N,N-dicyclohexylbenzothiazole-2-sulphenamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

• NOAEL = 100 mg/kg bw/day – OECD 422 – Ema (2007b)


• NOAEL = 1500 ppm (ca. 172 mg/kg bw/day) – pilot DRF for 2-generation reproductive toxicity – Ema (2007)


• NOAEL(maternal) = 291 mg/kg bw/day – OECD 416 – Ema (2008)

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Performed in 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD, 1990; Tobe et al, 1991)

Qualifier:

according to guideline

Guideline:

other: OECD guideline Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD, 1990; Tobe et al, 1991)

Principles of method if other than guideline:

There were 10 males and 10 females per group dosed at 0, 6, 25, 100 or 400 mg/kg bw/day once daily via oral gavage. Males were dosed until Study Day 44 and females were dosing through out gestation until Lactation Day 3. Both the males and females were dosed for 2 weeks prior to mating. Males were necropsied on Study Day 45 and female on Lactation Day 4 with their litters.
The following parameters were examined during the course of the study; clinical observations, body weight, food consumption, urinalysis (males only), haematology (males only), clinical chemistry (males only), mating performance, reproductive performance, gross necropsy, organ weighs and microscopic evaluation.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD[SD]) rats

Details on species / strain selection:

This strain was chose because it is the most commonly used strain in toxicity studies, including reproductive and developmental toxicity studies and historical control data are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Atsugi Breeding Center, Charles River Japan (10 males and females per group)
Females: non-pregnant
Age at study initiation: males were 8-9 weeks of age and the females were 7-8 week of age.
Fasting period before study: none reported
Housing: individually housed except during acclimatisation, mating and nursing periods
Diet: ad libitum
Water: ad libitum
Acclimation period: 12 days

Environmental conditions: The animals were maintained in an air-conditioned room at 22 ± 3°C, with a relative humidity of 55 ± 10%, a 12-h light/dark cycle, and ventilation with 10 and more air changes/hour.

Route of administration:

oral: gavage

Vehicle:

other: Sesame oil

Details on exposure:

DCBS was dissolved or suspended in sesame oil (lotion. A113, Miyazawa Yakuhin Co., Ltd., Tokyo, Japan) via oral gavage at a dose volume of 5mL/kg bw.

Details on mating procedure:

Males and females were mated on a 1:1 basis until copulation, there were no reports of second matings. Daily vaginal smears were examined for the presence of sperm, the presence of sperm in the vaginal smear and/or vaginal plug was considered evidence for successful mating. Pregnant females were housed individual after a successful mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of formulations has been confirmed for up to 7 days in a cool (4 C) and dark place. During use, the formulations were maintained under such conditions for less than 7 days, and the target concentration was 96.0-99.1%

Duration of treatment / exposure:

Exposure period: males: 44 days including 14 days before mating, females: from 14 days before mating, throughout the mating period and gestation until day 3 of lactation (total of 40-51 days).

Frequency of treatment:

daily, 7 days per week

Details on study schedule:

At mating the males were approximately 10-11 weeks old and the females were approximately 9-10 weeks old.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

6 mg/kg bw/day

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

400 mg/kg bw/day

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

Rational for dose selection: The dosage levels were determined based on the results of our previous dose-finding study, the 14-day repeated dose toxicity study in rats given DCBS by gavage at 0, 3, 10, 30, 100, or 300 mg/kg bw per day, at which the tendency to decrease the gain in body weight and to increase the weight of the kidney and adrenal gland were found at 300 mg/kg bw per day.

Animals were assigned randomly to groups.
No fasting period before blood sampling was reported for clinical biochemistry.

Parental animals: Observations and examinations:

Daily clinical observations

Oestrous cyclicity (parental animals):

oestrous cycles performed for the detection of mating during the mating period.

Litter observations:

Litter were observed from Lactation Day 0-4

Postmortem examinations (parental animals):

Gross Necropsy on Study Day 45 for males and Lactation Day 4 for females and their offspring.

Postmortem examinations (offspring):

Lactation Day 4

Statistics:

Performed on body weight, food consumption, haematology and blood chemistry data, organ weight, numbers of corpora lutea, implantations, pups, mortality of parent animals, incidences of toxicologic signs and histopathologic changes, mating index, fecundity index, gestation index, incidences of females with live born and with total litter loss, the sex ratio of pups, and the incidence of pups.

Reproductive indices:

Mating index, fecundity index, gestation index and incidences of females with live born and with total litter loss, implantation index, delivery index, live birth index, viability index LD0-4.

Offspring viability indices:

body weights and sex ratio.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males had clinical signs of salivation at 400 mg/kg bw/day.
Females had clinical signs of decreased locomotor activity, soil of the lower abdominal fur and reddish tears at 400 mg/kg bw/day, additionally one female was emaciation with fur loss.

Mortality:

mortality observed, treatment-related

Description (incidence):

3 females died on the day of expected parturition at 400 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights on days 7, 14, 21, 28, 35, 42, and 43 of the administration period were significantly lowered at 400 mg/kg bw per day in male rats. In female rats, the body weight on day 20 of pregnancy were significantly reduced at 400 mg/kg bw per day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption on the first day of administration was significantly lowered at 400 mg/kg bw per day in male rats. In female rats, food consumption on the first day of administration and day 20 of pregnancy were significantly reduced at 400 mg/kg bw per day.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Reduced platelet counts at 6 mg/kg bw per day, increased serum levels of total cholesterol at 25 and 100 mg/kg bw per day, and decreased serum levels of sodium at 6 and 400 mg/kg bw per day were found. However, changes in these parameters were thought to have no toxicologic meaning because these changes were relatively slight and were not dose-dependent.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly lower levels of glutamate pyruvate transaminase (GPT) and chloride at 400 mg/kg bw/day and sodium at 6 and 400 mg/kg bw/day, and higher levels of total cholesterol at 25 and 100 mg/kg bw/day and phosphorus at 400 mg/kg bw/day were noted.

Endocrine findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Significant increase in urinary ketones in males at 400 mg/kg bw/day.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the males, significantly higher incidences of rats with hyaline droplets in the proximal tubular epithelium in the kidney at 100 and 400 mg/ kg bw/day and with atrophy of the thymus at 400 mg/kg bw/day were noted. In females, the incidences of rats with fatty degeneration of the proximal tubular epithelium in the kidney, cortical cell vacuolization in the adrenal, and atrophy of the spleen were significantly higher at 400 mg/kg bw/day.

The α2u-globulin accumulation linked hyaline droplets in the proximal tubular epithelium in the kidney at 100 and 400 mg/kg bw per day in males were not considered as relevant to human health, although 100 mg/kg bw per day was an effect level in male rats. 

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

A lower, but not significantly lower, fecundity index was noted at 100 and 400 mg/kg bw/day. The gestation index at 400 mg/kg bw/day was significantly lower than that in the control group. Poor maternal behavior or nursing and total litter loss was found in three females at 400 mg/kg bw/day There was a significant decreases in the numbers of corpora lutea, implantations and pups born, and in the live birth index were detected at 400 mg/kg bw/day. A significantly higher number of stillborn was also observed at 400 mg/kg bw/day. At this 400 mg/kg bw/day no live pups were obtained on PND 4.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects

Remarks on result:

other: Deaths in females, and decreased body weight and changes in urinalysis, blood chemistry, and/or histopathology in both sexes at 400 mg/kg bw per day.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was a significant increase in the number of still borns (live birth index), there were no live pups by LD4 at 400 mg/kg bw/day

Body weight and weight changes:

no effects observed

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):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Reproductive effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Table 1: Absolute and relative organ weights of male and female rats given DCBS by gavage.

Dose (mg/kg bw/day)	0 (control)	6	25	100	400
No. of males	10	10	10	10	10
Terminal body weight (g)a	467 ± 30	469 ± 33	478 ± 17	476 ± 27	411 ± 18*
Liver (g)a	13.63 ± 1.27b 14.0 ± 0.11c	14.00 ± 1.96 2.97 ± 0.22	15.09 ± 0.92 3.16 ± 0.14	15.01 ± 0.92 3.14 ± 0.23	12.99 ± 1.19 3.16 ± 0.20
Kidney (g)a	3.06 ± 0.27 0.66 ± 0.06	2.98 ± 0.30 0.64 ± 0.04	3.09 ± 0.19* 0.65 ± 0.04	3.14 ± 0.15* 0.66 ± 0.04	3.19 ± 0.19* 0.78 ± 0.04*
Thymus (g)a	0.43 ± 0.07 0.09 ± 0.01	0.37 ± 0.08 0.08 ± 0.02	0.36 ± 0.08 0.08 ± 0.02	0.40 ± 0.10 0.08 ± 0.02	0.31 ± 0.09* 0.07 ± 0.02
Testis (g)a	3.30 ± 0.24 0.71 ± 0.07	3.25 ± 0.21 0.70 ± 0.06	3.27 ± 0.23 0.69 ± 0.06	3.25 ± 0.20 0.68 ± 0.05	3.25 ± 0.15 0.80 ± 0.05*
Epididymis (g)a	1.42 ± 0.09 0.30 ± 0.03	1.36 ± 0.16 0.29 ± 0.03	1.35 ± 0.09 0.28 ± 0.03	1.35 ± 0.13 0.28 ± 0.04	1.28 ± 0.11 0.31 ± 0.03
No. of females	10	10	10	9	5
Terminal body weight (g)a	323 ± 26	313 ± 23	320 ± 23	308 ± 26	263 ± 59*
Liver (g)a	13.16 ± 1.18b 4.08 ± 0.32c	13.72 ± 1.26 4.38 ± 0.30	12.88 ± 0.95 4.03 ± 0.24	13.14 ± 1.51 4.26 ± 0.36	12.48 ± 3.38 4.72 ± 0.35*
Kidney (g)a	1.98 ± 0.14 0.62 ± 0.06	1.89 ± 0.16 0.60 ± 0.03	1.94 ± 0.20 0.61 ± 0.05	2.01 ± 0.19 0.66 ± 0.11	1.96 ± 0.22 0.77 ± 0.17
Thymus (g)a	0.22 ± 0.07 0.07 ± 0.02	0.21 ± 0.07 0.07 ± 0.02	0.21 ± 0.08 0.06 ± 0.02	0.18 ± 0.05 0.06 ± 0.01	0.10 ± 0.04* 0.04 ± 0.01

^aValues are expressed as Mean ± SD.
^bAbsolute organ weight.
^cRelative organ weight = organ weight (g)/100 g body weight.

*Significantly different from the control, p < 0.05.

 

Table 2: Histopathological findings in male and female rats given DCBS by gavage.

Dose (mg/kg bw/day)		0 (control)	6	25	100	400
No. of males		10	10	10	10	10
Kidney
	Eosinophilic bodies in proximal tubule	3	2	2	2	1
	Focal tubular basophilic change	3	1	3	3	2
	Distal tubular dilatation	1	0	0	0	0
	Focal tubular dilatation with or without hyaline casts	2	1	1	2	1
	Hyalin droplets in proximal tubular epithelium	0	0	0	4*	8*
Thymus:	Atrophy	0	1	1	0	4*
No. of females		10	10	10	10	10
Liver
	Congestion	0	0	0	0	3
	Hepatocellular fatty acid change	0	0	0	1	1
Kidney
	Congestion	0	0	0	0	1
	Focal tubular basophilic change	3	0	1	2	1
	Focal tubular dilatation with hyaline casts	1	0	1	0	0
	Fatty degeneration of proximal tubular epithelium	0	0	0	3	4*
Adrenal
	Congestion	0	0	0	0	1
	Cortical cell vacuolization	0	0	0	1	9*
Thymus:
	Atrophy	2	2	2	3	7
Spleen:
	Atrophy	0	0	0	1	5

*Significantly different from the control, p < 0.05.

 

Fertility:

No test substance related changes were noted in the testis at 400 mg/kg per day, and in the pituitatry, testis, epididymis, seminal vesicle, and prostate of infertile males. In females, no administration-related changes were noted in the ovary, and mammary gland at 400 mg/kg bw and day. In in fertile females and females showing total litter loss no test substance-related changes were noted in ovary, mammary gland, uterus and vagina.No effects of DCBS were oberbed on the mating index and gestation lenght. Alower, but not significant lower, fecundity index was noted at 100 and 400 mg/kg bw/day. The gestation index at 400 mg/kg bw and day was significant lower than that of the control. Poor maternal behavior or nursing and total litter loss was found in three females at 400 mg/kg bw and day.

Significant decreases in the number of corpora lutea, implantations and pups born, and in live birth index were detected at 400 mg/kg bw and day. A significantly higher number of stillborn was observed at 400 mg/kg bw and day. At 400 mg/kg bw and day no live pups were obtained on PND 4.

 

Table 3: Reproductive findings in rats given DCBS by gavage.

Dose (mg/kg bw/day)	0 (control)	6	25	100	400
No. of pairs cohabitated	10	10	10	10	10
No. of pairs with confirmed mating	10	10	10	10	10
Mating index (%)a
Male	100	100	100	100	100
Female	100	100	100	100	100
No. of pregnant females	10	10	10	9	8
No. of non-pregnant females	0	0	0	1	2
Fecundity index (%)b	100	100	100	90	80
No. of dead pregnant females	0	0	0	0	3
No. of females with live born	10	10	10	10	3*
Gestation index (%)c	100	100	100	90	38*
Gestation length (day)	21.2 ±0.4	21.4 ±0.5	21.5 ±0.5	21.2 ±0.4	21.0 ±1.2
No. of females with total litter loss	0	0	0	0	3

^aMating index (%) = (no. of rats confirmed mating/no. of rats cohabitated) × 100.

^bFecundity index (%) = (no. of pregnant females/no. of females confirmed mating) × 100. ^cGestation index (%) = (no. of females with live born/no. of pregnant females) × 100. *Significantly different from the control, p < 0.05.

 

Developmental toxicity:

No significant differences in the incidences of pups with external malformation and internal variations between control and treatment groups

No fetuses with external malformation were observed in the control group and 25 mg/kg bw and day DCBS group.One pup showed micromelia and ectrodactyly at 6 mg/kg bw and day, at 100 mg/kg bw and day one pup had a short tail, at 400 mg/kg bw and day: five pups (of one litter) with cleft palate. A few pups with internal variations such as thymic remnants in the neck, left umbilical artery, and dilated renal pelvis were found in all groups, including control.

 

Table 4: Developmental findings in rats given DCBS by gavage.

Dose (mg/kg bw/day)	0 (control)	6	25	100	400
No. of litters	10	10	10	9	3
No. of corpora lutea^a	19.7 ± 2.0	18.0 ± 2.3	18.5 ± 2.4	17.6 ± 1.3	16.3 ± 1.2*
No. of implantations^a	18.8 ± 1.4	16.9 ± 2.3	17.4 ± 1.8	17.4 ± 1.4	15.3 ± 1.7*
Implantation index (%)^b	95.9	94.0	94.8	99.3	93.7
No. of pups born	17.0 ± 2.9	15.8 ± 1.9	15.5 ± 1.7	16.1 ± 1.9	12.0 ± 3.1*
No. of pups born alive^a	16.8 ± 2.9	15.4 ± 1.6	14.5 ± 2.0	15.4 ± 1.9	4.0 ± 3.8*
No. of still born^a	0.2 ± 0.4	0.4 ± 0.7	1.0 ± 2.0	0.7 ± 0.7	8.0 ± 4.0*
Delivery index^c	100	100	100	90	80
Live birth index (%)^d	98.8	97.7	94.0	95.9	31.4*
Viability index on postnatal day 4 (%)^e	97.1	99.3	93.6	88.9	0*
Sex ratio of pups born (males:females)	80/90	69/89	77/78	66/69	30/30
Bodyweight of male pups during lactation (g)^a
Day 0	6.4 ± 0.5	6.5 ± 0.5	6.7 ± 0.5	6.5 ± 0.2	5.6 ± 0.4
Day 4	9.5 ± 1.3	10.4 ± 1.2	10.9 ± 1.1	10.3 ± 0.6
Bodyweight of female pups during lactation (g)^a
Day 0	6.0 ± 0.5	6.2 ± 0.4	6.4 ± 0.6	6.2 ± 0.2	5.3 ± 0.3
Day 4	9.5 ± 1.3	10.4 ± 1.0	10.6 ± 1.2	10.0 ± 0.8
External examination of pups^f
Total no. of pups (litters) examined	170 (10)	158 (10)	154 (10)^h	145 (9)	57 (5)
Total no. of pups (litters) examined with malformations	0	1	0	1	5 (1)
Cleft palate	0	0	0	0	5 (1)
Micromelia and ectrodactyly	0	1	0	0	0
Short tail	0	0	0	1	0
Internal examination of pups^g
Total no. of pups (litters) examined	169 (10)^h	157 (10)^h	151 (10)^i	138 (9)^j	57 (5)^k
Total no. of pups (litters) examined with variations	1	6 (4)	2 (1)	4 (2)	1
Thymic remnants in neck	1	3 (3)	2 (1)	1	0
Left umbilical artery	0	3 (2)	0	1	0
Dilated renal pelvis	0	0	9	2 (1)	1

^a Values are expressed as mean ±S.D.

^b Implantation index (%) = (no. of implantations /no. of corpora lutea) x100

^c Delivery index (%) = (no. of pups born/no. of implantations) x100

^d Live birth index (%) = (no. of pups born alive/no. of pups born) x100
^e Viability index on postnatal day 4 (%) = (no. of live pups on postnatal day 4/no. of live pups on postnatal day 0) x100

^f External examinations were performed in all pups born (live born and stillborn) on postnatal day 0.

^g Internal examinations were performed in all pups (dead pups just after death and live pups on postnatal day 4).
^h One pup was not examined because of cannibalism.

ⁱ Seven pups were not examined because of cannibalism.

^j Three pups were not examined because of cannibalism.

^k Four pups were not examined because of cannibalism.

 

Summary

There were no effects indicative of toxicity on male reproductive performance. Toxic effects were revealed in female and pups at doses of 400 mg/kg. There was decreased number of the corpus lutea in accompany of decreases in number of implantation sites and litter size. One dam died during the delivery and another two had prolonged gestation length. All dams lost their litters at delivery or by day 4 of lactation. Therefore, there were decreases in reproduction/development parameters such as gestation index, number of live pups at birth, live birth index and viability index on day 4 of lactation. There were no effects on the mating and fertility, and morphogenesis in pups.

Executive summary:

Study design

A screening study for a vulcanization accelerator N,N-dicyclohexyl-2-benzothiazole- sulfenamide (DCBS) was performed in rats. The current study was conducted to obtain initial information about the possible general toxicity and reproductive and developmental toxicity of DCBS in male and female rats. DCBS was given to males during the premating and mating periods and to females during the premating, mating, and pregnancy periods and shortly after parturition.

 

Rats were given DCBS by gavage daily at 0, 6, 25, 100, or 400 mg/kg bw/day. The dosage used in the current study was sufficiently high that it should be expected to induce general toxic effects. Males were dosed for a total of 44 days beginning 14 days before mating. Females were dosed for a total of 40–51 days beginning 14 days before mating to day 3 of lactation. 

 

Results

Toxicologic changes were significantly noted only at 400 mg/kg. Three females died. An increased incidence of females showing decreased locomotor activity, soil of the lower abdominal fur, and reddish tears was observed (often associated with late pregnancy). A lowered body weight was found in males and females (for females this was only the case on day 20 pregnancy). The authors noted that such toxicologic signs during mid to late pregnancy and deaths during the periparturition period may suggest that female rats have a higher susceptibility to the toxicity of DCBS than male rats.

 

Increased urinary ketones and serum inorganic phosphorus and decreased serum glutamate pyruvate transaminase in males were found. Increased absolute and relative weights of the kidneys in males and decreased absolute weight of the thymus in both sexes were noted. Significant fatty degeneration of the renal tubular epithelia, vacuolation of the adrenocortical cells, and atrophy of the spleen were observed in females.

 

There were no effects indicative of toxicity on male reproductive performance. Toxic effects were revealed in female and pups at doses of 400 mg/kg. There was decreased number of the corpus lutea in accompany of decreases in number of implantation sites and litter size. One dam died during the delivery and another two had prolonged gestation length. All dams lost their litters at delivery or by day 4 of lactation. Therefore, there were decreases in reproduction/development parameters such as gestation index, number of live pups at birth, live birth index and viability index on day 4 of lactation. There were no effects on the mating and fertility, and morphogenesis in pups.

 

Conclusion

Based on significant decreases in the gestation index, numbers of corpura lutea, implantations, pups born and pups born alive, live birth index, and viability index were detected. It is concluded that the No Observed Adverse Effect Levels (NOAELs) for repeat dose and reproductive/developmental toxicity are 100 mg kg−1 day−1 in this screening study.

Reference 2

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

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

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

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

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.

Dose / conc.:

0 ppm

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

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

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

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).

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No significant changes in sperm counts, percentage of motile sperm and progressively motile sperm, swimming speed and pattern, or percentage of morphologically abnormal sperm in DCBS treated animals.

Reproductive performance:

no effects observed

Description (incidence and severity):

No significant differences in: copulation index, fertility index, gestation index, pre-coital interval, gestation length, number of implantations, delivery index, number of P1/F1 pups delivered, sex ratio of P1/F1 pups, or viability of P1/F1 pups during lactation between control and DCBS-treated groups; no malformed P1/F1 pups were found in any groups.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects

Remarks on result:

other: Generation: maternal

Key result

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

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no compound-related clinical signs of toxicity in either male or female rats during the pre-mating, mating, gestation, or lactation periods.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No compound-related mortality was noted.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

P1 males: 80 ppm significant decrease in food consumption during weeks 4 and 7, 600 ppm during week 6, and 4500 ppm during week 4.
P1 females: 80, 600, 4500 ppm: no effects.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

P1 males: no effects.
P1 females at 600 ppm: significant higher percent of lymphocytes.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

P1 males: no effects
P1 females at 600 ppm: significant higher percent of lymphocytes.
There were no significant changes in biochemistry parameters such as total protein, albumin and globulin in male and female adult rats.

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: oestrous cycle:

no effects observed

Description (incidence and severity):

Although one F1 female each in the control and 600 ppm groups displayed extended diestrous vaginal smears, no significant changes in the incidence of females having normal estrous cycles or length of the estrous cycles were observed.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

A significant decrease in the mean lateral head displacement was found at 4500 ppm in F1 males.

Reproductive performance:

no effects observed

Description (incidence and severity):

P1 parents/F2 offspring: No significant changes in the copulation index, fertility index, gestation index, pre-coital index, gestation lengh, number of implantations, delivery index, number of F2 pups delivered, sex ratio of F2 pups, or viability of F2 pups during lactation were observed.
(Two P1 males in the 600 ppm group did not copulate. One female of the control group and two females in the 80 and 600 ppm groups did not become pregnant. One female in the 600 ppm group did not deliver. One dam experienced a total litter loss by PND 3 at 4500 ppm),
(malformation: oligodactyly in one female of control group, one microphthalmia in one male at 80 ppm)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Remarks on result:

other: Generation: foetal

Clinical signs:

no effects observed

Description (incidence and severity):

There were no compound-related clinical signs of toxicity in either male or female rats during the pre-mating, mating, gestation, or lactation periods.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No compound-related mortality was noted.

Body weight and weight changes:

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.

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Remarks on result:

other: Generation: foetal

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

not 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

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

291 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Adults/weanlings

Mortality:

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

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

Clinical observations:

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

Body weight and body weight gain:

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

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

P1/F1 males and females: no effects

Food consumption:

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

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

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

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

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

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

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

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

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

Necropsy and histopathology:

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

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

Estrous cyclicity:

P0 females: no effects.

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

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

Reproductive effects:

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

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

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

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

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

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

Organ weights:

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

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

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

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

P1/F1 (weanlings and adults)

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

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

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

F2 (weanlings)

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

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

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

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

Hematological and blood biochemical parameters

P0 males at 4500 ppm: significant higher percent of lymphocytes

P0 females: no effects

P1/F1 males: no effects

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

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

Serum hormone levels (P0 and P1/F1 adults)

P0 male/female: no significant changes

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

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

Sperm parameter (P0 and P1/F1 adults)

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

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

Conclusions:

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

Executive summary:

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).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

291 mg/kg bw/day

Quality of whole database:

The endpoint is currently concluded based on a study assigned a Klimisch rating of 1: reliable without restriction. This has been undertaken according to GLP and an accepted OECD TG for this endpoint (TG 416). It is supported by further K1/K2 rated studies, also conducted to GLP and an accepted OECD TG (OECD 422 and equivalent).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are three relevant studies on toxicity toxicity to reproduction of DCBS.

 

1. Ema, 2007b: repeated dose study with reproduction/developmental toxicity screening test (similar to OECD TG 422)

 

Toxicity was evaluated in a repeated dose study with reproduction/developmental toxicity screening test (similar to OECD TG 422) (Ema 2007b). Male and female SD (Crj: CD) rats (10 per sex per group) were administered either 0, 6, 25, 100 and 400 mg/kg bw/day by oral gavage. Males were dosed for 44 days, beginning 14 days prior to mating, and females for a total of 40 to 51 days starting from 14 days prior to mating, then through mating, gestation and until day 3 of lactation.

 

At 400 mg/kg/day there were test substance related deaths of 3 females on the expected day of parturition or on the following day. There was also an increase in clinical observations at this dose level and sex. At 400 mg/kg/day there was lower body weight in the males from Study Day 8 to 44 and lower food consumption on Study Day 1. In the females there was a lower body weight gain on Gestation Day 20 and lower food consumption on Study Day 1 and on Gestation Day 20. Clinical chemistry and urinalysis parameters were altered in male animals (not assessed in female). For further discussion of systemic effects, see section ‘Repeated Dose Toxicity’.

 

No changes in absolute testis weights were noted in DCBS treated males compared to control, although there was an increase of relative testis weights noted at 400 mg/kg bw/day (ca. 13 %). This was considered to be secondary to the lower body weights rather than a direct effect of DCBS administration. In females, no administration-related changes were noted in the ovary and mammary gland at 400 mg/kg bw/day. Moreover, in infertile females and females showing total litter loss no DCBS-related changes were observed in the ovary, mammary gland, uterus or vagina.

 

No effects of DCBS were observed on the mating index and gestation length. No effects on reproduction were seen at 6, 25 and 100 mg/kg bw/day. At 400 mg/kg bw/day, the gestation index was significantly lower than control, resulting in a lower number of females giving birth to live pups. Poor maternal behavior or nursing resulted in total litter loss for all three females with live births at 400 mg/kg bw/day. This dose group also reported significantly decreased numbers of corpora lutea, implantations, pups born and live birth index and a higher number stillborn pups; there were no live pups by PND 4. For developmental findings, see section ‘Toxicity to Development’.

 

The most prominent effect of DCBS on reproduction and development is the marked decrease in the number of live pups and total loss of pups until PND 4 at 400 mg/kg bw/day, a dose that caused maternal death. The primary effects may be on the gestation index for dams and live birth index for pups, which appear to be affected at multiple points along the female reproductive process, as well as a viability of neonatal pups. In conclusion, the test substance DCBS caused mortalities (females only), decreased body weights and lower food consumption, changes in blood chemistry and urinalysis (males only) and histopathology at 400 mg/kg bw/day. At 400 mg/kg bw/day, all dams lost their litters at delivery or by day 4 of lactation. Thus, the authors concluded, that the NOAEL maternal/foetal is 100 mg/kg bw/day.

 

2. Ema, 2007: reproduction and developmental toxicity study (pilot study for a 2-generation reproductive toxicity study)

 

In another reproduction and developmental toxicity study (pilot study for a 2-generation reproductive toxicity study, Ema, 2007), male and female Crl: CD(SD) rats (6 per sex per group) were treated with 0, 1500, 3000, 6000 or 10000 ppm (ca. 0, 83, 172, 343 or 551 mg/kg bw/day in males and 0, 126, 264, 476, 707 mg/kg bw/day in females) by oral dietary administration. The male rats were treated for a total of 57 days beginning 16 days before mating. The female rats were treated for a total of 61 to 65 days from 16 days before mating, throughout the mating period, gestation and until day 21 of lactation in females. In the males body weight gains were reduced from Study Day 0-7 at 6000 and 10000 ppm and food consumption were reduced from Study Day 0-7 at 6000 ppm and Study Day 0-14 at 10000ppm. In females body weight gains were decreased on days 0-7 of premating at 6000 and 10000 ppm, on days 0-7 of gestation at 3000, 6000 and 10000 ppm and on days 0-4 of lactation at 10000 ppm. Food consumption was lower throughout dosing at 6000 and 10000 ppm, except between gestation day 7-20 of gestation at 6000 ppm. There was also a tendency towards decrease food consumption between day 0-7 of gestation at 3000 ppm. The weights of spleen at 6000 and 10000 ppm and of the thymus at 10000 ppm were decreased in females.

 

No changes in the estrous cyclicity, copulation index, fertility index, gestation index, delivery index, precoital interval or gestation length were observed at any dose of DCBS. The number of implantations at 6000 ppm and 10000 ppm and pups delivered at 10000 ppm were reduced. There were no changes in the sex ratio or viability of pups. No malformations were detected in pups in any group. The body weights of male and female pups were lowered at 6000 and 1000 ppm. The findings from this study indicate that DCBS caused adverse effects on reproduction and development in the range of maternal toxicity (6000 ppm and higher), indicated by a significantly lower number of implantations, pups delivered and decreased pup weights at 6000 ppm and 10000 ppm.

 

3. Ema, 2008: two-generation reproductive toxicity study (OECD 416)

 

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) was administered through the oral dietary route at 0, 80, 600 or 4500 ppm, 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 corresponded to ca. 0, 5.2, 39, 291 mg/kg bw/day in F0 males, 0, 7.2, 54, 416 mg/kg bw/day in F0 females, 0, 5.9, 44, 331 mg/kg bw/day in F1 males and 0, 7.4, 55, 417 mg/kg bw/day in F1 females. The dose levels were selected based on results of the previous reproduction and developmental toxicity study (Ema, 2007). Within this study there were reduced body weight gain at ³3000 ppm, reduced number of implantations at ³6000ppm, decreased absolute and relative weight of the spleen in females at ³6000 ppm, reduced number of pups born at 10000 ppm, lower pup weights at ³6000 ppm and decreased in the absolute and relative weight of the spleen in male weanlings at ³1500 ppm and female weanlings at ³3000 ppm. The author concluded that the dose level of 6000 ppm was potent enough to adversely affect the survival of the F1 pups. On review, it is considered that the dose level of 6000 ppm was therefore not selected as the high dose level as the study authors were concerned it was too high to ensure a sufficient number of pregnancies in the F0 and F1 generation and number of F1 pups surviving the lactation period to meet the objectives of a Two-Generation Study. As such, it is thought the high dose 4500 ppm was select as it is halfway between 6000 ppm (where toxicity was considered to be too high) and 3000 ppm (where there was little toxicity in the Dose Range Finding Study), as the highest level that could be used whilst ensuring there was a sufficient number of pregnancies and F1 weanlings to meet the objectives of a Two-Generation Study.

 

The deaths and clinical signs observed in the study were considered not to be related to the administration of DCBS. Decreased food consumption was noted in F0 males in Study Week 1-14 and females in Study Week 1 and Lactation Day 14-21 at 4500 ppm. At this dose level, body weight and body weight gain was also lower in the F0 males throughout the dosing period, in the females during in Study Week 1 and through gestation and lactation. A parallel lower food consumption was recorded in Study Week 4 in F1 males at 4500 ppm; no changes observed in the F0 females. There were no changes in body weights or body weight gains in the F1 males or females.

 

In the F1 males there was a slight but significant decrease in mean lateral head displacement was noted in F1 males of the highest group (20.5 µm ± 1.0 vs. control 21.3 ± 0.9 µm). No other 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 F1 adults between control and DCBS-treated groups. All F0 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 F1 female each in the control and 600 ppm groups displayed extended diestrus vaginal smears, no significant changes in the incidence of F1 females having normal oestrous cycles or length of the oestrous cycles were observed.  In the F0 and F1 generation although a few females had reproductive difficult within all groups there was considered to be 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.   In the F2 pups 0liodactyly in one female of the control group and microphthalmia in one male at 80 ppm were observed, no malformations were observed in the F1 generation therefore it was considered that there were no DCBS-related effects. In both the F1 and F2 pups there was a lower body weight between PND 7-21. In conclusion, no significant changes in reproductive indices were noted in any generation even at the highest dose 4500 ppm (ca. F0 males - 291 mg/kg bw/day, F0 females – 416 mg/kg bw/day, F1 males – 331 mg/kg bw/day and F1 females – 417 mg/kg bw/day).

 

Recap and Conclusion

The reproductive toxicity of the test substance DCBS was evaluated in three reproduction/developmental toxicity studies (Ema 2007, 2007b, 2008). In two studies adverse effects on reproduction were noted in treated animals in the range of maternal toxicity. In the reproduction/developmental study (Ema 2007b) adverse effects on reproduction was indicated by a significant lower number of implantations, pups delivered and decreased pup weights at 6000 ppm and 10000 ppm (ca. 476 and 707 mg/kg bw/day in females). These effects are in the range of maternal toxicity, indicated by reduced body weight gain and food consumption at 3000 ppm and higher (ca. 264 mg/kg bw/day). Adverse effects on reproduction was noted in the gavage study (Ema 2007) at 400 mg/kg bw/day in the range of severe maternal toxicity.

However, in the two-generation study no significant changes in reproductive indices were noted in any generation even at the highest dose 4500 ppm (ca. 291 mg/kg bw/day). Maternal toxicity was indicated by a reduced body weight, body weight gain and food consumption in F0 females at 4500 ppm.

The two-generation study is considered to be the most relevant study. Thus, based on the finding discussed above, the NOAEL fertility (foetal) is 4500 ppm (ca. 291 mg/kg bw/day) and the NOAEL fertility (maternal) is 600 ppm (ca. 54 mg/kg bw/day).

Effects on developmental toxicity

Description of key information

• NOAEL (foetal) is 4500 ppm (ca. 291 mg/kg bw/day) – OECD 416 rat – Ema (2008)


• NOAEL (maternal) is 600 ppm (ca. 54 mg/kg bw/day) – OECD 416 rat – Ema (2008)


• NOAEL (mat + foetal) = at least 1000 mg/kg bw/day – OECD 414 rabbit - Charles River Labs (2016)


• NOAEL = 100 mg/kg bw/day – OECD 422 rat – Ema (2007b)


• NOAEL = 1500 ppm (ca. 172 mg/kg bw/day) – pilot DRF for 2-generation reproductive toxicity rat – Ema (2007)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

54 mg/kg bw/day

Quality of whole database:

The endpoint is currently concluded based on studies assigned a Klimisch rating of 1: reliable without restriction, undertaken according to GLP and accepted OECD TG or similar for this endpoint.

Additional information

Four relevant studies are available:

 

1. Charles River Laboratories, 2016: OECD 414 in rabbit

An OECD Guideline 414 (Prenatal Developmental Toxicity Study) was conducted in rabbits (Charles River Laboratories, 2016). Eighty-eight mated female New Zealand White rabbits were assigned to four groups of 22 animals each. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 100, 300 and 1000 mg/kg bw/day (Groups 2, 3 and 4, respectively). Rabbits of the control group received the vehicle, 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, alone. No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups. No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for DCBS was established as being at least 1000 mg/kg bw/day.

 

2. Ema 2007b: repeated dose study with reproduction/developmental toxicity screening test (similar to OECD TG 422)

The toxicity for reproduction of DCBS was evaluated in a repeated dose study with reproduction/developmental toxicity screening test (similar to OECD TG 422) (Ema 2007b). Male and female SD (Crj: CD) rats (10 per sex per group) were administered either 0, 6, 25, 100 and 400 mg/kg bw/day by oral gavage. Males were dosed for 44 days, beginning 14 days prior to mating, and females for a total of 40 to 51 days starting from 14 days prior to mating, then through mating, gestation and until day 3 of lactation. The most prominent effect of DCBS on reproduction and development was the marked decrease in the number of live pups and total loss of pups until PND 4 at 400 mg/kg bw/day, a dose that caused maternal death (see section ‘Toxicity to Reproduction’). No pups with external malformations were observed in the control or 25 mg/kg bw/day groups. One pup showed micromelia and ectrodactyly at 6 mg/kg bw/day, at 100 mg/kg bw/day one pup had a short tail and at 400 mg/kg bw/day, five pups (of one litter) had cleft palate. A few pups with internal variations such as thymic remnants in the neck, left umbilical artery, and dilated renal pelvis were found in all groups, including control. These incidence malformation and variations were very low they were not considered to be significantly different from the control and where within the normal background variation for this strain of rat, therefore were considered not to be DCBS related. 

 

3. Ema, 2007: pilot study for a 2-generation reproductive toxicity study

In another reproduction and developmental toxicity study (pilot study for a 2-generation reproductive toxicity study, Ema, 2007), male and female Crl: CD(SD) rats (6 per sex per group) were treated with 0, 1500, 3000, 6000 or 10000 ppm (ca. 0, 83, 172, 343 or 551 mg/kg bw/day in males and 0, 126, 264, 476, 707 mg/kg bw/day in females) by oral dietary administration. The male rats were treated for a total of 57 days beginning 16 days before mating. The female rats were treated for a total of 61 to 65 days from 16 days before mating, throughout the mating period, gestation and until day 21 of lactation in females. No malformations were detected in pups in any group.

 

4. Ema, 2008: two-generation study (OECD TG 416)

In a two-generation study (OECD TG 416; Ema 2008) no substance-related mortality and clinical signs were noted across generations up to the highest concentration evaluated (ca. 416 mg/kg bw and day in F0 females), whereas reduction of body weight gain in parental animals and offspring was consistently observed throughout the generations in that dose group. The isolated observation of slight delays in preputial separation reported in male offspring and the slight delays in vaginal opening and transient reduced uterus weights in weanling females are of questionable relevance and mainly observed at the toxic dose. Based on the reported decrease in body weight and body weight gain in F1 and F2 offspring, body weight, body weight gain and food consumption in F0 animals at 4500 ppm maternal and developmental NOAEL values of 600 ppm (ca. 54 mg/kg bw and day) will be considered as starting point for the risk assessment.

 

Concluding Remarks

The two-generation study is considered to be the most relevant study. Thus, based on the finding discussed above, the NOAEL fertility (foetal) is 4500 ppm (ca. 291 mg/kg bw/day) and the NOAEL fertility (maternal) is 600 ppm (ca. 54 mg/kg bw/day).

 

A recent scientific evaluation of the Sulfenamides category data set was undertaken to identify testing requirements to ensure suitable coverage of required endpoints for all member substances. To further improve data coverage amongst category members, this category member was identified as having a data gap for prenatal developmental toxicity in a 1^st species (rat) (OECD 414), required in accordance with Annex IX of Regulation (EC) No 1907/2006.  A testing proposal has been submitted for an OECD 414 on DCBS (January 2023). The results of this study will be used to complete the dossier for DCBS, as well as form part of the overall category data set as a source study for read across.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 

 

Self-classification:

Within the current dataset, no relevant adverse effects were observed in the fertility studies in rats and developmental toxicity studies in rats and rabbits. Therefore, no classification is required according to the classification criteria of Regulation (EC) No. 1272/2008 (CLP). None of the category members is classified for reproductive or developmental toxicity.

An OECD 414 study in rat is planned on category member DCBS. An OECD 414 study in rabbit is planned on category member TBBS. An OECD 443 study (Cohort 1A + 1B) is planned on category member TBBS. The dossier will be updated as soon as the new studies become available.