Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is no reproduction/developmental toxicity screening study (OECD TG 421) and no two-generation reproduction toxicity study available for MBTS. A read across approach was conducted with study data from MBT (benzothiazole-2-thiol) (see discussion endpoint summary toxicokinetics).
The findings of the two-generation study in rats demonstrated that MBT did not have any adverse effects on reproductive functions of the F0 or Fl generation. Thus, a dosage level of 15000 ppm ( 15000 ppm = 1071 mg/kg bw/d) was determined to be a No Adverse Effect Level for reproductive toxicity. Minimal to mild toxic effects occurred in all treated groups in both F0 and Fl parental animals. The effects were more prominent in the Fl animals due to a greater intake and longer exposure to the test article.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
A read-across with the available pre-natal developmental toxicity study in the rabbit with the analogue substance benzothiazole-2-thiol (MBT) CAS-No. 149-30-4 is justified based on the ECHA decision, number: CCH-D-2114343759-36-01/F
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
15 000 ppm
Sex:
male/female
Basis for effect level:
other: no biological relevant effects on reproductive function
Dose descriptor:
LOAEL
Effect level:
2 500 ppm
Sex:
male/female
Basis for effect level:
other: body weight reduced
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
15 000 ppm
Sex:
male/female
Basis for effect level:
other: no biological relevant effects on reproductive function
Remarks on result:
other: 15000 ppm = 1071 mg/kg bw/d
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
15 000 ppm
Sex:
male/female
Basis for effect level:
other: no biological relevant effects on reproductive function
Remarks on result:
other: 15000 ppm = 1071 mg/kg bw/d
Reproductive effects observed:
not specified

Analytical Chemistry Data

Homogeneity and stability analyses determined that the test diets were

homogeneous and stable for at least eight days. Analyses for concentration revealed a recovery for the test article within ± 10% of the targeted concentration.

F0 Generation

Clinical Observations and Survival

There were no treatment related clinical signs observed in the MBT groups. Incidental findings occurred in all groups and included primarily loss of hair, scab, dark material around eyes, nose or mouth, and malaligned incisors. Survival was 100% in all study groups.

Body Weights F0 males

Mean body weights, calculated for the males in the 15000 ppm group, were slightly but significantly reduced beginning on study week 2 and continuing until sacrifice (study week 20). In the 8750 ppm group, significant reductions were noted beginning on study week 4 and continued until sacrifice. Mean body weights calculated for the males in the 2,500 ppm group were comparable with the control group throughout the study.

Mean body weight gain, however, did not parallel the reduction noted in the body weights. A slight but significantly reduced weight gain occurred during the first week of treatment in all MBT groups. Thereafter, significantly reduced gain occurred in the 15000 ppm group from study week 4 through week 8 and between weeks 17-18. Statistically significant reductions occurred inconsistently in the low and mid dosage groups (weeks 4-5, 7-8, 17-18 in the 8750 ppm group; weeks 2-3, 7-8, 17-18 in the 2500 ppm group). Body weight loss was noted in all groups between study weeks 14-16 when the males were used to breed females for a dominant lethal evaluation (see endpoint genetic toxicity in vivo, CMA 1989); during these two weeks, the food was removed for the time the females were with the males (from 4:00 p.m. to midnight).

Body Weights F0 females

Mean body weights, calculated for females, were slightly but significantly decreased in the 15000 ppm group during study weeks 3, 4, 6 and continuing until the breeding period (study week 11). Mean body weight was also slightly reduced in the high dosage group during gestation, but did not reach significance until day 20. Lactation body weights were slightly decreased throughout this period with statistical significance noted on days 1 and 14. Additionally, mean body weights were significantly reduced in the high dosage group following lactation and prior to sacrifice (study weeks 19 and 20). The female rats in the 8750 ppm group had body weights slightly but significantly reduced from week 4 to 8, and during week 20; gestation and lactation body weights in the mid dosage group were similar to those in the control group. Body weights in the 2500 ppm group were comparable to the control group throughout the study. Mean body weight gain calculated for the female rats did not parallel the pattern seen in body weights. Dose-dependent and significantly reduced weight gain occurred in the 8570 and 15000 ppm groups during the first week of treatment. Other significant reductions of weight gain occurred sporadically in all MBT groups and were not considered toxicologically significant between study weeks 7-8 in the 2500 and 8750 ppm groups, and between gestation days 0-7 in the 2500 and 15000 ppm groups. During lactation, body weight gains were, in general, higher in the MBT groups than in the control group; significant weight loss was noted, however, in the 15000 ppm group following lactation (study weeks 18-19).

Food Intake F0 males

Food consumption, calculated as g/animal/day, was slightly but significantly reduced for the males in the 15000 ppm group between study weeks 1-2 and from week 4 through week 11; significant reduction of food intake was also noted from week 18 until sacrifice (week 20). A slightly but significantly reduced food consumption was evident in the 8750 ppm group between study weeks 1-2 and 18-19 only. No statistically significant differences were observed in the 2500 ppm group. Calculation of food intake as g/kg/day exhibited a different pattern. Dose-dependent and significantly reduced food consumption occurred in the 8750 and 15000 ppm groups during the first week of treatment. Thereafter, food intake in the mid and high dosage groups was statistically significantly greater or similar to the control group.

Food Intake F0 females

Food intake, calculated as g/animal/day, was slightly reduced prior to breeding for females in the 15000 ppm group; these differences were statistically significant for weekly intervals 1-2, 3-4, 5-6 and continuing until breeding (weeks 10-11). Significant reductions also occurred between gestation days 0-7, lactation days 14-21, and following lactation until sacrifice (weeks 18 to 20). Food intake was comparable between the control, 2500 and 8750 ppm groups, when calculated as g/animal/day. When calculated as g/kg/day, on the other hand, mean food intake was significantly reduced in the 8750 and 15000 ppm groups during the first week of treatment, in a dose-dependent fashion. Thereafter, a statistically significant increase or decrease occurred inconsistently in these two groups as a result of body weight changes. Food consumption (g/kg/day) in the 2500 ppm group was comparable to the control group throughout the study.

Test article intake F0 males and females:

Test article intake, calculated as mg/kg/day based on the food consumption and body weights, decreased gradually for males from the first week of treatment to sacrifice (e.g., in the 15000 ppm group, test article intake during the first week was 1328 mg/kg/day decreasing to 778 mg/kg/day during the last week). A similar pattern was evident for the females prior to breeding (e.g., in the 15000 ppm group, test article intake decreased from 1,326 mg/kg/day to 945 mg/kg/day). During gestation, test article intake was slightly higher than the intake prior to breeding but remained constant throughout this period. Test article intake increased gradually during lactation (e.g., in the 15000 ppm group, intake increased from 1760 mg/kg/day during the first week to 2828 mg/kg/day the last week). The apparent increase in food intake in the second and the third weeks of lactation was due to the fact that the pups started feeding on the diet, therefore, the increased test article intake was not real for the dams. Following lactation (when the dams were without pups), the test article intake returned to the values prior to breeding.

Fertility Data (F0)

Copulation and fertility indices were comparable between the control and MBT groups for both males and females. The precoital interval (approximately 3 days) and gestation length (average of 22 days) were also similar in all study groups. There were no difficulties encountered at parturition in any of the study groups.

Pathology F0

There were no treatment-induced gross lesions observed in any MBT groups. Incidental findings were noted mainly in the kidneys, testes, pituitary, and thymus in one or two rats in various study groups. There was no evidence of infectious diseases which could have had an impact on the outcome of the study.

Treatment-related microscopic changes were seen in the kidneys of both males and females in the 8750 and 15000 ppm groups consisting of brown pigment in the lumen and epithelial cells of the proximal tubules.

Brown pigment occurred with a higher incidence in males than in females: 12 and 17 males in the 8750 and 15000 ppm groups, respectively, while only 1 and 4 females had the pigment in these same groups. In addition, male rats from all MBT groups had an increased incidence of basophilic tubules (2, 9, 10 and 13 in the control, 2500, 8750 and 15000 ppm groups, respectively) and alpha 2 µ-globulin inclusions in the proximal convoluted tubules (4, 10, 8 and 13 in the same groups), as evidenced by hematoxylin-eosin staining.

Histopathological changes seen in the kidneys of the males from the mid and high dosage groups correlated with a significantly increased organ weight, absolute and relative to final body weight. Absolute kidney weight measured for females in the MBT groups was comparable to the control groups. A significant increase in the relative kidney weight noted for the females in the mid and high dosage groups occurred as a result of a reduced final body weight in these two groups rather than a direct effect on the kidneys.

Mean absolute liver weight was comparable between the control and treated groups for both males and females. However, mean relative weight appeared significantly increased in the 8750 and 15000 ppm male and female groups. This increase is a result of reduced final body weights for both males and females in these two groups and not a direct effect on organ weight.

Mean absolute testes weight was significantly increased in the 2500 and 8750 ppm groups but not in the 15000 ppm group. These differences were not dose-dependent and similar effects were not evident in the males of the Fl generation. In addition, historical control data show a mean testes weight of 3.3 g with a range of 3.1 to 3.7 g.

Therefore, a mean testicular weight of 3.67 g in the low dosage group and 3.70 g in the mid dosage group were within the historical control range, indicating that the apparent testicular weight increase in these two groups is not toxicologically relevant. Mean testes weight relative to the final body weight appeared significantly increased in all MBT groups.

Since a dose-dependent response was not evident and similar effects were not seen in the Fl generation, increased testicular weight was not considered treatment-related. Furthermore, historical control data show a mean relative testes weight of 0.655, 0.689, and 0.665 in the low, mid and high dose groups, respectively, were well within the range of historical control data.

F1 generation

F1 Pup Viability Mean litter size was comparable between the control and MBT groups. Pup viability was similar in the control and treated groups with one exception: survival percentage was significantly reduced in the 8750 ppm group on day 4 prior to standardization of litter size. This occurrence was not considered related to treatment since a similar effect was not evident at the higher dosage level. There were no other significant differences noted between pup survival in the control and MBT groups throughout lactation.

F1 Pup Observations

There were no treatment-related findings noted in the MBT groups. Incidental findings included hypothermia, subcutaneous hemorrhage, laceration, and scab noted in all groups. Other findings, commonly seen in pups, included paleness in the mid dose group, apparent umbilical hernia in the low dose group, thin hair coat in the low, mid and high dose groups, and dome-shaped head in the control group.

F1 Pup Body Weight

Mean pup weight at birth was comparable between the control and treated groups. However, mean body weights were slightly reduced in the 8750 and 15000 ppm groups on lactation day 7 and significantly decreased on days 14 and 21. These reductions may be related to MBT effects on maternal animals and to a reduced pup food intake during the last weeks of lactation (e.g., food intake for F0 females was 72, 68, 69 and 66 g/animal/day between lactation days 14-21 in the control, low, mid and high dosage groups, respectively). Mean body weights in the 2500 ppm group were similar to the control group on lactation days 1, 4, 7 and 14 and slightly but not significantly reduced on lactation day 21.

F1 pups Necropsy Observations

There were no treatment-induced lesions noted in the pups which died during lactation or were necropsied after lactation day 21. Incidental findings occurred with low frequency and included distended ureter in the control and mid dose groups, apparent pulmonary atelectasis in all groups, pale liver in the control group, micrognathia and microphthalmia in the mid dose group, hydrocephaly and dilated renal pelvis in the control group.

Fl Generation Adults

F1 Clinical Observations and Survival

There were no treatment-related clinical signs observed in the MBT groups. Incidental findings were noted in all study groups. The most

common findings, observed in both males and females, included loss of hair, scabbing, dark material around the eyes, lacrimation, red ocular discharge and malaligned incisors. All animals in the control and treated groups survived to the scheduled sacrifice.

F1 males Body Weights

Weekly measurement of body weights and food consumption was initiated for the Fl generation approximately one week following weaning, which corresponds to week 18 of the study (from initiation of treatment for F0 generation). A statistically significant decrease of body weight was evident in the males in the 8750 and 15000 ppm groups throughout the study. It should be noted however, that mean body weight on study week 18 was dose-dependent and significantly reduced in these two groups. In the 2500 ppm group, significantly decreased body weights were noticeable from study week 20 through study week 25; thereafter, mean body weights in this group were similar to the control group. Mean body weight gains, on the other hand, were significantly reduced in the 8750 and 15000 ppm groups between study week 18 to week 23 and between weeks 31-32. For the remainder of weekly intervals, mean weight gains in the mid and high dosage groups were comparable to the control group with the exception of a significantly increased gain in both groups between study weeks 32-33 and in the 8750 ppm group between study weeks 36-37. Meanweight gain in the 2500 ppm group was similar to the control group,except for a single incidence of significantly reduced gain between study weeks 20-21.

F1 females Body Weights

Dose-dependent and statistically significant reduced mean body weights were also evident for the females in the 8750 and 15000 ppm groups for the entire treatment period prior to gestation (from study week 18 to week 30). Mean body weight continued to be reduced in the 15000 ppm group throughout gestation (study weeks 31-33) and nearly the entire lactation period (study weeks 34-36); mean body weights were comparable to the control group during the last week of lactation and immediately following lactation (study week 37). A statistically significant reduction of body weight occurred again in the high dosage group during the last week of treatment (study week 38). Mean body weights were reduced in the 8750 ppm group during gestation with statistical significance showing on days 0, 7 and 20 (study weeks 30-31, 31-32 and 33-34, respectively). Mean body weights were also reduced in the mid dosage group on lactation days 1 and 14 (study weeks 34-35 and 35-36), and the last week of treatment (study week 38). In the 2500 ppm group mean body weights were significantly reduced for nearly all weekly intervals prior to gestation. Mean body weights in the 2500 ppm group were similar to the control group throughout gestation, lactation and until sacrifice.

Mean body weight gain, on the other hand, were comparable between the control and treated groups prior to gestation with very few exceptions. A statistically significantly reduced gain in the 8750 ppm group was noted between study weeks 20-21 and in the 2500 ppm group between study weeks 23-24. Mean gestation weight gain was similar in the control and treated groups with the exception of a significant reduction noted in the 15000 ppm group during the last week of gestation (study weeks 33-34). During lactation, mean weight gain was significantly increased in the 15000 ppm group between days 14-21, in the 8750 ppm group between days 1-7 and 14-21 (study weeks 34-35, 36-37, respectively) and similar between the control and 2500 ppm groups. Following lactation (study weeks 37-38), a body weight loss was noticeable in all groups, including the control; mean weight loss was statistically significant in the low and mid dosage groups.

F1 males Food Intake

Mean food consumption, calculated as g/animal/day, was slightly but significantly reduced for males in the 8750 and 15000 ppm groups beginning on study week 21 and continuing until weeks 27 and 28, respectively. Mean food intake was comparable to the control group for the remaining weekly intervals. Mean food consumption in the 2500 ppm group was similar to the control group throughout the study. Food intake, calculated as g/kg/day, was dependent on body weight and revealed a different pattern. A slight but significant increase was evident in the 15000 ppm group for nearly all weekly intervals. Mean food intake in the 8750 ppm group was also slightly but significantly increased from study week 18 to week 21, and from weeks 24 to 25, 28 to 30, and 33 to 38. A single incidence of statistically increased food intake occurred in the 2500 ppm group between study weeks 28-29.

F1 females Food Intake

Food consumption, calculated as g/animal/day, was very slightly but significantly reduced for females in the 15000 ppm group prior to breeding, from study weeks 18 to 19, 21 to 23, and 29 to 30. Mean food intake was also very slightly but significantly reduced in the 8750 ppm group from study week 21 to week 23. Food consumption during gestation was similar between the mid, high and control groups. Lactation food intake in these two groups was, in general, comparable to the control group with the exception of a significant decrease in the high dosage group between days 14-21 (study weeks 36-37). Following lactation (study weeks 37-38), mean food intake was slightly but significantly reduced in the 8750 and 15000 ppm groups. Mean food intake in the 2500 ppm group was comparable to the control group except for a single incidence of significantly reduced food consumption between lactation days 14-21. Food intake, calculated as g/kg/day on the other hand, was significantly increased in the 15000 ppm group for nearly all weekly intervals prior to breeding. In the 8750 ppm group, mean food intake was also slightly increased with statistical significance noted from study weeks 18 to 20 and from week 24 to week 30. Gestation food intake was slightly increased in the mid and high dosage groups, with statistical significance showing between days 0 and 14 (study weeks 31 to 33) in the 15000 ppm group, and in the 8750 ppm group during the last week of gestation (study weeks 33-34). During lactation, food intake was slightly but significantly reduced in the 15000 ppm group between days 14-21 (study weeks 36-37), and increased in the 8750 ppm group from day 1 to 7 (study weeks 34-35).

Mean food intake in the 2500 ppm group was comparable to the control group with the exception of a significant increase between study weeks 24-25.

Test article intake F1 males and females:

Test article intake, calculated for males, decreased gradually from study week 18 until sacrifice on study week 38 (e.g., in the 15000 ppm group, test article intake decreased from 2633 mg/kg/day during week 18 to 779 mg/kg/day during week 38). A similar pattern was evident for females prior to breeding (e.g., in the 15000 ppm group, test article intake was 2615 mg/kg/day during week 18 decreasing to 980 mg/kg/day during week 30). Test article intake increased slightly during the first week of gestation and remained constant for the following two weeks.

During lactation, test article intake increased gradually in all groups (e.g., in the 15000 ppm group, intake increased from 1770 mg/kg/day in the first week to 2877 mg/kg/day in the last week). It should be noted that the pups began eating the diet at approximately two weeks of age, thus, the remarkably increased test article intake is not real for the dams.

Fertility Data F1 generation:

Copulation and fertility indices were similar in the control and treated groups for both males and females. The precoital interval was approximately three days in all groups and gestation length averaged 22 days.

Pathology F1 generation:

There were no treatment-induced gross lesions observed in the Fl parental animals. Gross lesions, commonly seen in rats, occurred in al groups with a very low frequency and included small epididymides, pitted kidneys and dilated renal pelvis, enlarged lymph nodes, corneal opacity, ovarian cyst, white splenic foci and enlarged thyroid.

Microscopic lesions were observed in the kidneys of males and females from the 8750 and 15000 ppm groups. Renal lesions were similar to those seen in the F0 parental animals. The incidence of brown pigment in males was 13 and 20 in the mid and high dosage groups, respectively, while in females, the incidence was much lower, 0 and 6 in the same groups. Cortical tubular basophilia occurred in all groups but the incidence was greater in the males of the high dosage group (0, 3, 4 and 10 in the 0, 2500, 8750, and 15000 ppm, respectively). Alpha 2 µ-globulin inclusions in the epithelial cells of the proximal convoluted tubules occurred in males of all MBT groups, with a higher incidence than in the control males. Histopathological findings correlated with an increase in absolute kidney weight noted in males from the 8750 and 15000 ppm groups, with statistical significance showing at the high level only. Relative kidney weight was significantly increased in both mid and high dosage groups. Absolute kidney weight for females in the treated groups was similar to the control group. Relative kidney weight on the other hand, was statistically significantly increased in all MBT groups due to a decreased final body weight in these groups, and thus was not considered toxicologically significant.

Other treatment-related changes, consisting of hepatic parenchymal hypertrophy, occurred in males and females from the 8750 and 15000 ppm groups. The incidence of this finding was greater in the males (0, 1, 22 and 23 in the 0, 2500, 8750 and 15000 ppm groups, respectively) than in the females (0, 0, 5 and 10 in the same groups). One male in the low dosage group exhibited hepatocyte hypertrophy. Since this change is seen occasionally in untreated rats and occurred in a single incidence, it was not considered related to MBT treatment. Histopathological changes correlated with a dose-dependent and a statistically significant increase in the absolute liver weight for males in the mid and high dosage group and for females in the high dosage group. Relative liver weight appeared statistically significantly increased for males in all MBT groups and for females in the mid and high dosage groups.

A statistically significant increase in relative testes weight was noted in the 15000 ppm group. This effect was related to reduced final body weight rather than a toxic effect on the testes. Furthermore, historical control data show a mean relative testes weight of 0.765 with a range of 0.637 to 1.023. The mean value in the high dosage group was 0.672, well within this range, while the mean control value of 0.610 was unusually low. Thus, the effect noted on relative testes weight was not toxicologically relevant. Relative ovaries weight was statistically significantly increased in the 8750 ppm group. The lack of a dose-response or an effect on absolute weight indicates that this increase is not treatment-related.

F2 Pup Viability

The number of dead pups on lactation day 0 was statistically significantly increased in the 15000 ppm group (2/22, 7/24, 8/23 and 9/23 dead pups per litter in the 0, 2500, 8750, and 15000 ppm groups, respectively). Historical control data show a mean number of dead pups per litter of 0.6 with a range of 0.2 to 1.2. It is evident that the number of dead pups per litter was unusually low in the control group (0.09). Furthermore, the number of dead pups per litter in the high dosage group (0.39) is well within the range of historical control data.

Thus, the statistically significant increased number of dead pups in the high dosage group is not indicative of reproductive toxicity.

F2 Pup Observations:

There were no treatment-related findings noted in the MBT groups. Incidental findings included mostly subcutaneous hemorrhage, hypothermia, laceration, and thin hair coat and occurred with similar incidences in the control or treated groups.

F2 Pup Body Weights:

Mean pup weight was similar in the control and MBT groups on lactation days 1, 4 and 7. Dose-dependent and significantly reduced body weights were noted in all treated groups on lactation days 14 and 21.

These effects may be related in part to the MBT effects on maternal animals and in part to reduced food intake by pups during the second half of lactation, when pups start eating the diet offered to dams (e.g., food intake between lactation days 14-21 was 71, 66, 68 and 63 g/anima]./day for the Fl females in the control, low, mid and high dosage groups, respectively).

F2 Necropsy Observations:

There were no treatment-related gross lesions seen in the pups which died during lactation or were sacrificed on lactation day 21.

Incidental findings included pulmonary atelectasis in all groups, distended ureter in the control and low dose groups, exencephaly and anophthalmia in the low dose group, and pale liver in the low and high dose groups.

In summary:

The potential reproductive toxicity of MBT was evaluated in this two-generation study in rats. There was no evidence of adverse reproductive effects in the F0 or Fl generation, following treatment with MBT for a minimum of 70 days prior to breeding, during breeding and continuing until sacrifice. Reproductive parameters, including precoital interval, and copulation and fertility indices, pregnancy percentage and gestation length, were similar in the control and treated groups of both the F0 and Fl generations. Litter size and litter viability were not affected by the administration of MBT to parental animals.

Nevertheless, mild toxic effects were noted in animals of the F0, Fl and F2 generations. A significant and dose-dependent reduction of body weight gain occurred during the first week of treatment in F0 males from all MBT groups and females from the 8750 and 15000 ppm groups.

Weight gain continued to be slightly reduced in F0 males for approximately ten weeks. F0 females from all MBT groups had a reduced weight gain during the first week of gestation, with statistical significance noted in the low and high dosage groups. Food intake, calculated as g/kg/day was significantly reduced in the 8700 and 15000 ppm groups of the F0 generation during the first week of treatment. Thereafter, food intake was equal to or greater than in the control group. Similar effects on body weights and food consumption were also evident in the Fl and F2 generations. Body weights were reduced in the Fl and F2 animals from all MBT groups in a dose-dependent fashion, beginning on lactation day 14.

Food consumption calculated as g/kg/day, was greater in the Fl parental animals from the treated groups than in the control group.

Histopathological changes occurred in the kidneys of males and females from both F0 and Fl generations. Brown pigment was observed in the lumen and epithelial cells of the proximal convoluted tubules with a greater incidence in males than in females. The presence of brown pigment in the lumen suggests a renal route of excretion rather than a toxic effect. Cortical tubular basophilia and alpha 2 µ-globulin inclusions were seen with higher frequency in the males from the treated groups than in the control group.

The authors suggested:

Tubular basophilia, commonly seen in repeated-dose studies, is most characteristic of chemically-induced nephropathies. The kidney, like the liver, is capable of metabolizing various chemicals and it is a major route of excretion of xenobiotics. Nuclear, and more frequently, cytoplasmic inclusions are relatively common changes seen in toxicological studies. In general, renal inclusions are not suggestive of degenerative or physiological alterations. The accumulation of alpha 2 µ-globulin in the proximal tubules, however, leads to necrosis. This type of change is unique to male rats and occurs following exposure to diverse hydrocarbons. It is possible that MBT metabolites are associated with alpha 2 µ-globulin, indicating a common mechanism for this phenomenon. Alpha 2 µ-globulin is synthesized by the liver of male rats under androgenic control. Nevertheless, alpha 2 µ-globulin is not found in humans and the male-specific nephrotoxicity does not predict the induction of similar nephropathy in humans.

Histopathological changes seen in the kidneys, correlated with a significantly increased organ weight, absolute and relative to final body weight. Other treatment-related lesions were seen in the liver from Fl males and females from the 8750 and 15000 ppm groups and consisted of hepatocyte hypertrophy. The incidence of hepatic parenchymal hypertrophy was greater in the males than in the females. Hepatocyte hypertrophy is a result of an increased number or size of various organellae, including mitochondria, endoplasmic reticulum, ribosomes, and occurs during biotransformation of xenobiotics (hydrocarbons, pesticides, diverse drugs) or endogenous compounds.

Hypertrophy appears to be a response to an increased workload due to continuous intake of MBT. It should be noted that the test article intake was greater in the Fl generation than in the F0 animals since the dietary consumption of MBT by Fl pups started at approximately 14 days of age.

Hepatic parenchymal hypertrophy correlated with a significant increase of absolute and relative organ weight for the males in both mid and high dosage groups, and for females in the high dosage group only. There were no other treatment-induced changes in any of the organs examined microscopically from rats in the MBT groups of either the F0 or Fl generation.

The authors stated that the results of this two-generation study in rats demonstrated that MBT did not have any adverse effects on reproductive functions of the F0 or Fl generation. Thus, a dosage level of 15,000 ppm was determined to be a no adverse effect level for reproductive toxicity. Minimal to mild toxic effects occurred in all groups in both F0 and Fl parental animals. The effects were more prominent in the Fl animals due to a greater intake and longer exposure to the test article.

Conclusions:
The authors stated that the results of this two-generation study in rats demonstrated that MBT did not have any adverse effects on reproductive functions of the F0 or F1 generation. Thus, a dosage level of 15,000 ppm was determined to be a no effect level for reproductive toxicity. Minimal to mild toxic effects occurred in all groups in both F0 and F1 parental animals. The effects were more prominent in the Fl animals due to a greater intake and longer exposure to the test article.
Executive summary:

The authors stated that the results of this two-generation study in rats demonstrated that MBT did not have any adverse effects on reproductive functions of the F0 or F1 generation. Thus, a dosage level of 15,000 ppm was determined to be a no effect level for reproductive toxicity. Minimal to mild toxic effects occurred in all groups in both F0 and F1 parental animals. The effects were more prominent in the Fl animals due to a greater intake and longer exposure to the test article.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 071 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP gudieline study
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 is no reproduction/developmental toxicity screening study (OECD TG 421) and no two-generation reproduction toxicity study available for MBTS. A read across approach was conducted with study data from MBT (benzothiazole-2-thiol) (see discussion endpoint summary toxicokinetics).

Read across with MBT

A two-generation reproductive toxicity study was performed to evaluate the potential effects of MBT on reproduction and development in Sprague-Dawley rats (CMA 1990). The study was designed to determine if MBT has any adverse effects on reproduction functions when fed to F0 and Fl parental animals for a minimum of 70 days prior to mating and continuing until sacrifice. Groups of 28 male and 28 female Sprague-Dawley rats were fed the basal diet or diet containing MBT at concentrations of 2500, 8750, and 15000 ppm. The food was provided ad libitum throughout the study. All F0 and Fl rats were observed daily for signs of overt toxicity, morbidity, or mortality. Body weights were measured weekly for the males throughout the study. For females, body weights were measured weekly prior to confirmation of mating and at specified intervals during gestation and lactation. Food intake was measured on the same days as body weights with the exception of periods of cohabitation when food intake was not measured. F0 and Fl parents were sacrificed and necropsied following weaning of their offspring. Selected tissues and organs were fixed in 10% neutral buffered formalin. Microscopic examination was performed on all tissues collected from the control and 15000 ppm groups. Kidneys from the F0 rats in the other treated groups and liver and kidneys from the Fl rats were also examined microscopically. Liver, kidneys, testes or ovaries from all F0 and Fl parents sacrificed for the scheduled necropsy were weighed. Fl and F2 pups were examined on lactation days 0, 4, 7, 14, and 21. Viability was determined daily. Litter size was reduced to eight pups (four males and four females when possible) on lactation day 4. Body weights were measured on lactation days 1, 4, 7, 14, and 21. Pups dying during lactation were necropsied with special attention given to morphological anomalies. Selection of the Fl generation was performed at weaning. Twenty-eight males and 28 females were selected randomly from each group. Non-selected pups were sacrificed and necropsied. F2 pups were sacrificed and necropsied on lactation day 21. Survival was 100% in all F0 and Fl parental animals. There were no treatment-related clinical signs observed in any of the MBT groups. Food intake, calculated as g/kg/day, was significantly reduced in the 8750 and 15000 ppm groups of the F0 generation during the first week of treatment. Thereafter, food intake was equal to or greater than in the control group. Body weight gain, however, was dose-dependent and significantly reduced in F0 males from all MBT groups and females from mid and high dosage groups during the first week of treatment. Weight gain continued to be slightly reduced for approximately ten weeks for males but not for females. Body weights were significantly reduced in the Fl pups from the mid and high dosage groups and in F2 pups from all MBT groups beginning on day 14 of lactation. Body weights were slightly reduced for Fl pups in the low dosage group and reached statistical significance following weaning. Treatment-related histopathological changes were seen in the kidneys of both F0 and Fl animals. Brown pigment was observed in the lumen and epithelial cells of the proximal convoluted tubules in males and females in the mid and high dosage groups, with a greater incidence in the males than in females. Cortical tubular basophilia and alpha 2 µ-globulin inclusions in the epithelial cells of the proximal convoluted tubules occurred in males from all groups with a higher incidence in the treated groups. In addition, absolute and relative kidney weights were significantly increased for F0 and F1 males in the mid and high dosage groups. Microscopic changes, consisting of hepatocyte hypertrophy, occurred in the livers of the Fl animals from the 15000 and 8750 ppm groups, at a higher incidence in males than in females. The increased workload due to continuous exposure to the test article, apparently led to hepatic hypertrophy. Furthermore, histopathological changes correlated with a significant increase of liver weight in the males in the mid and high dosage groups and for females in the high dosage group. Hepatic lesions, noted in the Fl animals are probably related to a greater intake of the test article, since Fl pups started to eat the test diet at approximately 14 days of age, while the F0 animals were administered MBT beginning at seven weeks of age. There were no other treatment-induced changes in any of the organs examined microscopically from rats in the MBT groups of either the F0 or F1 generation. There was no evidence of adverse reproductive effects in the F0 or Fl generation, following treatment with MBT for a minimum of 70 days prior to breeding, during breeding and continuing until sacrifice. Reproductive parameters, including precoital interval, and copulation and fertility indices, pregnancy percentage and gestation length, were similar in the control and treated groups of both the F0 and Fl generations. Litter size and litter viability were not affected by the administration of MBT to parental animals.

The authors concluded that a dosage level of 15000 ppm was considered a No Adverse Effect Level for reproductive toxicity. Minimal to mild toxic effects were observed in parental animals from all MBT groups. These effects were more prominent in the Fl generation due to a greater intake and longer exposure to the test article.


Short description of key information:
There is no reproduction/developmental toxicity screening study (OECD TG 421) and no two-generation reproduction toxicity study available for MBTS. A read across approach was conducted with study data from MBT (benzothiazole-2-thiol) (see discussion endpoint summary toxicokinetics).
The findings of the two-generation study in rats demonstrated that MBT did not have any adverse effects on reproductive functions of the F0 or Fl generation. Thus, a dosage level of 15000 ppm was determined to be a No Adverse Effect Level for reproductive toxicity. Minimal to mild toxic effects occurred in all treated groups in both F0 and Fl parental animals. The effects were more prominent in the Fl animals due to a greater intake and longer exposure to the test article.

Effects on developmental toxicity

Description of key information

The developmental toxicity potential of MBTS was evaluated in Wistar rats (Ema 1989). Pregnant rats (15 to 19 per dose) were given MBTS at a dosage of 0, 0.04, 0.2 or 1% in the diet from day 0 to day 20 of pregnancy. The dams were scheduled sacrificed on day 20 of pregnancy. In an additional approach dams (5 to 6 per dose group) were allowed to deliver spontaneously. These dams were killed on day 21 after birth. The offspring of these dams were scheduled sacrificed seven weeks after birth.
No significant compound–related effects on the incidences of pre- and post-implantation losses and the number, sex ratio and body weight of live foetuses were noted. Morphological examinations of the foetuses revealed no evidence of teratogenesis. In the postnatal development of the offspring from dams treated with MBTS, a high survival rate and good growth of the offspring were observed. The authors suggested a NOAEL developmental toxicity of 596 mg/kg bw/ day (highest dose evaluated). Maternal toxicity was indicated by a reduction in body weight gain during day 0 to day 14 of pregnancy at the highest dose group, thus a NOAEL of 127 mg/kg bw and day is suggested for maternal toxicity.

Read across with MBT

Additionally, a developmental toxicity study in rabbits for MBT, CAS no. 149-30-4 (read-across from supporting substance (structural analogue or surrogate)) is available.

The experimental design consisted of three MBT treated groups and a concurrent control group. Each group was comprised of 20 artificially inseminated New Zealand White rabbits. Dosage levels selected for this teratology study were 50, 150, and 300 mg/kg/day. MBT was suspended in 1% methylcellulose and administered at a volume of 2 ml/kg. Control animals received 1% aqueous solution of methylcellulose at an equivalent dosage volume. Treatment was performed from gestation day 6 through gestation day 18. All rabbits were observed daily for signs of overt toxicity. Body weights were measured on gestation days 0, 6, 9, 12, 15, 19, 24, and 29. Food consumption was measured daily and reported for the specified intervals. Cesarean section was performed on gestation day 29. Intrauterine survival was evaluated and fetuses were examined for external, visceral, and skeletal anomalies.

The oral administration of 50, 150, or 300 mg/kg/day (highest applied dose) of MBT to pregnant New Zealand White rabbits during major organogenesis did not induce any developmental toxicity or teratogenicity. Maternal toxicity was evident only at a level of 300 mg/kg/day as slightly decreased body weight gain and slightly elevated liver weight.


Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
596 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Acceptable well-documented publication which meets basic scientific principles.
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

The developmental toxicity potential of MBTS was evaluated in Wistar rats (Ema 1989). Pregnant rats (15 to 19 per dose) were given MBTS at a dosage of 0, 0.04, 0.2 or 1% in the diet from day 0 to day 20 of pregnancy. The dams were scheduled sacrificed on day 20 of pregnancy. The peritoneal cavity was open, and the numbers and positions of live and dead foetuses and resorption were recorded. The live foetuses were removed from the uterus by Caesarean section and were sexed, weighed and inspected for external anomalies and anomalies within the oral cavity using a stereoscopic microscope. The placental weight was also measured. About two-third of the live foetuses in each litter, randomly selected, were fixed in alcohol, cleared in KOH solution, stained with Alizarin Red S12 and examined for skeletal anomalies. The remaining one-third of the live foetuses in each litter were fixed in Bouin's solution, sectioned with a razor blade and examined for internal anomalies. Daily intakes of MBTS were 26 mg/ kg bw/day for the 0.04% group, 127 mg/kg bw/day for the 0.2% group and 596 mg/kg bw/day for the 1% group. No mortality occurred during the study. In addition, no clinical signs were noted in any of the treated dams and control dams. Maternal body weight gain during day 0 to day 14 of pregnancy was significant reduced (p< 0.05) in the 1% group compared to the corresponding control group. No significant differences between the MBTS-treated groups and the control group were noted in the pregnancy rate and food consumption during pregnancy. There were no significant differences in the incidences of pre- and post-implantation losses, the numbers of corpora lutea per litter, implantations per litter and live foetuses per litter, the sex ratio of live foetuses, the foetal body weight of both sexes and placental weight. No treatment-related effects for external, skeletal and internal malformations were noted. The incidences of the foetuses and litters that had skeletal variations or delayed ossifications were not significantly different between the MBTS treated animals and control animals. There was no significant difference between the MBTS treated animals and control animals in the degree of ossification, indicated by the numbers of the caudal vertebrae.

In an additional approach dams from all treatment groups and control (5 to six per group) were allowed to deliver spontaneously. The day of delivery was designated as day 0 after birth; the numbers of live and dead newborns were recorded. The live newborns were sexed weighed, inspected for external anomalies and anomalies within the oral cavity, marked for identification and allowed to suckle their own mother. The litter size was randomly adjusted to eight offspring, comprising four males and four females on day 0 after birth. The offspring were weighed weekly and weaned on day 21 after birth. The dams were killed on day 21 after birth and the number of implantation remnants was recorded. Thereafter, the offspring were reared until 7 weeks after birth and weight weekly. No significant differences between MBTS treated animals and control group animals were found in gestation length of maternal rats, the number of implantation per litter and live newborns per litter, and the live birth index. The survival rate of the offspring before and after weaning was very high and almost constant in all groups. Autopsy of the scheduled sacrificed offspring treated with MBTS revealed no treatment-related alterations. Body weights of pups treated with MBTS was not significant different compared to the corresponding control.

In conclusion, there were no significant compound–related effects on the incidences of pre- and post-implantation losses and the number, sex ratio and body weight of live foetuses. Morphological examinations of the foetuses revealed no evidence of teratogenesis. In the postnatal development of the offspring from dams treated with MBTS, a high survival rate and good growth of the offspring were observed. The authors suggested a NOAEL developmental toxicity of 596 mg/kg bw/ day (highest dose evaluated). Maternal toxicity was indicated by a reduction in body weight gain during day 0 to day 14 of pregnancy at the highest dose group, thus a NOAEL of 127 mg/kg bw and day is suggested for maternal toxicity.

The findings from this developmental toxicity study are in line with developmental toxicity study data from MBT.

Read across with MBT:

Additionally, a developmental toxicity study in rabbits for MBT, CAS no. 149-30-4 (read-across from supporting substance (structural analogue or surrogate)) is available.

The experimental design consisted of three MBT treated groups and a concurrent control group. Each group was comprised of 20 artificially inseminated New Zealand White rabbits. Dosage levels selected for this teratology study were 50, 150, and 300 mg/kg/day. MBT was suspended in 1% methylcellulose and administered at a volume of 2 ml/kg. Control animals received 1% aqueous solution of methylcellulose at an equivalent dosage volume. Treatment was performed from gestation day 6 through gestation day 18. All rabbits were observed daily for signs of overt toxicity. Body weights were measured on gestation days 0, 6, 9, 12, 15, 19, 24, and 29. Food consumption was measured daily and reported for the specified intervals. Cesarean section was performed on gestation day 29. Intrauterine survival was evaluated and fetuses were examined for external, visceral, and skeletal anomalies.

The oral administration of 50, 150, or 300 mg/kg/day (highest applied dose) of MBT to pregnant New Zealand White rabbits during major organogenesis did not induce any developmental toxicity or teratogenicity. Maternal toxicity was evident only at a level of 300 mg/kg/day as slightly decreased body weight gain and slightly elevated liver weight.

Toxicity to reproduction: other studies

Description of key information

No data.

Additional information

A read-across with the analogue substance benzothiazole-2-thiol (MBT) CAS-No. 149-30-4 is justified based on the ECHA decision, number: CCH-D-2114343759-36-01/F.

Justification for classification or non-classification

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification for toxicity to reproduction is not justified.