N,N-dicyclohexylbenzothiazole-2-sulphenamide

Endpoint summary

Description of key information

Repeated dose toxicity: oral

• NOAEL = 36.9 mg/kg bw/day – equiv. OECD 408 – Monsanto (1989)


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


• LOAEL = 186 mg/kg bw/day males: 201.9 mg/kg bw/day females - equiv. OECD 407 - Monsanto (1988)


• NOAEL = 83 mg/kg bw/day male; 126 mg/kg bw/day female – non-TG subacute – Ema (2007b)


• NOAEL (systemic) = 54 mg/kg bw/day male; 39 mg/kg bw/day female – OECD 416 – Ema (2008)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

36.9 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The endpoint is concluded based on a key subchronic toxicity study on DCBS conducted according to GLP and with minimal deviations by comparison to the most relevant version of the test guideline (OECD 408). It has been evaluated to be of good quality (Klimisch score = 1). Further supporting evidence is available from Supporting studies of good quality (Klimisch scores 1 & 2) conducted according to test guideline or good scientific principle, and well documented.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Additional information

Repeated dose toxicity: oral

The repeated dose toxicity was evaluated in several feeding studies and in a gavage study with Sprague Dawley rats. These are summarised below. 

 

1. Monsanto 1989: Dietary Subchronic toxicity study (key, K1)

In a 90-day feeding study according to OECD 408 male and female Sprague-Dawley rats were feed with 0, 540 ppm, 2600 ppm and 5400 ppm (study mean concentrations) in the diet (corresponding to mean compound consumption of 0, 36.9, 176.7 and 342.6 mg/kg bw/day) (Monsanto, 1989). Both sexes at the two highest exposure levels had reduced food consumption (males: -10.5 %, -12.4 %, and females: -9.7 %, -20.5 %) and reduced body weight gain compared to control (males: -6.4 %, -9 %, and females -10.0 %, -15.6 %). The lowest food intake values occurred during the first week of exposure, and the animals gradually began to approach control group feeding levels during the remainder of the study. The authors suggest that this pattern in food consumption after the initial exposure reveal that the animals acclimated themselves to the treated diets. As such, the lower weight gain appeared to have resulted from decreased food intake, rather than a direct toxic response to the test chemical. This interpretation was supported by the absence of any significant clinical, gross or microscopic pathologic findings.

 

Changes seen in some serum chemistry parameters (especially decreased glucose) and organ weights or their ratios may have resulted from the lower food intake and/ or the lower body weight. The authors concluded that the No Observed Effect Level (NOEL) for this study is 540 ppm (36.9 mg/kg bw/day).

 

2. Ema, 2008: dietary 2-generation reproductive toxicity, equiv. OECD 416 (supplementary, K1)

In a 2-generation reproductive toxicity study, equivalent in design to OECD 416 (Ema, 2008), male and female Crl: CD (SD) rats (24 per group and sex) were exposed to DCBS via the oral dietary route 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 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.

 

Any mortality and clinical observations in the study were not considered to be related to 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. Body weight and body weight gain were lower in the F0 males throughout the dosing period and in the female during in Study Week 1 and through gestation and lactation at 4500 ppm.  There was a lower food consumption in Study Week 4 in the F1 males at 4500 ppm, no changes observed in the F0 females. No changes in body weight or body weight gain in F1 males or females was observed.

 

No test-item related adverse changes were detected in clinical biochemistry, urinalysis or endocrine endpoints. Although some changes in organ weights were reported in parental and F1/F2 animals, these appeared to be without functional or microscopic correlate, were without clear dose-response or a clear pattern across generations. As such, they were considered not to be test item related. The parental generation NOAEL was considered to be 54 mg/kg bw/day in males and 39 mg/kg bw/day in females. The offspring NOAEL was 291 mg/kg bw/day in both F1 and F2.

 

3. Monsanto, 1989: dietary subacute toxicity, equiv. OECD 407 (supplementary, K2)

In a subacute feeding study, male and female Sprague Dawley rats were administered with 0, 2000, 3000, 5000, 7500 and 10000 ppm in the diet (corresponding for males to: 0, 186, 281.5, 457.2, 700.6, 933 mg/kg bw and day, and for females to 0, 201.9, 293.4, 473.5, 714.9, 946.1 mg/kg bw/day) (Monsanto, 1988). The study design was similar to that of OECD 407.

 

There were no treatment-related findings noted in mortality, physical observation, haematology, organ weight or gross pathology data. The mean body weights and body weight gains of the treated males and females at all dose levels were lower than control throughout the study (males: -4.5 %, -7.4 %, -10.1 %, -10.6 %, -12.0 %; females: -4.0 %, -7.1 %, -6.0 %, -12.6 %, -13.0% at 2000, 3000, 5000, 7500, and 10000 ppm respectively). These differences from control were generally dose related. During week 1 mean food consumption values were reduced in the treated males at all dose levels. Mean food consumption data were unremarkable in males during week 2, 3, and 4. Mean food consumption values were slightly reduced in treated females at 5000 ppm and higher during week 1, 2 and 3, and in the 2000 ppm and 3000 ppm treatment groups during week 2. The authors considered that this is indicative of a treatment-related effect.

 

Mean serum glutamic pyruvic transaminase levels were significantly reduced in males of the 7500 ppm and 10000 ppm groups at study termination. Since hepatic injury is usually associated with enzyme elevations; the significance of the reduction in serum glutamic pyruvic transaminase is unclear. The authors concluded that a no effect level for DCBS was not established, because of the mean body weight data generated. Sight effects were noted at 2000 ppm (LOAEL 186 mg/kg bw/day), the lowest dose level tested.

 

4. Ema, 2007: gavage subacute toxicity study, equiv. OECD 422 (supplementary, K2)

 

In an oral gavage study (similar to the OECD TG 422 combined repeated dose toxicity study with reproduction/developmental toxicity screening test) Sprague Dawley (Crj:CD) rats received doses of 0, 6, 25, 100 and 400 mg/kg bw/day via oral gavage (Ema 2007). Males were dosed for 44 days and females were dosed for 40-51 days from 14 days prior to mating to day 3 of lactation throughout the mating and pregnancy period.

 

At 400 mg/kg bw/day, 3 female animals died on the expected day of parturition. In the cage side observations, males had clinical signs of salivation at 400 mg/kg bw/day, and females had clinical signs of decreased locomotor activity, soil of the lower abdominal fur and reddish tears; one female presented with emaciation with fur loss.

 

Body weights on days 7, 14, 21, 28, 35, 42, and 43 of the administration period were significantly lowered at 400 mg/kg bw/day in male rats. In female rats, the body weight on day 20 of pregnancy were significantly reduced at 400 mg/kg bw/day. A significant reduction in food consumption was noted on the first day of administration at 400 mg/kg bw/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/day.

 

Clinical biochemistry identified 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. Urinalysis highlighted a significant increase in urinary ketones in males of the high dose group. Relating to haematology, although the platelet count was significantly decreased at 6 mg/kg bw per day, any other hematologic parameters were not changed by the administration of DCBS. On this basis, no effects were recorded as being adverse or treatment-related.

 

In males, a significant increase in the absolute weight of the kidneys at 25, 100, and 400 mg/kg bw/day and relative weight of the kidneys and testis at 400 mg/kg bw/day, and a decrease in the absolute weight of the thymus at 400 mg/kg bw/day were observed. In females, a significantly higher relative weight of the liver and decreased absolute weight of the thymus was found at 400 mg/kg bw/day. At histopathological analysis, significantly higher incidences of male rats with hyalin droplets in the kidney proximal tubular epithelium were identified at 100 and 400 mg/ kg bw/day; these were noted by the study authors as likely to be related to α2u-globulin accumulation and of little relevance. Atrophy of the thymus was noted at 400 mg/kg bw/day. 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 NOAEL for systemic toxicity was considered to be 100 mg/kg bw/day based on adverse effects on bodyweight and urine, blood chemistry and histopathology at the higher dose.

 

 

5. Ema, 2007b: dietary subacute toxicity study, non-TG (supplementary, K2)

In an oral feed study, used as a pilot for a 2-generation reproductive toxicity test, Sprague Dawley rats received doses / concentrations of 2000, 3000, 5000, 7500 or 10000 ppm DCBS (reported mean daily intakes - males: 83, 172, 343 and 551 mg/kg bw/day in males, and 126, 264, 476 and 707 mg/kg bw/day in females) (Ema, 2007b). The study was similar in design to the OECD 422 422 combined repeated dose toxicity study with reproduction/developmental toxicity screening test, exposing animals for between 57 and 65 days in total.

 

A dose-related depression in body weight gain and reduced feed consumption on a mg/kg bw/day basis was noted ≥ 6000 ppm in males, and ≥ 3000 ppm in females, in comparison to controls. The authors concluded that the body weight effects in males were associated with changes in food consumption, and that DCBS adversely affects body weight gain and food consumption at 6,000 ppm in male rats and 3000 ppm in female rats.

 

The report concluded that higher relative weights of the liver and kidney recorded at 10000 ppm in males seem were secondary effects of lowered body weight rather than direct effects of DCBS on the organs. More pronounced effects on organ weights were noted in females. Lower absolute and relative weights of the thymus at 10000 ppm and spleen at ≥ 6000 ppm were detected. Other changes in female organ weight such as the relative weights of the brain, pituitary, liver, kidney and adrenal gland, and absolute weight of the ovary were concluded to be unlikely to be adverse and test-item related, as the degree of changes was relatively small and no dose-dependency was shown. The apparent higher sensitivity in female rats was primarily thought to be related to pregnancy and lactation rather than a sex-specific general toxicity. The NOAEL for systemic toxicity was 3000 ppm in male animals, and 1500 ppm in females. The LOAEL for systemic toxicity was 6000 ppm / ca. 343 mg/kg bw/day in male animals and 3000 ppm / ca. 264 mg/kg bw/day in female animals (based on food consumption and body weight gain effects).

 

 

Conclusion:

In the available repeated dose toxicity studies consistently show effects on body weight, food consumption. Effects in further parameters are often linked by the study authors to alterations in food consumption. The subchronic toxicity study (Monsanto, 1989) reporting a NOEL of 540 ppm (36.9 mg/kg bw/day) has been selected as the most sensitive for hazard assessment.  

 

Repeated dose toxicity: dermal

Dermal repeated dose studies were conducted with CBS (Monsanto 1981), TBBS (Monsanto 1981) and MBS (Monsanto 1981). In general, no local or systemic effects were noted in CBS and MBS treated animals up to 2000 mg/kg bw and day (Monsanto 1981). For the TBBS no biological relevant systemic effects were noted up to 2000 mg/kg bw/day; whereas slight local effects were noted in treated animals of the highest dose group (LOAEL 2000 mg/kg bw/day, Monsanto 1981).

 

There is no dermal repeated dose study available for DCBS. Based on the similarities in effects and effect levels noted in oral and dermal acute toxicity studies and oral repeated dose studies it is suggested to use in a read across approach the data from CBS, TBBS and MBS for dermal DNEL calculation of DCBS (see category justification document, section 13). Thus, a starting point of 2000 mg/kg bw/day will be used for DNEL calculations.

 

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:

Based on the current dataset, no classification is required according to the classification criteria of regulation no. 1272/2008 (CLP). None of the category members is classified for repeated dose toxicity. An OECD 408 study is planned on CBS. The dossier will be updated as soon as the new study becomes available.