Di(benzothiazol-2-yl) disulphide

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Two generation reproduction study

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 (two-generation reprotoxicity 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

Reference 3

Reference Type:

review article or handbook

Title:

2-mercaptobenzothiazol(2-(3H)-Benzothiazolthion)

Author:

MAK

Year:

1999

Bibliographic source:

Maximale Arbeitsplatzkonzentrationen (MAK), 29 Lieferung 1999,Senatskommission zur Prüfung gesundheitsschädlicher Arbeitsstoffe

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:

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

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

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

F0 generation (males and females) received 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

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

Results and discussion

Effect levels

Dose descriptor:

LOAEL

Effect level:

2 500 ppm

Sex:

male/female

Basis for effect level:

other: body weight gain reduced

Target system / organ toxicity

Critical 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 2500

ppm group were comparable with the control group throughout the study.

Mean body weight gain, however, did not parallel the reduction noted in

the body weights. A slight but significantly reduced weight gain occurred

during the first week of treatment in all MBT groups. Thereafter,

significantly reduced gain occurred in the 15000 ppm group from study

week 4 through week 8 and between weeks 17-18. Statistically significant

reductions occurred inconsistently in the low and mid dosage groups

(weeks 4-5, 7-8, 17-18 in the 8750 ppm group; weeks 2-3, 7-8, 17-18 in

the 2500 ppm group). Body weight loss was noted in all groups between

study weeks 14-16 when the males were used to breed females for a dominant

lethal evaluation (see endpoint genetic toxicity in vivo, CMA 1989); during these two weeks, the food was removed for

the time the females were with the males (from 4:00 p.m. to midnight).

Body Weights F0 females

Mean body weights, calculated for females, were slightly but

significantly decreased in the 15000 ppm group during study weeks 3, 4, 6

and continuing until the breeding period (study week 11). Mean body

weight was also slightly reduced in the high dosage group during

gestation, but did not reach significance until day 20. Lactation body

weights were slightly decreased throughout this period with statistical significance noted on days 1 and 14. Additionally, mean body weights were

significantly reduced in the high dosage group following lactation and

prior to sacrifice (study weeks 19 and 20). The female rats in the 8750

ppm group had body weights slightly but significantly reduced from week 4

to 8, and during week 20; gestation and lactation body weights in the mid

dosage group were similar to those in the control group. Body weights in the 2500 ppm group were comparable to the control group throughout the

study. Mean body weight gain calculated for the female rats did not

parallel the pattern seen in body weights. Dose-dependent and

significantly reduced weight gain occurred in the 8570 and 15000 ppm

groups during the first week of treatment. Other significant reductions

of weight gain occurred sporadically in all MBT groups and were not

considered toxicologically significant between study weeks 7-8 in the

2500 and 8750 ppm groups, and between gestation days 0-7 in the 2500

and 15000 ppm groups. During lactation, body weight gains were, in

general, higher in the MBT groups than in the control group; significant

weight loss was noted, however, in the 15000 ppm group following

lactation (study weeks 18-19).

Food Intake F0 males

Food consumption, calculated as g/animal/day, was slightly but

significantly reduced for the males in the 15000 ppm group between study

weeks 1-2 and from week 4 through week 11; significant reduction of food

intake was also noted from week 18 until sacrifice (week 20). A slightly

but significantly reduced food consumption was evident in the 8750 ppm

group between study weeks 1-2 and 18-19 only. No statistically

significant differences were observed in the 2500 ppm group. Calculation

of food intake as g/kg/day exhibited a different pattern. Dose-dependent

and significantly reduced food consumption occurred in the 8750 and

15000 ppm groups during the first week of treatment. Thereafter, food

intake in the mid and high dosage groups was statistically significantly

greater or similar to the control group.

Food Intake F0 females

Food intake, calculated as g/animal/day, was slightly reduced prior

to breeding for females in the 15000 ppm group; these differences were

statistically significant for weekly intervals 1-2, 3-4, 5-6 and

continuing until breeding (weeks 10-11). Significant reductions also

occurred between gestation days 0-7, lactation days 14-21, and following

lactation until sacrifice (weeks 18 to 20). Food intake was comparable

between the control, 2500 and 8750 ppm groups, when calculated as

g/animal/day. When calculated as g/kg/day, on the other hand, mean food

intake was significantly reduced in the 8750 and 15000 ppm groups during

the first week of treatment, in a dose-dependent fashion. Thereafter, a

statistically significant increase or decrease occurred inconsistently in

these two groups as a result of body weight changes. Food consumption

(g/kg/day) in the 2500 ppm group was comparable to the control group

throughout the study.

Test article intake F0 males and females:

Test article intake, calculated as mg/kg/day based on the food

consumption and body weights, decreased gradually for males from the first

week of treatment to sacrifice (e.g., in the 15000 ppm group, test

article intake during the first week was 1328 mg/kg/day decreasing to 778

mg/kg/day during the last week). A similar pattern was evident for the females prior to breeding (e.g., in the 15000 ppm group, test article

intake decreased from 1,326 mg/kg/day to 945 mg/kg/day). During

gestation, test article intake was slightly higher than the intake prior

to breeding but remained constant throughout this period. Test article

intake increased gradually during lactation (e.g., in the 15000 ppm

group, intake increased from 1760 mg/kg/day during the first week to

2828 mg/kg/day the last week). The apparent increase in food intake in

the second and the third weeks of lactation was due to the fact that the

pups started feeding on the diet, therefore, the increased test article

intake was not real for the dams. Following lactation (when the dams were

without pups), the test article intake returned to the values prior to breeding.

Fertility Data (F0)

Copulation and fertility indices were comparable between the

control and MBT groups for both males and females. The precoital interval

(approximately 3 days) and gestation length (average of 22 days) were also

similar in all study groups. There were no difficulties encountered at

parturition in any of the study groups.

Pathology F0

There were no treatment-induced gross lesions observed in any MBT

groups. Incidental findings were noted mainly in the kidneys, testes,

pituitary, and thymus in one or two rats in various study groups. There

was no evidence of infectious diseases which could have had an impact on

the outcome of the study.

Treatment-related microscopic changes were seen in the kidneys of

both males and females in the 8750 and 15000 ppm groups consisting of

brown pigment in the lumen and epithelial cells of the proximal tubules.

Brown pigment occurred with a higher incidence in males than in females:

12 and 17 males in the 8750 and 15000 ppm groups, respectively, while

only 1 and 4 females had the pigment in these same groups. In addition,

male rats from all MBT groups had an increased incidence of basophilic

tubules (2, 9, 10 and 13 in the control, 2500, 8750 and 15000 ppm

groups, respectively) and alpha 2 µ-globulin inclusions in the proximal

convoluted tubules (4, 10, 8 and 13 in the same groups), as evidenced by

hematoxylin-eosin staining.

Histopathological changes seen in the kidneys of the males from the

mid and high dosage groups correlated with a significantly increased organ

weight, absolute and relative to final body weight. Absolute kidney

weight measured for females in the MBT groups was comparable to the

control groups.A significant increase in the relative kidney weight noted for the females in the mid and high dosage groups occurred as a

result of a reduced final body weight in these two groups rather than a

direct effect on the kidneys.

Mean absolute liver weight was comparable between the control and

treated groups for both males and females. However, mean relative weight

appeared significantly increased in the 8750 and 15000 ppm male and

female groups. This increase is a result of reduced final body weights

for both males and females in these two groups and not a direct effect on

organ weight.

Mean absolute testes weight was significantly increased in the

2500 and 8750 ppm groups but not in the 15000 ppm group. These

differences were not dose-dependent and similar effects were not evident

in the males of the Fl generation. In addition, historical control data

show a mean testes weight of 3.3 g with a range of 3.1 to 3.7 g.

Therefore, a mean testicular weight of 3.67 g in the low dosage group and

3.70 g in the mid dosage group were within the historical control range,

indicating that the apparent testicular weight increase in these two

groups is not toxicologically relevant. Mean testes weight relative to

the final body weight appeared significantly increased in all MBT groups.

Since a dose-dependent response was not evident and similar effects were

not seen in the Fl generation, increased testicular weight was not

considered treatment-related. Furthermore, historical control data show a

mean relative testes weight of 0.655, 0.689, and 0.665 in the low, mid and

high dose groups, respectively, were well within the range of historical

control data.

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 all

groups with a very low frequency and included small epididymides, pitted

kidneys and dilated renal pelvis, enlarged lymph nodes, corneal opacity,

ovarian cyst, white splenic foci and enlarged thyroid.

Microscopic lesions were observed in the kidneys of males and

females from the 8750 and 15000 ppm groups. Renal lesions were similar

to those seen in the F0 parental animals. The incidence of brown pigment

in males was 13 and 20 in the mid and high dosage groups, respectively,

while in females, the incidence was much lower, 0 and 6 in the same

groups. Cortical tubular basophilia occurred in all groups but the incidence was greater in the males of the high dosage group (0, 3, 4 and

10 in the 0, 2500, 8750, and 15000 ppm, respectively). Alpha 2 µ-globulin inclusions in the epithelial cells of the proximal convoluted

tubules occurred in males of all MBT groups, with a higher incidence than

in the control males.Histopathological findings correlated with an

increase in absolute kidney weight noted in males from the 8750 and

15000 ppm groups, with statistical significance showing at the high level

only. Relative kidney weight was significantly increased in both mid and

high dosage groups. Absolute kidney weight for females in the treated

groups was similar to the control group. Relative kidney weight on the

other hand, was statistically significantly increased in all MBT groups

due to a decreased final body weight in these groups, and thus was not

considered toxicologically significant.

Other treatment-related changes, consisting of hepatic parenchymal

hypertrophy, occurred in males and females from the 8750 and 15000 ppm

groups. The incidence of this finding was greater in the males (0, 1, 22

and 23 in the 0, 2500, 8750 and 15000 ppm groups, respectively) than in

the females (0, 5 and 10 in the same groups).One male in the low

dosage group exhibited hepatocyte hypertrophy. Since this change is seen

occasionally in untreated rats and occurred in a single incidence, it was

not considered related to MBT treatment. Histopathological changes

correlated with a dose-dependent and a statistically significant increase

in the absolute liver weight for males in the mid and high dosage group

and for females in the high dosage group.Relative liver weight appeared

statistically significantly increased for males in all MBT groups and for

females in the mid and high dosage groups.

A statistically significant increase in relative testes weight was

noted in the 15000 ppm group. This effect was related to reduced final

body weight rather than a toxic effect on the testes. Furthermore,

historical control data show a mean relative testes weight of 0.765 with a

range of 0.637 to 1.023 g. The mean value in the high dosage group was

0.672 g, well within this range, while the mean control value of 0.610g 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.

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 (for more details on reproduction toxicity see endpoint fertility)

Mild toxic effects were noted in animals of the F0,

and Fl 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 generations. Body weights were reduced in the Fl 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.