Di(benzothiazol-2-yl) disulphide

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Reference 2

Reference Type:

publication

Title:

Mercaptobenzothiazole (MBT): A two-generation study using Sprague-Dawley rats

Author:

Mercieca, M., D., et al.

Year:

1991

Bibliographic source:

The Toxicologist, vol. 11., no. 2, 112

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

Benzothiazole-2-thiol

EC Number:

205-736-8

EC Name:

Benzothiazole-2-thiol

Cas Number:

149-30-4

Molecular formula:

C7H5NS2

IUPAC Name:

1,3-benzothiazole-2-thiol

Details on test material:

MBT lot no.: N8F-228, purity: 98.2 % and 98.5 %

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: MBT contained in the diet

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.

Duration of treatment / exposure:

Exposure period: 10 weeks before mating, through gestation and lactation until sacrifice
Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks
Duration of test: approximately 88 days past weaning

Frequency of treatment:

ad libitum

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.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 2500, 8750, or 15000 ppm (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

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Sex:

male/female

Basis for effect level:

other: no biological relevant effects on reproductive function

2

Dose descriptor:

LOAEL

Effect level:

2 500 ppm

Sex:

male/female

Basis for effect level:

other: body weight reduced

Results: F1 generation

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 000 ppm

Sex:

male/female

Basis for effect level:

other: no biological relevant effects on reproductive function

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

15 000 ppm

Sex:

male/female

Basis for effect level:

other: no biological relevant effects on reproductive function

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Analytical Chemistry Data

Homogeneity and stability analyses determined that the test diets were

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

F0 Generation

Clinical Observations and Survival

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

Body Weights F0 males

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

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

Body Weights F0 females

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

Food Intake F0 males

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

Food Intake F0 females

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

Test article intake F0 males and females:

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

Fertility Data (F0)

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

Pathology F0

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

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

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

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

Mean absolute liver weight was comparable between the control andtreated groups for both males and females. However, mean relative weightappeared significantly increased in the 8750 and 15000 ppm male andfemale groups. This increase is a result of reduced final body weightsfor both males and females in these two groups and not a direct effect onorgan weight.

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

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

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

F1 generation

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

F1 Pup Observations

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

F1 Pup Body Weight

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

F1 pups Necropsy Observations

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

Fl Generation Adults

F1 Clinical Observations and Survival

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

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

F1 males Body Weights

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

F1 females Body Weights

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

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

F1 males Food Intake

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

F1 females Food Intake

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

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

Test article intake F1 males and females:

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

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

Fertility Data F1 generation:

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

Pathology F1 generation:

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

Microscopic lesions were observed in the kidneys of males and females from the 8750 and 15000 ppm groups. Renal lesions were similar to those seen in the F0 parental animals. The incidence of brown pigment in males was 13 and 20 in the mid and high dosage groups, respectively, while in females, the incidence was much lower, 0 and 6 in the same groups. Cortical tubular basophilia occurred in all groups but the incidence was greater in the males of the high dosage group (0, 3, 4 and 10 in the 0, 2500, 8750, and 15000 ppm, respectively). Alpha 2 µ-globulin inclusions in the epithelial cells of the proximal convoluted tubules occurred in males of all MBT groups, with a higher incidence than in the control males.Histopathological findings correlated with anincrease in absolute kidney weight noted in males from the 8750 and15000 ppm groups, with statistical significance showing at the high levelonly. Relative kidney weight was significantly increased in both mid andhigh dosage groups. Absolute kidney weight for females in the treatedgroups was similar to the control group. Relative kidney weight on theother hand, was statistically significantly increased in all MBT groupsdue to a decreased final body weight in these groups, and thus was notconsidered toxicologically significant.

Other treatment-related changes, consisting of hepatic parenchymalhypertrophy, occurred in males and females from the 8750 and 15000 ppmgroups. The incidence of this finding was greater in the males (0, 1, 22and 23 in the 0, 2500, 8750 and 15000 ppm groups, respectively) than inthe 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 appearedstatistically significantly increased for males in all MBT groups and forfemales in the mid and high dosage groups.

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

F2 Pup Viability

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

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

F2 Pup Observations:

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

F2 Pup Body Weights:

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

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

F2 Necropsy Observations:

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

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

In summary:

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

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

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

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

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

The authors suggested:

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

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

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

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

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

Applicant's summary and conclusion

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:

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