppm: testosterone levels in males were not significantly different from the control and there were no adverse effects with regard to the mating index or the fertility index. Furthermore, when testosterone levels were compared between the F0 and F1 males, the dosing was found to have had no biologically significant effect. The change observed in F0 males was judged to be due to the low value in one control male. With serum levels of FSH, LH and estradiol, no change related to the treatment was found in any dose groups of F0 and F1 animals of either sex. In addition, no abnormalities were found in the hormone levels measured in three F1 control and one 100 ppm F1, all of which were non-copulating or non-pregnant.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P1)

see above

Effect levels (P1)

open allclose all
Dose descriptor:
NOEL
Effect level:
< 100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOEL
Remarks:
concerning reproduction and endocrine system
Effect level:
> 2 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects at any dose level

Target system / organ toxicity (P1)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
2000 ppm: significant increase on PND 0 in males, decrease on PND 14-21 in males and females and also on PND 7-21 in males and females
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
2000 ppm: significantly elevated relative brain weights and lower absolute spleen weights in both males and females. These changes were considered due to inhibition of the body weight gain caused by the treatment.
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Description (incidence and severity):
Not done as no macroscopic changes were observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

see above

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
450 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
450 ppm: males showed significantly elevated body weights on PND 0
2000 ppm: significant elevation for body weights on PND 0 in males and females, decreases on PND 14-21 in males and females and on PND 7-21 in females
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
2000 ppm: significantly elevated relative brain weights in both males and females. These changes were considered due to inhibition of the body weight gain induced by the treatment.
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Description (incidence and severity):
Not done as no macroscopic changes were observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

see above

Effect levels (F2)

Key result
Dose descriptor:
NOEL
Generation:
F2
Effect level:
450 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F2)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

For details see background material section

Applicant's summary and conclusion

Conclusions:
With regard to the effects of benzophenone on the F0 and F1 parental animals, inhibition of body weight gain and food consumption, significantly elevated renal weights, dilatation of the renal proximal tubules, and regeneration of the proximal tubular epithelium were seen at doses of >= 450 ppm, along with an increase in hepatic weight and centrilobular hepatocytic hypertrophy, considered as vital adaptive changes, at doses of >= 100 ppm. Obvious effects on the endocrine system and reproductive toxicological effects were not found even at the dose of 2000 ppm in the F0 or F1 parent. There were no test substance related changes in the oestrous cycle, reproductive capability, delivery and lactation, sperm parameters, serum hormone levels, or necropsy findings. As for effects on the offspring, inhibition of body weight gain was observed in both the F1 and F2 males and females of the 2000 ppm group. No effects of benzophenone were seen in the number of male and female F1 or F2 pups delivered, viability, anogenital distance (AGD), physical development, the results of reflex and response tests, or on the observation results of external abnormalities.
Executive summary:

The reproductive toxicity of benzophenone was evaluated in a two generation test according to OECD 416, in which male and female Sprague-Dawley (SD) rats, parental (F0) and first generation (F1), were exposed by feeding diets containing 0, 100, 450 or 2000 ppm. From the present study the NOEL for the parental animals is concluded to be less than 100 ppm. Concerning the effects on the endocrine system and the reproductive toxicity in the parental animals, the NOEL is 2000 ppm. In terms of the effects on the offspring, the NOEL is considered to be 450 ppm.