N,N-dicyclohexylbenzothiazole-2-sulphenamide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

perform between 2006-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2008

Reference 2

Reference Type:

publication

Title:

Evaluation of two-generation reproductive toxicity of a vulcanization accelerator N,N-dicyclohexyl-2-benzothiazolesulfenamide (DCBS) in rats

Author:

Ema, M.; et al.

Year:

2007

Bibliographic source:

Toxicology Letters 172, Suppl., S184, Abstr. D18, 2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 416 (Two Generation Toxicity Study)

Version / remarks:

2

Deviations:

no

Principles of method if other than guideline:

Study performed in accordance to the OECD 416 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

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dicyclohexylbenzothiazole-2-sulphenamide

EC Number:

225-625-8

EC Name:

N,N-dicyclohexylbenzothiazole-2-sulphenamide

Cas Number:

4979-32-2

Molecular formula:

C19H26N2S2

IUPAC Name:

N-(1,3-benzothiazol-2-ylsulfanyl)-N-cyclohexylcyclohexanamine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CrL:CD (SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories Japan, Inc. (Yokohama, Japan)
- Females non pregnant.
- Age at study initiation: F0 males and females 5 weeks of age. F1 animals selected on PND 21-25.
- No fasting period before study.
- Housing: individually, except during acclimatisation, mating period and nursing.
- 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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: powdered basal diet (CRF-1, Oriental Yeast Co. Ltd., Tokyo, Japan)

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.

Diet was prepared at least every 21 days and stored at room temperature. Diet was replace every week within the animal cages

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

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: The presence of sperm in the vaginal smear and/or a vaginal plug was considered evidence for successful mating.
- F1 females that did not mate during the 3-week mating period were cohabited with other males from the same group who had been proven to copulate.
- Females were housed individually after the mating confirmed.

Duration of treatment / exposure:

P0 males: start dosing: 10 weeks before mating and were dosed for a total of 14-15 weeks.
P0 females: start dosing: 10 weeks before mating (day 0 of dosing), continuing throughout the mating period gestation and lactation on LD26 at the weaning of the F1 generation
P1 Males: Starting on postnatal day (PND) 21-25 (day 0 of dosing) males were dosed for 10 weeks before mating and were dosed for a total of 14-15 weeks.
P1 Females: Starting on postnatal day (PND) 21-25 (day 0 of dosing) females were dosed 10 weeks before mating (day 0 of dosing), continuing throughout the mating period gestation and lactation on LD26 at the weaning of the F2 generation

Frequency of treatment:

daily, 7 days each week

Duration of test:

F0: 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;
F1: 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

80 ppm

Remarks:

F0 Males: 5.2 mg/kg bw
F0 Females: 7.2 mg/kg bw
F1 Males: 5.9 mg/kg bw
F1 Females: 7.4 mg/kg bw

Doses / concentrations 3

Dose / conc.:

600 ppm

Remarks:

F0 Males: 39 mg/kg bw
F0 Females: 54 mg/kg bw
F1 Males: 44 mg/kg bw
F1 Females: 55 mg/kg bw

Doses / concentrations 4

Dose / conc.:

4 500 ppm

Remarks:

F0 Males: 291 mg/kg bw
F0 Females: 416 mg/kg bw
F1 Males: 331mg/kg bw
F1 Females: 417 mg/kg bw

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.
- Animals were randomly assigned to the dose groups.
- Animals were not fasted prior to haematology or clinical biochemistry.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males - weekly until termination, Females - weekly until positive mating then on 0, 7, 14 and 20 of gestation and 0, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Time schedule for examinations: Males - weekly until termination, Females - weekly until positive mating then on 0, 7, 14 and 20 of gestation and 0, 4, 7, 14 and 21 of lactation.
- Mean daily diet consumption and compound intake calculated.

Oestrous cycles

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.
- Screening for normal cycles during in pre-treatment was not performed.

Litter Observations
On PND 4, litters were randomly adjusted to eight pups comprising of four males and four females. No adjustment was made for litters of fewer than eight pups. Selected pups were assigned a unique number and limb tattooed on PND 4. Following adjustment of litter size on PND 4, culled pups were euthanized and subjected to a gross external and internal necropsy.
- Total litter size, number of live and dead pups, sexed, examined grossly, and individually weighed on PND 0, 4, 7, 14, 21
- All F1 and F2 pups were observed daily for pinna unfolding on PNDs 1–4, incisor eruption beginning on PND 8, and eye opening beginning on PND 12. One male and one female F1 and F2 pup selected from each dam was evaluated for the surface righting reflex on PND 5, negative geotaxis reflex on PND 8, and mid-air righting reflex on PND 18. All F1 offspring were observed daily for male preputial separation beginning on PND 35 or female vaginal opening beginning on PND 25. Body weight of the respective F1 rats was recorded on the day of preputial separation or vaginal opening. The AGD was measured using calipers on PND 4 in all F1 and F2 pups, and the AGD per cube root of body weight ratio was calculated
- Spontaneous locomotor activity was measured with a multi-channel actiiity monitoring system in 10 male and 10 female F1 rats selected from each group at 4 weeks of age. Rats were placed individually in transparent polycarbonate cages (27.6W × 44.5D × 20.4H cm, CL-0108-1) under an infrared sensor that detects thermal radiation from animals. Spontaneous motor activity was determined for 10 min intervals and for a total of 60 min.
A test in a water-filled multiple T-maze was conducted in 10 male and 10 female F1 rats selected from each group at 6 weeks of age. The water temperature of the maze was kept 22–23◦C. As a preliminary swimming ability test, each rat was allowed to swim three times in a straight channel on the day before the maze trial, and then tested in the maze with three trials per day for the next consecutive three days. The elapsed time between entry into the water at the starting point and touching the goal ramp as well as the number of errors were recorded. To prevent exhaustion of the rats, no animal was allowed to remain in the water for more than 3 min in any trial.

Ovaries and uterine content:

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

Blood sampling:

Haematology, clinical biochemistry and serum hormone blood samples taken on the day of necropsy from 10 males and 10 female F0 and F1 animals per group.

Statistics:

yes
Statistical analysis of offspring before weaning was carried out using the litter as the experimental unit.
Body weight, body weight gain, food consumption, length of estrous cycle, precoital interval, gestation length, numbers of implantations and pups delivered, delivery index, sperm parameters, hematological and blood chemical param- eters, hormone levels, organ weight, organ/body weight ratio (relative organ weight), reflex response time, age displayed pinna unfolding, incisor eruption, and eye opening, age and body weight at sexual maturation, parameters of behav- ioral tests, AGD, AGD/cube root of body weight ratio, and the viability of pups were analyzed for statistical significance in the following way. Bartlett’s test of homogeneity of variance was used to determine if the groups had equivalent vari- ances. If the variances were equivalent, the groups were compared by one-way analysis of variance (ANOVA). If significant differences were found, Dunnett’s multiple comparison test was performed. If the groups did not have equiva- lent variances, the Kruskal–Wallis test was used to assess the overall effects. Whenever significant differences were noted, pairwise comparisons were made by Mann–Whitney U-test. The incidence of pups with changes in clinical and gross internal observations, and reflex completion rate of pups were analyzed by Wilcoxon rank sum test. The number of primordial follicles in the control and highest dose groups was analyzed in the following way. Variance ratio was analyzed by F-test. Since the variance ratio was equivalent, the groups were compared by Student’s t-test. The incidence of females with normal estrous cycles, copulation index, fertility index, gestation index, neonatal sex ratio, and completion rate of the reflex response were analyzed by Fisher’s exact test.
The 0.05 level of probability was used as the criterion for significance.

Indices:

Reproductive Indices included length of oestrous cycles, copulation index, fertility index, gestation length, delivery index, sex ratio male and female pup weights (0, 4, 7, 14 and 21).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

P0 females: no effects

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

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):

P0 females: body weight at the scheduled terminal sacrifice was significant lower at 4500 ppm. There was a 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

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

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:

not examined

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Body weight and body weight gain in male (F0) and females (F0) of the 4500 ppm significant lower

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

54 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

not examined

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

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
lowered body weights of male and female pre-weaning F1 and F2 pups at 4500 ppm, slight delay of female vaginal opening at 600 and 4500 ppm (1.5 to 1.6 day delay), decreased weight of the uterus in F1 weanlings at 4500 ppm and F2 weanlings at 600 and 4500 ppm, transient longer elapsed times at 600 and 4500 ppm in females (F1) on day 2.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

54 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Adults/weanlings

Mortality:

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

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

Clinical observations:

F0 and F1: no compound-related clinical signs of toxicity in neither male or female F0 and F1 rats during the pre-mating, mating, gestation, or lactation period

Body weight and body weight gain:

F0 males: 80, 600 ppm: no effects, 4500 ppm significant decrease throughout the dosing period

F0 females: 80, 600 ppm: no effects, 4500 ppm significant decrease during first week of dosing and throughout pregnancy and lactation

F1 males and females: no effects

Food consumption:

F0 males: 80, 600 ppm no effects, 4500 ppm significant decrease during weeks 1 -8 and 13 -14

F0 females: 80, 600 ppm no effects, 4500 ppm significant decrease during week 1 of dosing and days 14 -21 of lactation

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

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

F0 males: 5.2, 39, 291 mg/kg bw

F0 females: 7.2, 54, 416 mg/kg bw

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

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

Necropsy and histopathology:

F0: 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

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 F1 females, did not significant differ in the number of primordial follicles between control and 4500 ppm groups

Estrous cyclicity:

F0 females: no effects

F1 females: no significant changes in the incidence of females having normal estrous cycles or length of the estrous cycles

(two F1 females showing abnormal estrous cycles remained in diestrus for 10 -11 days (1/24 control group, 1/24 600 ppm)

Reproductive effects:

F0 parents/F1 offspring: no significant differences in: copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of F1 pups delivered, sex ratio of F1 pups, or viability of F1 pups during lactation between control and DCBS-treated groups; no malformed F1 pups were found in any groups, a significant lower body weight was observed in male and female F1 pups at 4500 ppm on PND 4, 7 and 21

(2/24

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 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:

F0: body weight at the scheduled terminal sacrifice was significant lower at 4500 ppm

F0 males: 4500 ppm significant lower absolute organ weights: spleen, adrenal gland; increase in relative weight of: brain, thyroid, liver, kidney and testis

F0 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

F1 (weanlings and adults)

F1 males and females: body weight at the scheduled terminal sacrifice was significant lower at 4500 ppm

F1 males at 80 ppm: significant lower rel. and absolute thymus weight

F1 males: at 4500 ppm: significant decrease in absolute weights of brain, decreased absolute and rel. weights of seminal vesicle, increased rel. kidney weight, and increased absolute and rel. weights of the liver

F1 males at 4500 ppm: significant decrease in the absolute weights of the 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,

F1 females: significant increase in rel. weight of the kidney at 80 ppm, decrease absolute weight of the ovary at 600 ppm

F1 females at 4500 ppm: significant decrease absolute weights: brain, thymus, liver, kidney, spleen, adrenal, ovary and uterus and relative weight spleen weight;

F1 females at 4500 ppm: significant higher relative weights of brain and liver

F2 (weanlings)

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

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

F2 male at 80 ppm: significant decrease in the absolute and relative weight of the spleem

F2 males at 600 ppm: increase in rel weight of liver and kidney

F2 females at 80 ppm: decrease in rel thymus weight

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

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

Hematological and blood biochemical parameters

F0 males at 4500 ppm: significant higher percent of lymphocytes

F0 females: no effects

F1 males: no effects

F1 females at 600 ppm: significant higher percent of lymphocytes

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

Serum hormone levels (F0 and F1 adults)

F0 male/female: no significant changes

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

F1 female: no significant changes in any serum hormone levels

Sperm parameter (F0 and F1 adults)

F0 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

F1: at 4500 ppm a significant decrease in the mean lateral displacement

Developmental landmarks (F1 and F2)

Pinna unfolding and eye opening: no significant difference in the age of male and female F1 and F2 pups compared to control

Completion of incisor eruption: delay in male and female F1 pups at 80 ppm and in male and female F2 pups at 80 ppm and 4500 ppm

AGD and AGD per cube root of body weight ratio: male and female F1 and F2 pups were not significantly different between control and DCBS-treated groups

Reflex ontogeny:

F1 pups: all male and female F1 pups were not significant different in the reponse time of the surface righting reflex or the negative geotaxis between control and DCBS-treated groups

F2 pups: no significant difference between the control and DCBS-treated groups in the completion ratio and response time for these reflexes (F2 pups: one female did not complete the surface righting reflex and one male did not complete the mid-air righting reflex at 80 ppm, one female did not complete the mid-air righting reflex at 600 ppm, and one female did not complete the negative geotaxis at 4500 ppm)

Sexual development (F1)

F1 rats: significant delay in the age of preputial separation in males at 4500 ppm, but body weight was not significantly different between the control and DCBS-treated groups, in addition slight delay (1.5 days)

F1 females: at 600 and 4500 ppm a significantly delayed age of vaginal opening and a higher body weight at the age of vaginal opening

Behavioral effects (F1)

F1 rats: DCBS-treated animals were not significant different in spontaneous locomotor activity compared to control

Results of the water filled T-maze test: in F1 males, no significant differences were observed between control and DCBS-treated animals

F1 females: at 600 and 4500 ppm a significantly longer elapsed time at 600 and 4500 ppm and more errors at 4500 ppm noted on day 2 of the T-maze test; no significant differences in the elapsed time or number of errors on day 3 and 4 of the T-maze test

Absolute and Relative Uterus weight

F1 weanlings
DCBS ppm	0	80	600	4500
Absolute uterus weight	58.2 ± 14.4	55.8 ± 7.6	62.1 ± 12.3	48.4 ± 11.8*
Relative uterus weight	67.9 ± 15.8	67.9 ± 9.9	75.2 ± 14.1	65.0 ± 14.1
F2 weanlings
Absolute uterus weight	61.8  ± 18.9	58.1 ± 11.9	50.0 ± 10.0*	46.6 ± 12.9**
Relative uterus weight	73.3 ± 17.2	67.0  ± 13.5	60.7 ± 11.5*	62.3 ± 15.0*

*significantly different from control p<0.05

**significantly different from control p<0.01

Applicant's summary and conclusion

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:

Study design

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.

 

Results

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.

 

Conclusion

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/day).