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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Similar to OECD 416. No GLP, but peer reviewed. Only female reproductive indices reported.

Data source

Reference
Reference Type:
publication
Title:
TWO-GENERATION REPRODUCTIVE TOXICITY STUDY OF TRIBUTYLTIN CHLORIDE IN FEMALE RATS
Author:
Ogata et al
Year:
2001
Bibliographic source:
Journal of toxicology and environmental health, 63: 127-144.

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
Animals were bred in an air-conditioned room. In this room, the light cycle was 12 h light/12 h dark, the temperature was 24-26 °C, and the air humidity was 40-80 %. Because of the large number of animals involved, this study was carried out in three different stages. There were no discernible statistical differences observed between these stages. This experiment was reviewed by the Committee on the Ethics of Animal Experiments in the Faculty of Medicine, Kyushu University, and was carried out under the Guidelines for Animal Experiments in the Faculty of Medicine, Kyushu University, and the Law (No. 105) and Notification (No. 6) of the government of Japan.
Male and female Wistar rats (Kud: Wistar) were purchased at 9 wk of age from Kyudo Co., Ltd., Tosu, Japan. During the acclimation period, all rats were provided with CE-2 feed and tap water ad libitum. After a 2-wk acclimation period, the rats were housed as breeding pairs (one male and one female per cage) in polypropylene resin cages with wood shavings as bedding.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
ethanol
Details on exposure:
DIET PREPARATION
- To prepare the diets that contained the test material, the metal was dissolved in a small amount of ethanol (Kanto Chemical Co., Inc., 99.5 % pure) and homogenised with CE-2 feed (Clea Japan, Inc., Tokyo). Ethanol was evaporated by heating during the process of forming the feed pellet. Three test material diets containing 5, 25, or 125 µg/g test material (equivalent to 5, 25, or 125 ppm, respectively) were prepared. Based on the assumption that adult female rats eat 80 g diet/kg body weight/d, the concentrations of the test material chosen were 5, 25, or 125 ppm in the diet, and diets were prepared monthly. The test material diets were stored in vacuum plastic bags at room temperature. To verify the concentration of metal in the diets, the test material diets and the control diet (CE-2 feed) were collected at the beginning and termination of the use and stored at -80 °C until analysis. The concentrations of the test material in the diets were determined by a gas chromatograph equipped with a flame photometric detector (GC-FPD).
Tripentyltin chloride was used as the internal standard, and the concentrations of the test material in the diets were calculated based on the extraction efficiency. Extraction efficiencies, based on the amount of spiked tripentyltin, were 70 to 90 % and the detection limit of the test material concentration was 0.02 ppm.
Details on mating procedure:
After a 2-wk acclimation period, the rats were housed as breeding pairs (one male and one female per cage) in polypropylene resin cages with wood shavings as bedding. Copulation was examined every morning and was confirmed by the presence of a vaginal plug and/or sperm in a vaginal smear. The cohabitation period was 4 d, and only females that had confirmed vaginal plugs during this period were used as parental generation (P generation). From the day when vaginal plug was confirmed (d 0), female rats were housed individually in aluminum cages with wood shavings as bedding.
These rats were randomly assigned to the control or test material treatment group.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
The P generation rats in each metal group were provided with the prescribed the test material diet and tap water ad libitum from d 0 until termination of
the experiment.
The F1 & F2 rats were provided with the same maternal test materiaI diet until the termination of the experiment.
Frequency of treatment:
Ad libitum in diet.
Details on study schedule:
On PND 92, rats in the same treatment groups and from different litters were housed as breeding pairs (one male and one female per cage) in
polypropylene resin cages with wood shavings as bedding. The cohabitation period was 14 d, and females that did not copulate during this period
were euthanised.
Doses / concentrationsopen allclose all
Dose / conc.:
5 ppm (nominal)
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
125 ppm (nominal)
No. of animals per sex per dose:
10-14
Control animals:
yes

Examinations

Parental animals: Observations and examinations:
P generation
The P generation rats in each metal group were provided with the prescribed test material diet and tap water ad libitum from d 0 until termination of the experiment. During the gestational period, body weight was recorded on d 0, 7 and 14, and food consumption was measured between d 7 and 8, and between d 14 and 15. During the lactation period, body weight was recorded 7, 14, and 21 d after delivery, and food consumption was measured between 7 and 8 d after delivery, and between 14 and 15 d after delivery. On the day of weaning of the F1 generation rats (22 d after delivery), the P generation rats were euthanised.
Litter observations:
F1 Generation:
On the day of birth (postnatal d 0, PND 0), live/dead rats were counted, sexed, and examined for gross malformations. On PND 1, body weight and anogenital distance of the rats were recorded and litters were randomly culled to 4 males and 4 females, where possible. The body weight of the rats was recorded on PND 1, 7, 14, and 21, the anogenital distance was recorded on PND 1 and 4, and eye opening was examined from PND 14.
Rats were weaned on PND 22, and female rats were housed with litters in polypropylene resin cages with wood shavings as bedding. The number of rats in each cage was 4 until PND 35, and 2 thereafter. The rats were provided with the same maternal TBTCI diet until the termination of the experiment. Body weight and mean food consumption were recorded weekly. Vaginal opening was examined from PND 30. The body weight on the day of vaginal opening was also recorded, except for the first stage of this study. Estrous cycle was evaluated from PND 71 to PND 92.
On PND 92, rats in the same treatment groups and from different litters were housed as breeding pairs (one male and one female per cage) in polypropylene resin cages with wood shavings as bedding. The cohabitation period was 14 d, and females that did not copulate during this period were euthanized. The treatment 2nd examination of the rats during gestational and lactational periods were the same as those for the P generation rats. On the first day of the estrous stage from PND 148, the F1 generation rats were killed by inhalation of carbon dioxide. Blood was collected from the posterior vena cava, and serum was separated and stored at -80°C. The uterus and ovaries were removed and weighed.

F2 Generation:
The treatment and examination of the F2 generation rats were the same as those of the F1 generation except for the fact that rats in this generation were not mated. One female offspring randomly selected from each litter was killed on the first day of the oestrous stage from PND 92.

Anogenital Distance Evaluation:
Anogenital distance (ACD) was expressed by the absolute ACD (mm) divided by the cube root of body weight (g1/3). The absolute ACD/cube root of body weight was used as an index of ACD in order to remove the influence of body size as a confounding factor of the absolute ACD value.

Oestrous Cycle Evaluation:
A vaginal smear was examined every morning, and the stage of the oestrous cycle was determined based on cytology. The cycle was judged as normal when the cycle length was 4 or 5 d and 4 stages of the cycle (di-oestrous I, di-oestrous II, pro-oestrous, and oestrous) appeared sequentially. The number of cycles during the 21-d evaluation period was counted.

Tissue Preparation and Histopathology of the Ovary:
The ovary was fixed in 10% neutral buffered formalin solution, embedded in paraffin, thinly sectioned, and stained with haematoxylin and eosin. Five random sections of the ovary were examined for normal follicles (primordial, antral and growing follicles) and corpora lutea and follicular atresia. The three types of normal follicles were classified. The follicles with no surrounding granulosa cells or unilaver of granulosa cells were classified as primordial follicles, and the follicles with multilayer of granulosa cells but no antrum formation were classified as growing follicles. Antral follicles were defined as follicles that have apparent antrum formation.

Hormone Determination:
Serum concentrations of 17β-oestradiol and testosterone were measured by radioimmunoassay. The rest kits used were the DPC 17β-oestradiol kit (Diagnostic Products Corporation, Los Angeles, CA) and the DPC total testosterone kit (Diagnostic Products Corporation, Los Angeles, CA). The concentration detection limits of 17β-oestradiol and testosterone were 1.4 pg/mL and 4 ng/dL, respectively. The coefficients of variation for 13.6, 27.4, and 52.5 pg/mL of 17β-oestradiol (the standard solutions of this test kit) were 5.61, 6.59, and 5.85 % for the intraday assay and 11.60, 12.63, and 10.12 % for the interday assay, respectively, in this assay. For each assay, all the samples were randomised prior to analysis.
Statistics:
Statistical analysis of the offspring data during the lactational period was carried out using the litter as a unit. A chi-square test was used for the analysis of fertility index. Linear regression analysis was used to assess the dose dependency for the increase in ACD. Logarithmically transformed test materiall concentrations in diet were used as a representative value of metal dose. In this analysis, 0.02 ppm, the detection limit of TBTCl concentration in diet, was used as the control or background. With other data, statistical differences were analysed with Fisher's least significant difference procedure (Fisher's PLSD) after one-way analysis of variance (ANOVA). The results were interpreted as significant below a level of 0.05.

Results and discussion

Results: P0 (first parental animals)

Reproductive function / performance (P0)

Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The fertility index of females fed the test material diets was not markedly different from control in both generations. The total number of pups and the percentage of live pups decreased significantly in the 125 ppm test material group. Apparent gross malformations were not found in any of the pups. The mean body weight of the female pups on PND 1 also decreased significantly in the 125 ppm Test material group. Food consumption by the pregnant rats in the Test material groups did not differ from the control value in the P and F1 generations. However, gestational body weight gain in the 125 ppm Test material group was significantly less than for the control in both generations.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
5 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at 5 ppm

Results: P1 (second parental generation)

Reproductive function / performance (P1)

Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The fertility index of females fed the test material diets was not markedly different from control in both generations. The total number of pups and the percentage of live pups decreased significantly in the 125 ppm test material group. Apparent gross malformations were not found in any of the pups. The mean body weight of the female pups on PND 1 also decreased significantly in the 125 ppm Test material group. Food consumption by the pregnant rats in the Test material groups did not differ from the control value in the P and F1 generations. However, gestational body weight gain in the 125 ppm Test material group was significantly less than for the control in both generations.

Results: F1 generation

General toxicity (F1)

Description (incidence and severity):
The day (PND) of eye opening in the control, 5, 25, and 125 ppm Test material groups was 15.5 ± 0.5, 16 ± 0.6, 16 ± 0.6 and 16 ± 0.7, respectively, in the F1 generation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the lactational period, body weight gains in the 25 ppm and 125 ppm Test material groups were significantly less than control. However, after weaning, body weights in the 25 ppm Test material group were comparable to control. The body weight in the 125 ppm Test material group was consistently less than control even after weaning.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption in the 125 ppm Test material group initially increased from d 28 to 36 in F1 but returned to control thereafter.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
In the F1 generation, the AGD of all Test material groups was increased significantly compared with control on PND 1. At PND 4, a significant increase in AGD was found only in the 125 ppm Test material group. A dose-effect relationship was observed between the Test material concentration in diet and AGD based on linear regression analysis.
The day of vaginal opening was significantly delayed for approximately 6 d in the 125 ppm test material group compared with control in the F1 generation. However, the body weight on the day of vaginal opening was not increased in this group. In contrast, the body weight in this group was decreased compared with the control value.
The numbers of the oestrous cycle during a 21-d evaluation period were comparable in all groups in the F1 generation. In the control group, approximately 90 % of the oestrous cycle was normal. However, the percentage of the normal cycling was decreased significantly in the 125 ppm test material group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the 125 ppm Test material group, the weight of the ovary was decreased significantly in the F1 generation. In both groups, normal follicles and corpus lutea were observed. Histopathological changes were not found in the ovary of TBT-treated rats in the F1 generation.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no significant changes in serum 17β-oestradiol of F1 and F2 pups. The control 17β-oestradiol levels in serum were approximately 10 µg/mL; serum concentrations of testosterone in all female rats were below the detection limit (4 ng/dl) in this study.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
5 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at 5 ppm

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The day (PND) of eye opening in the control, 5, 25, and 125 ppm Test material groups was 15.1 ± 0.6, 15.2 ± 0.7, 15.4 ± 0.5 and 15.9 ± 0.8, respectively, in the F2 generation. The day of eye opening was significantly delayed in the 125 ppm Test material group in the F2 generation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption in F2 pups was not significantly altered.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
On PND 1 and PND 4, a significant increase in AGD was found only in the 125 ppm Test material group. A dose-effect relationship was observed between the Test material concentration in diet and AGD based on linear regression analysis.

The day of vaginal opening was significantly delayed for approximately 6 d in the 125 ppm test material group compared with control. However, the body weight on the day of vaginal opening was not increased in this group. In contrast, the body weight in this group was decreased compared with the control value.
The numbers of the oestrous cycle during a 21-d evaluation period were significantly decreased in the 125 ppm Test material group in the F2 generation. In the control group, approximately 90 % of the oestrous cycle was normal. However, the percentage of the normal cycling was decreased significantly in the 125 ppm Test material group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The weight of the uterus was increased significantly in the 125 ppm Test material group in the F2 generation. In both groups, normal follicles and corpus lutea were observed.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no significant changes in serum 17β-oestradiol of F1 and F2 pups. The control 17β-oestradiol levels in serum were approximately 10 µg/mL; serum concentrations of testosterone in all female rats were below the detection limit (4 ng/dL) in this study.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Test Material Concentrations in Diet:

The initial and final test material concentrations were similar and resembled the designated concentrations. The control diet contained approximately 0.03 ppm, which is more than 100-fold less than that of the 5 ppm group.

Applicant's summary and conclusion

Conclusions:
These results indicate that the whole-life exposure to the test material affects the sexual development and reproductive function of female rats.
Executive summary:

A two-generation reproductive toxicity study of the effects of the test material was conducted in female rats using dietary concentrations of 5, 25, and 125 ppm. Reproductive outcomes of dams (number and body weight of pups and the percentage of live pups) and the growth of female pups day of eye opening and body weight gain) were significantly decreased in the 125 ppm group. A delay in vaginal opening and impaired oestrous cyclicity were also observed in the 125 ppm group. However, an increase in anogenital distance was found in all test material groups on postnatal d 1. A dose-effect relationship was observed in the test material-induced changes in anogenital distance. These results indicate that the whole-life exposure to the test material affects the sexual development and reproductive function of female rats. In addition, the test material-induced increase in anogenital distance seems to suggest it may exert a masculinising effect on Female neonates.