Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are two key papers used to fulfil this endpoint as both papers were conducted in the same year, are similar in methodology (similar to OECD 416) and are considered to be of equal reliability.

A reliability rating of 2 was assigned to these studies, according to the criteria of Klimisch, 1997 as they are conducted according to comparable OECD methods with some deviations.

Link to relevant study records

Referenceopen allclose all

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

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.

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.

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 but with deviations. No GLP, but peer reviewed. Only male reproductive indices reported.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
not specified
Principles of method if other than guideline:
A 2-generation reproductive toxicity study of the test material was conducted in male rats using dietary concentrations of 5, 25, and 125 ppm to evaluate its effect on sexual development and the reproductive system.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
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 %. 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 Experiments in the Faculty of Medicine, Kyushu University and the Law (No. 105) and Notification (No. 6) of the Government of Japan.
Parent rats (Kud: Wistar) of F1 generation were purchased at 9 weeks of age from Kyudo Co., Ltd., Tosu, Japan. During the acclimation period, the parent rats were provided with CE-2 feed and tap water ad libitum. After a 2-week acclimation period, these rats were housed as breeding pairs (1 male and 1 female per cage) in polypropylene resin cages with wood shavings as bedding.
Route of administration:
oral: feed
Vehicle:
ethanol
Details on exposure:
DIET PREPARATION
To prepare the diets that contained the test material, the test material was dissolved in a small amount of ethanol (Kanto Chemical Co., Inc., 99.5% pure) and was homogenised with CE-2 feed (Clea Japan Inc., Tokyo, Japan). Ethanol was evaporated by heating during the process of forming the feed pellet. 3 test material diets were prepared, that contained 5, 25, or 125 µg of test material per g diet (5, 25, and 125 ppm, respectively). The test material diets were prepared monthly and were stored in vacuum plastic bags at room temperature. The test material was found to be stable in the diets over the duration of diet storage and Concentrations of the test material were within ± acceptable ranges. The concentrations of test material (mean ± SD) were 7.06 ± 4.68, 24.11 ± 4.01, and 123.75 ± 16.50 ppm in the 5, 25, and 125 ppm diets, respectively. The control diet also contained test materiall (< 0.02-0.06 ppm), although the concentration was less than 1/100 compared with that of the 5 ppm test material diet.
Details on mating procedure:
After a 2-week acclimation period, rats were housed as breeding pairs (1 male and 1 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 days. On the day when copulation was confirmed, pregnant female rats were moved into aluminum cages and were housed individually.
These rats were randomly assigned to the control group and the 3 test materiall groups and the rats in each test material group were provided with the prescribed test materiaI diet ad libitum until the day of weaning of the F1 generation rats.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Rats in each test material group were provided with the prescribed test material diet ad libitum until the day of weaning of the F1 generation rats.
The F1 and F2 rats were provided with the same test materiaI diet as their mother until the termination of the experiment.
Frequency of treatment:
Daily
Details on study schedule:
The F1 generation rats were weaned on PND 22 and the male rats were housed on litter bases 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 test material diet as their mother until the termination of the experiment. Body weight and mean food consumption were recorded weekly. On PND 92, rats in the same treatment groups and in the different litters were housed as breeding pairs to be parents of the F2 generation. The cohabitation period was 14 days and the exposure continued during this period. When copulation was confirmed or the 14-day cohabitation period was over, the male rats were housed by sex again and the exposure continued. On PND 319 the F1 generation male-rats were killed by inhalation of carbon dioxide. Blood was collecled from the posterior vena cava and serum was separated and stored at -80 °C. The testes, epididymis, ventral prostate, and seminal vesicle were removed and weighed. The seminal vesicle was weighed without fluid.

The exposure to F1 dams continued during gestation and lactation. The treatment and examination of the F2 generation male rats were the same as those of the F1 generation rats except that the rats in this generation were not mated. One F2 generation male rat randomly selected from each litter was killed on PND 91 and was examined as the F1 generation male rat.

Dose / conc.:
5 ppm (nominal)
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
125 ppm (nominal)
No. of animals per sex per dose:
7-18
Control animals:
yes
Parental animals: Observations and examinations:
On postnatal day 1 (PND 1), body weight and anogenital distance (AGD) of the F1 generation rats were recorded and litters were randomly reduced to 4 males and 4 females, where possible. The body weight of the rats was recorded on PNDs 1, 4, 14, and 21, AGD was recorded on PNDs 1 and 4, eye opening was examined from PND 14, and the testes descent was examined from PND 20.
Statistics:
The study was divided into 3 blocks, although no discernible block effects were observed. Statistical analysis of the offspring data during the lactational period was carried out using the litter as a unit. Cumulative chi-squares test was used for the analysis of the copulation index, fertility index, and the ratio of the rats with abnormal histopathologic finding of the testis. Regarding the other data, statistical differences were analyzed with Fisher's least significant difference procedure after 1-way ANOVA. The results were interpreted as significant below a level of 0.05.
Dose descriptor:
NOAEL
Effect level:
5 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects at 5 ppm
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the 125 ppm Test material group, significant delay of eye opening was also observed (Table 1). Eye opening was delayed for 0.6 day in the F1 generation. In the F1 generation, the body weight of the 25 ppm Test material group was also reduced significantly during the late lactation and eye opening was delayed in this group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight of the 125 ppm test material group was reduced significantly on PND 1 and was consistently reduced during the lactation period in both the F1 and F2 generations (Table 1). The body weight of the 125 ppm Test material group was approximately 70-80 % of the control value in the F1 generation.

Body weights of the F1 and F2 generations after weaning are shown in Figure 1 and food consumptions of the 2 generations are shown in Figure 2. In both the F1 and F2 generations, body weight gain in the 125 ppm Test material group was consistently suppressed. The body weight of the 125 ppm Test material group was approximately 70-80% of the control value in the F1 generation however, food consumption (g/kg body weight/day) was not decreased in this group (Fig. 2). In the F1 generation, body weight gain was also slightly suppressed in the 25 ppm Test material group.

Food consumption and compound intake (if feeding study):
no effects observed
Sexual maturation:
no effects observed
Description (incidence and severity):
AGD was measured on PND 1 and 4 (Table 1). AGD was expressed by the absolute AGD/cube root of body weight in order to correct the influence of body size on the absolute AGD value. In both the F1 and F2 generations, the AGD slightly increased in the 125 ppm test material group, although the increase was not significant. Testes descent was examined from PND 20 (Table 1). Irrespective of the groups, the testes descended into the scrotum on PND 20 in most of rats and there was no difference in the day of testes descent among 4 groups in both the F1 and F2 generations. Body weight gain was remarkably suppressed in the 125 ppm Test material group but testes descent was not delayed in this group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute organ weight rather than a relative organ weight was used to evaluate the testis and epididymis weights because these organ weights were independent of body weight. The weights of the testis, epididymis, and ventral prostate of the rats fed the 125 ppm Test material diet were significantly decreased compared with those of the control rats (Table 2). In addition, the testis weight was decreased significantly in the 5 and 25 ppm test material groups and the epididymis weight was decreased significantly in the 5 ppm test material group in the F1 generation, although the degree of the decreases were only a little (approximately 5 % of the control value). The weights of these organs in this group were more decreased in the F2 generation compared with those in the F1 generation. In the F2 generation, there was a significant decrease in the ventral prostate weight in the 25 ppm Test material group in addition to the 125 ppm Test material group and a dose-effect relationship was observed between the Test material concentration in the diet and the weight (Table 2). This dose-effect relationship was consistently observed in 3 blocks of this study (Fig. 3). In spite of the decrease in the ventral prostate weight, the weight of another accessory sex organ, the seminal vesicle, was not decreased in the Test material-treated rats.
Other effects:
no effects observed
Description (incidence and severity):
In the F1 generation, male rats were mated with female rats in the same treatment group and in the different litter to be parents of the F2 generation. Copulation indexes and fertility indexes in this generation are shown in Table 6. Neither of the indexes was affected by test material treatment.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
5 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects at 5 ppm
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the 125 ppm Test material group, significant delay of eye opening was observed (Table 1). Eye opening was delayed for 1.2 days in the F2 generation in this group.
Description (incidence and severity):
The body weight of the 125 ppm test material group was reduced significantly on PND 1 and was consistently reduced during the lactation period in both the F1 and F2 generations (Table 1). The body weight of the 125 ppm Test material group was approximately 65-75 % of the control value in the F2 generation during this period.
Body weights of the F1 and F2 generations after weaning are shown in Figure 1 and food consumptions of the 2 generations are shown in Figure 2. In both the F1 and F2 generations, body weight gain in the 125 ppm Test material group was consistently suppressed. The body weight of the 125 ppm Test material group was approximately 65-75 % of the control value in the F2 generation. However, food consumption (g/kg body weight/day) was not decreased in this group (Fig. 2).
Food consumption and compound intake (if feeding study):
no effects observed
Sexual maturation:
no effects observed
Description (incidence and severity):
AGD was measured on PND 1 and 4 (Table 1). AGD was expressed by the absolute AGD/cube root of body weight in order to correct the influence of body size on the absolute AGD value. In both the F1 and F2 generations, the AGD slightly increased in the 125 ppm test material group, although the increase was not significant. Testes descent was examined from PND 20 (Table 1). Irrespective of the groups, the testes descended into the scrotum on PND 20 in most of rats and there was no difference in the day of testes descent among 4 groups in both the F1 and F2 generations. Body weight gain was remarkably suppressed in the 125 ppm test material group but testes descent was not delayed in this group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute organ weight rather than a relative organ weight was used to evaluate the testis and epididymis weights because these organ weights were independent of body weight. The weights of the testis, epididymis, and ventral prostate of the rats fed the 125 ppm Test material diet were significantly decreased compared with those of the control rats (Table 2). In addition, the testis weight was decreased significantly in the 5 and 25 ppm test material groups and the epididymis weight was decreased significantly in the 5 ppm test material group in the F1 generation, although the degree of the decreases were only a little (approximately 5 % of the control value). The weights of these organs in this group were more decreased in the F2 generation compared with those in the F1 generation. In the F2 generation, there was a significant decrease in the ventral prostate weight in the 25 ppm Test material group in addition to the 125 ppm Test material group and a dose-effect relationship was observed between the Test material concentration in the diet and the weight (Table 2). This dose-effect relationship was consistently observed in 3 blocks of this study (Fig. 3). In spite of the decrease in the ventral prostate weight, the weight of another accessory sex organ, the seminal vesicle, was not decreased in the Test material-treated rats.
Reproductive effects observed:
not specified

 Sperm Parameters:

Homogenisation-resistant spermatid count was significantly decreased to approximately 80 % of the control value in the 125 ppm Test material group in both the F1 and F2 generations (Table 3). Homogenization-resistant spermatid count was also decreased significantly in the 25 ppm Test material group and this decrease demonstrated a dose-response relationship. In spite of the decrease in homogenisation-resistant spermatid count, sperm count in the cauda epididymidis was not decreased in the 125 ppm Test material group in the F1 generation. However, the sperm count in this group decreased significantly to approximately 80 % of the control value in the F2 generation (Table 3). Sperm motility and sperm morphology were not affected by Test material treatment (Table 3).

 

Histopathology of the Testis:

In the rats fed the control diet, histopathologic changes of seminiferous tubules were rare and Leydig cells appeared normal. Histopathologic changes of the tubules were occasionally observed in the rats of the Test material groups in the F1 generation and these included vacuolization of seminiferous epithelium, spermatid retention in the epithelium, delayed spermiation, and germ cell degeneration near the basement membrane in stages X-XIII (probably degeneration of spermatogonia or leptotene/zygotene spermatocytes). However, the frequencies were low and we did not regard the histopathologic finding of these rats as abnormal. In the F2 generation, more Test material-treated rats showed histopathologic changes of the tubules. The changes were minimal. However, the frequencies of the changes were rather high in some Test material-treated rats and the histopathologic findings of these rats were regarded as abnormal. Several vacuolisations of the epithelia are found. Step 19 spermatids still exist near the tubular lumen and spermiation was slightly delayed. Table 4 shows the frequency of histopathologic changes in the 4 groups. Analysed using cumulative chi-squares test, the increase in the ratio of the rats with abnormal histopathologic findings of the testis was not significant in Test material-treated groups in the F2 generation (p = 0.09). Leydig cell hyperplasia/hypertrophy was not evident even in the 125 ppm Test material group.

 

Serum Concentrations of Testosterone and LH:

Table 5 shows serum concentrations of testosterone, 17β-oestradiol, and LH in the 4 groups. There was a dose-dependent increase in serum testosterone concentration of the rats fed the Test material diets in the Fl generation but serum testosterone concentration did not increase in the F2 generation. Serum LH concentrations of the Test material-treated rats did not increase in the F1 generation but increased dose-dependently in the F2 generation. In spite of the absence of decreases in serum concentrations of LH and testosterone, serum concentration of 17β-oestradiol was decreased in the rats fed the 125 ppm Test material diet. In the F2 generation, serum 17β-oestradiol concentration decreased in the 25 ppm Test material group, too; however, the decrease was not significant (p = 0.09).

Conclusions:
The test material was proved to have male reproductive effects and might be an aromatase inhibitor in rats.
Executive summary:

A 2-generation reproductive toxicity study of the test material was conducted in male rats using dietary concentrations of 5, 25, and 125 ppm to evaluate its effect on sexual development and the reproductive system. F1 males were killed on postnatal day 119 and F2 males were killed on postnatal day 91. The test material affected the male reproductive system of rats. The weights of the testis and epididymis were decreased and homogenisation-resistant spermatid and sperm count were reduced mainly in the 125 ppm group. Histopathologic changes were also observed in the testis of this group and included vacuolisation of the seminiferous epithelium, spermatid retention, and delayed spermiation. However, the changes were minimal in nature. The weight of the ventral prostate was decreased to 84 % of the control value in the 125 ppm group in the F1 generation and decreased to 84 and 69 % of the control value in the 25 ppm and 125 ppm groups, respectively, in the F2 generation. The serum 17β-estradiol concentration was also decreased to 55 % of the control value in the 125 ppm group in the F1 generation and decreased to 78 and 57 % of the control value in the 25 ppm and 125 ppm groups, respectively, in the F2 generation. However, the serum concentrations of luteinising hormone (LH) and testosterone were not decreased in these groups. These changes corresponded with those caused by aromatase inhibition and therefore the test material might be a weak aromatase inhibitor in male rats.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5 mg/kg bw/day
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There are two key papers used to fulfil this endpoint as both papers were conducted in the same year, are similar in methodology (similar to OECD 416) and are considered to be of equal reliability.

The NOAEL in both papers is considered to be 5 ppm for both male and female reproductive indices.

In the Ogata, 2001 paper, 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 estrous 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.

In the Omura, 2001 paper, the test material affected the male reproductive system of rats. The weights of the testis and epididymis were decreased and homogenisation-resistant spermatid and sperm count were reduced mainly in the 125 ppm group. Histopathologic changes were also observed in the testis of this group and included vacuolisation of the seminiferous epithelium, spermatid retention, and delayed spermiation. The weight of the ventral prostate was decreased in the F1/F2 generations. The serum 17β-oestradiol concentration was also decreased in the F1/F2 generations. However, the serum concentrations of luteinising hormone (LH) and testosterone were not decreased in these groups. These changes corresponded with those caused by aromatase inhibition and therefore the test material might be a weak aromatase inhibitor in male rats.

Effects on developmental toxicity

Description of key information

The effects of exposure to low doses of the test material from day 8 of gestation until adulthood were examined. Pregnant rats were gavaged daily with 0, 0.025, 0.25 or 2.5 mg/kg body weight from day 8 of gestation until weaning. Stomach contents of suckling pups contained undetectable levels of TBT and dibutyltin (DBT) levels were detectable only in the highest dose used, indicating negligible lactational transfer to pups. Post weaning, pups were gavaged daily with the same dose of test material administered to their mothers and sacrificed on post-natal days (PND) 30 (males and females), 60 (females) and 90 (males). The test material had no effects on dams body weights, food consumption, litter size, sex ratio or survival of pups to weaning. However, all doses of the test material significantly affected parameters of the growth profile of the pups (mean body weights, average slope, curvature) and the ratio of weekly food consumption to weekly body weight gain indicated enhanced food conversion to body mass in females but a decreased conversion in males. Liver, spleen and thymus weights were also affected by the test material.

In male pups dosed at 2.5 mg/kg/day, reduced serum thyroxine levels were evident, indicating that the thyroid is a target for test material toxicity. No histopathological lesions were seen in the liver but elevated serum alanine aminotransferase, gamma-glutamyl transferase and amylase indicated hepatotoxicity. Significant decreases in liver weights in female pups exposed to 0.025 mg/kg/day were observed at PND 60. Decreases in spleen and thymus weights also pointed towards toxic effects of the test material on the immune system.

The 0.025 mg/kg/day dose should have been a no affect dose and yet this dose caused significant effects on growth profiles, decreased liver weights and elevated serum GGT levels in females, therefore the NOAEL for teratogenic effects is <0.025 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
0.003 mg/kg bw/day
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Oral toxicity:

In the key study (Cooke et al, 2004), the effects of exposure to low doses of the test material from day 8 of gestation until adulthood were examined.

The reliability rating for this study is 2, according to the criteria of Klimisch, 1997 as the study was conducted according to methods comparable to OECD 414 but with deviations.

This study is considered the most relevant and reliable study for this endpoint as the study was conducted most recently, was similar in methodology to OECD guidelines, and the dose levels tested were the lowest when comoared with all of the supporting studies, thus giving the most precautionary NOAEL value for classification purposes.

The following supporting studies are also available for this endpoint:

- In the Ema et al (1995) paper, pregnant rats were given either the test material at a dose of 40 or 80 mg/kg by gastric intubation on days 7 and 8 of pregnancy.

Treatment at 40 and 80 mg/kg caused a significantly decreased maternal weight gain and increased postimplantation embryolethality, no significantly increased incidence of malformed foetuses occurred. No increase in the incidence of foetal malformations was noted after treatment with the test material even at doses that caused a significant decrease in the maternal weight gain and an increase in the postimplantation embryolethality. These findings suggest that the test material administered on days 7 and 8 of pregnancy has no teratogenic effects even at doses that induced maternal toxicity and embryolethality.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was limited information on test material and no information on GLP, the methodolgy is well documented and the article was peer reviewed.

- In the Adeeko et al (2003) paper, the effects of the test material exposure on pregnancy outcome were investigated in the Sprague-Dawley rat. Timed pregnant rats were gavaged either with vehicle (olive oil) or tributyltin chloride (0.25, 2.5, 10, or 20 mg/kg) from days 0–19 or 8–19 of gestation.

Test material administration significantly reduced maternal weight gain only at the highest dose (20 mg/kg); a significant increase in post-implantation loss and decreased litter sizes, in addition to decreased foetal weights, was observed in this group. Test material exposure did not result in external malformations, nor was there a change in sex ratios. However, exposure to 0.25, 2.5, or 10 mg/kg from gestation days (GD) 0–19 resulted in a significant increase in normalised anogenital distances in male foetuses; exposure from days 8–19 had no effect. There was a dramatic increase in the incidence of low weight (<0.75 of the mean) foetuses after exposure to 20 mg/kg. Delayed ossification of the foetal skeleton was observed after in utero exposure to either 10 mg/kg or 20 mg/kg. Serum thyroxine and triiodothyronine levels were reduced significantly in dams exposed to 10 and 20 mg/kg throughout gestation; in dams treated with the test material from GD 8–19, serum thyroxine concentrations, but not triiodothyronine, were significantly decreased at both the 2.5 and 10 mg/kg exposures. Thus, maternal thyroid hormone homeostasis may be important in mediating the developmental toxicity of organotins.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was limited information on test material and no information on GLP, the methodolgy is well documented and the article was peer reviewed.

- In the Ema et al (1996) paper, pregnant rats were given the test material at a dose of 165 or 330 µmol/kg on days 13-15 of pregnancy. Treatment at 165 and 330 µmol/kg resulted in a significant decrease in the maternal weight gain. A significant decrease in the foetal weight was found at 330 µmol/kg. A significantly and markedly increased incidence of foetuses with cleft palate was noted in both groups treated with the test material.

The present study confirms that the test material produces foetal malformations in rats when administered during late organogenesis.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was limited information on test material and no information on GLP, the methodolgy is well documented and the article was peer reviewed.

- In the Ema and Harazono (2001) paper, developmental and reproductive toxicity of the test material was evaluated in rats. The test material was teratogenic when administered on day 8 and days 11-14 of pregnancy, and the most pronounced effect was seen after administration on day 13 of pregnancy. Cleft palate was predominantly observed.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was limited information on test material and no information on GLP, the methodolgy is well documented and the article was peer reviewed.

- In the Ema et al (1995) paper, the objective of this study was to further evaluate the development toxicity of the test material in rats. The manifestation of deviant development induced by the test material varies with the developmental stage at the time of administration and the test material possesses teratogenic potential with developmental phase specificity. The test material is embryolethal when administered on and prior to day 12 of pregnancy and is teratogenic when administered on and after day 10 of pregnancy.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was no information on GLP, the methodolgy is well documented and the article was peer reviewed.

- In the Ema et al (1997) paper, the objective of this study was to determine the susceptible day for the teratogenicity of the test material by a single administration on one of the days during organogenesis.

The results of the present study on the teratogenicity of the test material indicate that cleft palate is the most prevalent malformation induced by the test material in rats. In addition, the present data show that the test material possesses two critical periods for the production of fetal malformations, day 8 and days 11-14 of pregnancy.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was no information on GLP, the methodolgy is well documented and the article was peer reviewed.

- In the Harazono and Ema (2000) paper, the study was designed to assess the effects of the test material on uterine function as a cause of early embryonic loss in pseudopregnant rats. The test material suppresses the uterine decidual cell response and decreases progesterone levels, and these effects are responsible for early embryonic loss due to test material exposure.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was no information on GLP, the methodolgy is well documented and the article was peer reviewed.

- In the Harazono (1996) paper, the effect of the test material administered during early pregnancy on pregnancy maintenance was evaluated in rats. It appears that the test material causes pregnancy failure after administration during very early pregnancy.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was no information on GLP, the methodolgy is well documented and the article was peer reviewed.

- In the Harazono et al (1998) paper, the objective of the present study was to determine the susceptible period for the antifertility effect of the test material in rats.

The test material during early pregnancy induced antifertility and its susceptibility and manifestation depended on the stage when inseminated rats were exposed to the test material. The test material on days 0-3 caused implantation failure and dosage on days 4-7 adversely affected viability of implanted embryos. The preimplantation stage is more susceptible to the antifertility effects of the test material.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was no information on GLP, the methodolgy is well documented and the article was peer reviewed.

- In the Itami et al (1990) paper, the teratogenicity of the test material was examined in Wistar rats. Oral administration to pregnant rats produced statistically significant decreases in both maternal body weight (day 7-15) and food consumption (day 7-15) in the 25, 15, and 9 g/kg/day dose groups. This indicated that the maximum tolerated dose is between 9 and 5 mg/kg/day of the chemical and the maximum internal no-observable-effect-level (NOEL) is considered to be 5 mg/kg/day.

When administered orally to Wistar rats during days 7-15 of pregnancy produced related signs of foetal toxicity but no evidence of teratogenicity and induced a marked increase in placental weight.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was no information on GLP, the methodolgy is well documented and the article was peer reviewed.

- In the Garlund et al (1991) paper, the effects of prenatal administration of the test material (1 and 5 mg/kg) upon the development and behavioural repertoire of rats were studied. Test material treated offspring exhibited d-amphetamine-induced hyperactivity.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was limited information on test material and no information on GLP, the methodolgy is well documented and the article was peer reviewed.

Justification for classification or non-classification

The studies for reproductive and development toxicity indicate the following:

- Effects on female and male fertility (decreases in number of pups, pup bw, %live pups, delay in vaginal opening, impaired estrous cyclicity, effects on AGD in the female study, decreases in testes, epididymes and prostate wt, homogenisation-resistant spermatid and sperm count in the male study)

- Develpmental effects (implantation failure when exposure occurs very early during pregnancy, increase in post-implantation loss a little later, effects on AGD and malformations – mainly cleft palate for exposure later during pregnancy)

 

On these bases the classification for the substance would be considered to be: Repro. Cat. 1B; H360: May damage fertility or the unborn child, according to Regulation (EC) no 1272/2008.