N,N-dicyclohexylbenzothiazole-2-sulphenamide

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

TG study undertaken to GLP.

Justification for type of information:

Read across: repeated dose toxicity: dermal
There were no dermal repeated dose data and valid human skin sensitizing data available for the sulfonamide DCBS. To overcome this uncertainty a read across approach were conducted with the structure analogs sulfenamides CBS, TBBS and MBS.
Physicochemical parameter and toxicological parameters of these compounds were compared with each other to reveal a substantial toxicological profile of DCBS.
The substances CBS, DCBS, TBBS and MBS have a solid physical state and a powder-like appearance. The vapor pressure of the substances is very low; therefore inhalation exposure to the vapor might be negligible (see dossier: CBS, DCBS, TBBS and MBS).
The water solubility differs substantially, reflecting the different functional groups which are linked to the basic structure, the MBT. DCBS is practically insoluble in water (1.9 µg/l at 25°C), CBS (0.32 mg/l, Monsanto Co. 1980) and TBBS (0.3 mg/l, Monsanto Co. 1978) are also poorly soluble in water; whereas MBS (54.6 mg/l at RT, Monsanto Co. 1978) is soluble in water.
The molecular mass of the five substances range from 238.39 g/mol to 346.59 g/mol and the n-octanol/water coefficient (log Pow) is between 3.9 to ca. 4.93, thus suggest intestinal absorption subsequent to oral ingestion.
This assumption is confirmed by data from acute oral toxicity studies and repeated dose toxicity studies, which revealed systemic availability after oral application.
In acute oral toxicity studies with rats, clinical signs like reduced appetite and activity were seen in survivors; whereas increasing weakness, collapse and death were noted in decedents (CBS Monsanto Co. 1973, DCBS Monsanto 1985, TBBS Monsanto Co. 1973, MBS Monsanto Co. 1973). Systemic availability of the test substances were also noted in several repeated dose toxicity studies.
The acute oral toxicity of all five substances is low, indicated by oral LD50 values above 5000 mg/kg bw for the sulfenamides (CBS, DCBS, TBBS and MBS).
The sulfenamides (CBS, DCBS, TBBS, and MBS) were evaluated in several oral repeated dose toxicity studies.
The test substance CBS was evaluated in a 28-day gavage study with Crj: CD rats (MHWJ 1997). The NOAEL was suggested to be 80 mg/kg bw and day. CBS-related effects were present in males and female rats = 250 mg/kg bw and day (LOAEL). Suppression of food consumption and body weight gain were noted in treated rats. There were also signs of coagulopathy of the blood in both sexes and effects in the kidney of male rats.
Several repeated dose toxicity studies were performed to evaluate the toxicity of DCBS. The consistent finding made in all studies is a reduction of body weight, body weight gain and/or food consumption in treated animals. The NOAEL from the key study (TG 408) was suggested to be 36.9 mg/kg bw and day. DCBS-related effects were present in males and female rats = 176.7 mg/kg bw and day; indicated by a decrease in body weight gain (Monsanto Co. 1989).
Several repeated dose gavage studies were conducted to evaluate the toxicity of TBBS. Temporary salivation after test substance administration, a reduction of food consumption and body weight gain were noted in the highest dose groups evaluated. Moreover, an increase in absolute and/or relative kidney weights were noted in treated animals. The subchronic gavage study (Monsanto Co 1985) was used as key study. The NOAEL was suggested to be 100 mg/kg bw and day. A LOAEL of 300 mg/kg bw and day was suggested, which based on a decreased body weight gain and urine specific gravity in treated animals.
The repeated dose toxicity of MBS was evaluated in a chronic feeding study with Sprague-Dawley rats (Monsanto Co. 1982). The NOAEL was indicated at 50 mg/kg bw and day. A LOAEL of 400 mg/kg bw and day was suggested, which based on a reduction of body weight gain and increase in absolute and/or relative liver and kidney weights.
In summary, several oral repeated dose studies were conducted to evaluate the toxicity of the sulfenamides CBS, DCBS, TBBS and MBS. Mainly effects on body weight gain and effects on kidney and liver were noted in treated animals. The NOAEL values ranged from 37 mg/kg bw and day (DCBS Monsanto 1989) to 100 mg/kg bw and day (TBBS, Monsanto 1985) and thus are within the same dose range.
The acute dermal toxicity of the sulfenamides CBS, DCBS, TBBS and MBS is very low, indicated by dermal LD50 values in the rabbit above 5000 mg/kg bw. The effects noted after dermal application are slight and transient like reduced appetite and activity (CBS, MBS, Monsanto 1973) or nasal discharge and few occurrences of ocular irritation (DCBS, Monsanto 1985). Neither mortality nor gross pathological lesions were observed in any of the substances evaluated.
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 and day; whereas slight local effects were noted in treated animals of the highest dose group (LOAEL 2000 mg/kg bw and day, Monsanto 1981).

Table: read-across: see IUCLID chapter 7.5.3
 
 
 
 
 
 
 
 
 

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dicyclohexylbenzothiazole-2-sulphenamide

EC Number:

225-625-8

EC Name:

N,N-dicyclohexylbenzothiazole-2-sulphenamide

Cas Number:

4979-32-2

Molecular formula:

C19H26N2S2

IUPAC Name:

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

Details on test material:

no data

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No effects with the exception of a few spontaneous observations noted in all groups.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Dermal irritation: The majority of rabbits in both the control and test groups exhibited no dermal irritation except for the following observations:
control group: a few rabbits exhibited very slight erythema, atonia and desquamation, and red raised areas and dry chappped areas on the shaved backs.
125 mg/kg bw/d: a few rabbits exhibited very slight to slight erythema, very slight desquamation and dry chapped areas on the shaven backs.
500 mg/kg bw/d: a few rabbits exhibited very slight erythema, very slight to slight desquamation and red and chapped areas on the shaven backs 2000 mg/kg bw/d: very slight to slight erythema and desquamation and very slight edema were exhibited by a few rabbits.

Mortality:

no mortality observed

Description (incidence):

No mortality were observed.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Lymphocytes increased, neutrophiles, segmented decreased.
No other differences were noted in any of the treated animals compared to control (one male at 125/mg/kg bw/d and one male at 500 mg/kg bw/d had signs of aregenerative anemia)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant differences compared to control were noted in:
125 mg/kg bw/d (males): total bilirubin decreased
2000 mg/kg bw/d (males): total bilirubin decreased 2000 mg/kg bw/d (females): LDH decreased no other differences were noted in any treatment group when compared to control

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No statistically significant variations in the organ weights were noted in any of the test groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related effects on skin at the application site in any of the rabbits from the test groups.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

2000 mg/kg bw/d: slight to moderate acanthosis and hyperkeratosis.
125, 500 mg/kg bw/d: the intensity of the acanthosis and hyperkeratosis was much less as in the highest dose group or almost similar to that observed in the control group.

Control group and treated animals: dermal inflammatory cell infiltration in all animals control and treated animals; however severity of this lesion was judged to be slightly greater in the 2000 mg/kg bw/d group compared to control. In the other two lower dosage groups, the intensity of this lesion was almost simular to that in the control group

Authors concluded: compound related effects consisting of acanthosis, hyperkeratosis and dermal inflammatory cell infiltrate were seen in the treated skin of rabbits from the 2000 mg/kg bw/d group but not in the two lower dosage groups in which these lesions were judged to be almost similar to those occurring in the control group.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

> 2 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No biological relevant systemic effects

2

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Acanthosis, hyperkeratosis, and dermal inflammatory cell infiltration in the treated skin of rabbits from the 2000 mg/kg

3

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Acanthosis, hyperkeratosis, and dermal inflammatory cell infiltration in the treated skin of rabbits from the 2000 mg/kg

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

General observations:

no effects with the exception of a few spontaneous observations noted in all groups (control group: a few rabbits exhibited hair loss around neck in area of collar, right eye: red, swollen and clear discharge, possible anorexia, mucoid diarrhea and brown stain around anogenital region;

treated animals:signs of mucoid stool, brown stain around the anogenital region, hair loss on neck in area of collar and soft stool were observed for all of the dosage levels; possible nasal congestion, diarrhea, mucoid diarrhea, soft stool, clear ocular discharge, possible anorexia and a spontaneous injury to back (impaired use hind leg) were also exhibited in the test groups.

 

Mortality: No mortality were observed

 

Dermal irritation:

the majority of rabbits in both the control and test groups exhibited no dermal irritation except for the following observations:

control group: a few rabbits exhibited very slight erythema, atonia and desquamation, and red raised areas and dry chappped areas on the shaved backs.

125 mg/kg bw/d: a few rabbits exhibited very slight to slight erythema, very slight desquamation and dry chapped areas on the shaven backs.

500 mg/kg bw/d:a few rabbits exhibited very slight erythema, very slight to slight desquamation and red and chapped areas on the shaven backs

2000 mg/kg bw/d: very slight to slight erythema and desquamation and very slight edema were exhibited by a few rabbits.

 

Body weights: no effects

 

Hematology:

500 mg/kg bw/d (males): lymphocytes increased, neutrophiles, segmented decreased.

no other differences were noted in any of the treated animals compared to control (one male at 125/mg/kg bw/d and one male at 500 mg/kg bw/d had signs of aregenerative anemia).

 

Biochemistry:

Statistically significant differences compared to control were noted in:

125 mg/kg bw/d (males): total bilirubin decreased.

2000 mg/kg bw/d (males): total bilirubin decreased

2000 mg/kg bw/d (females): LDH decreased

no other differences were noted in any treatment group when compared to control

 

Macroscopic pathology:

No treatment related effects on skinat the application site in any of the rabbits from the test groups.

 

Organ weights:

no statistically significant variations in the organ weights were noted in any of the test groups.

 

Histopathology:

2000 mg/kg bw/d: slight to moderate acanthosis and hyperkeratosis

125, 500 mg/kg bw/d: the intensity of the acanthosis and hyperkeratosis was much less as in the highest dose group or almost similar to that observed in the control group.

 

Control group and treated animals: dermal inflammatory cell infiltration in all animals control and treated animals; however severity of this lesion was judged to be slightly greater in the 2000 mg/kg bw/d group compared to control. In the other two lower dosage groups, the intensity of this lesion was almost simular to that in the control group.

 

Authors concluded: compound related effects consisting of acanthosis, hyperkeratosis and dermal inflammatory cell infiltrate were seen in the treated skin of rabbits from the 2000 mg/kg bw/d group but not in the two lower dosage groups in which these lesions were judged to be almost similar to those occuring in the control group.

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Executive summary:

Study Design

Santocure NS (N-tert-butylbenzothiazole-2-sulphenamide) was administered by dermal application to 3 groups of 10 male and 10 female rabbits, one-half with intact skin and one-half with abraded skin, five days per week for 3 consecutive weeks at dose levels of 125, 500 or 2000 mg/kg. An identical control group was treated with saline. Criteria evaluated for treatment effect included mortality, pharmacotoxic signs, body weights, dermal irritation, hematological and clinical biochemical determinations, organ weights and macroscopic and selected microscopic evaluation of tissue.

 

Results

No mortality occurred in this study. The majority of rabbits in both the control and test groups appeared nomal with the exception of a few rabbits in all dose groups exhibiting occasional spontaneous pharmatoxic signs. No statistical significance was noted in body weights. No remarkable changes or differences in dermal irritation were observed between control and test groups. Statistically significant changes in hematological values occured in the 500 mg/kg males in lymphocytes and segmented neutrophils. A few statistically significant changes occurred in the biochemical values; 125 and 2000 mg/kg males showed a decrease in total bilirubin and 2000 mg/kg females showed a decrease in LDH.

 

No compound related macroscopic lesions or statistically significant organ weight variations were observed in this study.

 

Microscopically, compound related lesions consisting of acanthosis, hyperkeratosis, and dermal inflammatory cell infiltration were seen in the treated skin of rabbits from the 2000 mg/kg group. All other microscopic lesions observed were incidental in nature and not related to the application of the test substance.

 

Conclusion

Based on the results of the study, effects levels were determined as:

NOAEL for systemic effects: > 2000 mg/kg bw/day 

NOAEL for local effects: 500 mg/kg bw/day

LOAEL for local effects: 2000 mg/kg bw/day, based on the recording of acanthosis, hyperkeratosis, and dermal inflammatory cell infiltration in the treated skin of rabbits at this dose level.