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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2008
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:
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

Test material

Constituent 1
Chemical structure
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
Details on test material:
DCBS lot no. 508001, purity: 99.7%, supplier: Ouchishinko Chemical Industrial Co. Ltd. (Tokyo, Japan)
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.

Test animals

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 or test system 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

Administration / exposure

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.
Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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)

Effect levels (P1)

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

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

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

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

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.

Effect levels (F2)

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

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

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).

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:

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).