N,N-dicyclohexylbenzothiazole-2-sulphenamide

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

performed between 2006-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Cross-reference 2

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:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

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

Justification for study design:

- Premating exposure duration for parental (P0) animals - 10 weeks

- Basis for dose level selection - The dosage levels were determined based on the results of our previous dose-finding study in male and female rats fed a diet containing DCBS at 0, 1500, 3000, 6000 or 10,000 ppm (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. In that study, we found 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 the 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 the spleen in male weanlings at 1500 ppm and higher and female weanlings at 3000 ppm and higher.

For further discussion of the dose setting applied to this study, please reference the Endpoint Summary.

- Termination time for F2 - On or after Lactation Day 21.

- Route of administration - Oral Dietary

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 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)
- 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

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.

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 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

Details on study schedule:

-F0 animals were mated 10 weeks for the initiation of dosing, and were approximately 15 weeks of age at mating.
- F1 parental animals were mated 10 weeks after the selection from the F1 litters.
- Selections of parents from F1 generation when pups were PND 21-25.
- Animals were 13-14 weeks old at the starting of the mating period.

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 for both the P0 and P1 generation.

Control animals:

yes, plain diet

Details on study design:

- Dose Selection Rationale: 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.

Positive control:

no

Examinations

Parental animals: Observations and 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 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.
- Screening for normal cycles during in pre-treatment was not performed.

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:

- 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.

Postmortem examinations (parental animals):

Proestrous stage of the estrous cycle were performed for all females
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 and subjected to a gross external and internal necropsy.

For weanling a gross necropsy consisted of: external surface of the rats were examined, gross internal examination, For 1 male and female organ weights of brain, thymus, liver, kidney, spleen, adrenal,testis, epididymis, seminal vesicle, ventral prostate, uterus and ovary weights were collected.Histology was performed on the thymus, liver and spleen.

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.

Reproductive indices:

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).

Offspring viability indices:

Viability index( 0, 4 and 21).

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.

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

Histopathological findings: non-neoplastic:

no effects observed

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)

1

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

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects

Remarks on result:

other: Generation: paternal

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.

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):

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

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

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:

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):

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 examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

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):

no effects observed

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 examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

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 thymus was 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):

no effects observed

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

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:

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. 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).