Zinc di(benzothiazol-2-yl) disulphide

Endpoint summary

Description of key information

There are only limited repeated dose toxicity data from ZMBT available from an early cancer study in mice, which indicates that ZMBT was not carcinogenic under the experimental conditions used.  

A read across approach with data from MBT as source was conducted. There are several repeated dose toxicity studies available for MBT. In a two-generation toxicity study (key study with the source MBT, CMA 1990) a LOAEL of 2500 ppm (ca. 150 mg to 250 mg/kg bw) was observed. In a two year carcinogenicity study with Fischer 344 rats (NTP 1988) a LOAEL of 188 mg/kg bw and day for females and of 375 mg/kg bw and day for males was determined. In a subchronic toxicity study in rats (NTP 1988) a LOAEL of 187 mg/kg bw was noted. In mice a LOAEL of 375 mg/kg bw was suggested in a two-year carcinogenicity study (NTP 1988) and a NOAEL of 188 mg/kg bw in a subchronic toxicity study (NTP 1988). Thus, the NOAEL and LOAEL values from several MBT repeated dose studies are in the same range. The consistent findings in subchronic, chronic and reproduction/ developmental toxicity studies revealed a LOAEL in the range of 150 to 375 mg/kg bw and day. Following the recommendation given in the MAK publication (1999) an overall NOAEL of 50 mg/kg bw and day is suggested. This assumption based on data from the two-generation toxicity study in rats (key study with the source MBT) and is supported by the consistent findings in the subchronic and chronic toxicity studies. The overall NOAEL value of 50 mg/kg is used for DNEL calculation.  

There are also repeated dose data available for zinc, the second component of ZMBT (see discussion endpoint summary toxicokinetics). Data from human volunteers suggested an oral systemic NOAEL of 50 mg Zn2+/day (0.83 mg/kg bw/day), which based on a LOAEL of 150 mg Zn2+/day at which clinical signs and indications for disturbance of cupper homeostasis have been observed  (EU risk assessment 2004); whereas the MAK commission suggested a NOAEL of 0.43 mg Zn2+/kg bw/day, in a more recent study with a single exposure value (no LOAEL reported) (MAK 2009).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Two generation reproduction toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study (two-generation reprotoxicity 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:

other: 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 oral exposure:

Preparation of Test Diets
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.

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.

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:

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.

Frequency of treatment:

daily

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

No. of animals per sex per dose:

28 per sex and dose

Control animals:

yes

Positive control:

not adequate

Observations and examinations performed and frequency:

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 / No / Not specified
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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

OTHER:
Breeding
Following a minimum of 70 days of treatment for the FO 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.
Following completion of breeding, FO males from all study groups cohabitated with untreated females for a dominant lethal evaluation
During cohabitation, the diet was removed for the time the females with the males (from 4:00p.m. to midnight).
The Fl offspring were selected at weaning from different litters within the same dose level for cross matings to become parents of the F2 generation. during the breeding phase, sibling mating was avoided.

If copulation was not evident after ten days of mating, the female was paired with a proven male from the same group for five additional days. When mating was not successfully completed during the 15 days allocated for breeding, or when the pregnancy occurred without evidence of copulation, the females were separated from the males and placed in plastic cages with Bed-O-Cobs® laboratory animal bedding.

Gestation and Parturition
The rats were observed daily during gestation for signs of abortion or premature delivery. At the end of the gestation period, the rats were observed for signs of difficult or prolonged parturition at leat twice daily. The day parturition was completed was designated lactation day 0.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Parental FO and F1 animals were held until all litters were weaned and then sacrificed and necropsied. A complete examination with special attention given to the reproductive system, was performed on all FO and F1 adults. The following organ or tissue samples were taken and fixed in 10% neutral buffered formalin:
epididymides, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, testes, uterus, vagina, all gross lesions.

Organ Weights
The liver, kidneys, testes or ovaries from all FO and Fl adults were weighed at necropsy.

HISTOPATHOLOGY: Yes
Hematoxylin and eosin stained paraffin sections of all organs/tissues collected from the control and 15000 ppm groups were examined microscopically. Microscopic examination was limited to kidneys in the 8750 and 2500 ppm F0 groups and to the liver and kidneys in the Fl groups.

Other examinations:

F1 and F2 offspring
Standardization of Litter Size
On lactation day 4, litters were culled to a maximum of eight pups, four per sex whenever possible. The selection was random and the
remaining pups were weighed, sacrificed and discarded.

Standardization of Litter Size
Litter Data
Pup viability was determined daily throughout lactation. Detailed
examination of the pups was performed on lactation days 0, 4, 7, 14, and 21. The sex of the pups was determined on lactation day 0 and verified on lactation days 4 and 21. Individual pup weights were measured on lactation days 1, 4, 7, 14, and 21. Pups dying during lactation were necropsied. Emphasis was placed on morphological abnormalities. Following weaning, 28 Fl males and 28 Fl females were selected randomly from each group to breed the next generation. Nonselected Fl pups were sacrificed and examined. F2 pups were sacrificed and necropsied on lactation day 21.

Statistics:

Statistical analyses were performed by a Digital VAX 11/730 computer.
All analyses utilized two-tailed tests for a minimum significance level of 5%comparing the control group to each treatment group. Control group data were compared to the treated data using all groups or by an individual group by group comparison depending on the test. Quantitative data including maternal, fetal, and pup body weights, maternal body weight gains, maternal food consumption, gestation length and mean live litter size were analyzed by One-Way Analysis of Variance (ANOVA) [7]. If significance was detected, group by group comparisons were performed using Dunnett's test [8]. Qualitative data were analyzed using Chi-Square test for pup sex ratios, the number of dead pups per group on lactation day 0, and pup viability after lactation day 0.

Description (incidence and severity):

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

Mortality:

no mortality observed

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).

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):

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.

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):

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.

F1 generation:
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.

Description (incidence and severity):

F0 and F1 generation
There were no treatment-induced gross lesions observed in any MBT groups. Incidental findings were noted mainly in the kidneys, testes, pituitary, and thymus in one or two rats in various study groups. There was no evidence of infectious diseases which could have had an impact on the outcome of the study.

Description (incidence and severity):

F0 generation:
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.

F1 generation:
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.

Dose descriptor:

LOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight gain reduced

Critical effects observed:

not specified

Analytical Chemistry Data

Homogeneity and stability analyses determined that the test diets were

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

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. 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.

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.

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 relative 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.

The authors concluded that minimal to mild toxic effects (reduced body weights, increase in absolute and relative kidney and liver weights combined with histopathological changes in the kidneys and liver) 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.

Based on findings of this study (reduced body weight gain) a LOAEL of 2500 ppm (ca. 150 to 250 mg/kg bw MAK 1999) is suggested.

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Two generation reproduction toxicity study

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

Description (incidence and severity):

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

Mortality:

no mortality observed

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).

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):

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.

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):

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.

F1 generation:
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.

Description (incidence and severity):

F0 and F1 generation
There were no treatment-induced gross lesions observed in any MBT groups. Incidental findings were noted mainly in the kidneys, testes, pituitary, and thymus in one or two rats in various study groups. There was no evidence of infectious diseases which could have had an impact on the outcome of the study.

Description (incidence and severity):

F0 generation:
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.

F1 generation:
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.

Dose descriptor:

LOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight gain reduced

Critical effects observed:

not specified

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. 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.

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.

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 relative 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.

The authors concluded that minimal to mild toxic effects (reduced body weights, increase in absolute and relative kidney and liver weights combined with histopathological changes in the kidneys and liver) 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.

Based on findings of this study (reduced body weight gain) a LOAEL of 2500 ppm (ca. 150 to 250 mg/kg bw MAK 1999) is suggested.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

limited but acceptable documented publication, which meets basic scientific principles (This long-term carcinogenicity study is not in accordance with current testing procedures as proposed by guidelines on carcinogenicity and/or combined chronic toxicity carcinogenicity. however, this study is performed in accordance with generally accepted scientific standards.)

Principles of method if other than guideline:

other: Method: other early carcinogenicity study, oral application

GLP compliance:

no

Species:

mouse

Strain:

other: strains: B6C3F1 and B6AKF1

Sex:

male/female

Route of administration:

other: gavage (days 7-28 of age) and in the diet (after 28 days of age)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

18 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
1000 mg/kg (gavage) 3385 ppm (diet) (ca. 508 mg/kg bw/d)
Basis:

No. of animals per sex per dose:

18 per dose and sex and mice strain

Control animals:

yes

Dose descriptor:

NOAEL

Effect level:

3 385 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: ZMBT did not cause a significant increase in tumors

Critical effects observed:

not specified

ZMBT did not cause a significant increase in tumors

Endpoint conclusion

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are only limited data of repeated dose toxicity available for ZMBT. The carcinogenic potential of ZMBT was evaluated in an early cancer study in two mice strains (strains B6C3F1 and B6AKF1) (NCI 1968, Innes 1969). Even though the study was not performed according to present standards, it should be mentioned that ZMBT was not found to be tumorigenic when given orally to mice for 18 months at the maximal tolerated dose, which was fixed at 1000 mg/kg ZMBT by gavage (dissolved in 0.5 % gelatine) from day 7 to 28 of age and at 3385 ppm in the diet (ca. 508 mg/kg bw/day) from day 28 for additional 17 months.

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.

Read-across approach with MBT

Key study with MBT

A two-generation reproduction 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. 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. 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. 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 relative 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. The authors concluded that minimal to mild toxic effects (reduced body weights, increase in absolute and relative kidney and liver weights combined with histopathological changes in the kidneys and liver) 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. Based on findings of this study (reduced body weight gain) a LOAEL of 2500 ppm (ca. 150 to 250 mg/kg bw, MAK 1999) is suggested for this study.

Based on the recommendation of MAK (1999) the two-generation reproduction toxicity feeding study (CMA 1990) in rats with a LOAEL of 2500 ppm (ca. 150 mg to 250 mg/kg bw/day) is the most relevant study for the assessment of repeated dose toxicity of MBT. Following MAK (1999) an overall NOAEL of 50 mg MBT/kg bw and day is suggested for this study and this NOAEL is taken forward for DNEL derivation.

Supporting subchronic studies with MBT:

Subchronic studies were conducted with Fischer 344 rats and B6C3F1 mice to evaluate the cumulative toxic effects of repeated administration of MBT and to determine the doses to be used in the 2-year studies discussed below (NTP 1988). MBT was administered once daily via gavage. Animals were checked two times per day for clinical signs and mortality. Individual animal weights were recorded weekly. At the end of the 13-week studies, survivors were killed. A necropsy was performed on all animals except those excessively autolysis or cannibalized. Histological exams performed on some animals from all groups. No hematology and clinical biochemistry parameters were evaluated.

In the subchronic toxicity study with Fischer 344 rats groups of 10 animals of each sex were administered 0, 188, 375, 750, or 1500 mg/kg MBT in corn oil by gavage, 5 days per week for 13 weeks. The 13-week study in rats reported in this Technical Report was a second study. In the first study in rats, 3000 mg/kg groups all died during week 1. No substance-related deaths occurred in Fischer 344 rats treated up to 1500 mg/kg bw and day MBT; whereas in an early study all rats died within 2 days after receiving 3000 mg/kg bw and day. The body weight gain of males from the highest dose group and females of the both highest dose groups were decreased. Treated rats displayed irritable behavior that was more pronounced with increasing dose and was characterized as resistance to gavage. Liver weight and liver weight to body weight ratios were increased in dosed rats with the greatest change occurring at the two highest doses (750 and 1500 mg/kg). No gross or microscopic effects could be related to chemical administration in treated rats. Based on the findings of the study, increased liver weights, decreased body weight gain and irritable behavior after dosing, a LOAEL of 187 mg/kg bw and day is suggested.

In the subchronic toxicity study with B6C3F1 mice groups of 10 mice of each sex were administered 0, 94, 188, 375, 750 or 1500 mg/kg MBT. Deaths occurred in B6C3F1 mice of the highest dose groups; five of 10 males and 7/10 females that received 1500 mg/kg and 2/10 females dosed with 750 mg/kg bw and day died before the end of the studies. Two of the deaths were related to gavage technique. Chemical administration did not affect body weight gain of treated mice. Liver weights to body weight ratios of dosed groups were higher than those of the vehicle controls. Clonic seizures, lacrimation, and salivation were observed in the 750 and 1500 mg/kg groups. Lethargy and rough coats were observed in the 375 and 750 mg/kg groups. No compound-related gross or microscopic pathologic effects were observed. Based on the findings of the study a NOAEL of 188 mg/kg bw and day is suggested.

The ‘human biomonitoring’ commission of the German Federal Environmental Agency (2015) derived an overall NOAEL of 94 mg/kg bw/day for MBT, taking the subchronic study in mice (NTP 1988) as basis.

Supporting chronic studies with MBT:

In a carcinogenicity study (NTP 1988) male and female Fischer 344 rats were administered with MBT for 103 weeks. Groups of 50 male rats were administered 0, 375, or 750 mg/kg 2-mercaptobenzothiazole in corn oil by gavage, 5 days per week for 103 weeks. Groups of 50 female rats were administered 0, 188, or 375 mg/kg 2-mercaptobenzothiazole in corn oil by gavage on the same schedule. All animals were observed two times per day, and clinical signs were recorded once per week. Body weights by cage were recorded once per week for the first 12 week and once per month thereafter. A necropsy was performed on all animals including those found dead. During necropsy, all organs and tissues were examined for grossly visible lesions. Necropsy and histological exams were performed on all animals and all relevant tissues. No haematology and clinical biochemistry were performed. Survival of the low dose group of male rats was significantly lower (p < 0.001) than that of the vehicle controls after week 85 and of the high dose group after week 83 (mortality: low dose 28/50, high dose: 29/50; control: 8/50). Mortality of treated females were not significant different from control. Mean body weights of dosed male rats were similar to or greater than those of the vehicle controls. Mean body weights of dosed female rats were generally greater (up to 11%) than those of the vehicle controls. Rats were lethargic and showed prostration after they were dosed. The principal non-neoplastic lesions seen in these studies were nephropathy and inflammation and ulceration of the forestomach in rats. Nephropathy, characterized by tubular degeneration and regeneration, was present in all male rats and in more than 75% of the female rats; a severity grade from minimal to severe (1- 4) was recorded for each animal. The mean severity of nephropathy was increased in dosed male rats (vehicle control: mild-moderate; low dose and high dose: moderate-severe). However, these changes were later discussed as questionable (TSCA 1991-1993 cited in BG Chemie 2000). Ulcers and inflammation were observed at increased incidences in dosed rats, and epithelial hyperplasia and hyperkeratosis were observed at increased incidences in dosed male and low dose female rats. These effects were later discussed as treatment related effect (bolus-effect) but not as substance-related effect. The occurence of neoplatic lesions were noted in male and female rats and will be discussed in the chapter carcinogenicity. Overall, no clear carcinogenic activity can be concluded from this study.

Based on the findings of this study a LOAEL for males of 375 mg/kg bw and day (effects: decreased survival rate, lethargic and prostration after dosing) and for females of 188 mg/kg bw and day (effects: lethargic and prostration after dosing) was suggested for repeated dose toxicity.

In an additional NTP carcinogenicity study male and female B6C3F1 mice were administered with MBT for 103 weeks (NTP 1988). Groups of 50 male and 50 female mice were administered 0, 375, or 750 mg/kg 2-mercaptobenzothiazole in corn oil by gavage, 5 days per week for 103 weeks. The study design and schedule were similar to the Fischer 344 rat carcinogenicity study discussed. Survival of the high dose group of female mice was significantly lower than that of the vehicle controls after week 27. The survival rat of the female low dose group and the male treatment groups were comparable to control. Six high dose male and four high dose female mice died on the same day during week 13. Since they were mistakenly dosed twice within a 16-hour period, these mice were censored from the statistical incidence of survival after week 12. Mean body weights of high dose male mice were 6%-14% lower than those of the vehicle controls from week 3 to week 64; the mean body weights of low dose males were 4 to 8 % lower compared to control from week 6 to week 64. In females the mean body weights of the high dose group were within 6 % of the vehicle control, whereas the mean body weights from the low dose group were generally greater than those of the vehicle controls throughout the studies. Similar to the rat study, mice were lethargic and showed prostration after they were dosed; however, no histological alterations of the nerve system were revealed. No substance-related non-neoplastic lesions were indicated in any of the treated mice. The Pathological and histopathological incidences of non-neoplastic lesions were in the range of the vehicle control. Neoplastic lesions noted were within the historical control data range. Based on the findings of this study, the lethargy after dosing, a LOAEL for treated mice of 375 mg/kg bw and day is suggested.

Toxicological data for zinc:

Zn2+ is an essential trace element for human nutrition and ubiquitous in biological systems including humans. The human body has efficient mechanisms, both on systemic and cellular levels, to maintain zinc homeostasis over a broad exposure range. Consequently, zinc has a rather low toxicity. On the other hand, zinc deficiency is a condition with broad occurrence and potentially profound impact (Plum et al., 2010). Toxicity and impacts on human health of zinc has been extensively evaluated by the EU (EU RAR, 2004), the Agency for Toxic Substances and Disease Registry (ATSDR, 2006) and in reviews of Plum et al. (2010), and Chasapis et al. (2020). Zinc is thus included in the assessment of ZMBT based on these assessments.

As discussed in the EU risk assessment (2004) the acute oral and acute inhalation toxicity is very low indicated by an oral LD50 value >2000 mg/kg (zinc powder) and LC50 value > 5.41 g/m³ in rats (EU risk assessment 2004). No skin irritating potential of zinc oxide was noted. In addition, zinc oxide did not show any signs of upper airway irritation. No eye irritating effects of zinc dust and zinc powders were observed. No skin sensitizing effects were noted for zinc oxide in studies with guinea pigs. This was confirmed with human evidence (EU risk assessment report zinc oxide). No data are available on the potential for respiratory sensitization. Data from human volunteers suggested an oral systemic NOAEL of 50 mg Zn2+/day (0.83 mg/kg bw/day), which based on a LOAEL of 150 mg Zn2+/day at which clinical signs and indications for disturbance of cupper homeostasis have been observed (EU risk assessment 2004). Whereas the MAK commission suggested a NOAEL of 0.43 mg Zn2+/kg bw/day, in a more recent study with a single exposure value (no LOAEL reported) (MAK 2009). There are several in vitro and in vivo genotoxicity data available for several zinc compounds. The available data indicate that genotoxicity results vary widely (EU risk assessment 2004, MAK 2009). However, there is no clear evidence from the available data that zinc is genotoxic in vivo (EU risk assessment 2004). Neither fertility nor developmental toxicity are considered endpoints of concern for humans. Based on the available information there is no reason to classify neither metallic zinc nor any of the zinc compounds considered for reproductive toxicity (EU risk assessment 2004, MAK 2009).

Recommended daily zinc levels range from 5 mg/day for infants to 19 mg/day for women during lactation (EU risk assessment 2004). As cited in the ATSDR evaluation (2006) recommended dietary allowance (RDA) for zinc are given with 11 mg/day in men and 8 mg/day in women (IOM, 2002); these correspond to approximately 0.16 mg/kg per day for men and 0.13 mg/kg per day for women. Higher RDAs are recommended for women during pregnancy and lactation (12 mg/day). ATSDR (2006) established a Minimal Risk Level (MRL) of 0.3 mg zinc/kg bw per day for intermediate oral exposure that has also been accepted as the chronic oral MRL. The chronic oral MRL is expected to be without adverse effects when consumed on a daily basis over a long period of time; neither inducing nutritional deficiency in healthy, nonpregnant, adult humans ingesting the average American diet nor resulting in adverse effects from excess consumption (ATSDR, 2006).

In conclusion, Zinc plays a minor role in the toxicological assessment of ZMBT.

In conclusion:

There are only limited data of repeated dose toxicity available for ZMBT. The carcinogenic potential of ZMBT was evaluated in an early cancer study in two mice strains (strains B6C3F1 and B6AKF1) (NCI 1968, Innes 1969). ZMBT was not found to be tumorigenic when given orally to mice for 18 months at the maximal tolerated dose.

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.

There are several repeated dose toxicity studies available for MBT. Following the recommendation given in the MAK publication (1999) the most relevant study for repeated dose toxicity assessment of MBT is the two-generation toxicity study in rats (CMA 1990). Based on the (marginal) LOAEL of 2500 ppm (ca. 150 to 250 mg/kg bw, MAK 1999), MAK suggested an overall NOAEL of 50 mg MBT/kg bw and day.  Additionally, subchronic and two year carcinogenicity studies with rats and mice are available for MBT (NTP 1988). Consistent findings in subchronic, chronic and reproduction/ developmental toxicity studies revealed a LOAEL in the range of 150 to 375 mg/kg bw and day and support the overall NOAEL of 50 mg/kg bw/day determined by MAK (1999) as conservative value.  

Zn2+ is an essential trace element for human nutrition and ubiquitous in biological systems including humans. The human body has efficient mechanisms, both on systemic and cellular levels, to maintain zinc homeostasis over a broad exposure range. Consequently, zinc has a rather low toxicity and thus plays a minor role in the toxicological assessment of ZMBT.

The overall NOAEL of 50 mg MBT/kg bw/day is used as starting point for DNEL calculation.

Justification for classification or non-classification

No classification is required according to the classification criteria 67/548/EWG and regulation no. 1272/2008 (GHS).