Benzophenone

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test method similar to OECD 406. No data on GLP.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzophenone

EC Number:

204-337-6

EC Name:

Benzophenone

Cas Number:

119-61-9

Molecular formula:

C13H10O

IUPAC Name:

benzophenone

Details on test material:

- Name of test material (as cited in study report): Benzophenone (BZP)
- Source: benzophenone
- Analytical purity: > 99.98%
- Lot/batch No.: 112D2013

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: (P) 5 wks; (F1) 3 weeks
- Housing: Polycarbonate cages with bedding for laboratory animals.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-25.0 ºC
- Humidity (%): 35-75 %
- Air changes (per hr): 12
- Photoperiod: 12 hrs dark /12 hrs light

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 copulation: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- Poof of pregnancy: existence/absence of delivery or by investigating implantation sites at the time of necropsy.
- Further matings after two unsuccessful attempts: no

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Administration of F0 parental animals started from the 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 (Postnatal day: PND 21). Administration to F1 parental animals was started from the time of weaning (3 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 (Postnatal day: PND 21). Administration to the non-delivery F0/F1 animals continued until necropsy, which was conducted at least 26 days after confirmation of copulation.

Frequency of treatment:

Continous by feeding.

Details on study schedule:

For the F1 parental animals, one male and female each were selected randomly from each litter on postnatal day 21.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 450 and 2000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

24 rats per sex and per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: A dose-range finding was performed. Benzophenone was administered at doses of 0 (control), 600, 2000, 6000 or 20000 ppm for 28 days. Emaciation, reduction in body weight and inhibition of food consumption were observed in the 20000 ppm group. The changes observed in the 6000 ppm group or less included lower values for body weights and food consumption. Elevated hepatic weights were recognized in the 600 ppm or higher groups, with higher renal weights and liver enlargements. The 2000 and 6000 ppm groups showed elevated values for testicular weights. The 6000 ppm group demonstrated lower prostatic weights and higher epididymal weights. A small uterus was found in one animal of the 6000 ppm group. Based on the results the highest dose for the definite study was set at 2000 ppm. The intermediate and lowest doses were defined with a 4.5 ratio (450 and 100 ppm).

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT AND FOOD CONSUPTION: Yes
- Time schedule for examinations: on gestation days 0, 7, 14 and 20, on lactation days 0, 4, 7, 14 and at necropsy.

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):

Sperm motility was measured in 10 animals of the parental animals in each F0 or F1 group. 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. Smear specimens were prepared and examined for morphologically abnormal sperm to calculate the appearance rate.

Litter observations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily throughtout the lactation period

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- Maximum of 8 pups/litter (4)/sex/litter as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F0 / F1 / F2] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other: reflex tests (pain responses, geotaxis, air righting, pinna reflexes)

GROSS EXAMINATION OF DEAD PUPS: Yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced
- Maternal animals: All surviving animals after the last litter of each generation was weaned

GROSS NECROPSY
- All the parental animals sacrified or found dead were necropsied.

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.

HISTOPATHOLOGY
- All males and females of the control and 2000 ppm groups in the F0 and F1 parents: 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.
- All males and females of the 100 and 450 ppm groups in the F0 and F1 parental animals: liver and kidneys.
- Macroscopically abnormal sites
- Any animals that died during the course of the study or sacrificed upon becoming moribund were examined to investigate the causes.

SERUM HORMONE LEVELS
- 6 males in each treatment group of the F0 and F1 parental animals were randomly selected for hormony measurement at necropsy. 6 females in each treatment group in the proestrous stage were randomly selected, left for ca. 1 hour, and were sacrified to collect blood samples by decapitation. Using sera separated from the blood, testosterone, FSH and LH in males and estradiol, FSH and LH in females were measured by the RIA method.

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
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

ORGAN WEIGTHS
- At weaning, 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 set at 5%.

Reproductive indices:

Number. of days until copulation (days), Mating index (%), Fertility index (%), Gestation length (days), Gestation index (%), Sex ratio.

Offspring viability indices:

Birth index (%), Viability rate on PND 21 (weaning rate)(%), Viability on PND 4

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No effects were observed at any dose level.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At 450 ppm or 2000 ppm inhibited body weight gain and food consumption in both F0 and F1 males and females.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The mean daily intakes of the test substance in the group receiving the concentrations (doses) of 0, 100, 450, and 2000 ppm were equivalent to 0, 6.445, 29.01, and 130.0 mg/kg for the F0 male parents, 0, 8.379, 38.15, and 166.5 mg/kg for the F0 female parents, 0, 7.785, 34.60, and 159.4 mg/kg for the F1 male parents, and 0, 8.776, 40.52, and 179.2 mg/kg for the F1 female parents, respectively.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No effects were observed at any dose level.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects were observed at any dose level.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects were observed at any dose level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In the 2000 ppm group, elevated weights of live and kidneys were observed in F0 and F1 males and females. In the 450 ppm group, increase was evident for hepatic and renal weights in F0 males and females and liver and kidney weights in F1 males and females. In the 100 ppm group hepatic weights increased in F0 and F1 females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No changes related to BZP treatment were found in either F0 or F1 males or females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In the 100 ppm group, hypertrophy of centrilobular hepatocytes in the parental F0/F1 males and females was observed. In the 450 ppm or 2000 ppm groups, dilation of renal proximal tubules was observed in both F0 and F1 males and females. Regeneration of the proximal tubular epithelium was also apparent in all groups of F0 and F1 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.

SERUM HORMONE LEVELS (PARENTAL ANIMALS)
In the 100 ppm group (or higher), testosterone levels in the F0 males showed a tendency to be elevated, while values for F1 males were not significantly different from the control, so that no consistent trend was apparent. The change observed 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 BZP treatment was found in any dose groups of F0 and F1 animals of either sex. 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.

Effect levels (P0)

1

Dose descriptor:

LOEC

Remarks:

(Parental toxicity)

Effect level:

100 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: (Equivalent to measured chemical intake of 6.4 to 8.7 mg/kg bw/day for F0 and F1) (Basis for effect: centrilobular hypertrophy at 100 ppm)

Remarks on result:

other: Generation: F0 and F1 (migrated information)

2

Dose descriptor:

NOEL

Remarks:

(Reproductive system)

Effect level:

>= 2 000 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: (Equivalent to measured chemical intake of 130 to 179.2 mg/kg bw/day for F0 and F1) (Basis for effect: No effects were observed at the highest dose)

Remarks on result:

other: Generation: F0, F1, F2 (migrated information)

3

Dose descriptor:

NOEL

Remarks:

(Offspring toxicity)

Effect level:

450 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: (Equivalent to measured chemcial intake of 29 to 40.5 mg/kg bw/day for F0 and F1) (Basis for effect: inhibition of body weights at 2000 ppm)

Remarks on result:

other: Generation: F1 and F2 (migrated information)

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

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Details on results (F1)

VIABILITY (OFFSPRING)
No effects were observed at any dose level.

CLINICAL SIGNS (OFFSPRING)
No effects were observed at any dose level.

BODY WEIGHT (OFFSPRING)
In the 450 ppm group, the F2 male offspring showed significantly elevated body weights on PND 0. In the 2000 ppm group, a significant elevation was noted for body weights on PND 0 in F1 males and F2 male and females offspring. Moreover, significantly low values were found for body weights on PNDs 14-21 in the F1 and F2 male and females offspring and for body weight gain on PNDs 7-21 in F1 male and females and F2 females offspring.

SEXUAL MATURATION (OFFSPRING)
No effects were observed at any dose level.

ORGAN WEIGHTS (OFFSPRING)
In the 2000 ppm group, significantly elevated relative brain weights were observed in both male and female F1/F2 pups, and lower absolute spleen weights were found in both F1 sexes. These changes were considered due to inhibition of the body weight gain. No other effects were observed.

GROSS PATHOLOGY (OFFSPRING)
No effects were observed at any dose level.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In the two-generation reproduction toxicity test in rats, the LOAEL for parental toxicity was determined to be 100 ppm, based on the dose-dependent histopathological findings in liver. Reproductive toxicity was not observed in this study (NOAEL > 2000 ppm) and th effects on the offspring (decreased fetal body weight) were observed at the highest dose only (NOAEL = 450 ppm).

Executive summary:

A two-generation reproduction toxicity test was performed with benzophenone in accordance with an equivalent test method to OECD 416. Male and female Sprague-Dawley rats, parental (F0) and first generation (F1), were exposed to the test item by feeding diet containing at concentrations of 0 (control), 100, 450 or 2000 ppm (corresponding approximately to doses of 6-9, 29-40 and 130-179 mg/kg body weight/day, respectively) . In 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 recognized at doses of 450 ppm and 2000 ppm, along with an increase in hepatic weight and centrilobular hepatocytic hypertrophy. Obvious effects on the endocrine system and reproductive toxicological effects were not observed up to the highest dose of 2000 ppm in the F0 or F1 parent animals (no test substance related changes in the estrous 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, but no other treatment-related effects were observed (in the number of male and female F1 or F2 pups delivered, viability, anogenital distance, physical development, the results of reflex and response tests, or on the observation results of external abnormalities). Based on the dose-dependent histopathological findings in liver of adult rats a LOAEL of 100 ppm (~ 6 mg/kg bw/day) was derived. Reproductive toxicity was not observed in this study (NOAEL for reprotox > 2000 ppm, ~ 130 mg/kg bw/day) and the effects on the offspring (decreased fetal body weight) were only observed at the highest dose (NOAEL = 450 ppm, ~29 mg/kg bw/day).