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Key value for chemical safety assessment

Effects on fertility

Description of key information

A two-generation reproductive toxicity study was performed to evaluate the potential effects of DCBS on reproduction and development in rats (Ema 2008). Based on the category justification for CBS (CAS 95-33-0), DCBS (CAS 4979-32-2), TBBS (CAS 95-31-8) and MBS (CAS 102-77-2) a read-across with the 2-generation study with DCBS is justified and conducted.

Male and female Crl: CD (SD) rats (24 per group and sex) were fed a diet containing DCBS at 0, 80, 600 or 4500 ppm throughout the study beginning at the onset of a 10-week pre-mating period and continuing through mating, gestation, and lactation periods for two generations. The test substance intake correspond to ca. 0, 5.2, 39, 291 mg/kg bw in P0 males, 0, 7.2, 54, 416 mg/kg bw/day in P0 females, 0, 5.9, 44, 331 mg/kg bw/day in P1/F1 males and 0, 7.4, 55, 417 mg/kg bw/day in P1/F1 females. The deaths and clinical signs observed in the present study are not related to the administration of DCBS. Decreased food consumption was noted in P0 males and females at 4500 ppm and was accompanied by reduced body weight, body weight gain and food consumption. However, no consistent lowered food consumption accompanied by lower body weights were noted in P1/F1 adults. No significant changes in sperm counts, percentage of motile sperm and progressively motile sperm, swimming speed and pattern, or percentage of morphologically abnormal sperm were noted in F0 and P1/F1 adults between control and DCBS-treated groups. However, a slight but significant decrease in mean lateral head displacement was noted in P1/F1 males of the highest group (20.5 µm ± 1.0 vs. control 21.3 ± 0.9 µm). All P0 females showed normal oestrous cycles in all groups, and the length of the oestrous cycles was not different between the control and DCBS-treated groups. Although one P1/F1 female each in the control and 600 ppm groups displayed extended diestrus vaginal smears, no significant changes in the incidence of females having normal oestrous cycles or length of the oestrous cycles were observed. There were no significant differences in the copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of P1/F1 pups delivered, sex ratio of P1/F1 pups, or viability of P1/F1 pups during lactation between control and DCBS-treated groups. No malformed P1/F1 pups were found in any groups. Two P1/F1 males in the 600 ppm group did not copulate. One female in the control group and two females each in the 80 and 600 ppm groups did not become pregnant. One pregnant female in the 600 ppm group did not deliver. One dam in the control group died on day 5 of lactation, and her pups were euthanized. One dam experienced a total litter loss by PND 3 at 4500 ppm. No significant changes in the copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of F2 pups delivered, sex ratio of F2 pups, or viability of F2 pups during lactation were observed. Oliodactyly in one female of the control group and microphthalmia in one male at 80 ppm were observed. Although a few P0 and P1/F1 adults showed reproductive difficulties, necropsy and the histopathology of reproductive organs revealed no evidence of reproductive failure in these rats. In conclusion, no significant changes in reproductive indices were noted in any generation even at the highest dose 4500 ppm (ca. 291 mg/kg bw and day).

From an oral 28-day repeated dose toxicity test with CBS in rats data on reproductive organ toxicity were available (MHWJ 1997). Atrophy of seminiferous tubuli, hyperplasia of interstitial cells and decrease in epididymal sperm (each of 1/6 males, respectively were found at a dose of 800 mg/kg bw and day (14 day recovery period). No histopathological abnormalities were detected in ovary from females of 800 mg/kg groups. Based on the findings from this subacute toxicity study, the NOAEL for males concerning adverse effects on the reproductive organs is considered to be 250 mg/kg bw and day and for females 800 mg/kg bw and day (highest dose tested).

Link to relevant study records

Referenceopen allclose all

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:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Male and female Crl:CD(SD) rats were fed a diet containing rubber accelerator N,N-dicyclohexyl-2-benzothiazolesulfenamide (DCBS) at 0, 80, 600 or 4500 ppm (P0 males: 0, 5.2, 39, 291 mg/kg bw, P0 females: 0, 7.2, 54, 416 mg/kg bw, P1/F1 males: 0, 5.9, 44, 331 mg/kg bw, P1/F1 females: 0, 7.4, 55, 417 mg/kg bw) throughout the study beginning at the onset of a 10-week pre-mating period and continuing through the mating, gestation, and lactation periods for two generations.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
N,N-Dicyclohexyl-2-benzothiazolesulfenamide (DCBS, CAS No. 4979-32-2) was obtained from Ouchishinko Chemical Industrial Co. Ltd. (Tokyo, Japan). DCBS in the form of off white to tan granules is very slightly soluble in water and methanol but soluble in oil, and its melting point is 100–105°C, density at 21°C is 1230 kg/m³, and molecular weight is 347. The DCBS (Lot no. 508001) used in this study was 99.7% pure.
Species:
rat
Strain:
other: Crl:CD (SD)
Details on species / strain selection:
Rats of this strain were chosen because they are the most commonly used in reproductive and developmental toxicity studies and historical control data are available.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories Japan, Inc. (Yokohama, Japan)
- Age at study initiation: (P) x wks; (P1) 5 wks
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/-3°C
- Humidity: 50 +/- 20 %
- Air changes :10-15 times per hr
- Photoperiod: 12hrs dark / 12hrs light
Route of administration:
oral: feed
Vehicle:
other: DCBS contained in the diet
Details on exposure:
DIET PREPARATION: Dosed diet preparations were formulated by mixing DCBS into an appropriate amount of powdered basal diet (CRF-1, Oriental Yeast Co. LTd., Tokyo Japan) for each dietary concentration.


Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until copulation or mating period had elapsed (3 weeks)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purity and stability of DCBS were verified by analysis using HPLC before and after the study.
Analysis showed that DCBS was homogeneous in the diet and stable for at least 21 days at room temperature
Duration of treatment / exposure:
P0: start dosing: 10 weeks before mating (day 0 of dosing), continuing throughout the matting period, administration was continued throughout gestation and lactation, age at scheduled terminal sacrifice: males: 19-20 wks, females: 21-22 wks;
P1: start: Postnatal day (PND) 21-25 (day 0 of dosing), starting dosing: 10 weeks prior mating, continuing throughout the matting period, administration was continued throughout gestation and lactation
Frequency of treatment:
daily
Details on study schedule:
Selection of parents from P1 generation when pups were PNDs 21-25.
Remarks:
Doses / Concentrations:
0, 80, 600, 4500 ppm in diet
Basis:

No. of animals per sex per dose:
24 per sex and group
Control animals:
yes, plain diet
Details on study design:
Dose range finding study: 0, 1500, 3000, 6000, 10000 ppm in diet (0, 83, 172, 343 or 551 mg/kg bw per day in males and 0, 126, 264, 476 or 707 mg/kg bw per day in females) for a total of eight weeks beginning 16 days before mating in males and a total of nine weeks in females throughout the mating, gestation and lactation periods beginning 16 days before mating; results: reduced body weight gain in males at 6000 ppm and higher and females at 3000 ppm and higher, reduced number of implantations at 6000 ppm and higher, decreased absolute and relative weight of spleen in females at 6000 ppm and higher, reduced number of pups born at 10000 ppm, lowered body weight of pups at 6000 ppm and higher, and decreased absolute and relative weight of spleen in male weanlings at 1500 ppm and higher and female weanlings at 3000 ppm and higher
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Time schedule: weekly
Oestrous cyclicity (parental animals):
Daily vaginal lavage samples of each F0 and F1 female were evalauted for estrous cyclicity throughout the 2-week pre-cohabitation period and during cohabitation until evidence of copulation was detected.
Sperm parameters (parental animals):
Parameters examined in all P0 and P1 male parental generations:
Right testis weight, right cauda epididymis weight, sperm count in epididymides, sperm motility, sperm count per gram of epididymal tissue, sperm morphology.
Litter observations:
Total litter size, number of live and dead pups, sexed, examined grossly, and individually weighed on PND 0, 4, 7, 14, 21
Postmortem examinations (parental animals):
Proestrous stage of the estrous cycle,
Complete necropsy all animals: external surface of the rats were examined, gross internal examination, weights of brain, pituitary, thyroid, thymus, liver, kidney, spleen, adrenal,testis, epididymis, seminal vesicle, ventral prostate, uterus and ovary.
Histopathological evaluations in P0 and P1 adults: liver, pituitary,thymus, thyroid, kidney, spleen, adrenal,bone marrow, mesenteric lymph node, Peyer's patcjes, testis, epidiymis, seminal vesicle, coagulation gland, ventral prostate, ovary, uterus, vagina and mammary gland, in addition any organs or tissues of P0 and P1 adults showing gross alterations.
Postmortem examinations (offspring):
SACRIFICE
- The P1 offspring not selected as parental animals and all F1 offspring were sacrificed at PND 4 days of age.

Gross necropsy consisted of: external surface of the rats were examined, gross internal examination,

Organ weights: of brain, pituitary, thyroid, thymus, liver, kidney, spleen, adrenal,testis, epididymis, seminal vesicle, ventral prostate, uterus and ovary weights (of pituitats and throid were not determnied).

HISTOPATHOLOGY: tymus, liver, spleen
Statistics:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
There were no compound-related clinical signs of toxicity in either male or female rats during the pre-mating, mating, gestation, or lactation periods.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No compound-related mortality was noted.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
P0 males: 80, 600 ppm: no effects, 4500 ppm significant decrease throughout the dosing period.
P0 females: 80, 600 ppm: no effects, 4500 ppm significant decrease during first week of dosing and throughout pregnancy and lactation.
P0: body weight at the scheduled terminal sacrifice was significant lower at 4500 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
P0 males: 80, 600 ppm no effects, 4500 ppm significant decrease during weeks 1 -8 and 13 -14
P0 females: 80, 600 ppm no effects, 4500 ppm significant decrease during week 1 of dosing and days 14 -21 of lactation
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
P0 males at 4500 ppm: significant higher percent of lymphocytes
P0 females: no effects
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no significant changes in biochemistry parameters such as total protein, albumin and globulin in male and female P0 and P1 adult rats.
No significant changes in any serum hormone levels of male and female P0 adults were noted between the control and DCBS treated groups.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No compoundrelated gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs, were noted in males and females in the highest dose group and dead animals before the scheduled terminal sacrifice.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No significant changes in sperm counts, percentage of motile sperm and progressively motile sperm, swimming speed and pattern, or percentage of morphologically abnormal sperm in DCBS treated animals.
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant differences in: copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of P1/F1 pups delivered, sex ratio of P1/F1 pups, or viability of P1/F1 pups during lactation between control and DCBS-treated groups; no malformed P1/F1 pups were found in any groups.
Key result
Dose descriptor:
NOAEL
Effect level:
54 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects
Remarks on result:
other: Generation: maternal
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no compound-related clinical signs of toxicity in either male or female rats during the pre-mating, mating, gestation, or lactation periods.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No compound-related mortality was noted.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
P1 males: 80 ppm significant decrease in food consumption during weeks 4 and 7, 600 ppm during week 6, and 4500 ppm during week 4.
P1 females: 80, 600, 4500 ppm: no effects.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
P1 males: no effects.
P1 females at 600 ppm: significant higher percent of lymphocytes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
P1 males: no effects
P1 females at 600 ppm: significant higher percent of lymphocytes.
There were no significant changes in biochemistry parameters such as total protein, albumin and globulin in male and female adult rats.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
P1 males: at 4500 ppm: significant decrease in absolute weights of brain, thymus, liver, kidney, adrenal gland, epididymis, ventral prostate, decrease in both relative and absolute weights: of spleen, increase in relative weights of the brain, liver and testis, decrease in rel. and absolute seminal vesicle, increased relative weight of kidney, increase of relative and absolute liver weights.
P1 females: significant decrease at 4500 ppm at scheduled sacrifice; at 4500 ppm: significant decrease absolute weights: brain, thymus, liver, kidney, spleen, adrenal, ovary and uterus and relative weight spleen, brain, liver, kidney.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group. Histopathological examinations of the ovary in P1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group. Histopathological examinations of the ovary in F1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Delayed preputial separation at 4500 ppm as well as delayed vaginal opening and higher body weight at the age of vaginal opening at 600 and 4500 ppm were found in the F1 generation.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Although one F1 female each in the control and 600 ppm groups displayed extended diestrous vaginal smears, no significant changes in the incidence of females having normal estrous cycles or length of the estrous cycles were observed.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in the mean lateral head displacement was found at 4500 ppm in F1 males.
Reproductive performance:
no effects observed
Description (incidence and severity):
P1 parents/F2 offspring: No significant changes in the copulation index, fertility index, gestation index, pre-coital index, gestation lengh, number of implantations, delivery index, number of F2 pups delivered, sex ratio of F2 pups, or viability of F2 pups during lactation were observed.
(Two P1 males in the 600 ppm group did not copulate. One female of the control group and two females in the 80 and 600 ppm groups did not become pregnant. One female in the 600 ppm group did not deliver. One dam experienced a total litter loss by PND 3 at 4500 ppm),
(malformation: oligodactyly in one female of control group, one microphthalmia in one male at 80 ppm)
Key result
Dose descriptor:
NOAEL
Effect level:
291 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Remarks on result:
other: Generation: foetal
Clinical signs:
no effects observed
Description (incidence and severity):
There were no compound-related clinical signs of toxicity in either male or female rats during the pre-mating, mating, gestation, or lactation periods.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No compound-related mortality was noted.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effects.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1 males: 80 ppm significant decrease in food consumption during weeks 4 and 7, 600 ppm during week 6, and 4500 ppm during week 4
F1 females: 80, 600, 4500 ppm: no effects
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 males: no effects.
F1 females at 600 ppm: significant higher percent of lymphocytes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
F1 males: no effects
F1 females at 600 ppm: significant higher percent of lymphocytes.
There were no significant changes in biochemistry parameters such as total protein, albumin and globulin in male and female adult rats.
Urinalysis findings:
not specified
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 males: at 4500 ppm: significant decrease in absolute weights of brain, thymus, liver, kidney, adrenal gland, epididymis, ventral prostate, decrease in both relative and absolute weights: of spleen, increase in relative weights of the brain, liver and testis, decrease in rel. and absolute seminal vesicle, increased relative weight of kidney, increase of relative and absolute liver weights.
F1 females: significant decrease at 4500 ppm at scheduled sacrifice; at 4500 ppm: significant decrease absolute weights: brain, thymus, liver, kidney, spleen, adrenal, ovary and uterus and relative weight spleen, brain, liver, kidney.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group. Histopathological examinations of the ovary in F1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups.
Histopathological findings:
no effects observed
Description (incidence and severity):
No compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group. Histopathological examinations of the ovary in F1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Delayed preputial separation at 4500 ppm as well as delayed vaginal opening and higher body weight at the age of vaginal opening at 600 and 4500 ppm were found in the F1 generation.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
291 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Remarks on result:
other: Generation: foetal
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of F2 pups at 4500 ppm were significantly lowered on PNDs 7,14 and 21 in males and PNDs 14 and 21 in females.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males: A decrease in the absolute and relative weight of the spleen was observed at 80 ppm. The relative weights of the liver and kidney were higher at 600 ppm. At 4500 ppm,a decreased absolute weight of the adrenal gland, decreased absolute and relative weights of the thymus and spleen, and increased relative weights of the brain, liver, and kidney were noted in males.
Females: A significant decrease in the body weight at sacrifice was found at 4500 ppm. The relative weight of the thymuswas lower at 80 ppm. An increased relative weights of the liver and kidney, and reduced absolute and relative weights of the uterus were found at 600 ppm. At 4500 ppm, decreased absolute weights of the brain and spleen, and absolute and relative weights of the thymus and uterus, and increased relative weights of the brain, liver and kidney were noted in females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no compound-related gross lesions or microscopic alterations in male and female F1 and F2 pups, including pups that died before weaning.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no compound-related gross lesions or microscopic alterations in male and female F1 and F2 pups, including pups that died before weaning.
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
There were no compound-related gross lesions or microscopic alterations in male and female P1/F1 and F2 pups, including pups that died before weaning.
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
291 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Adults/weanlings

Mortality:

P0: no compound-related mortality was noted in any of the animals of the F0 generation during the pre-mating, mating, gestation or lactation period

P1/F1: no compound-related mortality was noted in any of the animals of the F1 generation during the pre-mating, mating, gestation or lactation period

Clinical observations:

P0 and P1/F1: no compound-related clinical signs of toxicity in neither male or female P0 and P1/F1 rats during the pre-mating, mating, gestation, or lactation period

Body weight and body weight gain:

P0 males: 80, 600 ppm: no effects, 4500 ppm significant decrease throughout the dosing period

P0 females: 80, 600 ppm: no effects, 4500 ppm significant decrease during first week of dosing and throughout pregnancy and lactation

P1/F1 males and females: no effects

Food consumption:

P0 males: 80, 600 ppm no effects, 4500 ppm significant decrease during weeks 1 -8 and 13 -14

P0 females: 80, 600 ppm no effects, 4500 ppm significant decrease during week 1 of dosing and days 14 -21 of lactation

P1/F1 males: 80 ppm significant decrease in food consumption during weeks 4 and 7, 600 ppm during week 6, and 4500 ppm during week 4

P1/F1 females: 80, 600, 4500 ppm: no effects

Mean daily intake (corresponding to 80, 600, 4500 ppm in diet)

P0 males: 5.2, 39, 291 mg/kg bw

P0 females: 7.2, 54, 416 mg/kg bw

P1/F1 males: 5.9, 44, 331 mg/kg bw

P1/F1 females: 7.4, 55, 417 mg/kg bw

Necropsy and histopathology:

P0: no compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group

P1/F1: no compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group. Histopathological examinations of the ovary in P1/F1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups.

Estrous cyclicity:

P0 females: no effects.

P1/F1females: no significant changes in the incidence of females having normal estrous cycles or length of the estrous cycles

(two P1/F1 females showing abnormal estrous cycles remained in diestrus for 10 -11 days).

Reproductive effects:

P0 parents/P1/F1 offspring: no significant differences in: copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of P1/F1 pups delivered, sex ratio of P1/F1 pups, or viability of P1/F1 pups during lactation between control and DCBS-treated groups; no malformed P1/F1 pups were found in any groups, a significant lower body weight was observed in male and female P1/F1 pups at 4500 ppm on PND 4, 7 and 21.

(P0 parent animals, all pairs in all groups copulated, although two females in the control group did not become pregnant, and all pregnant females in all groups delivered live pups)

P1/F1 parents/F2 offspring: No significant changes in the copulation index, fertility index, gestation index, pre-coital index, gestation lengh, number of implantations, delivery index, number of F2 pups delivered, sex ratio of F2 pups, or viability of F2 pups during lactation were observed.

(Two P1/F1 males in the 600 ppm group did not copulate. One female of the control group and two females in the 80 and 600 ppm groups did not become pregnant. One female in the 600 ppm group did not deliver. One dam experienced a total litter loss by PND 3 at 4500 ppm),

(malformation: oligodactyly in one female of control group, one microphthalmia in one male at 80 ppm).

Body weights of F2 pups at 4500 ppm were significant lowered on PND 7, 14, and 21 in males and PNDs 14 to 21 in females.

Organ weights:

P0: body weight at the scheduled terminal sacrifice was significant lower at 4500 ppm

P0males: 4500 ppm significant lower absolute organ weights: spleen, adrenal gland; increase in relative weight of: brain, thyroid, liver, kidney and testis

P0 females: significant increase in absolute weights of: brain (80 ppm, 600 ppm), pituitary (80 ppm); decrease in relative weights: spleen (80 ppm and 600 ppm),

significant decrease absolute weight of: spleen and increase relative weights of brain, kidney, adrenal gland at 4500 ppm

P1/F1 (weanlings and adults)

P1/F1 males: at 4500 ppm: significant decrease in absolute weights of brain, thymus, liver, kidney, adrenal gland, epididymis, ventral prostate, decrease in both relative and absolute weights: of spleen, increase in relative weights of the brain, liver and testis, decrease in rel. and absoluteseminal vesicle, increased relative weight of kidney, increase of relativeand abolute liver weights.

P1/F1 females: significant decrease at 4500 ppm at scheduled sacrifice;

at 4500 ppm: significant decrease absolute weights: brain, thymus, liver, kidney, spleen, adrenal, ovary and uterus and relative weight spleen, brain, liver, kidney.

F2 (weanlings)

F2 males/females: at 4500 ppm body weight significant reduced

F2 males at 4500 ppm: signifcant decrease in absolute weights: adrenal gland, decrease in absolute and relative weights of: thymus, spleen; increase in rel. weights in brain, liver, kidney

F2 females: at 600 ppm: significantly increase in rel weights: liver, kidney, reduced absolute and rel. weights: uterus

at 4500 ppm: significant decrease in absolute weights: brain, spleen, and absolute and rel. weights of thymus, uterus, and increased rel. weights of brain, liver and kidney

Hematological and blood biochemical parameters

P0 males at 4500 ppm: significant higher percent of lymphocytes

P0 females: no effects

P1/F1 males: no effects

P1/F1 females at 600 ppm: significant higher percent of lymphocytes

F0/P1,F1: no significant changes in biochemistry parameters such as total protein, albumin and globulin

Serum hormone levels (P0 and P1/F1 adults)

P0 male/female: no significant changes

P1/F1 male: significant higher levels of testosterone at 80 and 600 ppm, no significant changes at 4500 ppm

P1/F1 female: no significant changes in any serum hormone levels

Sperm parameter (P0 and P1/F1 adults)

P0 males: no significant changes in sperm counts, percentage of motile sperm and progressively motile sperm, swimming speed and pattern, or percentage of morphologically abnormal sperm in DCBS treated animals

P1/F1: at 4500 ppm a significant decrease in the mean lateral displacement (20.5 µm ± 1.0 vs. control 21.3 ± 0.9 µm).

Conclusions:
Although a few P0 and P1/F1 adults showed reproductive difficulties, necropsy and the histopathology of reproductive organs revealed no evidence of reproductive failure in these rats. In conclusion, no significant changes in reproductive indices were noted in any generation even at the highest dose 4500 ppm (ca. 291 mg/kg bw and day).
Executive summary:

A two-generation reproductive toxicity study was performed to further evaluate the potential effects of DCBS on reproduction and development in rats (Ema 2008). Male and female Crl: CD (SD) rats (24 per group and sex) were fed a diet containing DCBS at 0, 80, 600 or 4500 ppm throughout the study beginning at the onset of a 10-week pre-mating period and continuing through mating, gestation, and lactation periods for two generations. The test substance intake correspond to ca. 0, 5.2, 39, 291 mg/kg bw in P0 males, 0, 7.2, 54, 416 mg/kg bw/day in P0 females, 0, 5.9, 44, 331 mg/kg bw/day in P1/F1 males and 0, 7.4, 55, 417 mg/kg bw/day in P1/F1 females. The deaths and clinical signs observed in the present study are not related to the administration of DCBS. Decreased food consumption was noted in P0 males and females at 4500 ppm and was accompanied by reduced body weight, body weight gain and food consumption. However, no consistent lowered food consumption accompanied by lower body weights were noted in P1/F1 adults. No significant changes in sperm counts, percentage of motile sperm and progressively motile sperm, swimming speed and pattern, or percentage of morphologically abnormal sperm were noted in F0 and P1/F1 adults between control and DCBS-treated groups. However, a slight but significant decrease in mean lateral head displacement was noted in P1/F1 males of the highest group (20.5 µm ± 1.0 vs. control 21.3 ± 0.9 µm). All P0 females showed normal oestrous cycles in all groups, and the length of the oestrous cycles was not different between the control and DCBS-treated groups. Although one P1/F1 female each in the control and 600 ppm groups displayed extended diestrus vaginal smears, no significant changes in the incidence of females having normal oestrous cycles or length of the oestrous cycles were observed. There were no significant differences in the copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of P1/F1 pups delivered, sex ratio of P1/F1 pups, or viability of P1/F1 pups during lactation between control and DCBS-treated groups. No malformed P1/F1 pups were found in any groups. Two P1/F1 males in the 600 ppm group did not copulate. One female in the control group and two females each in the 80 and 600 ppm groups did not become pregnant. One pregnant female in the 600 ppm group did not deliver. One dam in the control group died on day 5 of lactation, and her pups were euthanized. One dam experienced a total litter loss by PND 3 at 4500 ppm. No significant changes in the copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of F2 pups delivered, sex ratio of F2 pups, or viability of F2 pups during lactation were observed. Oliodactyly in one female of the control group and microphthalmia in one male at 80 ppm were observed. Although a few P0 and P1/F1 adults showed reproductive difficulties, necropsy and the histopathology of reproductive organs revealed no evidence of reproductive failure in these rats. In conclusion, no significant changes in reproductive indices were noted in any generation even at the highest dose 4500 ppm (ca. 291 mg/kg bw and day).

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See attached justification: Read-across Justification Document for the Category of four Sulfenamides
Specific details on test material used for the study:
N,N-Dicyclohexyl-2-benzothiazolesulfenamide (DCBS, CAS No. 4979-32-2) was obtained from Ouchishinko Chemical Industrial Co. Ltd. (Tokyo, Japan). DCBS in the form of off white to tan granules is very slightly soluble in water and methanol but soluble in oil, and its melting point is 100–105°C, density at 21°C is 1230 kg/m³, and molecular weight is 347. The DCBS (Lot no. 508001) used in this study was 99.7% pure.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no compound-related clinical signs of toxicity in either male or female rats during the pre-mating, mating, gestation, or lactation periods.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No compound-related mortality was noted.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
P0 males: 80, 600 ppm: no effects, 4500 ppm significant decrease throughout the dosing period.
P0 females: 80, 600 ppm: no effects, 4500 ppm significant decrease during first week of dosing and throughout pregnancy and lactation.
P0: body weight at the scheduled terminal sacrifice was significant lower at 4500 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
P0 males: 80, 600 ppm no effects, 4500 ppm significant decrease during weeks 1 -8 and 13 -14
P0 females: 80, 600 ppm no effects, 4500 ppm significant decrease during week 1 of dosing and days 14 -21 of lactation
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
P0 males at 4500 ppm: significant higher percent of lymphocytes
P0 females: no effects
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no significant changes in biochemistry parameters such as total protein, albumin and globulin in male and female P0 and P1 adult rats.
No significant changes in any serum hormone levels of male and female P0 adults were noted between the control and DCBS treated groups.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No compoundrelated gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs, were noted in males and females in the highest dose group and dead animals before the scheduled terminal sacrifice.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No significant changes in sperm counts, percentage of motile sperm and progressively motile sperm, swimming speed and pattern, or percentage of morphologically abnormal sperm in DCBS treated animals.
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant differences in: copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of P1/F1 pups delivered, sex ratio of P1/F1 pups, or viability of P1/F1 pups during lactation between control and DCBS-treated groups; no malformed P1/F1 pups were found in any groups.
Key result
Dose descriptor:
NOAEL
Effect level:
54 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects
Remarks on result:
other: Generation: maternal
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no compound-related clinical signs of toxicity in either male or female rats during the pre-mating, mating, gestation, or lactation periods.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No compound-related mortality was noted.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
P1 males: 80 ppm significant decrease in food consumption during weeks 4 and 7, 600 ppm during week 6, and 4500 ppm during week 4.
P1 females: 80, 600, 4500 ppm: no effects.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
P1 males: no effects.
P1 females at 600 ppm: significant higher percent of lymphocytes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
P1 males: no effects
P1 females at 600 ppm: significant higher percent of lymphocytes.
There were no significant changes in biochemistry parameters such as total protein, albumin and globulin in male and female adult rats.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
P1 males: at 4500 ppm: significant decrease in absolute weights of brain, thymus, liver, kidney, adrenal gland, epididymis, ventral prostate, decrease in both relative and absolute weights: of spleen, increase in relative weights of the brain, liver and testis, decrease in rel. and absolute seminal vesicle, increased relative weight of kidney, increase of relative and absolute liver weights.
P1 females: significant decrease at 4500 ppm at scheduled sacrifice; at 4500 ppm: significant decrease absolute weights: brain, thymus, liver, kidney, spleen, adrenal, ovary and uterus and relative weight spleen, brain, liver, kidney.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group. Histopathological examinations of the ovary in P1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group. Histopathological examinations of the ovary in F1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Delayed preputial separation at 4500 ppm as well as delayed vaginal opening and higher body weight at the age of vaginal opening at 600 and 4500 ppm were found in the F1 generation.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Although one F1 female each in the control and 600 ppm groups displayed extended diestrous vaginal smears, no significant changes in the incidence of females having normal estrous cycles or length of the estrous cycles were observed.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in the mean lateral head displacement was found at 4500 ppm in F1 males.
Reproductive performance:
no effects observed
Description (incidence and severity):
P1 parents/F2 offspring: No significant changes in the copulation index, fertility index, gestation index, pre-coital index, gestation lengh, number of implantations, delivery index, number of F2 pups delivered, sex ratio of F2 pups, or viability of F2 pups during lactation were observed.
(Two P1 males in the 600 ppm group did not copulate. One female of the control group and two females in the 80 and 600 ppm groups did not become pregnant. One female in the 600 ppm group did not deliver. One dam experienced a total litter loss by PND 3 at 4500 ppm),
(malformation: oligodactyly in one female of control group, one microphthalmia in one male at 80 ppm)
Key result
Dose descriptor:
NOAEL
Effect level:
291 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Remarks on result:
other: Generation: foetal
Clinical signs:
no effects observed
Description (incidence and severity):
There were no compound-related clinical signs of toxicity in either male or female rats during the pre-mating, mating, gestation, or lactation periods.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No compound-related mortality was noted.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effects.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1 males: 80 ppm significant decrease in food consumption during weeks 4 and 7, 600 ppm during week 6, and 4500 ppm during week 4
F1 females: 80, 600, 4500 ppm: no effects
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 males: no effects.
F1 females at 600 ppm: significant higher percent of lymphocytes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
F1 males: no effects
F1 females at 600 ppm: significant higher percent of lymphocytes.
There were no significant changes in biochemistry parameters such as total protein, albumin and globulin in male and female adult rats.
Urinalysis findings:
not specified
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 males: at 4500 ppm: significant decrease in absolute weights of brain, thymus, liver, kidney, adrenal gland, epididymis, ventral prostate, decrease in both relative and absolute weights: of spleen, increase in relative weights of the brain, liver and testis, decrease in rel. and absolute seminal vesicle, increased relative weight of kidney, increase of relative and absolute liver weights.
F1 females: significant decrease at 4500 ppm at scheduled sacrifice; at 4500 ppm: significant decrease absolute weights: brain, thymus, liver, kidney, spleen, adrenal, ovary and uterus and relative weight spleen, brain, liver, kidney.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group. Histopathological examinations of the ovary in F1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups.
Histopathological findings:
no effects observed
Description (incidence and severity):
No compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group. Histopathological examinations of the ovary in F1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Delayed preputial separation at 4500 ppm as well as delayed vaginal opening and higher body weight at the age of vaginal opening at 600 and 4500 ppm were found in the F1 generation.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
291 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Remarks on result:
other: Generation: foetal
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of F2 pups at 4500 ppm were significantly lowered on PNDs 7,14 and 21 in males and PNDs 14 and 21 in females.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males: A decrease in the absolute and relative weight of the spleen was observed at 80 ppm. The relative weights of the liver and kidney were higher at 600 ppm. At 4500 ppm,a decreased absolute weight of the adrenal gland, decreased absolute and relative weights of the thymus and spleen, and increased relative weights of the brain, liver, and kidney were noted in males.
Females: A significant decrease in the body weight at sacrifice was found at 4500 ppm. The relative weight of the thymuswas lower at 80 ppm. An increased relative weights of the liver and kidney, and reduced absolute and relative weights of the uterus were found at 600 ppm. At 4500 ppm, decreased absolute weights of the brain and spleen, and absolute and relative weights of the thymus and uterus, and increased relative weights of the brain, liver and kidney were noted in females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no compound-related gross lesions or microscopic alterations in male and female F1 and F2 pups, including pups that died before weaning.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no compound-related gross lesions or microscopic alterations in male and female F1 and F2 pups, including pups that died before weaning.
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
There were no compound-related gross lesions or microscopic alterations in male and female P1/F1 and F2 pups, including pups that died before weaning.
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
291 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Adults/weanlings

Mortality:

P0: no compound-related mortality was noted in any of the animals of the F0 generation during the pre-mating, mating, gestation or lactation period

P1/F1: no compound-related mortality was noted in any of the animals of the F1 generation during the pre-mating, mating, gestation or lactation period

Clinical observations:

P0 and P1/F1: no compound-related clinical signs of toxicity in neither male or female P0 and P1/F1 rats during the pre-mating, mating, gestation, or lactation period

Body weight and body weight gain:

P0 males: 80, 600 ppm: no effects, 4500 ppm significant decrease throughout the dosing period

P0 females: 80, 600 ppm: no effects, 4500 ppm significant decrease during first week of dosing and throughout pregnancy and lactation

P1/F1 males and females: no effects

Food consumption:

P0 males: 80, 600 ppm no effects, 4500 ppm significant decrease during weeks 1 -8 and 13 -14

P0 females: 80, 600 ppm no effects, 4500 ppm significant decrease during week 1 of dosing and days 14 -21 of lactation

P1/F1 males: 80 ppm significant decrease in food consumption during weeks 4 and 7, 600 ppm during week 6, and 4500 ppm during week 4

P1/F1 females: 80, 600, 4500 ppm: no effects

Mean daily intake (corresponding to 80, 600, 4500 ppm in diet)

P0 males: 5.2, 39, 291 mg/kg bw

P0 females: 7.2, 54, 416 mg/kg bw

P1/F1 males: 5.9, 44, 331 mg/kg bw

P1/F1 females: 7.4, 55, 417 mg/kg bw

Necropsy and histopathology:

P0: no compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group

P1/F1: no compound-related gross lesions or remarkable microscopic alterations of tissues and organs, including the reproductive organs were noted in males and females of the highest dose group. Histopathological examinations of the ovary in P1/F1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups.

Estrous cyclicity:

P0 females: no effects.

P1/F1females: no significant changes in the incidence of females having normal estrous cycles or length of the estrous cycles

(two P1/F1 females showing abnormal estrous cycles remained in diestrus for 10 -11 days).

Reproductive effects:

P0 parents/P1/F1 offspring: no significant differences in: copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of P1/F1 pups delivered, sex ratio of P1/F1 pups, or viability of P1/F1 pups during lactation between control and DCBS-treated groups; no malformed P1/F1 pups were found in any groups, a significant lower body weight was observed in male and female P1/F1 pups at 4500 ppm on PND 4, 7 and 21.

(P0 parent animals, all pairs in all groups copulated, although two females in the control group did not become pregnant, and all pregnant females in all groups delivered live pups)

P1/F1 parents/F2 offspring: No significant changes in the copulation index, fertility index, gestation index, pre-coital index, gestation lengh, number of implantations, delivery index, number of F2 pups delivered, sex ratio of F2 pups, or viability of F2 pups during lactation were observed.

(Two P1/F1 males in the 600 ppm group did not copulate. One female of the control group and two females in the 80 and 600 ppm groups did not become pregnant. One female in the 600 ppm group did not deliver. One dam experienced a total litter loss by PND 3 at 4500 ppm),

(malformation: oligodactyly in one female of control group, one microphthalmia in one male at 80 ppm).

Body weights of F2 pups at 4500 ppm were significant lowered on PND 7, 14, and 21 in males and PNDs 14 to 21 in females.

Organ weights:

P0: body weight at the scheduled terminal sacrifice was significant lower at 4500 ppm

P0males: 4500 ppm significant lower absolute organ weights: spleen, adrenal gland; increase in relative weight of: brain, thyroid, liver, kidney and testis

P0 females: significant increase in absolute weights of: brain (80 ppm, 600 ppm), pituitary (80 ppm); decrease in relative weights: spleen (80 ppm and 600 ppm),

significant decrease absolute weight of: spleen and increase relative weights of brain, kidney, adrenal gland at 4500 ppm

P1/F1 (weanlings and adults)

P1/F1 males: at 4500 ppm: significant decrease in absolute weights of brain, thymus, liver, kidney, adrenal gland, epididymis, ventral prostate, decrease in both relative and absolute weights: of spleen, increase in relative weights of the brain, liver and testis, decrease in rel. and absoluteseminal vesicle, increased relative weight of kidney, increase of relativeand abolute liver weights.

P1/F1 females: significant decrease at 4500 ppm at scheduled sacrifice;

at 4500 ppm: significant decrease absolute weights: brain, thymus, liver, kidney, spleen, adrenal, ovary and uterus and relative weight spleen, brain, liver, kidney.

F2 (weanlings)

F2 males/females: at 4500 ppm body weight significant reduced

F2 males at 4500 ppm: signifcant decrease in absolute weights: adrenal gland, decrease in absolute and relative weights of: thymus, spleen; increase in rel. weights in brain, liver, kidney

F2 females: at 600 ppm: significantly increase in rel weights: liver, kidney, reduced absolute and rel. weights: uterus

at 4500 ppm: significant decrease in absolute weights: brain, spleen, and absolute and rel. weights of thymus, uterus, and increased rel. weights of brain, liver and kidney

Hematological and blood biochemical parameters

P0 males at 4500 ppm: significant higher percent of lymphocytes

P0 females: no effects

P1/F1 males: no effects

P1/F1 females at 600 ppm: significant higher percent of lymphocytes

F0/P1,F1: no significant changes in biochemistry parameters such as total protein, albumin and globulin

Serum hormone levels (P0 and P1/F1 adults)

P0 male/female: no significant changes

P1/F1 male: significant higher levels of testosterone at 80 and 600 ppm, no significant changes at 4500 ppm

P1/F1 female: no significant changes in any serum hormone levels

Sperm parameter (P0 and P1/F1 adults)

P0 males: no significant changes in sperm counts, percentage of motile sperm and progressively motile sperm, swimming speed and pattern, or percentage of morphologically abnormal sperm in DCBS treated animals

P1/F1: at 4500 ppm a significant decrease in the mean lateral displacement (20.5 µm ± 1.0 vs. control 21.3 ± 0.9 µm).

Conclusions:
Although a few P0 and P1/F1 adults showed reproductive difficulties, necropsy and the histopathology of reproductive organs revealed no evidence of reproductive failure in these rats. In conclusion, no significant changes in reproductive indices were noted in any generation even at the highest dose 4500 ppm (ca. 291 mg/kg bw and day).
Executive summary:

A two-generation reproductive toxicity study was performed to further evaluate the potential effects of DCBS on reproduction and development in rats (Ema 2008). Male and female Crl: CD (SD) rats (24 per group and sex) were fed a diet containing DCBS at 0, 80, 600 or 4500 ppm throughout the study beginning at the onset of a 10-week pre-mating period and continuing through mating, gestation, and lactation periods for two generations. The test substance intake correspond to ca. 0, 5.2, 39, 291 mg/kg bw in P0 males, 0, 7.2, 54, 416 mg/kg bw/day in P0 females, 0, 5.9, 44, 331 mg/kg bw/day in P1/F1 males and 0, 7.4, 55, 417 mg/kg bw/day in P1/F1 females. The deaths and clinical signs observed in the present study are not related to the administration of DCBS. Decreased food consumption was noted in P0 males and females at 4500 ppm and was accompanied by reduced body weight, body weight gain and food consumption. However, no consistent lowered food consumption accompanied by lower body weights were noted in P1/F1 adults. No significant changes in sperm counts, percentage of motile sperm and progressively motile sperm, swimming speed and pattern, or percentage of morphologically abnormal sperm were noted in F0 and P1/F1 adults between control and DCBS-treated groups. However, a slight but significant decrease in mean lateral head displacement was noted in P1/F1 males of the highest group (20.5 µm ± 1.0 vs. control 21.3 ± 0.9 µm). All P0 females showed normal oestrous cycles in all groups, and the length of the oestrous cycles was not different between the control and DCBS-treated groups. Although one P1/F1 female each in the control and 600 ppm groups displayed extended diestrus vaginal smears, no significant changes in the incidence of females having normal oestrous cycles or length of the oestrous cycles were observed. There were no significant differences in the copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of P1/F1 pups delivered, sex ratio of P1/F1 pups, or viability of P1/F1 pups during lactation between control and DCBS-treated groups. No malformed P1/F1 pups were found in any groups. Two P1/F1 males in the 600 ppm group did not copulate. One female in the control group and two females each in the 80 and 600 ppm groups did not become pregnant. One pregnant female in the 600 ppm group did not deliver. One dam in the control group died on day 5 of lactation, and her pups were euthanized. One dam experienced a total litter loss by PND 3 at 4500 ppm. No significant changes in the copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of F2 pups delivered, sex ratio of F2 pups, or viability of F2 pups during lactation were observed. Oliodactyly in one female of the control group and microphthalmia in one male at 80 ppm were observed. Although a few P0 and P1/F1 adults showed reproductive difficulties, necropsy and the histopathology of reproductive organs revealed no evidence of reproductive failure in these rats. In conclusion, no significant changes in reproductive indices were noted in any generation even at the highest dose 4500 ppm (ca. 291 mg/kg bw and day).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
291 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available fertility studies were conducted equivalent or similar to guideline studies.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
adverse effect observed
Additional information

A two-generation reproductive toxicity study was performed to evaluate the potential effects of DCBS on reproduction and development in rats (Ema 2008). Based on the category justification for CBS (CAS 95-33-0), DCBS (CAS 4979-32-2), TBBS (CAS 95-31-8) and MBS (CAS 102-77-2) a read-across with the 2-generation study with DCBS is justified and conducted.

Male and female Crl: CD (SD) rats (24 per group and sex) were fed a diet containing DCBS at 0, 80, 600 or 4500 ppm throughout the study beginning at the onset of a 10-week pre-mating period and continuing through mating, gestation, and lactation periods for two generations. The test substance intake correspond to ca. 0, 5.2, 39, 291 mg/kg bw in P0 males, 0, 7.2, 54, 416 mg/kg bw/day in P0 females, 0, 5.9, 44, 331 mg/kg bw/day in P1/F1 males and 0, 7.4, 55, 417 mg/kg bw/day in P1/F1 females. The deaths and clinical signs observed in the present study are not related to the administration of DCBS. Decreased food consumption was noted in P0 males and females at 4500 ppm and was accompanied by reduced body weight, body weight gain and food consumption. However, no consistent lowered food consumption accompanied by lower body weights were noted in P1/F1 adults. No significant changes in sperm counts, percentage of motile sperm and progressively motile sperm, swimming speed and pattern, or percentage of morphologically abnormal sperm were noted in F0 and P1/F1 adults between control and DCBS-treated groups. However, a slight but significant decrease in mean lateral head displacement was noted in P1/F1 males of the highest group (20.5 µm ± 1.0 vs. control 21.3 ± 0.9 µm). All P0 females showed normal oestrous cycles in all groups, and the length of the oestrous cycles was not different between the control and DCBS-treated groups. Although one P1/F1 female each in the control and 600 ppm groups displayed extended diestrus vaginal smears, no significant changes in the incidence of females having normal oestrous cycles or length of the oestrous cycles were observed. There were no significant differences in the copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of P1/F1 pups delivered, sex ratio of P1/F1 pups, or viability of P1/F1 pups during lactation between control and DCBS-treated groups. No malformed P1/F1 pups were found in any groups. Two P1/F1 males in the 600 ppm group did not copulate. One female in the control group and two females each in the 80 and 600 ppm groups did not become pregnant. One pregnant female in the 600 ppm group did not deliver. One dam in the control group died on day 5 of lactation, and her pups were euthanized. One dam experienced a total litter loss by PND 3 at 4500 ppm. No significant changes in the copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of F2 pups delivered, sex ratio of F2 pups, or viability of F2 pups during lactation were observed. Oliodactyly in one female of the control group and microphthalmia in one male at 80 ppm were observed. Although a few P0 and P1/F1 adults showed reproductive difficulties, necropsy and the histopathology of reproductive organs revealed no evidence of reproductive failure in these rats. In conclusion, no significant changes in reproductive indices were noted in any generation even at the highest dose 4500 ppm (ca. 291 mg/kg bw and day).


From a subacute oral (gavage) 28-day repeated dose toxicity study data on organ weights and histopathology of reproductive organs are available (MHWJ 1997). As discussed and confirmed by recent literature (Mangelsdorf et al. 2003, Ulbrich & Palmer 1995, Janer et al. 2007, Dent 2007, Sanbuissho et al. 2008) in rodents histopathological examinations in repeated dose toxicity studies of reproductive tissues are of high value and sensitivity for evaluation of reproductive toxicity in males and females. Histopathological changes on the reproductive organs in repeated dose toxicity studies are indicative of effects on fertility. With this respect repeated dose toxicity studies should be considered sensitive and reliable information to evaluate toxicity on fertility if histological examination of the reproductive organs is covered.

In a subacute gavage study groups of Crj: CD (SD) rats were treated with 0, 25, 80, 250 and 800 mg/kg bw and day CBS (MHWJ 1997). Systemic toxicity of CBS was noted in treated rats at >= 250 mg/kg bw and day indicated by adverse effects on hematology and kidneys, suppression of food consumption and body weight gain and loss of general conditions (for more details see chapter repeated dose toxicity). No treatment related changes in absolute and relative weights of ovaries and absolute weights of testis were observed. Relative weights of testis were significant higher in animals treated with 800 mg/kg bw and day than in control animals concomitant to decrease body weights. In the recovery group in one out of 6 rats histopathological changes were seen after a 14 day recovery period at 800 mg/kg bw and day (moderate diffuse atrophy of seminiferous tubuli, slight diffuse interstitial cell hyperplasia and market decreased sperm content on the epididymis). No such effects were observed in male terminated immediately after the administration period. No histopathological abnormalities were detected in ovary from females of 800 mg/kg groups. Based on the findings from this subacute toxicity study, the NOAEL for males concerning adverse effects on the reproductive organs is considered to be 250 mg/kg bw and day and for females 800 mg/kg bw and day (highest dose tested).

Effects on developmental toxicity

Description of key information

The findings from the three available oral developmental toxicity studies in rats consistently demonstrated that CBS induces maternal toxicity in terms of impairment of maternal weight gain during gestation and signs of foetal growth retardation in terms of reduced mean foetal body weight. Foetal body weight impairment, however, was exclusively observed at oral dosages associated with significantly reduced maternal weight. A substance-related specific embryotoxic and or/ teratogenic potential was not revealed from available studies. The guideline-consistent developmental toxicity study (Monsanto 1981) was considered as the most relevant developmental toxicity study. A NOAEL derived from this study for maternal toxicity is 100 mg/kg bw and day and is based on a decrease in mean body weight gain. The NOAEL for the offspring is 300 mg/kg bw and day and is based on a decrease mean foetal body weight.
However, the findings from another developmental toxicity study (Ema 1989) indicated obviously lower NOAELs (NOAEL maternal: 7.1 mg/kg bw and day, NOAEL fetal: 69.6 mg/kg bw and day). At present there is no reasonable explanation to the unexpected low LOAEL for weight gain impairment in the dietary study of Ema (1989). The two other developmental toxicity studies with gavage administration and repeated dose toxicity study via gavage (28 day study MHWJ 1997) consistently gave considerable higher LOAEL values. The lower maternal NOAEL in the study of Ema (1997) with a 20 day dietary exposure is thus supposed to be not related to a higher sensitivity of pregnant rats but rather be explained from the administration route or probably rat strain (Wistar rats (Kar: Wistar, Keari Co., Osaka).

An OECD Guideline 414 study was performed to evaluate the potential effects of DCBS on prenatal developmental toxicity in rabbits. Based on the category justification for CBS (CAS 95-33-0), DCBS (CAS 4979-32-2), TBBS (CAS 95-31-8) and MBS (CAS 102-77-2) a read-across with the prenatal developmental toxicity study with DCBS is justified and conducted.

Eighty-eight mated female New Zealand White rabbits were assigned to four groups of 22 animals each. The test item, DCBS (N,N-dicyclohexylbenzothiazole-2-sulphenamide), was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 100, 300 and 1000 mg/kg/day (Groups 2, 3 and 4, respectively). Rabbits of the control group received the vehicle, 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals.

On Day 29 post-coitum, all animals were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. All fetuses were dissected and examined for visceral anomalies. Skeletons of all fetuses of all groups were examined.

No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups. No developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for DCBS (N,N-dicyclohexylbenzo- thiazole-2- sulphenamide) was established as being at least 1000 mg/kg/day.


Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Groups of 25 pregnant females were treated by gavage with CBS at dose levels of 100, 300, 500, and 900 mg/kg bw and day during gestation day 6 to 15. Cesarean sections were performed on all surviving dams on gestation day 20.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Charles River COBS CD
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
M/F ratio per cage: 1:1
Duration of treatment / exposure:
6th to 15th day of pregnancy
Frequency of treatment:
Daily
Duration of test:
GD 20 (scheduled sacrifice)
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
900 mg/kg bw/day
No. of animals per sex per dose:
25 females per dose
Control animals:
yes
Details on study design:
Sex: female
Maternal examinations:
appearance and behaviour, body weights, Cesarean section observations, fetal morphology observations.
Ovaries and uterine content:
Immediately following sacrifice, the uterus was excised and weighed prior removal of the fetuses. The umber and location of viable and nonviable fetuses, early and late resorptions and number of total inplantations and corpora lutea were recorded.
Fetal examinations:
All fetuses were individually weighed and examined for external malformations and variations, including palate and eyes. Approximaetly one half of the fetuses were used for visceral examinations and the other for skeletal examinations.
Statistics:
Male and female sex distribution and the number of litters with malformations were compared using the Chi-square test criterion, Fisher's exact probability test; number of early and late resorptions and postimplantation loss were compared by the Mann-Whitney U-test; the mean number of viable fetuses, total implantations, corpora lutea and mean fetal body weights were compared by anaylsis of variance (one-way classification).
Historical control data:
Yes.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Due to the early deaths of 5 rats and due to signs of excessive maternal toxicity and weight loss in the remaining animals, the rats in the 900 mg/kg7day dosage group were sacrificed and discarded.
Mortality:
mortality observed, treatment-related
Description (incidence):
Due to the early deaths of 5 rats and due to signs of excessive maternal toxicity and weight loss in the remaining animals, the rats in the 900 mg/kg7day dosage group were sacrificed and discarded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Due to the early deaths of 5 rats and due to signs of excessive maternal toxicity and weight loss in the remaining animals, the rats in the 900 mg/kg7day dosage group were sacrificed and discarded. A severe decrease in in mean maternal body weight occurred in the 500 mg/kg/day group and a slight to moderate decrease in mean maternal body weight was noted in the 300 mg/kg/day dosage group during the treatment period.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no remarkable necropsy findings in those females surviving to the scheduled sacrifice with the exception of one non-gravid rat with hdyrometra. in the 300 mg/kg/day dosage group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
a slight decrese in mean fetal body weight was observed in the 100 and 300 mg/kg/day dosage groups when compared to the control group, with a statistical significant decrease in the 500 mg/kg/day dosage group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There were no meaningful differences in the mean numbers of corpora lutea, total implantations, postimplantation loss, vialble fetuses or the fetal sex distribution in rats in any of the treated groups when compared to the control group.
External malformations:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the number of litters with malformations in any of the treated groups when compared to the control group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the number of litters with malformations in any of the treated groups when compared to the control group.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the number of litters with malformations in any of the treated groups when compared to the control group.
Description (incidence and severity):
a very slight increase in the number of fetuses and litters with malformations was noted in the 500 mg/kg/day dosage group when compared to the control group. This increase was due primarily to 3 fetuses, each from a different litter, with a thread-like tail and a smal anus. The only malformed fetus in the 100 mg/kg/day dosage group also had a thread-like tail and a small anus.
there were no biologically meaningful differences or dose-related trends in the number of fetuses or litters with genetic and developmental variations in any treated group when compared to the control group.
Details on embryotoxic / teratogenic effects:
treatment with Santocure did not produce a teratogenic response when administered to pregant Charles River COBS CD rats at a dosage level of 500 mg/kg/day or less.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
LOEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Maternal toxicity

900 mg/kg bw/day:

Appearance and behavior: Matting and yellow or red staining of the anogenital region were observed in 23 of 25 rats in the 900 mg/kg bw/day group. Red and chalky urine, a severe red nasal discharge, mucoid ocular discharge, soft stool or hair loss were noted on many rats.

Mortality: 5/25 rats died between gestation days 10 and 12, three of these showing signs of convulsions prior to death. Observations at necropsy on these five rats included: two rats with mucoid ocular discharge and hyperemic bladder mucosa or cecum mucosa; one with slightly congested brain vessels; two with congested lungs; one with mucoid contents in the small intestine and two with a severe, red nasal discharge. Pneumonia was considered a cause of death for 1 dam. A cause of death could not be determined for the remaining animals that died. The remaining 20 rats in the 900 mg/kg bw dosage group were sacrificied between gestation days 11 and 14 due to excessive maternal toxicity.

Body weight gain: serve decreased prior to sacrifice

500 mg/kg bw/day:

mortality: 1/25 (one animal died on gestation day 9. Observations at necropsy revealed a congested thymus and black, fluid contents in the stomach. (No cause of death could be determined for this female).

Matting and/or staining of the vantral surface or anogenital region (noted also in the control (2/25) but with the greatest incidence occuring in the 300 and 500 mg/kg bw/day groups (17/25 rats), hair loss occured in all treatment groups but most frequently at 500 mg/kg bw/day

Body weight gain: serve decrease during the treatment period, slightly reduced body weight gain folowing the treatment period and severely reduced adjusted body weights on gestation day 20

Cesaren section observations: no biologically relevant differences in postimplantation loss or the fetal sex distribution compared to control; however statistically significant decrease in the mean number of corpora lutea, mean number of total implantations and viable fetuses. These findings were considered as not biologically relevant since implantation occurred before test article administration began and the values were within the range established in the historical control data.

300 mg/kg bw/day:

survival: 100 %

Matting and/or staining of the vantral surface or anogenital region (noted also in the control (2/25) but with the greatest incidence occuring in the 300 and 500 mg/kg bw/day groups (12/25 rats)

Necropsy: there were no remarkable necropsy findings in those females surviving to the scheduled sacrifice, with one exception (1/25 non- gravid rats with hydrometra)

Body weight gain: a slight to moderate decrease during the treatment period; adjusted body weights on gestation day 20 were comparable to control

Cesaren section observations: no biologically relevant differences in the mean number of corpora lutea, total implantations, postimplantation loss or viable fetuses or compared to control; however statistically significant differences in the fetal sex distribution; however was attributed to random occurence and was not considered compound relevant.

100 mg/kg bw/day:

Survival: 100 %

No meaningful difference in the appearance or behavior of rats of the 100 mg/kg bw/day group compared to control

Matting and/or staining of the vantral surface or anogenital region (2/25; control 2/25)

Necropsy: there were no remarkable necropsy findings

Body weight gain: no effects

Cesaren section observations: no biologically relevant differences in the mean number of corpora lutea, total implantations, postimplantation loss, viable fetuses or the fetal sex distribution compared to control

Fetal toxicity:

500 mg/kg bw/day:

Statistically significant decrease in mean fetal body weight; these findings were considered as biologically relevant (values were less than of both control groups and historical control data)

Fetal morphological observations:

There were no statistically significant differences in the number of litters with malformation, however a very slight increase in the number of fetuses and litters with malformation was noted; due primarly to 3 fetuses, each from a different litter, with a thread-like tail and a small anus.

300 mg/kg bw/day:

Slight decrease in mean fetal body weight

Fetal morphological observations:

There were no statistically significant differences in the number of litters with malformation

100 mg/kg bw/day:

Slight decrease in mean fetal body weight

Fetal morphological observations:

There were no statistically significant differences in the number of litters with malformation, however 1 fetus had a thread-like tail and a small anus

Remarks:

There were no biologically relevant differences or dose-related trends in the number of fetuses or litters with genetic and developmental variations in any treated group when compared to control group. The occurence of fetuses with a thred-like tail and small anus has previously been observed in litters of control animals in this laboratory. The author concluded that the increase in frequency of fetuses with a thred-like tail and small anus reported in the study may be due to an increase incidence of genetic expression of this condition among female rats used in this study and occurred at a level which exhibited frank materal toxicity.

Conclusions:
The NOAEL derived from this study for maternal toxicity is 100 mg/kg bw and day and is based on a decrease in mean body weight gain. The NOAEL for the offspring is 300 mg/kg bw and day and is based on a decrease mean foetal body weight.
Executive summary:

In a guideline-consistent developmental toxicity study (Monsanto 1981) which was performed with Charles River COBS CD rats, groups of 25 pregnant females were treated by gavage with CBS at dose levels of 100, 300, 500, and 900 mg/kg bw and day during gestation day 6 to 15. The control group received corn oil. Due to excessive toxicity and death the 900 mg/kg bw and day dose group had to be ceased before end of the study. One maternal death and severe decrease in mean maternal body weight gain and adjusted body weight on gestation day 29 were observed at the 500 mg/kg dose level. A slight to moderate decrease in mean maternal body weight gain was noted in the 300 mg/kg dose group. Hair-loss was noted in all groups, most frequently in the 500 mg/kg dose group. There were no dose-dependent and biologically meaningful effects on mean numbers of corpora lutea, total implantations, post-implantation loss, viable foetuses, or foetal sex distribution in any of the treated groups when compared to controls. There were a very slight increase in the number of foetuses and litters with malformations in the 500 mg/kg dose group due to primarily to three foetuses, each from a different litter, with a thread-like tail and small anus. However, overall, there were no statistically significant differences in the number of litters with malformations in any of the treated groups when compared to the control groups. Further effects observed in the offspring were related to foetal body weight. A slight decrease in mean foetal body weight was observed in the 100 and 300 mg/kg dose group which was statistically significant different from controls for the 500 mg/kg bw and day dose group. The NOAEL derived from this study for maternal toxicity is 100 mg/kg bw and day and is based on a decrease in mean body weight gain. The NOAEL for the offspring is 300 mg/kg bw and day and is based on a decrease mean foetal body weight.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Developmental toxicity study:
The teratogenicity of N-cyclohexyl-2-benzothiazylsulfenamide (CBS) was studied in Wistar rats. Pregnant rats were given CBS at a dosage of 0.001, 0.01, 0.1 or 0.5 % ((corresponding to 0, 0,7; 7,1; 69,6 or 288,8 mg/kg bw/d)) in the diet from Day 0 to day 20 of pregnancy.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Wistar rats (Kar: Wistar, Keari Co., Osaka).
Route of administration:
oral: feed
Vehicle:
other: CBS contained in the diet
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Females were mated overnight with males.
Duration of treatment / exposure:
Day 0 to day 20 of pregnancy.
Frequency of treatment:
Daily.
Duration of test:
Test determination on day 20 of pregnancy.
Dose / conc.:
0.001 other: %
Remarks:
= 0.7 mg/kg
Dose / conc.:
0.01 other: %
Remarks:
= 7.1 mg/kg
Dose / conc.:
0.1 other: %
Remarks:
= 69.6 mg/kg
Dose / conc.:
0.5 other: %
Remarks:
= 288.8 mg/kg
No. of animals per sex per dose:
10-17 animals
Control animals:
yes
Details on study design:
Sex: female
Maternal examinations:
No. of rats (pregnant/copulated), body weight gain during pregancy, food consumption during pregancy, daily intake of CBS.
Ovaries and uterine content:
No. of litters, No. of corpora lutea per litter, No. of implantations per litter, Pre-implantation loss per litter, Post-implantation loss per litter, No. of live fetuses per litter, sex ratio of live fetuses (male/female), body weight of live fetuses, placental weight.
Fetal examinations:
external examination, skeletal examination, internal examination.
Statistics:
Student's t-test, Wilcoxon's rank sum test, chi square test, or Fisher's exact probability test.
Clinical signs:
no effects observed
Description (incidence and severity):
Neither death nor clinical signs of toxicity were found in the pregnant females of any group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Neither death nor clinical signs of toxicity were found in the pregnant females of any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Maternal body weight gain during pregnancy was lower in all CBS-treated animals than in the control group (108 +/- 26 g), the difference was significant (p<0.05) in the 0.1% (91 +/- 9 g) and 0.5% (73 +/- 15 g) dose groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption during pregnancy in the CBS-treated groups did not differ from that in the control (355 +/- 52 g), except for the 0.5% group (302 +/- 30 g), significant different p <0.05) from control.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No signifcant differences between the CBS-treated animals and control in the incidences in pre- and post-implantation losses, the number of corpora lutea per litter, implantations per litter, live fetuses per litter and the sex ratio of live fetuses.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No signifcant differences between the CBS-treated animals and control in the incidences in pre- and post-implantation losses, the number of corpora lutea per litter, implantations per litter, live fetuses per litter and the sex ratio of live fetuses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No signifcant differences between the CBS-treated animals and control in the incidences in pre- and post-implantation losses, the number of corpora lutea per litter, implantations per litter, live fetuses per litter and the sex ratio of live fetuses.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
No signifcant differences between the CBS-treated animals and control in the incidences in pre- and post-implantation losses, the number of corpora lutea per litter, implantations per litter, live fetuses per litter and the sex ratio of live fetuses.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Percentage of pregnant females in CBS-treated groups was approximately the same as in the control.
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
7.1 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
significant(p <0.05)lower body weights of male (3.88 +/- 0.29 g)and female (3.77 +/- 0.34 g) fetuses and placentae (0.42 +/- 0.07 g)in the 0.5 % group (control: males: 4.43 +/- 0.38, females: 4.17 +/- 0.36 g, placenta: 0.60 +/- 0.15 g. Fetal weights and placenta weight not significant different in any other CBS-treated groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
External examinations revealed no fetus with anomalies; no skeletal anomalies were detected in any group. Several types of skeletal variations in the vertebrae, sternebrae and/or ribs were observed in the CBS-treated groups. Delayed ossification in the sternebrae were seen in all groups. The incidence of fetuses and litters with skeletal variation or delayed ossification was not significantly different between the CBS-treated and control groups in the degree of ossification indicated by the numbers of the caudal vertebrae. Dilatation of the renal pelvis occured in one fetus in the control group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
External examinations revealed no fetus with anomalies; no skeletal anomalies were detected in any group. Several types of skeletal variations in the vertebrae, sternebrae and/or ribs were observed in the CBS-treated groups. Delayed ossification in the sternebrae were seen in all groups. The incidence of fetuses and litters with skeletal variation or delayed ossification was not significantly different between the CBS-treated and control groups in the degree of ossification indicated by the numbers of the caudal vertebrae. Dilatation of the renal pelvis occured in one fetus in the control group.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Morphological examinations of the fetuses revealed no evidence of teratogenesis. It could be concluded that CBS possesses no adverse effects on the prenatal development of the offspring in rats at doses employed in the present study.
Dose descriptor:
NOAEL
Effect level:
69.6 mg/kg bw/day
Basis for effect level:
other: fetotoxicity
Abnormalities:
no effects observed
Developmental effects observed:
no

Maternal toxicity:

Body weight gain:

Maternal body weight gain during pregnancy was lower in all CBS-treated animals than in the control group (108 +/- 26 g), the difference was significant (p<0.05) in the 0.1% (91 +/- 9 g) and 0.5% (73 +/- 15 g) dose groups.

Food consumption:

Food consumption during pregnancy in the CBS-treated groups did not differ from that in the control (355 +/- 52 g), except for the 0.5% group (302 +/- 30 g), significant different p <0.05) from control.

Percentage of pregnant females in CBS-treated groups was approximately the same as in the control.

Mortality, clinical signs:

Neither death nor clinical signs of toxicity were found in the pregnant females of any group.

Fetal toxicity:

Significant (p <0.05) lower body weights of male (3.88 +/- 0.29 g)and female (3.77 +/- 0.34 g) fetuses and placentae (0.42 +/- 0.07 g)in the 0.5 % group (control: males: 4.43 +/- 0.38, females: 4.17 +/- 0.36 g, placenta: 0.60 +/- 0.15 g. Fetal weights and placenta weight not significant different in any other CBS-treated groups.

No signifcant differences between the CBS-treated animals and control in the incidences in pre- and post-implantation losses, the number of corpora lutea per litter, implantations per litter, live fetuses per litter and the sex ratio of live fetuses.

Morphological examinations (92 to 150 animals per dose groups):

External examinations revealed no fetus with anomalies; no skeletal anomalies were detected in any group. Several types of skeletal variations in the vertebrae, sternebrae and/or ribs were observed in the CBS-treated groups. Delayed ossification in the sternebrae were seen in all groups. The incidence of fetuses and litters with skeletal variation or delayed ossification was not significantly different between the CBS-treated and control groups in the degree of ossification indicated by the numbers of the caudal vertebrae. Dilatation of the renal pelvis occured in one fetus in the control group.

Conclusions:
Morphological examinations of the fetuses revealed no evidence of teratogenesis. It could be concluded that CBS possesses no adverse effects on the prenatal development of the offspring in rats at doses employed in the present study.
Executive summary:

Morphological examinations of the fetuses revealed no evidence of teratogenesis. It could be concluded that CBS possesses no adverse effects on the prenatal development of the offspring in rats at doses employed in the present study. At the highest applied dose (0.5 % = 288.8 mg/kg/day) a lowered weight in fetuses and placentae were observed.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Publication with limitations (e.g. no historical control data given for the specific rat strain used, which showed in this study a generally elevated number of fetuses with abnormalities) As cited in the EU risk assessment, the observations of this study are considered to be not indicative for a substance specific teratogenic potential.
Principles of method if other than guideline:
Developmental toxicity study.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
sulfenamide TS was purchased from the I Institute of Rubber Industry (Piastow Poland). No further information.
Species:
rat
Strain:
other: Institute's own breeding colony (Imp: DAK)
Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
Sulfenamide TS (N-cyclohxyl-2-benzothiazolesulfenamide) was administered to female rats by gavage at doses of 50, 150, or 450 mg/kg during organogenesis.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Females were mated overnight with males.
Duration of treatment / exposure:
Days 6 to 15 of gestation.
Frequency of treatment:
Once a day.
Duration of test:
From days 6 to 15 of gestation.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
450 mg/kg bw/day
No. of animals per sex per dose:
17-22 animals per group.
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: day 20 of gestation
Maternal examinations:
Mortality, appearance/behaviour, body weight gain, organ weights (liver, kidneys, ovaries, spleen).
Ovaries and uterine content:
Live/dead fetuses, Litters with (early) resorptions, Corpora lutea, total implants, Pre-/post implantation losses, placental weight.
Fetal examinations:
External, skeletal and visceral observations.
Statistics:
Yes.
Historical control data:
No information about histrorical control data.
Description (incidence and severity):
No significant differences in the appearance and behaviour were found between the test and control pregnant females.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The toxic activity of sulfenamide TS at the highest dose of 450 mg/kg was reflected in decreased maternal body weight gain.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the dose of 450 mg/kg a significant increase of relative weight of kidneys, and a decrease of absolute weight of spleen compared to controls were observed. the placental weight in female rats exposed to 450 mg/kg was found to be much lower than in unexposed rats.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
No significant differences in the appearance and behaviour were noted between test and control pregnant females. Dams in the high-dose group (450 mg/kg bw and day) exhibited significantly decreased body weight gain and a significant increase in relative kidney weights and in absolute spleen weights. Also the placental weight was found to be significantly lower than in unexposed rats. The high-dose group (450 mg/kg bw) also exhibited embryo-/fetotoxic effects in terms of a significant increase in the frequency of litters with early resorptions, an increase of mean value of late resorptions per litter and of postimplantation losses as well as decreased foetal body weight and crown-rump length. 192 to 213 foetuses were examined for external, skeletal and visceral abnormalities. From visceral examinations an increased number of fetuses with internal hydocephalus were diagnosed for the high dose group but also for the 150 mg/kg dose group.

However, the numbers of foetuses with enlarged cerebral ventricles (about 20 % of all foetuses affected) and/or renal pelvis were obviously elevated in this study across all treatment groups including the controls. This kind of observations, however, was not obtained from the two other studies and is difficult to put into perspective, since there are no historical data available for this particular rat strain used in this study. Therefore, these observations are not considered as being indicative for a substance specific teratogenic potential.
Dose descriptor:
NOAEL
Basis for effect level:
other: fetotoxicity
Abnormalities:
effects observed, treatment-related
Localisation:
other: From visceral examinations an increased number of fetuses with internal hydocephalus were diagnosed for the high dose group but also for the 150 mg/kg dose group.
Developmental effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes

Maternal toxicity:

450 mg/kg: decreased body weight gain, increased relative kidney weights, decreased absolute spleen weights of the dams;

Fetal toxicity:

Body weight:

450 mg/kg bw signifcant (p< 0.05) decreased compared to control

150 and 450 mg/kg: dose dependent increased number of fetuses with internal hydrocephalus; the number of fetuses with enlarged cerebral ventricles and/or renal pelvis was not dose dependent increased

No significant differences in the appearance and behaviour were noted between test and control pregnant females. Dams in the high-dose group (450 mg/kg bw and day) exhibited significantly decreased body weight gain and a significant increase in relative kidney weights and in absolute spleen weights. Also the placental weight was found to be significantly lower than in unexposed rats. The high-dose group (450 mg/kg bw) also exhibited embryo-/fetotoxic effects in terms of a significant increase in the frequency of litters with early resorptions, an increase of mean value of late resorptions per litter and of postimplantation losses as well as decreased foetal body weight and crown-rump length. 192 to 213 foetuses were examined for external, skeletal and visceral abnormalities. From visceral examinations an increased number of fetuses with internal hydocephalus were diagnosed for the high dose group but also for the 150 mg/kg dose group.

However, the numbers of foetuses with enlarged cerebral ventricles (about 20 % of all foetuses affected) and/or renal pelvis were obviously elevated in this study across all treatment groups including the controls. This kind of observations, however, was not obtained from the two other studies and is difficult to put into perspective, since there are no historical data available for this particular rat strain used in this study. Therefore, these observations are not considered as being indicative for a substance specific teratogenic potential.

Conclusions:
No significant differences in the appearance and behaviour were noted between test and control pregnant females. Dams in the high-dose group (450 mg/kg bw and day) exhibited significantly decreased body weight gain and a significant increase in relative kidney weights and in absolute spleen weights. Also the placental weight was found to be significantly lower than in unexposed rats. The high-dose group (450 mg/kg bw) also exhibited embryo-/fetotoxic effects in terms of a significant increase in the frequency of litters with early resorptions, an increase of mean value of late resorptions per litter and of postimplantation losses as well as decreased foetal body weight and crown-rump length. 192 to 213 foetuses were examined for external, skeletal and visceral abnormalities. From visceral examinations an increased number of fetuses with internal hydocephalus were diagnosed for the high dose group but also for the 150 mg/kg dose group.

However, the numbers of foetuses with enlarged cerebral ventricles (about 20 % of all foetuses affected) and/or renal pelvis were obviously elevated in this study across all treatment groups including the controls. This kind of observations, however, was not obtained from the two other studies and is difficult to put into perspective, since there are no historical data available for this particular rat strain used in this study. Therefore, these observations are not considered as being indicative for a substance specific teratogenic potential.
Executive summary:

No significant differences in the appearance and behaviour were noted between test and control pregnant females. Dams in the high-dose group (450 mg/kg bw and day) exhibited significantly decreased body weight gain and a significant increase in relative kidney weights and in absolute spleen weights. Also the placental weight was found to be significantly lower than in unexposed rats. The high-dose group (450 mg/kg bw) also exhibited embryo-/fetotoxic effects in terms of a significant increase in the frequency of litters with early resorptions, an increase of mean value of late resorptions per litter and of postimplantation losses as well as decreased foetal body weight and crown-rump length. 192 to 213 foetuses were examined for external, skeletal and visceral abnormalities. From visceral examinations an increased number of fetuses with internal hydocephalus were diagnosed for the high dose group but also for the 150 mg/kg dose group.

However, the numbers of foetuses with enlarged cerebral ventricles (about 20 % of all foetuses affected) and/or renal pelvis were obviously elevated in this study across all treatment groups including the controls. This kind of observations, however, was not obtained from the two other studies and is difficult to put into perspective, since there are no historical data available for this particular rat strain used in this study. Therefore, these observations are not considered as being indicative for a substance specific teratogenic potential.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Eighty-eight mated female New Zealand White rabbits were assigned to four groups of 22 animals each. The test item, N,N-dicyclohexylbenzothiazole-2-sulphenamide, was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 100, 300 and 1000 mg/kg/day (Groups 2, 3 and 4, respectively). Rabbits of the control group received the vehicle, 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity.
On Day 29 post-coitum, all animals were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. Approximately, one half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol. Following staining with Alizarin Red S, skeletons of all fetuses of all groups were examined.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Identification: N,N-dicyclohexylbenzothiazole-2-sulphenamide
Appearance: Beige solid
Batch: Vulkacit DZ Filtercake DCBS 05.08.2015
Purity/Composition: 93.9%
Test item storage: At room temperature

Purity/composition correction factor: Yes, correction factor is 1.065 according to purity
Test item handling: No specific handling conditions required
Stability at higher temperatures: Not available
Chemical name (IUPAC), synonym
or trade name: N,N-dicyclohexylbenzothiazole-2-sulphenamide
CAS Number: 4979-32-2
Molecular formula: C19H26N2S2
Molecular weight: 346.55

Stability in vehicle:
• 1% CMC + 0.1% Tween-80 Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the
concentration range 1 to 200 mg/g, Test Facility Study No. 510548
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Test System:
Test System Rabbit: female albino rabbits, New Zealand White (NZW) strain (SPF-Quality), from a non-inbred laboratory colony. Nulliparous, non-pregnant and untreated females were used at initiation of the study.
Stock male NZW rabbits were used for mating with the females. These males were adult and proven fertile. After mating they were placed back in their stock for possible use in further studies.
Rationale: This species and strain of rabbit has been recognized as appropriate for developmental toxicity studies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Source: Charles River, Chatillon sur Chalaronne, France.
Number of animals: F0-generation: 88 females.
F1-generation: 768 fetuses.
Age at delivery: Females were approximately 17-18 weeks.
Acclimatization: At least 5 days prior to pairing.
Health inspection: Upon receipt of the animals.
Randomization: The animals were allocated to the groups by computergenerated random algorithm according to body weight, with all animals within ± 20% of the mean.
Upon observation of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups.
Females which were mated on the same day were classified in the same subgroup.
Identification: By tattoo in the ear.
Mating procedures: One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 postcoitum.
Veterinary examination: No veterinary inspection of the rabbits was performed during the experimental phase of this study.

Allocation:
Group Dose level Number of females Animal numbers
(mg/kg/day)
1 0 22 1-22
2 100 22 23-44
3 300 22 45-66
4 1000 22 67-88

Animal Husbandry:

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation: Females were individually housed in labelled cages with perforated floors
Diet: Free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy). In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) and
wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were provided during the study period.
Water: Free access to tap-water.

Diet and water evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous carboxymethyl cellulose with 0.1% Tween
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method (Test Facility Study No. 510548). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Details on mating procedure:
One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
From Days 6 to 28 post-coitum, inclusive.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Duration of test:
Start mating: 09 May 2016
Start treatment: 15 May 2016
Start necropsy: 07 June 2016
Experimental completion date: 14 June 2016 (end in-life phase)
Dose / conc.:
0 mg/kg bw/day
Remarks:
control
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
22 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
In a dose range finding study (Test Facility Study No. 510544) in which dose levels of 100, 300 and 1000 mg/kg/day were tested in pregnant females, no relevant toxicity was observed up to 1000 mg/kg/day. In the preceding tolerability study with non-pregnant females (Test Facility Study No. 510543) dose levels up to 1000 mg/kg/day did not result in toxicity. Based on these findings dose levels of 100, 300 and 1000 mg/kg/day were selected for the main study.
Maternal examinations:
Observations:
Mortality / Viability: At least twice daily.
Clinical signs: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Cage debris was examined to detect abortion or premature birth, if applicable.
Body weights: Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.
Food consumption: Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Pathology/Necropsy:
All animals were euthanized on Day 29 post-coitum by intravenous injection of pentobarbital and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.
All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).

Ovaries and uterine content:
Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The weight of each fetus.
• The sex of each fetus (during further fetal examination).
• Externally visible macroscopic fetal abnormalities.

Fetal examinations:
Fetal Examination:
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (=60mg) of sodium pentobarbital into the oral cavity using a small flexible plastic or metal feeding tube. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed (if possible). Late resorption A043-13 with malformations was fixed in 96% aqueous ethanol.

Visceral (Internal)
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was determined by internal examination.
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.
All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal
All the eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S. Subsequently, the skeletal examination was done on all fetuses. Based on possible treatment related effects in the high dose group, skeletal examination was extended to all fetuses from the low and mid dose group.
All specimens were archived in glycerin with bronopol as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Statistics:
The following statistical methods were used to analyze the data:

• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
• Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
For each litter the following calculations were performed:
Pre-implantation loss (%) = [(number of corpora lutea - number of implantation sites) : (number of corpora lutea)] x 100

Post-implantation loss (%) = [(number of implantation sites - number of live fetuses) : (number of implantation sites)] x 100

The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%) = [(number of viable fetuses affected/litter : (number of viable fetuses/litter)] x 100
Historical control data:
Historical control data are available.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In 16/22, 19/22, 17/22 and 19/22 females treated at 0, 100, 300 and 1000 mg/kg/day, respectively, faeces production was reduced, varying in duration and severity. Additionally, incidental diarrhea, faeces containing mucus or pale faeces was noted in the control, mid and/or high dose groups. As the majority of these findings were noted in all groups, this is not related to treatment with the test item.
Three female (no. 28, 53 and 83 in Groups 2, 3 and 4, respectively) had red staining of the vagina and/or red fluid on the manure tray at the end of the treatment period. All three females had normal litters and therefore these finding were not considered adverse effects of treatment.
The lean appearance of female 83 (1000 mg/kg), corresponds with a body weight loss of approximately 7% (20.5 % when corrected for weight of the uterus). This female was the most sensitive animal in this group.
The incidence of all other observed clinical signs remained within the range of background findings to be expected for rabbits of this strain and age.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
From Day 16 post-coitum onwards, body weight gain was slightly reduced in females treated at 1000 mg/kg/day, compared to the control animals. As this effect was very slight, not reaching statistical significance and body weight corrected for weight of the uterus was unaffected, it was considered not toxicologically relevant.
At 100 and 300 mg/kg/day, body weight remained within the same range as the control group.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related macroscopic findings were observed up to 1000 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Remarks on result:
other: Purity/composition correction factor: Correction factor is 1.065 according to purity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no treatment related effect on mean fetal body weights.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There was no treatment related effect on mean fetal body weights.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no treatment related effects on litter size for any group.
The mean litter size in the high dose group was slightly lower compared to the control group (8.4 compared to 9.8 for the 1000 mg/kg/day and control groups, respectively). The lower litter size in the high dose group could be explained by the litters of females 75 and 76, which both had only two viable fetuses. For female no. 76, this was caused by a relatively high pre-implantation loss, which cannot be contributed to treatment, as this starts after implantation. The relatively high number of early resorptions and consequently high post-implantation loss, resulting in the low number of viable fetuses for female no. 75, was considered a chance finding. Taken together, the lower litter size at 1000 mg/kg/day was of no toxicological relevance. Mean litter sizes were 9.9 and 9.3 for the 100 and 300 mg/kg/day groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 1000 mg/kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment related effects on litter size for any group.
The mean litter size in the high dose group was slightly lower compared to the control group (8.4 compared to 9.8 for the 1000 mg/kg/day and control groups, respectively). The lower litter size in the high dose group could be explained by the litters of females 75 and 76, which both had only two viable fetuses. For female no. 76, this was caused by a relatively high pre-implantation loss, which cannot be contributed to treatment, as this starts after implantation. The relatively high number of early resorptions and consequently high post-implantation loss, resulting in the low number of viable fetuses for female no. 75, was considered a chance finding. Taken together, the lower litter size at 1000 mg/kg/day was of no toxicological relevance. Mean litter sizes were 9.9 and 9.3 for the 100 and 300 mg/kg/day groups, respectively.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to 1000 mg/kg/day.
Three malformations were observed in this study. One 1000 mg/kg/day fetus A082-02 had an omphalocele and fetus A063-08 in the 300 mg/kg/day group was totally malformed externally, which was substantiated viscerally and skeletally. The third affected fetus was the dead fetus in the 100 mg/kg/day group (A040-07) which had a distended abdomen; at visceral examination this appeared to be ascites accompanied with a large heart. All these cases occurred singly at different dose levels and therefore were considered to be chance findings and not related to treatment.
There were no external variations seen for any fetus in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 1000 mg/kg/day.
Malformations occurred in 0 (0), 2 (2), 9 (7) and 7 (4) fetuses (litters) in the control, 100, 300 and 1000 mg/kg/day groups, respectively.
In total, seven different types of malformations were noted of which three were seen in the 1000 mg/kg/day group only. These were a rib anomaly in fetus A072-04 and A086-02, a caudal vertebral anomaly in fetus A072-01 and fused skull bones in fetus A072-02.
It is remarkable that these were not observed in the other groups, but all three malformations occurred previously in historical controls. Moreover, it is noteworthy that three of the aforementioned fetuses are from litter A072 and that in this litter, fetus 09 was skeletally affected as well. This fetus had a vertebral anomaly with associated rib anomaly and was found to have multiple cardiovascular malformations viscerally. Also it should be noted that the mean fetal body weights of litter A072 are the lowest of the group and far below the group mean value (30.1 versus 38.2 grams, respectively), despite normal litter size (9 viable fetuses and 1 early resorption). Taken these aspects into account, it is considered that all skeletal malformations in litter A072 were a result of general defective fetal development and were not related to treatment.
The fetus in the 300 mg/kg/day that was totally malformed externally and viscerally (A063-08) was also totally malformed skeletally. At the single incidence, it was considered a chance finding.
Remaining skeletal malformations of this study were a vertebral anomaly with or without associated rib anomaly (A028-09, A051-02, A057-06 and A074-10), fused sternebrae (A035-01, A050-06, A59-09, A085-03), split skull bones (A060-04, A064-02) and bent limb bones (A064 fetus 06 and 07). These occurred once or twice in one or more dose groups and all were noted previously in historical controls; thus, these were considered not to be treatment related.
Skeletal variations that were noted in this study occurred at low incidences, in the absence of a dose-related incidence and/or at frequencies that were within the range of available historical control data.

Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 1000 mg/kg/day.
In the 1000 mg/kg/day group, four fetuses had a visceral malformation. Multiple cardiovascular malformations (interrupted aortic arch, ventricular septum defect, retroesophageal ductus arteriosus and right sided descending aorta) were noted in fetus
A072-09, Tetralogy of Fallot was found in fetus A067-09 and absence of an accessory lung lobe in litter mates A072-06 and -08. The latter two malformations also occurred in other dose groups, namely Tetralogy of Fallot at 300 mg/kg/day (A045-04) and a missing accessory lung lobe in the control group (A001-06 and A016-01) and at 100 mg/kg/day (A030-02 and A040-08). The very low incidence and group distribution of these malformations does not indicate a treatment relationship. Moreover, all but two (retroesophageal ductus arteriosus and right sided aorta descending) were noted previously in historical controls.
The fetus in the 300 mg/kg/day that was totally malformed externally (A063-08) was also totally malformed viscerally. At the single incidence, it was considered a chance finding.
All variations noted, were considered not treatment related as they occurred infrequently, occurred at frequencies that were within the range of available historical control data, were observed in the absence of a dose-incidence trend or were limited to control fetuses only.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
No developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: Purity/composition correction factor: Correction factor is 1.065 according to purity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS

 Dose Group:  1     2     3     4   
 No.  %  No.  %  No.  %  No.  %
 Females on study  22    22    22    22  

 Females that oborted

or delievered

 0  0.0  0  0.0  0  0.0  0  0.0
 Females that died  0  0.0  0  0.0  0  0.0  0  0.0
 Females that aborted  0  0.0  0  0.0  0  0.0  0  0.0
 Nongravid  0  0.0  0  0.0  0  0.0  0  0.0
 Gravid  0  0.0  0  0.0  0  0.0  0  0.0
                 
 Females that were euthanized  0  0.0  0  0.0  0  0.0  0  0.0
 Nongravid  0  0.0  0  0.0  0  0.0  0  0.0
 Gravid  0  0.0  0  0.0  0  0.0  0  0.0
                 
 Females examined at                
 Scheduled necropsy  22  100.0  22  100.0  22  100.0  22  100.0
 Nongravid  2  9.1  1  4.5  1  4.5  2  9.1
 Gravid  20  90.9  21  95.5  21  95.5  20  90.9
 With resorptions only  2  0.0  0  0.0  0  0.0  0  0.0
 with viable fetuses  20  100.0  21  100.0  21  100.0  20  100.0
                 
 Total females gravid  20  90.9  21  95.5  21  95.5  20  90.9

1- 0 mg/kg/day 2- 100 mg/kg/day 3- 300 mg/kg/day 4- 1000 mg/kg/day

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

 Group

 

 Sex

M

Sex

F

  Viable

Fetuses

Dead

Fetuses

 Resorptions

 Early

Resporptions

Late

 Post

Implantation

Loss

Implantation

Sites 

Corpora

Lutea

  Pre

implantation

Loss 

Fetal

Weights

in grams

No. of

gravid

females

 1

 Total

 98

 98

 196

 0

 6

 0

 6

 202

 216

 14

 NA

 20

 1

 Mean

 4.9

 4.9

 9.8

 0 .0

 0.3

 0.0

 0.3

 10.1

 10.8

 0.7

 38.2

 

 1

 S.D.

 2.31

 1.80

 1.99

 0.00

 0.57

 0.00

 0.57

 1.80

 1.77

 0.73

 4.55

 

 2

 Total

 100

 107

 207

 1

 3

 5

 9

 216

 221

 5

 Na

 21

 2

 Mean

 4.8

 5.1

 9.9

 0.0

 0.1

 0.2

 0.4

 10.3

 10.5

 0.2

 37.6

 

 2

 S.D.

 1.55

 1.37

 1.49

 0.22

 0.36

 0.54

 0.81

 1.35

 1.44

 0.44

 4.63

 

 3

 Total

 96

 100

 196

 0

 3

 3

 6

 202

 211

 9

 NA

 21

 3

 Mean

 4.6

 4.8

 9.3

 0.0

 0.1

 0.1

 0.3

 9.6

 10.0

 0.4

 37.3

 

 3

 S.D.

 1.75

 1.30

 2.22

 0.00

 0.48

 0.48

 0.78

 2.48

 2.44

 0.81

 4.76

 

 4

 Total

 82

 86

 168

 0

 12^

 1^

 13^

 173^

 189^

 16^

 NA

 20

 4

 Mean

 4.1

 4.3

 8.4

 0.0

 0.6^

 0.1^

 0.7^

 9.1^

 9.9^

 0.8^

 38.2

 

 4

 S.D.

 2.10

 1.89

 2.91

 0.00

1.42^

 0.23^

 1.42^

 2.62^

 2.01^

 1.57^

 5.67

 

-----------------------------

------------------------------------------------------------------------------------------------------

None significantly different from control group

NA = NOT APPLICABLE MEAN NUMBER OF VIABLE FETUSES,

MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA, FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

^ Calculated based on n=19 litters, due to missing data for dam# A085 -----------------------------------------------------------------------------------------------------------------------------------

1- 0 MG/KG/DAY 2- 100 MG/KG/DAY 3- 300 MG/KG/DAY 4- 1000 MG/KG/DAY

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY [% PER LITTER]

 Group  0 mg/kg/day   100 mg/kg/day   300 mg/kg/day   1000 mg/kg/day

 Corproa Lutea

       
 Mean  10.8  10.5  10.0  9.9
 S.D.  1.77  1.44  2.44  2.01
 N  20  21  21  19
 Implantation sites        
 Mean  10.1  10.3  9.6  9.1
 S.D.  1.80  1.35  2.48  2.62
 N  20  21  21  19
 Viable fetuses (%)        
 Mean  96.8  95.9  97.7  92.3
 S.D.  5.97  7.83  6.08  17.46
 N  20  21  21  19
 Early resprptions        
 Mean  3.2  1.5  1.2  7.3
 S.D.  5.97  3.72  4.16  17.55
 N  20  21  21  19
 Late resorptions        
 Mean  0.0  2.2  1.2  0.4
 S.D.  0.00  4.81  3.69  1.77
 N  20  21  21  19
 Total resorptions        
 Mean  3.2  3.6  2.3  7.7
 S.D.  5.97  6.35  6.08  17.46
 N  20  21  21  19
 Pre-implantation loss (%)        
 Mean  6.4  3.6  2.3  7.7
 S.D.  6.97  6.35  6.08  17.46
 N  20  21  21  19
 Post-implantation loss (%)        
 Mean  3.2  4.1  2.3  8.9
 S.D.  5.97  3.90  6.08  17.54
 N  20  21  21  19
 Males (%)        
 Mean  48.9  48.0  47.9  46.9
 S.D.  19.53  13.62  11.52  18.47
 N  20  21  21  20
 Females (%)        
 Mean  51.1  52.0  52.1  53.1
 S.D.  19.53  13.62  11.52  18.47
 N  20  21  21  20
 Male fetal weights (g)        
 Mean  39.0  38.7  38.0  38.0
 S.D.  5.35  4.39  4.88  5.34
 N  20  21  21  19
 Female fetal weights (g)        
 Mean  37.6  36.6  36.6  37.8
 S.D.  4.96  5.34  4.96  5.67
 N  20  21  21  20
 Combined fetal weights (g)        
 Mean  38.2  37.6  37.3  38.2
 S.D.  4.55  4.63  4.76  5.67
 N  20  21  21  20

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO.]

              Fetuses           Litters
   Dose group:  1  2  3  4  1  2  3  4
 Number examined externally    196  207  196  168  20  21  21  20
 Fetus totally malformed    0  0  1  0  0  0  1  0
 Trunk omphalocele    0  0  0  1  0  0  0  1
                   
Number examined viscerally     196  207  196  168  20  21  21  20
 Teratology of fallot    0  0  1  1  0  0  1  1
 Lung - absent lobe(s)    2  2  0  2  2  2  0  1
 Aortic arch - interrupted    0  0  0  1  0  0  0  1
Ventricular septum defect     0  0  0  1  0  0  0  1
 Ductus arteriosus retroesophagal    0  0  0  1  0  0  0  1
 Aorta descending - right sides    0  0  0  1  0  0  0  1
 Fetus totally malformed    0  0  1  0  0  0  1  0
                   
 Number examined skeletally    196  207  196  168  20  21  21  20

 Vertebral anomaly with or without

associated rib anomaly

   0  1  2  2  0  1  2  2
 Rib anomaly    0  0  0  2  0  0  0  2
  Caudal vertebral anomaly    0  0  0  1  0  0  0  1
   Skull bones - fused    0  0  0  1  0  0  0  1
  Sternebrae fused    0  1  2  1  0  1  2  1
  Skull bone(s) - split    0  0  2  0  0  0  2  0
  Bent limb bones    0  0  2  0  0  0  1  0
  Fetus totally malformed    0  0  1  0  0  0  1  0
                   
  Total number with malformations                  
  External    0  0  1  1  0  0  1
  Soft tissue    2  2  2  4  2  2  2  2
  Skeletal    0  2  9  7  0  2  7  4
  Combined    2  4  10  11  2  4  8  6

1- 0 MG/KG/DAY 2- 100 MG/KG/DAY 3- 300 MG/KG/DAY 4- 1000 MG/KG/DAY

SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS % PER LITTER

   Dose group    1  2  3  4
 Number of litters examined            
 Total malformations  

 

       
 Percent per litter with external malformations  

 Mean

S.D.

 0.0

0.00

 0.0

0.00

0.3

1.56 

 1.0

4.47

 Percent per litter with soft tissuel malformations  

 Mean

S.D.

 1.0

2.94

 1.3

4.04

 0.8

2.62

 2.2

7.67

 Percent per litter with skeletal malformations  

 Mean

S.D.

 0.0

0.00

1.0

3.01 

 5.5

10 .97

 4.0

10.50

 Total percent per litter with malformations  

 Mean

S.D.

 1.0

2.94

2.2

4.78 

 6.3

10.85

 6.6

15.41

1- 0 MG/KG/DAY 2- 100 MG/KG/DAY 3- 300 MG/KG/DAY 4- 1000 MG/KG/DAY

None significantly different from control group

SUMMARY OF LITTER PROPORTIONS OF VARIATIONS % PER LITTER

 Dose group    1  2  3  4
 Number of litters examined      20  21  21  20
 Total variations  

 

       
 Percent per litter with external variations  

 Mean

S.D.

 0.0

0.00

 0.0

0.00

00

0.00

 0.0

0.00

 Percent per litter with soft tissuel variations  

 Mean

S.D.

 6.4

9.24

 3.5

6.20

 5.0

6.23

 7.1

8.18

 Percent per litter with skeletal variations  

 Mean

S.D.

 72.6

26.43

74.8

19.14 

 76.1

24.04

 83.7

21.04

 Total percent per litter with variations  

 Mean

S.D.

 73.9

25.31

75.7

18.79 

 77.3

23.95

 85.2

19.54

1- 0 MG/KG/DAY 2- 100 MG/KG/DAY 3- 300 MG/KG/DAY 4- 1000 MG/KG/DAY

None significantly different from control group

Conclusions:
In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for N,N-dicyclohexylbenzothiazole-2-sulphenamide was established as being at least 1000 mg/kg/day.
Executive summary:

Title

Prenatal developmental toxicity study of N,N-dicyclohexylbenzothiazole-2-sulphenamide in rabbits by oral gavage.

Guidelines

The study procedures described in this report were based on the following guidelines:

•Organization of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, January 2001.

•Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.31: "Prenatal Developmental Toxicity Study". Official Journal of the European Union No. L142, May 2008.

•The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998.

Study outline

Eighty-eight mated female New Zealand White rabbits were assigned to four groups of 22 animals each. The test item, N,N-dicyclohexylbenzothiazole-2-sulphenamide, was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 100, 300 and 1000 mg/kg/day (Groups 2, 3 and 4, respectively). Rabbits of the control group received the vehicle, 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity.

On Day 29 post-coitum, all animals were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. Approximately, one half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol. Following staining with Alizarin Red S, skeletons of all fetuses of all groups were examined.

Results

Accuracy and homogeneity of formulations were demonstrated by analyses.

Maternal findings:

No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.

Developmental findings:

No developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.

Conclusion

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for N,N-dicyclohexylbenzothiazole-2-sulphenamide was established as being at least 1000 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
See attached justification: Read-across Justification Document for the Category of four Sulfenamides
Specific details on test material used for the study:
Identification: N,N-dicyclohexylbenzothiazole-2-sulphenamide
Appearance: Beige solid
Batch: Vulkacit DZ Filtercake DCBS 05.08.2015
Purity/Composition: 93.9%
Test item storage: At room temperature

Purity/composition correction factor: Yes, correction factor is 1.065 according to purity
Test item handling: No specific handling conditions required
Stability at higher temperatures: Not available
Chemical name (IUPAC), synonym
or trade name: N,N-dicyclohexylbenzothiazole-2-sulphenamide
CAS Number: 4979-32-2
Molecular formula: C19H26N2S2
Molecular weight: 346.55

Stability in vehicle:
• 1% CMC + 0.1% Tween-80 Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the
concentration range 1 to 200 mg/g, Test Facility Study No. 510548
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Test System:
Test System Rabbit: female albino rabbits, New Zealand White (NZW) strain (SPF-Quality), from a non-inbred laboratory colony. Nulliparous, non-pregnant and untreated females were used at initiation of the study.
Stock male NZW rabbits were used for mating with the females. These males were adult and proven fertile. After mating they were placed back in their stock for possible use in further studies.
Rationale: This species and strain of rabbit has been recognized as appropriate for developmental toxicity studies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Source: Charles River, Chatillon sur Chalaronne, France.
Number of animals: F0-generation: 88 females.
F1-generation: 768 fetuses.
Age at delivery: Females were approximately 17-18 weeks.
Acclimatization: At least 5 days prior to pairing.
Health inspection: Upon receipt of the animals.
Randomization: The animals were allocated to the groups by computergenerated random algorithm according to body weight, with all animals within ± 20% of the mean.
Upon observation of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups.
Females which were mated on the same day were classified in the same subgroup.
Identification: By tattoo in the ear.
Mating procedures: One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 postcoitum.
Veterinary examination: No veterinary inspection of the rabbits was performed during the experimental phase of this study.

Allocation:
Group Dose level Number of females Animal numbers
(mg/kg/day)
1 0 22 1-22
2 100 22 23-44
3 300 22 45-66
4 1000 22 67-88

Animal Husbandry:

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation: Females were individually housed in labelled cages with perforated floors
Diet: Free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy). In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) and
wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were provided during the study period.
Water: Free access to tap-water.

Diet and water evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous carboxymethyl cellulose with 0.1% Tween
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method (Test Facility Study No. 510548). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Details on mating procedure:
One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
From Days 6 to 28 post-coitum, inclusive.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Duration of test:
Start mating: 09 May 2016
Start treatment: 15 May 2016
Start necropsy: 07 June 2016
Experimental completion date: 14 June 2016 (end in-life phase)
Dose / conc.:
0 mg/kg bw/day
Remarks:
control
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
22 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
In a dose range finding study (Test Facility Study No. 510544) in which dose levels of 100, 300 and 1000 mg/kg/day were tested in pregnant females, no relevant toxicity was observed up to 1000 mg/kg/day. In the preceding tolerability study with non-pregnant females (Test Facility Study No. 510543) dose levels up to 1000 mg/kg/day did not result in toxicity. Based on these findings dose levels of 100, 300 and 1000 mg/kg/day were selected for the main study.
Maternal examinations:
Observations:
Mortality / Viability: At least twice daily.
Clinical signs: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Cage debris was examined to detect abortion or premature birth, if applicable.
Body weights: Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.
Food consumption: Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Pathology/Necropsy:
All animals were euthanized on Day 29 post-coitum by intravenous injection of pentobarbital and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.
All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).

Ovaries and uterine content:
Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The weight of each fetus.
• The sex of each fetus (during further fetal examination).
• Externally visible macroscopic fetal abnormalities.

Fetal examinations:
Fetal Examination:
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (=60mg) of sodium pentobarbital into the oral cavity using a small flexible plastic or metal feeding tube. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed (if possible). Late resorption A043-13 with malformations was fixed in 96% aqueous ethanol.

Visceral (Internal)
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was determined by internal examination.
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.
All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal
All the eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S. Subsequently, the skeletal examination was done on all fetuses. Based on possible treatment related effects in the high dose group, skeletal examination was extended to all fetuses from the low and mid dose group.
All specimens were archived in glycerin with bronopol as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Statistics:
The following statistical methods were used to analyze the data:

• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
• Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
For each litter the following calculations were performed:
Pre-implantation loss (%) = [(number of corpora lutea - number of implantation sites) : (number of corpora lutea)] x 100

Post-implantation loss (%) = [(number of implantation sites - number of live fetuses) : (number of implantation sites)] x 100

The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%) = [(number of viable fetuses affected/litter : (number of viable fetuses/litter)] x 100
Historical control data:
Historical control data are available.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In 16/22, 19/22, 17/22 and 19/22 females treated at 0, 100, 300 and 1000 mg/kg/day, respectively, faeces production was reduced, varying in duration and severity. Additionally, incidental diarrhea, faeces containing mucus or pale faeces was noted in the control, mid and/or high dose groups. As the majority of these findings were noted in all groups, this is not related to treatment with the test item.
Three female (no. 28, 53 and 83 in Groups 2, 3 and 4, respectively) had red staining of the vagina and/or red fluid on the manure tray at the end of the treatment period. All three females had normal litters and therefore these finding were not considered adverse effects of treatment.
The lean appearance of female 83 (1000 mg/kg), corresponds with a body weight loss of approximately 7% (20.5 % when corrected for weight of the uterus). This female was the most sensitive animal in this group.
The incidence of all other observed clinical signs remained within the range of background findings to be expected for rabbits of this strain and age.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
From Day 16 post-coitum onwards, body weight gain was slightly reduced in females treated at 1000 mg/kg/day, compared to the control animals. As this effect was very slight, not reaching statistical significance and body weight corrected for weight of the uterus was unaffected, it was considered not toxicologically relevant.
At 100 and 300 mg/kg/day, body weight remained within the same range as the control group.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related macroscopic findings were observed up to 1000 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Remarks on result:
other: Purity/composition correction factor: Correction factor is 1.065 according to purity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no treatment related effect on mean fetal body weights.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There was no treatment related effect on mean fetal body weights.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no treatment related effects on litter size for any group.
The mean litter size in the high dose group was slightly lower compared to the control group (8.4 compared to 9.8 for the 1000 mg/kg/day and control groups, respectively). The lower litter size in the high dose group could be explained by the litters of females 75 and 76, which both had only two viable fetuses. For female no. 76, this was caused by a relatively high pre-implantation loss, which cannot be contributed to treatment, as this starts after implantation. The relatively high number of early resorptions and consequently high post-implantation loss, resulting in the low number of viable fetuses for female no. 75, was considered a chance finding. Taken together, the lower litter size at 1000 mg/kg/day was of no toxicological relevance. Mean litter sizes were 9.9 and 9.3 for the 100 and 300 mg/kg/day groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 1000 mg/kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment related effects on litter size for any group.
The mean litter size in the high dose group was slightly lower compared to the control group (8.4 compared to 9.8 for the 1000 mg/kg/day and control groups, respectively). The lower litter size in the high dose group could be explained by the litters of females 75 and 76, which both had only two viable fetuses. For female no. 76, this was caused by a relatively high pre-implantation loss, which cannot be contributed to treatment, as this starts after implantation. The relatively high number of early resorptions and consequently high post-implantation loss, resulting in the low number of viable fetuses for female no. 75, was considered a chance finding. Taken together, the lower litter size at 1000 mg/kg/day was of no toxicological relevance. Mean litter sizes were 9.9 and 9.3 for the 100 and 300 mg/kg/day groups, respectively.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to 1000 mg/kg/day.
Three malformations were observed in this study. One 1000 mg/kg/day fetus A082-02 had an omphalocele and fetus A063-08 in the 300 mg/kg/day group was totally malformed externally, which was substantiated viscerally and skeletally. The third affected fetus was the dead fetus in the 100 mg/kg/day group (A040-07) which had a distended abdomen; at visceral examination this appeared to be ascites accompanied with a large heart. All these cases occurred singly at different dose levels and therefore were considered to be chance findings and not related to treatment.
There were no external variations seen for any fetus in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 1000 mg/kg/day.
Malformations occurred in 0 (0), 2 (2), 9 (7) and 7 (4) fetuses (litters) in the control, 100, 300 and 1000 mg/kg/day groups, respectively.
In total, seven different types of malformations were noted of which three were seen in the 1000 mg/kg/day group only. These were a rib anomaly in fetus A072-04 and A086-02, a caudal vertebral anomaly in fetus A072-01 and fused skull bones in fetus A072-02.
It is remarkable that these were not observed in the other groups, but all three malformations occurred previously in historical controls. Moreover, it is noteworthy that three of the aforementioned fetuses are from litter A072 and that in this litter, fetus 09 was skeletally affected as well. This fetus had a vertebral anomaly with associated rib anomaly and was found to have multiple cardiovascular malformations viscerally. Also it should be noted that the mean fetal body weights of litter A072 are the lowest of the group and far below the group mean value (30.1 versus 38.2 grams, respectively), despite normal litter size (9 viable fetuses and 1 early resorption). Taken these aspects into account, it is considered that all skeletal malformations in litter A072 were a result of general defective fetal development and were not related to treatment.
The fetus in the 300 mg/kg/day that was totally malformed externally and viscerally (A063-08) was also totally malformed skeletally. At the single incidence, it was considered a chance finding.
Remaining skeletal malformations of this study were a vertebral anomaly with or without associated rib anomaly (A028-09, A051-02, A057-06 and A074-10), fused sternebrae (A035-01, A050-06, A59-09, A085-03), split skull bones (A060-04, A064-02) and bent limb bones (A064 fetus 06 and 07). These occurred once or twice in one or more dose groups and all were noted previously in historical controls; thus, these were considered not to be treatment related.
Skeletal variations that were noted in this study occurred at low incidences, in the absence of a dose-related incidence and/or at frequencies that were within the range of available historical control data.

Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 1000 mg/kg/day.
In the 1000 mg/kg/day group, four fetuses had a visceral malformation. Multiple cardiovascular malformations (interrupted aortic arch, ventricular septum defect, retroesophageal ductus arteriosus and right sided descending aorta) were noted in fetus
A072-09, Tetralogy of Fallot was found in fetus A067-09 and absence of an accessory lung lobe in litter mates A072-06 and -08. The latter two malformations also occurred in other dose groups, namely Tetralogy of Fallot at 300 mg/kg/day (A045-04) and a missing accessory lung lobe in the control group (A001-06 and A016-01) and at 100 mg/kg/day (A030-02 and A040-08). The very low incidence and group distribution of these malformations does not indicate a treatment relationship. Moreover, all but two (retroesophageal ductus arteriosus and right sided aorta descending) were noted previously in historical controls.
The fetus in the 300 mg/kg/day that was totally malformed externally (A063-08) was also totally malformed viscerally. At the single incidence, it was considered a chance finding.
All variations noted, were considered not treatment related as they occurred infrequently, occurred at frequencies that were within the range of available historical control data, were observed in the absence of a dose-incidence trend or were limited to control fetuses only.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
No developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: Purity/composition correction factor: Correction factor is 1.065 according to purity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS

 Dose Group:  1     2     3     4   
 No.  %  No.  %  No.  %  No.  %
 Females on study  22    22    22    22  

 Females that oborted

or delievered

 0  0.0  0  0.0  0  0.0  0  0.0
 Females that died  0  0.0  0  0.0  0  0.0  0  0.0
 Females that aborted  0  0.0  0  0.0  0  0.0  0  0.0
 Nongravid  0  0.0  0  0.0  0  0.0  0  0.0
 Gravid  0  0.0  0  0.0  0  0.0  0  0.0
                 
 Females that were euthanized  0  0.0  0  0.0  0  0.0  0  0.0
 Nongravid  0  0.0  0  0.0  0  0.0  0  0.0
 Gravid  0  0.0  0  0.0  0  0.0  0  0.0
                 
 Females examined at                
 Scheduled necropsy  22  100.0  22  100.0  22  100.0  22  100.0
 Nongravid  2  9.1  1  4.5  1  4.5  2  9.1
 Gravid  20  90.9  21  95.5  21  95.5  20  90.9
 With resorptions only  2  0.0  0  0.0  0  0.0  0  0.0
 with viable fetuses  20  100.0  21  100.0  21  100.0  20  100.0
                 
 Total females gravid  20  90.9  21  95.5  21  95.5  20  90.9

1- 0 mg/kg/day 2- 100 mg/kg/day 3- 300 mg/kg/day 4- 1000 mg/kg/day

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

 Group

 

 Sex

M

Sex

F

  Viable

Fetuses

Dead

Fetuses

 Resorptions

 Early

Resporptions

Late

 Post

Implantation

Loss

Implantation

Sites 

Corpora

Lutea

  Pre

implantation

Loss 

Fetal

Weights

in grams

No. of

gravid

females

 1

 Total

 98

 98

 196

 0

 6

 0

 6

 202

 216

 14

 NA

 20

 1

 Mean

 4.9

 4.9

 9.8

 0 .0

 0.3

 0.0

 0.3

 10.1

 10.8

 0.7

 38.2

 

 1

 S.D.

 2.31

 1.80

 1.99

 0.00

 0.57

 0.00

 0.57

 1.80

 1.77

 0.73

 4.55

 

 2

 Total

 100

 107

 207

 1

 3

 5

 9

 216

 221

 5

 Na

 21

 2

 Mean

 4.8

 5.1

 9.9

 0.0

 0.1

 0.2

 0.4

 10.3

 10.5

 0.2

 37.6

 

 2

 S.D.

 1.55

 1.37

 1.49

 0.22

 0.36

 0.54

 0.81

 1.35

 1.44

 0.44

 4.63

 

 3

 Total

 96

 100

 196

 0

 3

 3

 6

 202

 211

 9

 NA

 21

 3

 Mean

 4.6

 4.8

 9.3

 0.0

 0.1

 0.1

 0.3

 9.6

 10.0

 0.4

 37.3

 

 3

 S.D.

 1.75

 1.30

 2.22

 0.00

 0.48

 0.48

 0.78

 2.48

 2.44

 0.81

 4.76

 

 4

 Total

 82

 86

 168

 0

 12^

 1^

 13^

 173^

 189^

 16^

 NA

 20

 4

 Mean

 4.1

 4.3

 8.4

 0.0

 0.6^

 0.1^

 0.7^

 9.1^

 9.9^

 0.8^

 38.2

 

 4

 S.D.

 2.10

 1.89

 2.91

 0.00

1.42^

 0.23^

 1.42^

 2.62^

 2.01^

 1.57^

 5.67

 

-----------------------------

------------------------------------------------------------------------------------------------------

None significantly different from control group

NA = NOT APPLICABLE MEAN NUMBER OF VIABLE FETUSES,

MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA, FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

^ Calculated based on n=19 litters, due to missing data for dam# A085 -----------------------------------------------------------------------------------------------------------------------------------

1- 0 MG/KG/DAY 2- 100 MG/KG/DAY 3- 300 MG/KG/DAY 4- 1000 MG/KG/DAY

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY [% PER LITTER]

 Group  0 mg/kg/day   100 mg/kg/day   300 mg/kg/day   1000 mg/kg/day

 Corproa Lutea

       
 Mean  10.8  10.5  10.0  9.9
 S.D.  1.77  1.44  2.44  2.01
 N  20  21  21  19
 Implantation sites        
 Mean  10.1  10.3  9.6  9.1
 S.D.  1.80  1.35  2.48  2.62
 N  20  21  21  19
 Viable fetuses (%)        
 Mean  96.8  95.9  97.7  92.3
 S.D.  5.97  7.83  6.08  17.46
 N  20  21  21  19
 Early resprptions        
 Mean  3.2  1.5  1.2  7.3
 S.D.  5.97  3.72  4.16  17.55
 N  20  21  21  19
 Late resorptions        
 Mean  0.0  2.2  1.2  0.4
 S.D.  0.00  4.81  3.69  1.77
 N  20  21  21  19
 Total resorptions        
 Mean  3.2  3.6  2.3  7.7
 S.D.  5.97  6.35  6.08  17.46
 N  20  21  21  19
 Pre-implantation loss (%)        
 Mean  6.4  3.6  2.3  7.7
 S.D.  6.97  6.35  6.08  17.46
 N  20  21  21  19
 Post-implantation loss (%)        
 Mean  3.2  4.1  2.3  8.9
 S.D.  5.97  3.90  6.08  17.54
 N  20  21  21  19
 Males (%)        
 Mean  48.9  48.0  47.9  46.9
 S.D.  19.53  13.62  11.52  18.47
 N  20  21  21  20
 Females (%)        
 Mean  51.1  52.0  52.1  53.1
 S.D.  19.53  13.62  11.52  18.47
 N  20  21  21  20
 Male fetal weights (g)        
 Mean  39.0  38.7  38.0  38.0
 S.D.  5.35  4.39  4.88  5.34
 N  20  21  21  19
 Female fetal weights (g)        
 Mean  37.6  36.6  36.6  37.8
 S.D.  4.96  5.34  4.96  5.67
 N  20  21  21  20
 Combined fetal weights (g)        
 Mean  38.2  37.6  37.3  38.2
 S.D.  4.55  4.63  4.76  5.67
 N  20  21  21  20

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO.]

              Fetuses           Litters
   Dose group:  1  2  3  4  1  2  3  4
 Number examined externally    196  207  196  168  20  21  21  20
 Fetus totally malformed    0  0  1  0  0  0  1  0
 Trunk omphalocele    0  0  0  1  0  0  0  1
                   
Number examined viscerally     196  207  196  168  20  21  21  20
 Teratology of fallot    0  0  1  1  0  0  1  1
 Lung - absent lobe(s)    2  2  0  2  2  2  0  1
 Aortic arch - interrupted    0  0  0  1  0  0  0  1
Ventricular septum defect     0  0  0  1  0  0  0  1
 Ductus arteriosus retroesophagal    0  0  0  1  0  0  0  1
 Aorta descending - right sides    0  0  0  1  0  0  0  1
 Fetus totally malformed    0  0  1  0  0  0  1  0
                   
 Number examined skeletally    196  207  196  168  20  21  21  20

 Vertebral anomaly with or without

associated rib anomaly

   0  1  2  2  0  1  2  2
 Rib anomaly    0  0  0  2  0  0  0  2
  Caudal vertebral anomaly    0  0  0  1  0  0  0  1
   Skull bones - fused    0  0  0  1  0  0  0  1
  Sternebrae fused    0  1  2  1  0  1  2  1
  Skull bone(s) - split    0  0  2  0  0  0  2  0
  Bent limb bones    0  0  2  0  0  0  1  0
  Fetus totally malformed    0  0  1  0  0  0  1  0
                   
  Total number with malformations                  
  External    0  0  1  1  0  0  1
  Soft tissue    2  2  2  4  2  2  2  2
  Skeletal    0  2  9  7  0  2  7  4
  Combined    2  4  10  11  2  4  8  6

1- 0 MG/KG/DAY 2- 100 MG/KG/DAY 3- 300 MG/KG/DAY 4- 1000 MG/KG/DAY

SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS % PER LITTER

   Dose group    1  2  3  4
 Number of litters examined            
 Total malformations  

 

       
 Percent per litter with external malformations  

 Mean

S.D.

 0.0

0.00

 0.0

0.00

0.3

1.56 

 1.0

4.47

 Percent per litter with soft tissuel malformations  

 Mean

S.D.

 1.0

2.94

 1.3

4.04

 0.8

2.62

 2.2

7.67

 Percent per litter with skeletal malformations  

 Mean

S.D.

 0.0

0.00

1.0

3.01 

 5.5

10 .97

 4.0

10.50

 Total percent per litter with malformations  

 Mean

S.D.

 1.0

2.94

2.2

4.78 

 6.3

10.85

 6.6

15.41

1- 0 MG/KG/DAY 2- 100 MG/KG/DAY 3- 300 MG/KG/DAY 4- 1000 MG/KG/DAY

None significantly different from control group

SUMMARY OF LITTER PROPORTIONS OF VARIATIONS % PER LITTER

 Dose group    1  2  3  4
 Number of litters examined      20  21  21  20
 Total variations  

 

       
 Percent per litter with external variations  

 Mean

S.D.

 0.0

0.00

 0.0

0.00

00

0.00

 0.0

0.00

 Percent per litter with soft tissuel variations  

 Mean

S.D.

 6.4

9.24

 3.5

6.20

 5.0

6.23

 7.1

8.18

 Percent per litter with skeletal variations  

 Mean

S.D.

 72.6

26.43

74.8

19.14 

 76.1

24.04

 83.7

21.04

 Total percent per litter with variations  

 Mean

S.D.

 73.9

25.31

75.7

18.79 

 77.3

23.95

 85.2

19.54

1- 0 MG/KG/DAY 2- 100 MG/KG/DAY 3- 300 MG/KG/DAY 4- 1000 MG/KG/DAY

None significantly different from control group

Conclusions:
In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for N,N-dicyclohexylbenzothiazole-2-sulphenamide was established as being at least 1000 mg/kg/day.
Executive summary:

Title

Prenatal developmental toxicity study of N,N-dicyclohexylbenzothiazole-2-sulphenamide in rabbits by oral gavage.

Guidelines

The study procedures described in this report were based on the following guidelines:

•Organization of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, January 2001.

•Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.31: "Prenatal Developmental Toxicity Study". Official Journal of the European Union No. L142, May 2008.

•The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998.

Study outline

Eighty-eight mated female New Zealand White rabbits were assigned to four groups of 22 animals each. The test item, N,N-dicyclohexylbenzothiazole-2-sulphenamide, was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 100, 300 and 1000 mg/kg/day (Groups 2, 3 and 4, respectively). Rabbits of the control group received the vehicle, 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity.

On Day 29 post-coitum, all animals were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. Approximately, one half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol. Following staining with Alizarin Red S, skeletons of all fetuses of all groups were examined.

Results

Accuracy and homogeneity of formulations were demonstrated by analyses.

Maternal findings:

No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.

Developmental findings:

No developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.

Conclusion

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for N,N-dicyclohexylbenzothiazole-2-sulphenamide was established as being at least 1000 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Equivalent or similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study).
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a guideline-consistent developmental toxicity study (Monsanto 1981) which was performed with Charles River COBS CD rats, groups of 25 pregnant females were treated by gavage with CBS at dose levels of 100, 300, 500, and 900 mg/kg bw and day during gestation day 6 to 15. The control group received corn oil. Due to excessive toxicity and death the 900 mg/kg bw and day dose group had to be ceased before end of the study. One maternal death and severe decrease in mean maternal body weight gain and adjusted body weight on gestation day 29 were observed at the 500 mg/kg dose level. A slight to moderate decrease in mean maternal body weight gain was noted in the 300 mg/kg dose group. Hair-loss was noted in all groups, most frequently in the 500 mg/kg dose group. There were no dose-dependent and biologically meaningful effects on mean numbers of corpora lutea, total implantations, post-implantation loss, viable foetuses, or foetal sex distribution in any of the treated groups when compared to controls. There were a very slight increase in the number of foetuses and litters with malformations in the 500 mg/kg dose group due to primarily to three foetuses, each from a different litter, with a thread-like tail and small anus. However, overall, there were no statistically significant differences in the number of litters with malformations in any of the treated groups when compared to the control groups. Further effects observed in the offspring were related to foetal body weight. A slight decrease in mean foetal body weight was observed in the 100 and 300 mg/kg dose group which was statistically significant different from controls for the 500 mg/kg bw and day dose group. The NOAEL derived from this study for maternal toxicity is 100 mg/kg bw and day and is based on a decrease in mean body weight gain. The NOAEL for the offspring is 300 mg/kg bw and day and is based on a decrease mean foetal body weight.

In another developmental toxicity study (Ema 1989) groups of 10 to 17 mated female Wistar rats were administered CBS via diet at dosage levels of 0, 0.001, 0.01, 0.1 and 0.5% from day 0 to day 20 of pregnancy. According to the authors the average daily intake amounted to 0, 0.7, 7.1, 69.6 and 288.8 mg/kg body weight and day, respectively. Significantly lower maternal body weight gain during pregnancy was noted in the 0.5% group (91 ± 9 g) and 0.1 % (73 ± 15 g) in comparison to the control group (108 ± 26 g). Also food consumption in the 0.5 % group (302 ± 30 g) differed significant from that of controls (355 ± 52 g). Neither death nor clinical signs of toxicity were reported for the pregnant females of any group. There were no significant compound related effects on pre- and post-implantation losses, the number of the live foetuses per litter or sex ratio of live foetuses. However, significantly lower body weights of male (3.89 ± 0.29 g in comparison to 4.43 ± 0.38 g in the controls) and female (3.77 ± 0.034 g in comparison to 4.17 ± 0.36 in the controls) foetuses and of placenta (0.42 ± 0.07 in comparison to 0.60 ± 0.15 in the controls) were noted at the highest dose level. Extensive external, internal and skeletal examinations (of 92 to 150 foetuses per dose group) did neither reveal any malformations nor any changes in the incidence of skeletal variations at any dosage level. The NOAEL derived from the results of this study for dams is 7.1 mg/kg bw and day (0.01 % CBS in diet) based on reductions in the body weight gain. The NOAEL(foetal) is 69.6 mg/kg bw and day (0.1 % CBS in the diet) and is based on decreased mean foetal body weights.

In a further developmental toxicity study (Sitarek 1996) which was performed with Imp: DAK rats (Institute's own breeding colony) groups of 17 to 22 mated females were treated by gavage with CBS at dose levels of 50, 150, and 450 mg/kg bw and day during gestation days 6 to 15. The control group received sunflower oil. No significant differences in the appearance and behaviour were noted between test and control pregnant females. Dams in the high-dose group (450 mg/kg bw and day) exhibited significantly decreased body weight gain and a significant increase in relative kidney weights and in absolute spleen weights. Also the placental weight was found to be significantly lower than in unexposed rats. The high-dose group (450 mg/kg bw) also exhibited embryo-/fetotoxic effects in terms of a significant increase in the frequency of litters with early resorptions, an increase of mean value of late resorptions per litter and of postimplantation losses as well as decreased foetal body weight and crown-rump length. 192 to 213 foetuses were examined for external, skeletal and visceral abnormalities. From visceral examinations an increased number of fetuses with internal hydocephalus were diagnosed for the high dose group but also for the 150 mg/kg dose group. However, the numbers of foetuses with enlarged cerebral ventricles (about 20 % of all foetuses affected) and/or renal pelvis were obviously elevated in this study across all treatment groups including the controls. This kind of observations, however, was not obtained from the two other studies and is difficult to put into perspective, since there are no historical data available for this particular rat strain used in this study. Therefore, these observations are not considered as being indicative for a substance specific teratogenic potential.

An OECD Guideline 414 study was performed to evaluate the potential effects of DCBS on prenatal developmental toxicity in rabbits. Based on the category justification for CBS (CAS 95-33-0), DCBS (CAS 4979-32-2), TBBS (CAS 95-31-8) and MBS (CAS 102-77-2) a read-across with the prenatal developmental toxicity study with DCBS is justified and conducted.

Eighty-eight mated female New Zealand White rabbits were assigned to four groups of 22 animals each. The test item, DCBS (N,N-dicyclohexylbenzothiazole-2-sulphenamide), was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 100, 300 and 1000 mg/kg/day (Groups 2, 3 and 4, respectively). Rabbits of the control group received the vehicle, 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals.

On Day 29 post-coitum, all animals were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. All fetuses were dissected and examined for visceral anomalies. Skeletons of all fetuses of all groups were examined.

No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups. No developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for DCBS (N,N-dicyclohexylbenzo- thiazole-2- sulphenamide) was established as being at least 1000 mg/kg/day.

Toxicity to reproduction: other studies

Description of key information

From an oral 28-day repeated dose toxicity test with CBS in rats data on reproductive organ toxicity were available (MHWJ 1997). Atrophy of seminiferous tubuli, hyperplasia of interstitial cells and decrease in epididymal sperm (each of 1/6 males, respectively were found at a dose of 800 mg/kg bw and day (14 day recovery period). No histopathological abnormalities were detected in ovary from females of 800 mg/kg groups. Based on the findings from this subacute toxicity study, the NOAEL for males concerning adverse effects on the reproductive organs is considered to be 250 mg/kg bw and day and for females 800 mg/kg bw and day (highest dose tested).

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction: other studies
Remarks:
subacute oral toxicity study
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: subacute oral toxicity study (weight of sex organs, and histological examination of the testis documented)
Qualifier:
equivalent or similar to
Guideline:
other: Guidelines for 28-day repeated dose toxicity testing of chemicals (Japan)
Principles of method if other than guideline:
N-cyclohexyl-2-benzothiazolesulfenamide was studied for oral toxicity in rats in a 28-day repeat dose toxicity test at doses of 0, 25, 80, 250 or 800 mg/kg/day. At termination of the study weight of organs ovaries, testis, epididymis were determined, and a and histological examination of the testis and epidididymis conducted.
GLP compliance:
yes
Type of method:
in vivo
Specific details on test material used for the study:
Purity: 98.8 %
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on exposure:
Exposure period: 28 days
Duration of treatment / exposure:
Exposure period: 28 days.
Frequency of treatment:
Daily.
Duration of test:
28 days with a recovery period of 14 days.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
80 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
800 mg/kg bw/day
No. of animals per sex per dose:
6 per dose and sex
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects on testis, but dose range with systemic adverse effects (signs of a coagulopathy, adverse effects in the kidney
Dose descriptor:
LOAEL
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: increase of the relative testis weights, 1/6 moderate diffuse atrophy of the seminiferous tubule, a slight diffusse interstitial cell hyperplasia and a market decreased sperm content on the epididymis
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remarks'
Remarks on result:
other:
Remarks:
No substance-related effects on absolute and relative ovary weights, no abnormalities were detected in ovary from animals of 800 mg/kg groups, but dose range with systemic adverse effects (e.g. signs of a coagulopathy, reduced body weight gain, reduced food consumption)

All animals survived until the end of the study. Body weight gain and food consumption were reduced in females of the 250  and 800 mg/kg-groups and in males of the 800 mg/kg-group Clinical signs: Deterioration of general condition (piloerection, soiled fur) in females  of the 800 mg/kg-group (for more details see chapter repeated dose toxicity: oral  Relative organ weights of ovary and epididymis were comparable to the control; the testis weight was increased in males of the 800 mg/kg-group (P < 0.01). In one male of the 800 mg/kg/recovery group each there was  observed a moderate diffuse atrophy of the seminiferous tubule, a slight diffuse interstitial cell hyperplasia and a marked decreased sperm content in the epididymis. In the ovary there were no substance-related effects seen.

Histopathological findings in male rats

Organ  Findings  Grade  Dose (mg/kg bw and day)         
     

 after administration period   

 after recovery period   
 Testis      0  800  0  800
   Atrophy, seminiferous tubule, diffuse

++ 

 0/6  0/6  0/6  1/6
   Hyperplasia, interstitial cell, diffuse  +  0/6  0/6  0/6  1/6
   Epididymis: decreased sperm  +++  0/6  0/6  0/6  1/6

+: slight, ++: moderate, +++: market  

The NOAEL for males concerning adverse effects on the reproductive organs   was 250 mg/kg bw/day; the NOAEL for females concerning adverse effects on the reproductive organs was 800 mg/kg bw/day (highest dose tested).

Executive summary:

In a subacute gavage study groups of Crj:CD (SD) rats were treated with 0, 25, 80, 250 and 800 mg/kg bw and day CBS (MHWJ 1997). No treatment related changes in absolute and relative weights of ovaries and absolute weights of testis were observed. Relative weights of testis were significant higher in animals treated with 800 mg/kg bw and day than in control animals concomitant to decrease body weights. In the recovery group in one out of 6 rats histopathological changes were seen after a 14 day recovery period at 800 mg/kg bw and day (moderate diffuse atrophy of seminiferous tubuli, slight diffuse interstitial cell hyperplasia and market decreased sperm content on the epididymis). No such effects were observed in male terminated immediately after the administration period. No histopathological abnormalities were detected in ovary from females of 800 mg/kg groups.

Additional information

From a subacute oral (gavage) 28-day repeated dose toxicity study data on organ weights and histopathology of reproductive organs are available (MHWJ 1997). As discussed and confirmed by recent literature (Mangelsdorf et al. 2003, Ulbrich & Palmer 1995, Janer et al. 2007, Dent 2007, Sanbuissho et al. 2008) in rodents histopathological examinations in repeated dose toxicity studies of reproductive tissues are of high value and sensitivity for evaluation of reproductive toxicity in males and females. Histopathological changes on the reproductive organs in repeated dose toxicity studies are indicative of effects on fertility. With this respect repeated dose toxicity studies should be considered sensitive and reliable information to evaluate toxicity on fertility if histological examination of the reproductive organs is covered.

In a subacute gavage study groups of Crj: CD (SD) rats were treated with 0, 25, 80, 250 and 800 mg/kg bw and day CBS (MHWJ 1997). Systemic toxicity of CBS was noted in treated rats at >= 250 mg/kg bw and day indicated by adverse effects on hematology and kidneys, suppression of food consumption and body weight gain and loss of general conditions (for more details see chapter repeated dose toxicity). No treatment related changes in absolute and relative weights of ovaries and absolute weights of testis were observed. Relative weights of testis were significant higher in animals treated with 800 mg/kg bw and day than in control animals concomitant to decrease body weights. In the recovery group in one out of 6 rats histopathological changes were seen after a 14 day recovery period at 800 mg/kg bw and day (moderate diffuse atrophy of seminiferous tubuli, slight diffuse interstitial cell hyperplasia and market decreased sperm content on the epididymis). No such effects were observed in male terminated immediately after the administration period. No histopathological abnormalities were detected in ovary from females of 800 mg/kg groups. Based on the findings from this subacute toxicity study, the NOAEL for males concerning adverse effects on the reproductive organs is considered to be 250 mg/kg bw and day and for females 800 mg/kg bw and day (highest dose tested).

Justification for classification or non-classification

No relevant adverse effects were observed in the fertility studies in rats and developmental toxicity studies in rats and rabbits. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.