Zinc di(benzothiazol-2-yl) disulphide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no data for the assessment of reproduction toxicity available for ZMBT.

ZMBT is composed of two 2-Mercaptobenzothiazole (MBT) molecules (CAS 149-30-4, EC 205-736-8) associated with zinc ion (mass content ZMBT: 84% MBT, 16% Zn2+). It was shown in hydrolysis studies that under acidic conditions (pH 3) a rapid degradation of ZMBT to MBT and zinc ions Zn2+ was observed. A read-across with toxicological data for MBT as source is thus considered adequate. For justification of the read-across see separate Read-Across Justification Document attached to the IUCLID.

The findings of a two-generation study in rats demonstrated that MBT did not have any adverse effects on reproductive functions of the F0 or Fl generation (CMA 1990). Thus, the highest tested dose of level of 15000 ppm was determined to be a No Adverse Effect Level for reproductive toxicity. Based on the calculation of test item intake at 2500 ppm with ca. 150 mg to 250 mg/kg bw/day, the NOAEL of 15000 ppm for reproductive toxicity can be considered to be well above 700 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA Final Test Rule FR 53 No. 173, PP. 34514 - 34531, September 7, 1988.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Final Test Guidelines 40 CFR Part 798.4700. FR 50 No. 188, pp. 39432 - 39434, September 27, 1985.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Test Guidelines Amendments. FR 52 No. 97, pg. 19077, May 20, 1987.

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

oestrus cycle, sperm parameters, pup behavior were not evaluated

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: MBT contained in the diet

Details on exposure:

The appropriate amount of the test article was weighed and added to a small amount of rodent meal. This premix was blended in a Hobart blender for approximately ten minutes. The premix was then transferred to a twin shell blender and mixed with the diet in sufficient quantity to achieve the specified concentration for each group. The diets were mixed for 30 minutes. The basal diet was also blended in a twin shell blender. The test diets were prepared fresh weekly. Due to an error made during test diet preparation, all Fl and F2 MBT-treated groups received 27.8 % less test article than intended during study week 36-37 (lactation week 3). Therefore, the actual dose concentrations for this week were 1805 ppm, 6318 ppm and 10830 ppm for groups 2, 3 and 4, respectively. This occurrence should not have had an impact on the study.

Details on mating procedure:

Following a minimum of 70 days of treatment for the F0 generation and 88 days for the Fl generation, the rats were cohabitated (one male and one female from the same group). Each mating pair was observed daily for evidence of copulation. The day the copulation was noted was designated day 0 of gestation, and the females were returned to their cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of the test diets was determined prior to study initiation. Samples were taken from the top, middle, and bottom of the prepared diet and analyzed. Stability analyses were also performed prior to the initiation of treatment. The test diets were analyzed for concentration weekly for the first four weeks and monthly thereafter.

Homogeneity and stability analyses determined that the test diets were homogeneous and stable for at least eight days. Analyses for concentration revealed a recovery of the test article within ± 10% of the targeted concentration.

Duration of treatment / exposure:

Exposure period: 10 weeks before mating, through gestation and lactation until sacrifice
Premating exposure period (males): 10 weeks
Duration of test: approximately 88 days past weaning
MBT was administered in the diet. Oral route of administration was recommended in the EPA guidelines mentioned previously. The test diets were prepared fresh weekly and provided ad libitum throughout the study. The FO generation (males and females) received the test diet for a minimum of ten weeks (70 days) prior to mating and continuing until sacrifice. FO rats were approximately seven weeks of age when the treatment was initiated.
Fl generation may have been exposed to the test article in utero. During lactation, the rats were possibly exposed to the compound through the milk in the first 2 weeks of lactation and in the diet after 14 days of age. Following selection and separation of Fl parental rats (approximately 28 days of age) the treatment continued in the diet for a minimum of 88 days (at weaning) prior to breeding and until sacrifice.

Frequency of treatment:

ad libitum via food

Details on study schedule:

F0 generation (males and females) reveived the test diet for a minimum of ten weeks (70 days) prior mating and continuing until sacrifice. F0 rats were ca. seven weeks of age when the treatment was initiated.
F1 generation may have been exposed to the test article in utero. During lactation, the rats were possibly exposed to the compound through the milk in the first 2 weeks of lactation and in the diet after 14 days of age. Following selection and separation of F1 parental rats (approximately 28 days of age) the treatment continued in the diet for a minimum of 88 days (at weaning) prior to breeding and until sacrifice.

Dose / conc.:

2 500 ppm

Dose / conc.:

8 750 ppm

Dose / conc.:

15 000 ppm

Remarks:

15000 ppm correspond to:
ca. 778 to 1328 mg/kg bw/d F0 males,
779 to 2633 mg/kg bw/d F1 males,
745 to 1760 mg/kg bw/d F0 females,
980 to 1770 mg/kg bw/d F1 females)
Basis:

No. of animals per sex per dose:

28 per sex and dose

Control animals:

yes, plain diet

Positive control:

not adequate

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
The rats were observed daily for clinical signs and behavioral anomalies. Mortality checks were performed twice daily, in the morning

BODY WEIGHT: Yes
Individual body weights were measured as presented below:
F0 males and F0 females prior to breeding: weekly, beginning on the day of study initiation;
Fl males and Fl females prior to breeding: weekly. A day was arbitrarily selected (3 to 9 days following weaning) and designated as initiation of weekly measurements
F0 and Fl females once evidence of copulation was noted:
gestation days: 0, 7, 14, and 20; lactation days: 1, 7, 14, and 21.
Individual body weights were recorded until sacrifice. Body weight gain was calculated for the respective intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Individual food consumption was measured on the same days the body weights were measured. Food consumption was not measured while the rats were paired for breeding. Food intake was calculated and reported as g/animal/day and g/kg/day for any given interval.

Description (incidence and severity):

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.

Description (incidence):

Survival was 100% in all study groups.

Description (incidence and severity):

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).

Description (incidence and severity):

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 1326 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.

Description (incidence and severity):

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 g in the low, mid and high dose groups, respectively, were well within the range of historical control data.

Reproductive performance:

no effects observed

Description (incidence and severity):

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.

Dose descriptor:

LOAEL

Remarks:

parental toxicity

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight reduced

Remarks on result:

other: according to 150 to 200 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

not specified

Description (incidence and severity):

There were no treatment-related clinical signs observed in the F1 adult 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.

Description (incidence):

All animals in the control and treated groups survived to the scheduled sacrifice.

Description (incidence and severity):

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 gains on the other hand, was 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.

Description (incidence and severity):

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.

Description (incidence and severity):

There were no treatment-induced gross lesions observed in the F1 parental animals. Gross lesions, commonly seen in rats, occurred in all 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.

Description (incidence and severity):

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 group. Cortical tubular basophilia occurred in all groups but the incidence was greater in the males of the high dosage group (0, 3, 4 and10 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 0.765g with a range of 0.637 to 1.023g. The mean value in the high dosage group was 0.672 g, 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.

Reproductive performance:

no effects observed

Description (incidence and severity):

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.

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

not specified

Description (incidence and severity):

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.

Description (incidence and severity):

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.

Description (incidence and severity):

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.

Description (incidence and severity):

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.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: related to maternal effects F0

Critical effects observed:

not specified

Description (incidence and severity):

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.

Description (incidence and severity):

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.

Description (incidence and severity):

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).

Description (incidence and severity):

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.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no biological relevant effects on reproductive function

Critical effects observed:

not specified

Reproductive effects observed:

no

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 of the test article within ± 10% of the targeted concentration.

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 F1 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, F1 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 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. Similar effects on body weights and food consumption were also evident in the F1 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 F1 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 F1 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 F1 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 F1 generation.

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 Fl 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 Fl 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:

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.

Reference 2

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:

Analogue Approach
“The read-across hypothesis is that different substances give rise to (the same) common compounds to which the organism is exposed.”

The read-across approach is intended to fill data-gaps for the target molecule Zinc 2-Mercaptobenzothiazole (ZMBT, CAS 155-04-4, EC 205-840-3) with studies available for the source molecule 2-Mercaptobenzothiazole (MBT, CAS 149-30-4, EC 205-736-8 ) for humane toxicological endpoints following the oral route of exposure. Source and target compounds show a high grade of similarity. In production processes the target compound ZMBT is synthesized from the source compound MBT by deprotonation and precipitation with zinc ions Zn2+. Vice versa, ZMBT is easily reprotonated under acidic conditions to form MBT and Zn2+ ions. Following this rationale, the intend to read-across toxicity studies following ingestion is very reasonable.
For further information see attached document:
Justification for a read-across from 2-Mercaptobenzothiazole (CAS 149-30-4, EC 205-736-8) [Source] to Zinc 2-Mercaptobenzothiazole (CAS 155-04-04, EC 205-840-3) [Target]

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Description (incidence and severity):

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.

Description (incidence):

Survival was 100% in all study groups.

Description (incidence and severity):

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).

Description (incidence and severity):

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 1326 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.

Description (incidence and severity):

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 g in the low, mid and high dose groups, respectively, were well within the range of historical control data.

Reproductive performance:

no effects observed

Description (incidence and severity):

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.

Dose descriptor:

LOAEL

Remarks:

parental toxicity

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight reduced

Remarks on result:

other: according to 150 to 200 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

not specified

Description (incidence and severity):

There were no treatment-related clinical signs observed in the F1 adult 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.

Description (incidence):

All animals in the control and treated groups survived to the scheduled sacrifice.

Description (incidence and severity):

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 gains on the other hand, was 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.

Description (incidence and severity):

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.

Description (incidence and severity):

There were no treatment-induced gross lesions observed in the F1 parental animals. Gross lesions, commonly seen in rats, occurred in all 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.

Description (incidence and severity):

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 group. Cortical tubular basophilia occurred in all groups but the incidence was greater in the males of the high dosage group (0, 3, 4 and10 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 0.765g with a range of 0.637 to 1.023g. The mean value in the high dosage group was 0.672 g, 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.

Reproductive performance:

no effects observed

Description (incidence and severity):

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.

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

not specified

Description (incidence and severity):

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.

Description (incidence and severity):

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.

Description (incidence and severity):

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.

Description (incidence and severity):

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.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: related to maternal effects F0

Critical effects observed:

not specified

Description (incidence and severity):

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.

Description (incidence and severity):

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.

Description (incidence and severity):

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).

Description (incidence and severity):

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.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no biological relevant effects on reproductive function

Critical effects observed:

not specified

Reproductive effects observed:

no

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 of the test article within ± 10% of the targeted concentration.

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 F1 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, F1 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 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. Similar effects on body weights and food consumption were also evident in the F1 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 F1 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 F1 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 F1 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 F1 generation.

Conclusions:

Based on the available toxicological information for ZMBT and for the hydrolysis product MBT it can be concluded that the same biological targets are affected and the same toxicological effects were observed for MBT and ZMBT. No classification is warranted for reproductive toxicity (fertility).

A read-across with data for MBT as source to fill data gaps (repeated dose toxicity, reproduction toxicity and developmental toxicity) of ZMBT as target is justified.

Executive summary:

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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.

The findings of a two-generation study in rats demonstrated that MBT did not have any adverse effects on reproductive functions of the F0 or Fl generation (CMA 1990). Thus, the highest tested dose of level of 15000 ppm was determined to be a No Adverse Effect Level for reproductive toxicity. Based on the calculation of test item intake at 2500 ppm with ca. 150 mg to 250 mg/kg bw/day, the NOAEL of 15000 ppm for reproductive toxicity can be considered to be well above 700 mg/kg bw/day.

Based on the available information there is no reason to classify neither metallic zinc nor any of the zinc compounds considered by the EU risk assessment (2004) or MAK (2009) for reproductive toxicity.

Effects on developmental toxicity

Description of key information

There are no data for the assessment of reproduction toxicity available for ZMBT.

ZMBT is composed of two 2-Mercaptobenzothiazole (MBT) molecules (CAS 149-30-4, EC 205-736-8) associated with zinc ion (mass content ZMBT: 84% MBT, 16% Zn2+). It was shown in hydrolysis studies that under acidic conditions (pH 3) a rapid degradation of ZMBT to MBT and zinc ions Zn2+ was observed. A read-across with toxicological data for MBT as source is thus considered adequate. For justification of the read-across see separate Read-Across Justification Document attached to the IUCLID.

In the developmental toxicity study with pregnant Sprague-Dawely rats (CMA 1989), the oral administration of MBT during the critical period of organogenesis (gestation days 6 through 15, caesarean section on day 20) induced moderate maternal toxicity at a level of 1800 mg/kg bw/day, expressed by clinical signs, reduced body weight gain, and reduced food intake. Clinical signs were the only indication of treatment-related effects at a dose level of 1200 mg/kg bw/day. The dose of 300 mg/kg bw/day was determined to be a NOAEL for maternal toxicity. MBT administered during major organogenesis at dosage levels of 300, 1200, or 1800 mg/kg/day did not induce any developmental toxicity or teratogenicity in treated rats. Thus, the NOAEL for developmental toxicity was determined with 1800 mg/kg bw/day for rats.

In the developmental toxicity study with pregnant New Zealand White rabbits (CMA 1989b) oral administration of MBT during the critical period of organogenesis (gestation days 6 through 18, cesarean section on day 29) induced moderate maternal toxicity at a level of 300 mg/kg bw/day expressed by reduced body weight gain and slightly enhanced liver weight. Oral administration of 50, 150, or 300 mg/kg/day 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 the highest dose tested of 300 mg/kg bw/day as slightly decreased body weight gain and slightly elevated liver weight. Thus, the NOAEL for developmental toxicity was determined with 300 mg/kg bw/day for rabbits.

Based on these an overall NOAEL for developmental toxicity of 300 mg/kg bw and day is suggested.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA regulation (TSCA) guidelines (40 CFR Part 798.4700, September 1985, and revised edition May 1987)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazelton Research Products Inc., Denver, PA
- Age at study initiation: approximately 6 months
- Weight at study initiation: 3-5 kg
- Housing: individually according to AAALAC
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 30 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68°F +- 5°F
- Humidity (%): 55% +- 15%

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous solution of methylcellulose

Details on exposure:

administration volume: 2 mL/kg bw
Dosage levels of 50, 150, and 300 mg/kg/day were selected based upon results of a range-finding study conducted also on New Zealand white rabbits (CMA 1989). Treatment-related deaths occurred in the range-finding study at levels of 600 mg/kg/day and higher, while dose-dependent body weight loss was evident in all treated groups (150, 300, 600, 1000, and 1500 mg/kg/day) during the treatment period. A dosage level of 600 mg/kg/day as the highest dosage level was considered excessive for a definitive teratology study due to the magnitude of body weight loss and induced mortality. Similarly, dosage levels greater than 300 mg/kg/day were also considered inappropriate due to an anticipated excessively high magnitude of body weight loss and possible mortality. Therefore, 300 mg/kg/day of MBT was selected as the highest dosage level knowing that two does out of five had severe body weight loss during the treatment period. A dosage level of 150 mg/kg/day was expected to induce minimal maternal toxicity and a dosage level of 50 mg/kg/day was selected to determine a no effect level for maternal and developmental toxicity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity in 1% aqueous solution of methylcellulose and stabilty for seven days were determined prior to test article administration.
The HPLC analyses determined that MBT was stable in 1 % aqueous methycellulose for a period of 7 days. Dosage suspensions were found to be homogeneous at concentrations similar to the lowest and highest levels. Analyses for concentration of the test article in the dosage suspensions revealed a recovery of the test article within ± 10 % of targeted concentration.

Details on mating procedure:

Artificial insemination:
Proven resident New Zealand White male rabbits, purchased from the same supplier, were used as the semen donors. Semen was collected from seven male rabbits, using an artificial vagina and evaluated for volume, motility, and concentration. Prior to insemination, the semen was diluted with 0.9 % physiologic saline and maintained in a water bath at approximately 37 °C during the insemination procedure. Approximately 0.5 ml of the diluted semen was introduced into the doe s vagina. Semen collected from one male on a given day was used to inseminate an equal number of females in each study group. Immediately following the insemination, the females were administered human chorionic gonadotropin, via the marginal ear vein. The day of insemination was considered day 0 of gestation.

Duration of treatment / exposure:

Day 6-18 of gestation

Frequency of treatment:

daily

Duration of test:

up to gestation day 29

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

No. of animals per sex per dose:

20 per dose

Control animals:

yes

Details on study design:

Sex: pregnant female s
Duration of test: sacrifice on to Day 29 of gestation

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0, 6, 9, 12, 15, 19, 24, and 29

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: emphasis on morphological changes that could interfere with intrauterine survival and fetal development; trimmed liver weights were recorded and livers were fixed for histopathological evaluation

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]

Statistics:

two-tailed analyses for a significance level of 5%; comparison of treated groups with control group 1 or by an individual group by group comparison;
one way analyses of variance followed by Dunnett's test was used to analyse maternal and fetal data including body weights, food consumption, number of viable fetuses, implanation sites, and corpora lutea.
Man-Whighney U test used for postimplantation loss, dead fetuses and resorptions.
Fetal sex ratios were analysed using the Chi-Square test. A one-tailed Fisher's Exact test was used in analyses of fetuses and litters with malformations and vairations.

Description (incidence and severity):

There were no treatment-induced clinical signs observed in this study. Incidental findings occurred in all groups and included primarily soft stools, or mucoid stools, reduced feces, urine or fecal staining, and loss of hair.

Description (incidence):

No treatment-related deaths occurred during the conduct of this study. One doe in the 150 mg/kg/day group died on gestation day 13. Mottled lungs and yellow semi-solid contents in the trachea indicated that this death was a result of intubation error. A single incidence of abortion was noted in the 150 mg/kg/day group on gestation day 19. One doe in the 300 mg/kg/day group delivered prematurely on gestation day 29 before the scheduled cesarean section.

Description (incidence and severity):

Mean maternal body weights and net maternal body weight gain were comparable between the groups. There were no statistically significant differences observed when body weight changes were calculated for the specified intervals. Treatment-related body weight losses were evident however, during the last days of treatment (gestation days 15 -19); several does lost in excess of 150 grams; this pattern was also observed in the range finding study. This weight loss attributed to much of the reduced mean weight gains during the treatment period (gestation days 6 - 19 300 mg/kg bw/d group - 63 % vs. control).

Description (incidence and severity):

Food consumption, calculated as g/animal/day or g/kg/day was comparable between groups for the duration of the study.

Description (incidence and severity):

Absolute and relative liver weights from gravid does were slightly, increased in the 300 mg/kg/day group (absolute liver weight: 107 %, rel. liver weight: 104% vs. control)
gravid uterine weights were similar in all groups

Description (incidence and severity):

There were no treatment-induced lesions noted in the MBT groups. Incidental findings included hydrothorax and umbilical hernia in the control group, mottled lungs and semi-solid material in the trachea in the 150 mg/kg/day group (the doe died on gestation day 13).

Description (incidence and severity):

Pregnancy percentages were 95, 90, 85, and 100 in the control, 50, 150, and 300 mg/kg/day groups, respectively.

Details on maternal toxic effects:

Cesarean section observations
Parameters measured at cesarean section, including gravid uterine weight, the number of viable fetuses, early and late resorptions, fetal sex ratio and fetal weights were similar in all groups.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Fetal morphologic observations
No statistically or biologically significant differences were noted between the control and MBT groups with regard to malformations or variations. The malformations that occurred in this study are commonly seen in rabbits. Morphological variations were similar and occurred with comparable frequency in the control and treated groups

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Executive summary:

In summary, the present study was conducted to evaluate maternal and developmental toxicity of MBT in New Zealand White rabbits. Dosage levels of 50, 150, and 300 mg/kg/day were selected based upon results of a range-finding study conducted also on New Zealand white rabbits (CMA 1989). Treatment-related deaths occurred in the range-finding study at levels of 600 mg/kg/day and higher, while dose-dependent body weight loss was evident in all treated groups (150, 300, 600, 1000, and 1500 mg/kg/day) during the treatment period. A dosage level of 600 mg/kg/day as the highest dosage level was considered excessive for a definitive teratology study due to the magnitude of body weight loss and induced mortality. Similarly, dosage levels greater than 300 mg/kg/day were also considered inappropriate due to an anticipated excessively high magnitude of body weight loss and possible mortality. Therefore, 300 mg/kg/day of MBT was selected as the highest dosage level knowing that two does out of five had severe body weight loss during the treatment period. A dosage level of 150 mg/kg/day was expected to induce minimal maternal toxicity and a dosage level of 50 mg/kg/day was selected to determine a no effect level for maternal and developmental toxicity.

Oral administration of MBT to pregnant rabbits at a level of 300 mg/kg/day induced maternal toxicity as expressed by slightly reduced body weight gain during treatment and slightly elevated liver weight, absolute or relative to final body weight. This toxicity became apparent upon visual inspection of individual body weight gain data particularly during the last days of treatment (gestation days 15-19) for this group when compared to the control group. During this period, six treated does had body weight losses greater than 150 grams while no control dam lost this magnitude of body weight. Lower dosage levels of MBT (50 and 150 mg/kg/day) did not produce any evidence of maternal toxicity. A single abortion was noted in the 150 mg/kg/day group on gestation day 19 and a premature delivery occurred in the 300 mg/kg/day group on gestation day 29, prior to the scheduled cesarean section. A single incidence of abortion was noted in the range-finding study at levels of 600 and 1000 mg/kg/day. However, the biological and toxicological significance of the abortion and premature delivery in the present study is equivocal, knowing that: abortions or premature deliveries are common in rabbits; a dose-related pattern was not evident (in particular, the time of occurrence); abortions were not noted in the range-finding study at levels of 150 or 300 mg/kg/day (2). One rabbit in the 300 mg/kg/day group died on gestation day 13. Postmortem findings indicated that the death occurred following an intubation error (semi-solid yellow contents in trachea).

The administration of MBT during the critical period organogenesis at dose levels of 50, 150, or 300 mg/kg/day did not induce any developmental toxicity or teratogenicity, in that all fetal data, including viability, mean body weight, malformations, and variations were comparable between the MBT and control groups.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Analogue Approach
“The read-across hypothesis is that different substances give rise to (the same) common compounds to which the organism is exposed.”

The read-across approach is intended to fill data-gaps for the target molecule Zinc 2-Mercaptobenzothiazole (ZMBT, CAS 155-04-4, EC 205-840-3) with studies available for the source molecule 2-Mercaptobenzothiazole (MBT, CAS 149-30-4, EC 205-736-8 ) for humane toxicological endpoints following the oral route of exposure. Source and target compounds show a high grade of similarity. In production processes the target compound ZMBT is synthesized from the source compound MBT by deprotonation and precipitation with zinc ions Zn2+. Vice versa, ZMBT is easily reprotonated under acidic conditions to form MBT and Zn2+ ions. Following this rationale, the intend to read-across toxicity studies following ingestion is very reasonable.
For further information see attached document:
Justification for a read-across from 2-Mercaptobenzothiazole (CAS 149-30-4, EC 205-736-8) [Source] to Zinc 2-Mercaptobenzothiazole (CAS 155-04-04, EC 205-840-3) [Target]

Reason / purpose for cross-reference:

read-across source

Duration of test:

up to gestation day 29

Description (incidence and severity):

There were no treatment-induced clinical signs observed in this study. Incidental findings occurred in all groups and included primarily soft stools, or mucoid stools, reduced feces, urine or fecal staining, and loss of hair.

Description (incidence):

No treatment-related deaths occurred during the conduct of this study. One doe in the 150 mg/kg/day group died on gestation day 13. Mottled lungs and yellow semi-solid contents in the trachea indicated that this death was a result of intubation error. A single incidence of abortion was noted in the 150 mg/kg/day group on gestation day 19. One doe in the 300 mg/kg/day group delivered prematurely on gestation day 29 before the scheduled cesarean section.

Description (incidence and severity):

Mean maternal body weights and net maternal body weight gain were comparable between the groups. There were no statistically significant differences observed when body weight changes were calculated for the specified intervals. Treatment-related body weight losses were evident however, during the last days of treatment (gestation days 15 -19); several does lost in excess of 150 grams; this pattern was also observed in the range finding study. This weight loss attributed to much of the reduced mean weight gains during the treatment period (gestation days 6 - 19 300 mg/kg bw/d group - 63 % vs. control).

Description (incidence and severity):

Food consumption, calculated as g/animal/day or g/kg/day was comparable between groups for the duration of the study.

Description (incidence and severity):

Absolute and relative liver weights from gravid does were slightly, increased in the 300 mg/kg/day group (absolute liver weight: 107 %, rel. liver weight: 104% vs. control)
gravid uterine weights were similar in all groups

Description (incidence and severity):

There were no treatment-induced lesions noted in the MBT groups. Incidental findings included hydrothorax and umbilical hernia in the control group, mottled lungs and semi-solid material in the trachea in the 150 mg/kg/day group (the doe died on gestation day 13).

Description (incidence and severity):

Pregnancy percentages were 95, 90, 85, and 100 in the control, 50, 150, and 300 mg/kg/day groups, respectively.

Details on maternal toxic effects:

Cesarean section observations
Parameters measured at cesarean section, including gravid uterine weight, the number of viable fetuses, early and late resorptions, fetal sex ratio and fetal weights were similar in all groups.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Fetal morphologic observations
No statistically or biologically significant differences were noted between the control and MBT groups with regard to malformations or variations. The malformations that occurred in this study are commonly seen in rabbits. Morphological variations were similar and occurred with comparable frequency in the control and treated groups

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Based on the available toxicological information for ZMBT and for the hydrolysis product MBT it can be concluded that the same biological targets are affected and the same toxicological effects were observed for MBT and ZMBT. No classification is warranted for reproductive toxicity (fertility).

A read-across with data for MBT as source to fill data gaps (repeated dose toxicity, reproduction toxicity and developmental toxicity) of ZMBT as target is justified.

Executive summary:

In summary, the present study was conducted to evaluate maternal and developmental toxicity of MBT in New Zealand White rabbits. Dosage levels of 50, 150, and 300 mg/kg/day were selected based upon results of a range-finding study conducted also on New Zealand white rabbits (CMA 1989). Treatment-related deaths occurred in the range-finding study at levels of 600 mg/kg/day and higher, while dose-dependent body weight loss was evident in all treated groups (150, 300, 600, 1000, and 1500 mg/kg/day) during the treatment period. A dosage level of 600 mg/kg/day as the highest dosage level was considered excessive for a definitive teratology study due to the magnitude of body weight loss and induced mortality. Similarly, dosage levels greater than 300 mg/kg/day were also considered inappropriate due to an anticipated excessively high magnitude of body weight loss and possible mortality. Therefore, 300 mg/kg/day of MBT was selected as the highest dosage level knowing that two does out of five had severe body weight loss during the treatment period. A dosage level of 150 mg/kg/day was expected to induce minimal maternal toxicity and a dosage level of 50 mg/kg/day was selected to determine a no effect level for maternal and developmental toxicity.

Oral administration of MBT to pregnant rabbits at a level of 300 mg/kg/day induced maternal toxicity as expressed by slightly reduced body weight gain during treatment and slightly elevated liver weight, absolute or relative to final body weight. This toxicity became apparent upon visual inspection of individual body weight gain data particularly during the last days of treatment (gestation days 15-19) for this group when compared to the control group. During this period, six treated does had body weight losses greater than 150 grams while no control dam lost this magnitude of body weight. Lower dosage levels of MBT (50 and 150 mg/kg/day) did not produce any evidence of maternal toxicity. A single abortion was noted in the 150 mg/kg/day group on gestation day 19 and a premature delivery occurred in the 300 mg/kg/day group on gestation day 29, prior to the scheduled cesarean section. A single incidence of abortion was noted in the range-finding study at levels of 600 and 1000 mg/kg/day. However, the biological and toxicological significance of the abortion and premature delivery in the present study is equivocal, knowing that: abortions or premature deliveries are common in rabbits; a dose-related pattern was not evident (in particular, the time of occurrence); abortions were not noted in the range-finding study at levels of 150 or 300 mg/kg/day (2). One rabbit in the 300 mg/kg/day group died on gestation day 13. Postmortem findings indicated that the death occurred following an intubation error (semi-solid yellow contents in trachea).

The administration of MBT during the critical period organogenesis at dose levels of 50, 150, or 300 mg/kg/day did not induce any developmental toxicity or teratogenicity, in that all fetal data, including viability, mean body weight, malformations, and variations were comparable between the MBT and control groups.

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Analogue Approach
“The read-across hypothesis is that different substances give rise to (the same) common compounds to which the organism is exposed.”

The read-across approach is intended to fill data-gaps for the target molecule Zinc 2-Mercaptobenzothiazole (ZMBT, CAS 155-04-4, EC 205-840-3) with studies available for the source molecule 2-Mercaptobenzothiazole (MBT, CAS 149-30-4, EC 205-736-8 ) for humane toxicological endpoints following the oral route of exposure. Source and target compounds show a high grade of similarity. In production processes the target compound ZMBT is synthesized from the source compound MBT by deprotonation and precipitation with zinc ions Zn2+. Vice versa, ZMBT is easily reprotonated under acidic conditions to form MBT and Zn2+ ions. Following this rationale, the intend to read-across toxicity studies following ingestion is very reasonable.
For further information see attached document:
Justification for a read-across from 2-Mercaptobenzothiazole (CAS 149-30-4, EC 205-736-8) [Source] to Zinc 2-Mercaptobenzothiazole (CAS 155-04-04, EC 205-840-3) [Target]

Reason / purpose for cross-reference:

read-across source

Vehicle:

corn oil

Description (incidence and severity):

Salivation, dark material around mouth, and urine staining were the most prevalent signs noted in the 1200 and 1800 mg/kg/day group following dosing. Several rats in the 1800 mg/kg/day group also displayed decreased activity. Daily observations, made prior to dosing, included soft stools, fecal and urine staining, hair loss, and dark material around nose in the control group; hair loss and dark material around nose and eyes in the low dosage group; umbilical hernia, hair loss, urine staining, and dark material around nose in the mid dosage group, and increased frequency of urine staining and dark material around nose and mouth in the high dosage group.

Description (incidence):

Survival was 100% in the control, 300, and 1200 mg/kg/day groups. One rat in the 1800 mg/kg/day group died on gestation day 16.
Pregnancy percentages were 92.3, 88.5, 96.2, and 84.6 in the control, 300, 1200, and 1800 mg/kg/day groups, respectively.

Description (incidence and severity):

Mean maternal body weights were significantly (p < 0.05) reduced in the 1800 mg/kg/day group on gestation day 9 and comparable to the corresponding control values for the remaining gestation days.
Calculation of body weight changes revealed significant weight loss in the high dose group between gestation days 6 - 9. Body weights and body weight changes were in general comparable between the control, 300, and 1200 mg/kg/day groups throughout gestation. A significantly increased body weight gain occurred during treatment in the low dosage group and was attributed to random occurrence. Net maternal terminal body weight gain was comparable between the control and MBT treated groups.

Description (incidence and severity):

Food consumption, calculated as g/animal/day or g/kg/day was significantly reduced (p < 0.01) in the 1800 mg/kg/day group between gestation days 6 - 9. Food intake, calculated as g/kg/day was significantly
(p < 0.05) increased in the 1200 and (p < 0.01) in the 1800 mg/kg/day groups between gestation days 9 -12. There were no other remarkable differences noted between the groups.

Description (incidence and severity):

No treatment-related morphopathological changes occurred in this study. One rat in the 1800 mg/kg/day group died on gestation day 16 due to a congenital malformation. Necropsy findings included umbilical hernia and intestines with brown mucoid contents.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

1 200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Description (incidence and severity):

The mean number of viable fetuses in the treated groups was slightly greater than in the control group. However, the mean number of early resorptions was increased in all treated groups, resulting in a significantly (p < 0.05) increased post-implantation loss in the 300 and 1800 mg/kg/day groups. There was no statistically significant reduction of the fetal weights in the MBT groups in relation to the control group.

Description (incidence and severity):

Fetal sex ratios were similar in all groups.

Description (incidence and severity):

No statistically or biologically significant differences were noted between the control and MBT groups with regard to fetal malformations or variations. The malformations that occurred in this study are commonly seen in rats. Morphological variations were similar and occurred with comparable frequency in the control and treated groups.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

1 800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

no

In summary:

Maternal toxicity was manifested by pharmacotoxic signs, reduced body weights, and food intake. Pharmacotoxic signs, mainly salivation, dark material around mouth, and urine staining were observed in the 1200 and 1800 mg/kg/day groups following treatment. Decreased activity was noted also in several rats in the 1800 mg/kg/day group. No treatment-related deaths occurred in this experiment. One rat in the high dosage group died on gestation day 16 due to a congenital anomaly (umbilical hernia) which was aggravated by pregnancy. Treatment-related effects on body weights and food consumption were apparent in the 1800 mg/kg/day group. Significant body weight loss occurred in this group between gestation days 6 and 9, and food intake was significantly reduced during the same interval, correlating with the body weight loss. However, body weight gains and food consumption were similar to the control group for the remainder of gestation. Body weight gains and food intake were comparable between the control, 300, and 1200 mg/kg/day groups throughout gestation. There were no morphopathological changes seen in the rats examined at the time of cesarean section.

The number of viable fetuses in the treated groups was slightly greater than in the control group. However, the mean number of post- implantation loss (early resorptions) was increased in all MBT groups, with statistical significance noted in the 300 and 1800 mg/kg/day groups. Although increased, the post-implantation loss in the 300 and 1200 mg/kg/day groups did not appear to be treatment-related in that: (1) the increase was not dose-dependent; (2) it was statistically significant at the lover dosage level but not at the 1200 mg/kg/day level; (3) the mean values were within the range of the historical control data; (4) similar effects were not apparent in the range-finding study (CMA 1989) at any of the dosage levels. Mean post-implantation loss in the 1800 mg/kg/day group (1.7) was slightly outside the range of our historical control data (0.6- 1.4). However, reports on post-implantation loss in Sprague-Dawley

CD rats determined a range of 0.3 to 1.8 Lang (1988*) and Woo and Hoar (1979**) averaged the data obtained from 157 studies on CD rats and found an average resorption rate of 7%. In the present study, resorption rate in the control group was 5.3%, an unusually low value. In the light of these findings, significantly increased post-implantation loss in the 1800 mg/kg/day group appears toxicologically equivocal while that of the 300 mg/kg/day group was deemed biologically insignificant. No statistically significant differences occurred between groups with respect to fetal weights. Mean fetal weight was slightly reduced (approximately 5%) in the 1800 mg/kg/day group: however, the mean value for this group (3.5 g) is well within the range of the historical control data (3.4-3.7 g) for this parameter. The type and incidence of malformations and variations did not demonstrate a teratogenic effect produced by MBT. The malformations and variations which occurred in this study are commonly seen in the rat fetuses.

The authors conclude that oral administration of MBT to pregnant Sprague-Dawley rats induced frank maternal toxicity at a level of 1800 mg/kg/day expressed by pharmacotoxic signs, reduced body weight gains, and food intake. Pharmacotoxic signs were the only indication of treatment-related effects at a dosage level of 1200 mg/kg/day. A dosage level of 300 mg/kg/day was determined to be a no effect level for maternal toxicity. MBT administered during major organogenesis at dosage levels of 300, 1200, or 1800 mg/kg/day did not induce any developmental toxicity or teratogenicity.

* Lang, P. L., Embryo and fetal developmental toxicity (teratology) control data in the Charles River Crl:CD®BR rat, (1988). Charles River, The International Standard from the Hand of the Veterinarian to

Research.

**Woo, D. C., and R. M. Hoar. Reproductive performance and spontaneous malformations in control Charles River CD®rats: A joint study by MARTA (1979). Teratology, 19:54A.

RS-Freetext:
1) 300 mg/kg bw/d:

PI loss.

RS-Freetext:
2) 1200 mg/kg bw/d:
salivation, F;
urine staining, F;
dark red material around mouth, F.

RS-Freetext:
3) 1800 mg/kg bw/d:
PI loss;
salivation, F;
urine staining, F;
dark red material around mouth, F;
activity, decr, F;
bw, decr, F;
food consumption, decr, F, Days 6-9.

Conclusions:

Based on the available toxicological information for ZMBT and for the hydrolysis product MBT it can be concluded that the same biological targets are affected and the same toxicological effects were observed for MBT and ZMBT. No classification is warranted for reproductive toxicity (fertility).

A read-across with data for MBT as source to fill data gaps (repeated dose toxicity, reproduction toxicity and developmental toxicity) of ZMBT as target is justified.

Executive summary:

The developmental toxicity of MBT was evaluated in a teratology study with pregnant Sprague-Dawley rats (CMA 1989a). The test substance was orally administered during the critical period of organogenesis (gestation days 6 through 15). Groups of 26 rats, presumed pregnant, were treated with MBT at dosage levels of 300, 1200, and 1800 mg/kg/day. MBT was suspended in corn oil and administered by gavage at a volume of 10 ml/kg. A concurrent control group, consisting of 26 rats, received corn oil at an equivalent dosage volume. All rats were observed daily for signs of toxicity. Body weights and food consumption were measured on gestation days 0, 6, 9, 12, 16, and 20. Caesarean sections were performed on gestation day 20. Foetuses were weighed and examined for external, visceral, and skeletal anomalies.

Maternal toxicity was evident in the 1800 mg/kg/day group and manifested by clinical signs (salivation, dark red material around mouth, urine staining, and decreased activity), body weight losses and reduced food consumption between gestation days 6 and 9. Clinical signs (salivation, urine staining, and dark material around mouth) were the only indication of treatment-related effects in the 1200 mg/kg/day group. There were no treatment-related effects noted in the 300 mg/kg/day group.

Foetal viability was not affected by the administration of MBT during the critical period of organogenesis, in that the mean number of viable foetuses was slightly greater in the treated groups in relation to the control group. The only possible effect of treatment seen in the reproductive or foetal parameters was a statistically significant increase in post-implantation loss in the 300 and 1800 mg/kg/day groups. The biological significance of this finding was deemed equivocal for the 1800 mg/kg/day dosage group and biologically insignificant for the 300 mg/kg/day group. Substantiating and concurrent observations included no statistically significant effect of treatment on post-implantation loss in the 1200 mg/kg/day group as well as no treatment-related effect on foetal weights, sex ratio and morphological variations or malformations.

The authors concluded, that oral administration of 1800 mg/kg/day of MBT to pregnant Sprague-Dawley rats induced maternal toxicity exhibited by clinical signs, reduced body weight gain and food intake. Clinical signs of toxicity were also evident in the 1200 mg/kg/day group. A dosage level of 300 mg/kg/day was determined to be a No Adverse Effect Level for maternal toxicity. MBT was neither developmentally toxic nor teratogenic at dosage levels of 300, 1200, or 1800 mg/kg/day, thus a NOAEL for developmental toxicity of 1800 mg/kg/day is suggested.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: approximately 3 months
- Weight at study initiation: 240 to 315 g on gestation day 0
- Housing: individually (except during mating)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72°F +- 4°F
- Humidity (%): 55% +- 15%

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

dosage volume: 10 mL/kg bw
MBT was suspended in corn oil at specified concentrations using a stir plate and stir bars. Dosage suspensions were stirred until homogeneous. All dosage suspersions were prepared weekly, stored refrigerated and dispensed daily for dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations analyses were performed on samples from each dosage level on the first day of dosing, midway through dosing and near conclusion of dosing.
HPLC analyses determined that MBT was proven to be stable in corn oil for a period of 7 days prior to initiation of study. Dosage suspensions were found to be homogeneous at concentrations similar to the lowest and highest levels. Analyses for concentration of the test article in the dosage suspensions revealed a recovery of the test article within ± 10 % of the targeted concentration.

Details on mating procedure:

At the conclusion of acclimation, the animals were determined to be suitable test subjects based on age, healthy external appearance, and body weight of a minimum 220 g. The rats were approximately three months old at study initiation and body weights ranged from 240 g to 316 g on gestation day 0. Selected females were cohabited with proven resident Sprague-Dawley male rats which were six to nine months old and had a healthy appearance. The evidence of mating was determined by the presence of a copulatory plug in the vagina or a sperm positive vaginal smear. The day evidence of copulation was found was designated day 0 of gestation

Duration of treatment / exposure:

Day 6-15 of gestation

Frequency of treatment:

daily

Duration of test:

up to day 20 of gestation

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 200 mg/kg bw/day

Dose / conc.:

1 800 mg/kg bw/day

No. of animals per sex per dose:

26 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: sacrifice on Day 20 of gestation

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0, 6, 9, 12, 16, and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
on gestation days 0, 6, 9, 12, 16, and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: necropy examination included evaluation of the external surfaces, orifices, and viscera. Emphasis was placed on morphopathological changes that could interfere with intrauterine survival and fetal development

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Uteri with no evidence of implants were placed in 10% aqueous ammonium sulfide solution and examined macroscopically for detection of early embryonic deaths.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]

Statistics:

two-tailed analyses for a significance level of 5%; comparison of treated groups with control group 1 or by an individual group by group comparison;
one way analyses of variance followed by Dunnett's test was used to analyse maternal and fetal data including body weights, food consumption, numter of viable fetuses, implanation sites, and corpora lutea.
Man-Whighney U test used for postimplantation loss, dead fetuses and resorptions.
Fetal sex ratios were analysed using the Chi-Square test. A one-tailed Fisher's Exact test was used in analyses of fetuses and litters with malformations and vairations.

Description (incidence and severity):

Salivation, dark material around mouth, and urine staining were the most prevalent signs noted in the 1200 and 1800 mg/kg/day group following dosing. Several rats in the 1800 mg/kg/day group also displayed decreased activity. Daily observations, made prior to dosing, included soft stools, fecal and urine staining, hair loss, and dark material around nose in the control group; hair loss and dark material around nose and eyes in the low dosage group; umbilical hernia, hair loss, urine staining, and dark material around nose in the mid dosage group, and increased frequency of urine staining and dark material around nose and mouth in the high dosage group.

Description (incidence):

Survival was 100% in the control, 300, and 1200 mg/kg/day groups. One rat in the 1800 mg/kg/day group died on gestation day 16.
Pregnancy percentages were 92.3, 88.5, 96.2, and 84.6 in the control, 300, 1200, and 1800 mg/kg/day groups, respectively.

Description (incidence and severity):

Mean maternal body weights were significantly (p < 0.05) reduced in the 1800 mg/kg/day group on gestation day 9 and comparable to the corresponding control values for the remaining gestation days.
Calculation of body weight changes revealed significant weight loss in the high dose group between gestation days 6 - 9. Body weights and body weight changes were in general comparable between the control, 300, and 1200 mg/kg/day groups throughout gestation. A significantly increased body weight gain occurred during treatment in the low dosage group and was attributed to random occurrence. Net maternal terminal body weight gain was comparable between the control and MBT treated groups.

Description (incidence and severity):

Food consumption, calculated as g/animal/day or g/kg/day was significantly reduced (p < 0.01) in the 1800 mg/kg/day group between gestation days 6 - 9. Food intake, calculated as g/kg/day was significantly
(p < 0.05) increased in the 1200 and (p < 0.01) in the 1800 mg/kg/day groups between gestation days 9 -12. There were no other remarkable differences noted between the groups.

Description (incidence and severity):

No treatment-related morphopathological changes occurred in this study. One rat in the 1800 mg/kg/day group died on gestation day 16 due to a congenital malformation. Necropsy findings included umbilical hernia and intestines with brown mucoid contents.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

1 200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Description (incidence and severity):

The mean number of viable fetuses in the treated groups was slightly greater than in the control group. However, the mean number of early resorptions was increased in all treated groups, resulting in a significantly (p < 0.05) increased post-implantation loss in the 300 and 1800 mg/kg/day groups. There was no statistically significant reduction of the fetal weights in the MBT groups in relation to the control group.

Description (incidence and severity):

Fetal sex ratios were similar in all groups.

Description (incidence and severity):

No statistically or biologically significant differences were noted between the control and MBT groups with regard to fetal malformations or variations. The malformations that occurred in this study are commonly seen in rats. Morphological variations were similar and occurred with comparable frequency in the control and treated groups.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

1 800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

In summary:

Maternal toxicity was manifested by pharmacotoxic signs, reduced body weights, and food intake. Pharmacotoxic signs, mainly salivation, dark material around mouth, and urine staining were observed in the 1200 and 1800 mg/kg/day groups following treatment. Decreased activity was noted also in several rats in the 1800 mg/kg/day group. No treatment-related deaths occurred in this experiment. One rat in the high dosage group died on gestation day 16 due to a congenital anomaly (umbilical hernia) which was aggravated by pregnancy. Treatment-related effects on body weights and food consumption were apparent in the 1800 mg/kg/day group. Significant body weight loss occurred in this group between gestation days 6 and 9, and food intake was significantly reduced during the same interval, correlating with the body weight loss. However, body weight gains and food consumption were similar to the control group for the remainder of gestation. Body weight gains and food intake were comparable between the control, 300, and 1200 mg/kg/day groups throughout gestation. There were no morphopathological changes seen in the rats examined at the time of cesarean section.

The number of viable fetuses in the treated groups was slightly greater than in the control group. However, the mean number of post- implantation loss (early resorptions) was increased in all MBT groups, with statistical significance noted in the 300 and 1800 mg/kg/day groups. Although increased, the post-implantation loss in the 300 and 1200 mg/kg/day groups did not appear to be treatment-related in that: (1) the increase was not dose-dependent; (2) it was statistically significant at the lover dosage level but not at the 1200 mg/kg/day level; (3) the mean values were within the range of the historical control data; (4) similar effects were not apparent in the range-finding study (CMA 1989) at any of the dosage levels. Mean post-implantation loss in the 1800 mg/kg/day group (1.7) was slightly outside the range of our historical control data (0.6- 1.4). However, reports on post-implantation loss in Sprague-Dawley

CD rats determined a range of 0.3 to 1.8 Lang (1988*) and Woo and Hoar (1979**) averaged the data obtained from 157 studies on CD rats and found an average resorption rate of 7%. In the present study, resorption rate in the control group was 5.3%, an unusually low value. In the light of these findings, significantly increased post-implantation loss in the 1800 mg/kg/day group appears toxicologically equivocal while that of the 300 mg/kg/day group was deemed biologically insignificant. No statistically significant differences occurred between groups with respect to fetal weights. Mean fetal weight was slightly reduced (approximately 5%) in the 1800 mg/kg/day group: however, the mean value for this group (3.5 g) is well within the range of the historical control data (3.4-3.7 g) for this parameter. The type and incidence of malformations and variations did not demonstrate a teratogenic effect produced by MBT. The malformations and variations which occurred in this study are commonly seen in the rat fetuses.

The authors conclude that oral administration of MBT to pregnant Sprague-Dawley rats induced frank maternal toxicity at a level of 1800 mg/kg/day expressed by pharmacotoxic signs, reduced body weight gains, and food intake. Pharmacotoxic signs were the only indication of treatment-related effects at a dosage level of 1200 mg/kg/day. A dosage level of 300 mg/kg/day was determined to be a no effect level for maternal toxicity. MBT administered during major organogenesis at dosage levels of 300, 1200, or 1800 mg/kg/day did not induce any developmental toxicity or teratogenicity.

* Lang, P. L., Embryo and fetal developmental toxicity (teratology) control data in the Charles River Crl:CD®BR rat, (1988). Charles River, The International Standard from the Hand of the Veterinarian to

Research.

**Woo, D. C., and R. M. Hoar. Reproductive performance and spontaneous malformations in control Charles River CD®rats: A joint study by MARTA (1979). Teratology, 19:54A.

RS-Freetext:
1) 300 mg/kg bw/d:

PI loss.

RS-Freetext:
2) 1200 mg/kg bw/d:
salivation, F;
urine staining, F;
dark red material around mouth, F.

RS-Freetext:
3) 1800 mg/kg bw/d:
PI loss;
salivation, F;
urine staining, F;
dark red material around mouth, F;
activity, decr, F;
bw, decr, F;
food consumption, decr, F, Days 6-9.

Executive summary:

The developmental toxicity of MBT was evaluated in a teratology study with pregnant Sprague-Dawley rats (CMA 1989a). The test substance was orally administered during the critical period of organogenesis (gestation days 6 through 15). Groups of 26 rats, presumed pregnant, were treated with MBT at dosage levels of 300, 1200, and 1800 mg/kg/day. MBT was suspended in corn oil and administered by gavage at a volume of 10 ml/kg. A concurrent control group, consisting of 26 rats, received corn oil at an equivalent dosage volume. All rats were observed daily for signs of toxicity. Body weights and food consumption were measured on gestation days 0, 6, 9, 12, 16, and 20. Caesarean sections were performed on gestation day 20. Foetuses were weighed and examined for external, visceral, and skeletal anomalies.

Maternal toxicity was evident in the 1800 mg/kg/day group and manifested by clinical signs (salivation, dark red material around mouth, urine staining, and decreased activity), body weight losses and reduced food consumption between gestation days 6 and 9. Clinical signs (salivation, urine staining, and dark material around mouth) were the only indication of treatment-related effects in the 1200 mg/kg/day group. There were no treatment-related effects noted in the 300 mg/kg/day group.

Foetal viability was not affected by the administration of MBT during the critical period of organogenesis, in that the mean number of viable foetuses was slightly greater in the treated groups in relation to the control group. The only possible effect of treatment seen in the reproductive or foetal parameters was a statistically significant increase in post-implantation loss in the 300 and 1800 mg/kg/day groups. The biological significance of this finding was deemed equivocal for the 1800 mg/kg/day dosage group and biologically insignificant for the 300 mg/kg/day group. Substantiating and concurrent observations included no statistically significant effect of treatment on post-implantation loss in the 1200 mg/kg/day group as well as no treatment-related effect on foetal weights, sex ratio and morphological variations or malformations.

The authors concluded, that oral administration of 1800 mg/kg/day of MBT to pregnant Sprague-Dawley rats induced maternal toxicity exhibited by clinical signs, reduced body weight gain and food intake. Clinical signs of toxicity were also evident in the 1200 mg/kg/day group. A dosage level of 300 mg/kg/day was determined to be a No Adverse Effect Level for maternal toxicity. MBT was neither developmentally toxic nor teratogenic at dosage levels of 300, 1200, or 1800 mg/kg/day, thus a NOAEL for developmental toxicity of 1800 mg/kg/day is suggested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Quality of whole database:

NOAEL based on studies with rats and rabbits

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

Read-across with MBT:

The developmental toxicity potential of MBT was evaluated in Sprague-Dawely rats (CMA 1989) and in New Zealand White rabbits (CMA 1989a).

The developmental toxicity of MBT was evaluated in a teratology study with pregnant Sprague-Dawley rats (CMA 1989). The test substance was orally administered to pregnant Sprague-Dawely rats during the critical period of organogenesis. Groups of 26 rats, presumed pregnant, were treated with MBT at dosage levels of 300, 1200, and 1800 mg/kg/day. MBT was suspended in corn oil and administered by gavage at a volume of 10 ml/kg. A concurrent control group, consisting of 26 rats, received corn oil at an equivalent dosage volume. The treatment was performed from gestation day 6 through gestation day 15. All rats were observed daily for signs of toxicity. Body weights and food consumption were measured on gestation days 0, 6, 9, 12, 16, and 20. Caesarean sections were performed on gestation day 20. Foetuses were weighed and examined for external, visceral, and skeletal anomalies. Maternal toxicity was evident in the 1800 mg/kg/day group and manifested by clinical signs (salivation, dark red material around mouth, urine staining, and decreased activity), body weight losses and reduced food consumption between gestation days 6 and 9. Clinical signs (salivation, urine staining, and dark material around mouth) were the only indication of treatment-related effects in the 1200 mg/kg/day group. There were no treatment-related effects noted in the 300 mg/kg/day group. Foetal viability was not affected by the administration of MBT during the critical period of organogenesis, in that the mean number of viable foetuses was slightly greater in the treated groups in relation to the control group. The only possible effect of treatment seen in the reproductive or foetal parameters was a statistically significant increase in post-implantation loss in the 300 and 1800 mg/kg/day groups. The biological significance of this finding was deemed equivocal for the 1800 mg/kg/day dosage group and biologically insignificant for the 300 mg/kg/day group. Substantiating and concurrent observations included no statistically significant effect of treatment on post-implantation loss in the 1200 mg/kg/day group as well as no treatment-related effect on foetal weights, sex ratio and morphological variations or malformations. The authors concluded, that oral administration of 1800 mg/kg/day of MBT to pregnant Sprague-Dawley rats induced maternal toxicity exhibited by clinical signs, reduced body weight gain and food intake. Clinical signs of toxicity were also evident in the 1200 mg/kg/day group. A dosage level of 300 mg/kg/day was determined to be a No Adverse Effect Level for maternal toxicity. MBT was neither developmentally toxic nor teratogenic at dosage levels of 300, 1200, or 1800 mg/kg/day, thus a NOAEL for developmental toxicity of 1800 mg/kg/day is suggested.

Another teratology study was performed with New Zealand White rabbits (CMA 1989). The objective of this study was to evaluate potential toxic and teratogenic effects of 2-mercaptobenzothiazole (MBT) when administered orally to pregnant rabbits during the critical period of organogenesis. 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. Caesarean section was performed on gestation day 29. Intrauterine survival was evaluated and foetuses were examined for external, visceral, and skeletal anomalies. Oral administration of MBT to pregnant rabbits at a level of 300 mg/kg/day induced maternal toxicity as expressed by reduced body weight gain during treatment (day 6 to 19: -63 % vs. control) and slightly elevated liver weight, absolute (+ 7 %) or relative (+ 4 %) to final body weight. This toxicity became apparent upon visual inspection of individual body weight gain data particularly during the last days of treatment (gestation days 15-19) for this group when compared to the control group. During this period, six treated does had body weight losses greater than 150 grams while no control dam lost this magnitude of body weight. Lower dosage levels of MBT (50 and 150 mg/kg/day) did not produce any evidence of maternal toxicity. A single abortion was noted in the 150 mg/kg/day group on gestation day 19 and a premature delivery occurred in the 300 mg/kg/day group on gestation day 29, prior to the scheduled caesarean section. A single incidence of abortion was noted in the range-finding study at levels of 600 and 1000 mg/kg/day (CMA 1989). However, the biological and toxicological significance of the abortion and premature delivery in the present study is equivocal, knowing that abortions or premature deliveries are common in rabbits; a dose-related pattern was not evident (in particular, the time of occurrence); abortions were not noted in the range-finding study at levels of 150 or 300 mg/kg/day. One rabbit in the 300 mg/kg/day group died on gestation day 13. Post-mortem findings indicated that the death occurred following an intubation error (semi-solid yellow contents in trachea).

The administration of MBT during the critical period organogenesis at dose levels of 50, 150, or 300 mg/kg/day did not induce any developmental toxicity or teratogenicity, in that all foetal data, including viability, mean body weight, malformations, and variations were comparable between the MBT and control groups. The authors concluded that, oral administration of 50, 150, or 300 mg/kg/day of MBT to pregnant New Zealand White rabbits during major organogenesis did not induce any developmental toxicity or teratogenicity. Thus, based on the selected dose range evaluated and the findings of this study a NOAEL developmental toxicity of 300 mg/kg bw and day is suggested. Maternal toxicity was evident only at a level of 300 mg/kg/day as slightly decreased body weight gain and slightly elevated liver weight.

Based on the available information there is no reason to classify neither metallic zinc nor any of the zinc compounds considered by the EU risk assessment (2004) or MAK (2009) for reproductive toxicity.