Registration Dossier

Administrative data

Description of key information

Several repeated dose gavage toxicity studies were conducted to evaluate the toxicity of the test substance TBBS. The subchronic gavage study (Monsanto Co. 1985) is considered to be the most relevant study for risk assessment and thus based on the findings of this study a NOAEL oral of 100 mg/kg bw and day is suggested.
The inhalation toxicity of TBBS was examined in a subacute inhalation study (Monsanto Co. 1981). The study is limited concerning the recommended particle-size distribution given in current guidelines and thus an exposure of all relevant regions of the respiratory tract might not have been given. The biologically relevance of sporadic findings reported, especially systemic effects are questionable; whereas local effects noted in the inhalation study will be used as supporting evidence. Based on the fact that signs of nasal irritation observed at clinical examination were reversible, only short in duration and could not be correlated to histopathological effects, no toxicological significance was attached to this finding and thus, no relevant local effects were observed up to 0.084 mg/l.
The dermal toxicity of the test substance TBBS was evaluated in a 21-day dermal toxicity study (Monsanto Co. 1981). No death or systemic toxicity was indicated in any of the treated animals. No effects on body weight or dermal irritation were noted. Some incidental changes in haematology and biochemistry was observed. No compound related macroscopic lesions or statistically significant organ weight variations were found in this study. However, 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 dose group. Thus, a NOAEL dermal systemic of 2000 mg/kg bw and day and a NOAEL dermal local of 500 mg/kg bw and day were considered.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
Twenty male and twenty female Sprague-Dawley rates received 0, 100, 300 or 1000 mg N-tert-butylbenzothiazole-2-sulphenamide/kg bw by gavage. The animals were examined for mortality, overt signs of toxicity, body weight and food consumption. Clinical chemistry, haematological and urinalysis determinations were performed. At sacrifice animals were subjected to a complete gross necropsy and a microscopic examination for the control and high dose group performed.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animals were ca. six week of age and weighed 175-213 g for males and 130-159 g for females at the start.
Route of administration:
oral: gavage
Details on route of administration:
Dosing suspensions were perparted weekly. All animals were administered a constant volume of suspensions which was 0.5 ml of suspension per 100 g of body weight.
Vehicle:
corn oil
Details on oral exposure:
All animals were administered a constant volume of suspensions which was 0.5 ml of suspension per 100 g of body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were analysed weekly using a liquid chromatographic procedure.
Duration of treatment / exposure:
90 d
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 300, and 1000 mg/kg bw/d
Basis:

No. of animals per sex per dose:
20 per dose and sex
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: no
Positive control:
No.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals in all treatment groups salivated after dosing and mid and high dose animals salivated prior to dosing or sometimes resisted dosing. High dose females exhibited urine stained abdomens. Other than the above, no clinical signs or abnormal behavior related to treatment were observed. Due to the lack of gross or microscopic findings of gastrointestinal effects, the behavioral reactions were considered to be related to poor palatability of the test material rather than a toxic response.
Mortality:
no mortality observed
Description (incidence):
All animals survived three months of exposure to the test substance.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weights were noted for males at the middle and highest dose groups (300 and 1,000 mg/kg/day, respectively). Most affected were the high dose group males with 13 weeks reduction at 99% statistical confidence. Middle group males were affected the final 11 out of 13 weeks, primarily at 95% statistical confidence. Body weights were unaffected for the low dose males (100 mg/kg/day) and all three female treatment groups as compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Similar amounts of food were consumed by both sexes from all three treatment groups when compared to their control groups.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urine specific gravity was increased in males from Group 3 (1000 mg/kg bw) and females from Groups 2 and 3 (300 and 1000 mg/kg bw/d). The increased values for males and females from Group 3 were statistically significant.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The only significant changes in absolute organ weights were decreased mean brain weight in females from Group 3 (1000 mg/kg bw/d) and increased mean hepatic weight in females from Group 3 (1000 mg/kg bw/d). Organ to body weight ratios were increased in a dose related manner for all male organs for which weights were obtained (statistically significant in most instances) for Groups 2 and 3. Kidneys to body weight ratios were increased in females from Group 3 (statistically significant) as were liver to body weight ratios in females from Groups 2 and 3 (300 and 1000 mg/kg bw/d).
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross lesions associated with chemical administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no microscopic lesions with an apparent association with chemical administration.
Histopathological findings: neoplastic:
not specified
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no adverse effects
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: decreased body weight in males, increase in urine specific gravity in females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
other: decreased body weight in males, increase in urine specific gravity in females
Organ:
other: decreased body weight in males, increase in urine specific gravity in females
Treatment related:
yes
Dose response relationship:
yes

Mortality: All animals survived three months of exposure to the test substance

Body Weight:

Reduced body weights were noted for males at the middle and highest dose groups (300 and 1,000 mg/kg/day, respectively). Most affected were the high dose group males with 13 weeks reduction at 99% statistical confidence. Middle group males were affected the final 11 out of 13 weeks, primarily at 95% statistical confidence. Body weights were unaffected for the low dose males (100 mg/kg/day) and all three female treatment groups as compared to controls.

Food Consumption:

Similar amounts of food were consumed by both sexes from all three treatment groups when compared to their control groups. Two exceptions were mid dose group males (300 mg/kg/day) at week 9 and high dose males (1,000 mg/kg/day) at week 1, however, these exceptions appear random and are probably not related to SANTOCURE NS exposure.

Clinical Observations:

Animals in all treatment groups salivated after dosing and mid and high dose animals salivated prior to dosing or sometimes resisted dosing. High dose females exhibited urine stained abdomens. Other than the above, no clinical signs or abnormal behavior related to treatment were observed. Due to the lack of gross or microscopic findings of gastrointestinal effects, the behavioral reactions were considered to be related to poor palatability of the test material rather than a toxic response.

Hematology and chemical data:

There were several statistically significant changes in mean values for hematologic or serum chemistry parameters. Statistically significant increases in reticulocyte counts occurred in Group 2 males (300 mg/kg bw/d) and Group 3 (1000 mg/kg bw/d) females. These values remained well within normal limits and were of no toxicological significance. Depressions of SGOT and SGPT at 100 and 1000 mg/kg bw in females were probably artifactual due to high group mean values for these enzymes in control females. When clearly abnormal values for NFOO4 and NFO15 are deleted, a minimal (p 0.05) depression was found affecting only Group 1 females (100 mg/kg bw/d). These changes were not considered to be relevant. Minimal depressions in sodium affected all dose level of females and Group 2 (300 mg/kg bw/d) males. These were within normal range and were not considered to be toxicologically relevant. The only change which may have been biologically relevant was a mild increase in the mean cholesterol value for females from Group 3 (1000 mg/kg bw /d). Urine specific gravity was increased in males from Group 3 (1000 mg/kg bw) and females from Groups 2 and 3 (300 and 1000 mg/kg bw/d). The increased values for males and females from Group 3 were statistically significant. There were increases in the occurrence of mild ketonuria in male Groups 1, 2 and 3.

Gross Pathology:

There were no gross lesions associated with chemical administration. There was a linear decrease in terminal body weights of males from Groups 1, 2 and 3. There were, however, essentially no changes in weights of females from treated groups as compared to controls. The only significant changes in absolute organ weights were decreased mean brain weight in females from Group 3 (1000 mg/kg bw/d) and increased mean hepatic weight in females from Group 3 (1000 mg/kg bw/d). Organ to body weight ratios were increased in a dose related manner for all male organs for which weights were obtained (statistically significant in most instances) for Groups 2 and 3. Kidneys to body weight ratios were increased in females from Group 3 (statistically significant) as were liver to body weight ratios in females from Groups 2 and 3 (300 and 1000 mg/kg bw/d).

Microscopic Pathology:

There were no microscopic lesions with an apparent association with chemical administration.

No test substance-related adverse effects on the reproductive organs were indicated.

Conclusions:
The lowest dose leve (100 mg/kg/day) produced no toxicologically significant changes.
Executive summary:

The test substance was administered orally by gavage to four groups of 20 male and 20 female Sprague-Dawley rats at doses of 0, 100, 300 or 1000 mg/kg/day for 3 months. All animals survived the treatment period. changes appearently associated with the test substance exposure at 1000 mg/kg7day included increased urine specific gravity and decreased body weights among males. Females at this dose level had stained abdomens, increased urine specific gravity, elevated cholesterol values, and increased liver weights. Middle dose level (300 mg/kg/day) males had decreased body weight and females had increased urine specific gravity. The lowest dose leve (100 mg/kg/day) produced no toxicologically significant changes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP study, comparable to guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Accpetable documented study report with restriction (test substance purity not indicated; the study is limited concerning the recommended particle-size distribution given in current guidelines and thus an exposure of all relevant regions of the respiratory tract was presumably not given. The biologically relevance of findings from this study, especially systemic effects are questionable, whereas local effects noted in the inhalation study could be used for supporting purpose.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:
In a four-week pilot inhalation study male and female rats were exposed to the dust atmosphere of Santocure NS 6 (N-tert-butylbenzothiazole-2-sulphenamide) hours per day, 5 days per week for 20 or 21 exposures during a 29-day experimental period. The mean nominal exposure concentrations for groups III, IV and V were 0.058, 0.172 or 0.524 mg/liter, respectively. The equivalent aerodynamic diameter was 8.4 micrometers.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
8.4 µm
Remarks on MMAD:
MMAD / GSD: aerodynamic mass medium diameter: 8.4 micrometer with a geometric standard deviation of 4.31
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the airborne dusts in the chamber atmosphere was determined analytically using the fiberglass filter sampling technique.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0.0, 0.0024, 0.029, and 0.084 mg/l
Basis:

No. of animals per sex per dose:
10 animals per dose and sex
Control animals:
yes
Details on study design:
Post-exposure period: no
Positive control:
None.
Observations and examinations performed and frequency:
Clinical signs (ocular and nasal irritation, respiratory distress), body weights, food consumption, clinical chemistry, hematology, biochemistry, urinalysis.
Sacrifice and pathology:
At the termination of the study the rats from all groups were sacrificed for gross and histopathological examinations.
Statistics:
All statistical analysis compared the treatment group with the control group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Nasal irritation was observed in the rats and ranged in severity from slight to market. The symptom was usually present after exposure and had generally disappeared by the following morning. The severity of nasal irritation appeared to be concentration dependent during the first half of the study, several rats in the highest dose group exhibited an urogenital discharge. This symptom was observed after exposure and was still apparent the following morning. Alopecia was observed in two rats of the highest dose group.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No effects.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
at 0.029 and 0.084 mg/l: SGOT values for males and females were significant elevated compared to control (II).
at 0.0024 mg/l: SGOT values were comparable to control all other parameters appeared comparable to one or both control groups.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No effects.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Some variations in absolute and rel. weights, but the variations did not appear to be dose dependent and there were no compound related microscopic alterations associated with the involved tissue, therefore the biological signifcance of the variations could not be determined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross lesions interpreted as compound induced observed in any animal at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
0.084 mg/l: brown pigments within sinusoidal macrophages in lymph nodes (6/20)was slightly increased when compared to controls (2 control I, 1 control II)concurrent erythrophagocytosis in the lymph nodes was also slightly increased in the Santocure NS treated group (4/20) as compared to controls (0 control T, 1 control II) Very slight to moderate focal to multifocal hepatocellular necrosis was observed in 3/20 animals; an associated acute inflammatory infiltrate was present in 2 of the 3 affected animals. This lesions was not present in controls While the previously described lesions may represent a compound effect, they have been observed in control rats at the facility and therefore the possibility that they are spontaneous in origin cannot be excluded.

The occurence and incidence of all other microscopic lesions were similar to that observed in controls.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
other:
Effect level:
0.029 mg/L air
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified
Critical effects observed:
not specified

Nominal concentrations: 0.058, 0.17 and 0.52 mg/l

Analytical concentrations: mean analytical concentrations: 0.0024, 0.029 and 0.084 mg/l

Gravimetric concentrations: mean gravimetric concentrations: 0.0024, 0.028, 0.088 mg/l

Dust particle size distribution analyses: the equivalent aerodynamic mass medium diameter: 8.4 µm , geometric standard deviation of 4.31

Clinical studies Appearance and general behavior: Nasal irritation was observed in the rats and ranged in severity from slight to market. The symptom was usually present after exposure and had generally disappeared by the following morning. The severity of nasal irritation appeared to be concentration dependent during the first half of the study, several rats in the highest dose group exhibited an urogenital discharge. This symptom was observed after exposure and was still apparent the following morning. Alopecia was observed in two rats of the highest dose group.

Body weights: no effects.

Food consumption: no time or test material related differences were observed.

Hematology: no effects.

Blood chemistry:

at 0.029 and 0.084 mg/l: SGOT values for males and females were significant elevated compared to control (II).

at 0.0024 mg/l: SGOT values were comparable to control all other parameters appeared comparable to one or both control groups.

Urinalysis: no effects.

Gross pathology: No gross lesions interpreted as compound induced observed in any animal at necropsy.

Organ weights: Some variations in absolute and rel. weights, but the variations did not appear to be dose dependent and there were no compound related microscopic alterations associated with the involved tissue, therefore the biological signifcance of the variations could not be determined.

Histopathology: 0.084 mg/l: brown pigments within sinusoidal macrophages in lymph nodes (6/20)was slightly increased when compared to controls (2 control I, 1 control II)concurrent erythrophagocytosis in the lymph nodes was also slightly increased in the Santocure NS treated group (4/20) as compared to controls (0 control T, 1 control II) Very slight to moderate focal to multifocal hepatocellular necrosis was observed in 3/20 animals; an associated acute inflammatory infiltrate was present in 2 of the 3 affected animals. This lesions was not present in controls While the previously described lesions may represent a compound effect, they have been observed in control rats at the facility and therefore the possibility that they are spontaneous in origin cannot be excluded.

The occurence and incidence of all other microscopic lesions were similar to that observed in controls.

Histological examinations of treated animals (highest dose group) of the nasal turbinate area, trachea, lung and olfactory bulb revealed no biologically relevant differences compared to the controls.

Conclusions:
Study is limited concerning the recommended particle-size distribution given in current guidelines and thus an exposure of all relevant regions of the respiratory tract was presumably not given. The biologically relevance of findings from this study, especially systemic effects are questionable.
Executive summary:

In a four-week pilot inhalation study male and female rats were exposed to the dust atmosphere of Santocure NS 6 (N-tert-butylbenzothiazole-2-sulphenamide) hours per day, 5 days per week for 20 or 21 exposures during a 29-day experimental period. The mean nominal exposure concentrations for groups III, IV and V were 0.058, 0.172 or 0.524 mg/liter, respectively. The equivalent aerodynamic diameter was 8.4 micrometers.

The study is limited concerning the recommended particle-size distribution given in current guidelines and thus an exposure of all relevant regions of the respiratory tract was presumably not given. The biologically relevance of findings from this study, especially systemic effects are questionable.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
29 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Accpetable documented study report with restriction (test substance purity not indicated; the study is limited concerning the recommended particle-size distribution given in current guidelines and thus an exposure of all relevant regions of the respiratory tract was presumably not given. The biologically relevance of findings from this study, especially systemic effects are questionable, whereas local effects noted in the inhalation study could be used for supporting purpose.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Accpetable documented study report with restriction (test substance purity not indicated; the study is limited concerning the recommended particle-size distribution given in current guidelines and thus an exposure of all relevant regions of the respiratory tract was presumably not given. The biologically relevance of findings from this study, especially systemic effects are questionable, whereas local effects noted in the inhalation study could be used for supporting purpose.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:
In a four-week pilot inhalation study male and female rats were exposed to the dust atmosphere of Santocure NS 6 (N-tert-butylbenzothiazole-2-sulphenamide) hours per day, 5 days per week for 20 or 21 exposures during a 29-day experimental period. The mean nominal exposure concentrations for groups III, IV and V were 0.058, 0.172 or 0.524 mg/liter, respectively. The equivalent aerodynamic diameter was 8.4 micrometers.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
8.4 µm
Remarks on MMAD:
MMAD / GSD: aerodynamic mass medium diameter: 8.4 micrometer with a geometric standard deviation of 4.31
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the airborne dusts in the chamber atmosphere was determined analytically using the fiberglass filter sampling technique.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0.0, 0.0024, 0.029, and 0.084 mg/l
Basis:

No. of animals per sex per dose:
10 animals per dose and sex
Control animals:
yes
Details on study design:
Post-exposure period: no
Positive control:
None.
Observations and examinations performed and frequency:
Clinical signs (ocular and nasal irritation, respiratory distress), body weights, food consumption, clinical chemistry, hematology, biochemistry, urinalysis.
Sacrifice and pathology:
At the termination of the study the rats from all groups were sacrificed for gross and histopathological examinations.
Statistics:
All statistical analysis compared the treatment group with the control group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Nasal irritation was observed in the rats and ranged in severity from slight to market. The symptom was usually present after exposure and had generally disappeared by the following morning. The severity of nasal irritation appeared to be concentration dependent during the first half of the study, several rats in the highest dose group exhibited an urogenital discharge. This symptom was observed after exposure and was still apparent the following morning. Alopecia was observed in two rats of the highest dose group.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No effects.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
at 0.029 and 0.084 mg/l: SGOT values for males and females were significant elevated compared to control (II).
at 0.0024 mg/l: SGOT values were comparable to control all other parameters appeared comparable to one or both control groups.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No effects.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Some variations in absolute and rel. weights, but the variations did not appear to be dose dependent and there were no compound related microscopic alterations associated with the involved tissue, therefore the biological signifcance of the variations could not be determined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross lesions interpreted as compound induced observed in any animal at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
0.084 mg/l: brown pigments within sinusoidal macrophages in lymph nodes (6/20)was slightly increased when compared to controls (2 control I, 1 control II)concurrent erythrophagocytosis in the lymph nodes was also slightly increased in the Santocure NS treated group (4/20) as compared to controls (0 control T, 1 control II) Very slight to moderate focal to multifocal hepatocellular necrosis was observed in 3/20 animals; an associated acute inflammatory infiltrate was present in 2 of the 3 affected animals. This lesions was not present in controls While the previously described lesions may represent a compound effect, they have been observed in control rats at the facility and therefore the possibility that they are spontaneous in origin cannot be excluded.

The occurence and incidence of all other microscopic lesions were similar to that observed in controls.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
other:
Effect level:
0.029 mg/L air
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified
Critical effects observed:
not specified

Nominal concentrations: 0.058, 0.17 and 0.52 mg/l

Analytical concentrations: mean analytical concentrations: 0.0024, 0.029 and 0.084 mg/l

Gravimetric concentrations: mean gravimetric concentrations: 0.0024, 0.028, 0.088 mg/l

Dust particle size distribution analyses: the equivalent aerodynamic mass medium diameter: 8.4 µm , geometric standard deviation of 4.31

Clinical studies Appearance and general behavior: Nasal irritation was observed in the rats and ranged in severity from slight to market. The symptom was usually present after exposure and had generally disappeared by the following morning. The severity of nasal irritation appeared to be concentration dependent during the first half of the study, several rats in the highest dose group exhibited an urogenital discharge. This symptom was observed after exposure and was still apparent the following morning. Alopecia was observed in two rats of the highest dose group.

Body weights: no effects.

Food consumption: no time or test material related differences were observed.

Hematology: no effects.

Blood chemistry:

at 0.029 and 0.084 mg/l: SGOT values for males and females were significant elevated compared to control (II).

at 0.0024 mg/l: SGOT values were comparable to control all other parameters appeared comparable to one or both control groups.

Urinalysis: no effects.

Gross pathology: No gross lesions interpreted as compound induced observed in any animal at necropsy.

Organ weights: Some variations in absolute and rel. weights, but the variations did not appear to be dose dependent and there were no compound related microscopic alterations associated with the involved tissue, therefore the biological signifcance of the variations could not be determined.

Histopathology: 0.084 mg/l: brown pigments within sinusoidal macrophages in lymph nodes (6/20)was slightly increased when compared to controls (2 control I, 1 control II)concurrent erythrophagocytosis in the lymph nodes was also slightly increased in the Santocure NS treated group (4/20) as compared to controls (0 control T, 1 control II) Very slight to moderate focal to multifocal hepatocellular necrosis was observed in 3/20 animals; an associated acute inflammatory infiltrate was present in 2 of the 3 affected animals. This lesions was not present in controls While the previously described lesions may represent a compound effect, they have been observed in control rats at the facility and therefore the possibility that they are spontaneous in origin cannot be excluded.

The occurence and incidence of all other microscopic lesions were similar to that observed in controls.

Histological examinations of treated animals (highest dose group) of the nasal turbinate area, trachea, lung and olfactory bulb revealed no biologically relevant differences compared to the controls.

Conclusions:
Study is limited concerning the recommended particle-size distribution given in current guidelines and thus an exposure of all relevant regions of the respiratory tract was presumably not given. The biologically relevance of findings from this study, especially systemic effects are questionable.
Executive summary:

In a four-week pilot inhalation study male and female rats were exposed to the dust atmosphere of Santocure NS 6 (N-tert-butylbenzothiazole-2-sulphenamide) hours per day, 5 days per week for 20 or 21 exposures during a 29-day experimental period. The mean nominal exposure concentrations for groups III, IV and V were 0.058, 0.172 or 0.524 mg/liter, respectively. The equivalent aerodynamic diameter was 8.4 micrometers.

The study is limited concerning the recommended particle-size distribution given in current guidelines and thus an exposure of all relevant regions of the respiratory tract was presumably not given. The biologically relevance of findings from this study, especially systemic effects are questionable.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
84 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Accpetable documented study report with restriction (test substance purity not indicated; the study is limited concerning the recommended particle-size distribution given in current guidelines and thus an exposure of all relevant regions of the respiratory tract was presumably not given. The biologically relevance of findings from this study, especially systemic effects are questionable, whereas local effects noted in the inhalation study could be used for supporting purpose.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
animals tested: one-half with intact skin, one-half with abraded skin
Principles of method if other than guideline:
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.
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Type of coverage:
semiocclusive
Vehicle:
physiological saline
Details on exposure:
The test article, Santocure NS (N-tert-butylbenzothiazole-2-sulphenamide), was ground with a mortar and pesle and then moinstened with saline and mixed into a spreadable paste. It was applied evenly over each test site with a glass stirring rod at dosage levels of 125, 500 or 2000 mg/kg bw.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
21 d
Frequency of treatment:
Five days per week for 3 consecutive weeks.
Remarks:
Doses / Concentrations:
0, 125, 500, or 2000 mg/kg bw/d
Basis:

No. of animals per sex per dose:
Five per sex and dose with intact skin, 5 per sex and dose with abraded skin.
Control animals:
yes
Details on study design:
Post-exposure period: no
Observations and examinations performed and frequency:
Dosing was repeated once daily, five days per week for three consecutive weeks. Individual doses were adjusted weekly based on the body weight obtained at the beginning of each study week.
General observations: rabbits were observed once daily for signs of dermal irritation, daily observations for pharmacotoxic signs and other findings, the rabbits were observed for mortality twice daily.
Body weight: recorded weekly.
Hematology: hemoglobin, hemocrit, erythrocyte count, total leucocyte count, platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocytes, differential leucocyte count.
Biochemistry: glucose, blood urea nitrogen, serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase, albumin, serum glutamic pyruvic transaminase (SGPT), total protein, cacium, cholesterol, total bilirubin, creatinine, lactic dehydrogenase (LDH), sodium, potassium, chloride and globulin.
Sacrifice and pathology:
Pathology: gross pathology.
Organ weights: liver, kidneys, heart, testes, ovaries, thyroid/parathyroid, adrenals and brain, pituitary.
Histopathology: treated and untreated skin, liver, kidneys and any gross lesions.
Recorded: erythema (score 0 to 3), edema (0 to3), atonia (0 to 3), coriaceousness (0 to 3), fissuring (0 to 3), eschar, exfoliation
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
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):
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:
not examined
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 occuring in the control group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
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
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Acanthosis, hyperkeratosis, and dermal inflammatory cell infiltration in the treated skin of rabbits from the 2000 mg/kg
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
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 mg/kg bw/day
System:
other: skin
Organ:
skin
Treatment related:
yes
Dose response relationship:
yes

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 skin at 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.


Conclusions:
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 ant not related to the application of the test substance.
Executive summary:

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.

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 ant not related to the application of the test substance.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 000 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
GLP and guideline study

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
animals tested: one-half with intact skin, one-half with abraded skin
Principles of method if other than guideline:
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.
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Type of coverage:
semiocclusive
Vehicle:
physiological saline
Details on exposure:
The test article, Santocure NS (N-tert-butylbenzothiazole-2-sulphenamide), was ground with a mortar and pesle and then moinstened with saline and mixed into a spreadable paste. It was applied evenly over each test site with a glass stirring rod at dosage levels of 125, 500 or 2000 mg/kg bw.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
21 d
Frequency of treatment:
Five days per week for 3 consecutive weeks.
Remarks:
Doses / Concentrations:
0, 125, 500, or 2000 mg/kg bw/d
Basis:

No. of animals per sex per dose:
Five per sex and dose with intact skin, 5 per sex and dose with abraded skin.
Control animals:
yes
Details on study design:
Post-exposure period: no
Observations and examinations performed and frequency:
Dosing was repeated once daily, five days per week for three consecutive weeks. Individual doses were adjusted weekly based on the body weight obtained at the beginning of each study week.
General observations: rabbits were observed once daily for signs of dermal irritation, daily observations for pharmacotoxic signs and other findings, the rabbits were observed for mortality twice daily.
Body weight: recorded weekly.
Hematology: hemoglobin, hemocrit, erythrocyte count, total leucocyte count, platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocytes, differential leucocyte count.
Biochemistry: glucose, blood urea nitrogen, serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase, albumin, serum glutamic pyruvic transaminase (SGPT), total protein, cacium, cholesterol, total bilirubin, creatinine, lactic dehydrogenase (LDH), sodium, potassium, chloride and globulin.
Sacrifice and pathology:
Pathology: gross pathology.
Organ weights: liver, kidneys, heart, testes, ovaries, thyroid/parathyroid, adrenals and brain, pituitary.
Histopathology: treated and untreated skin, liver, kidneys and any gross lesions.
Recorded: erythema (score 0 to 3), edema (0 to3), atonia (0 to 3), coriaceousness (0 to 3), fissuring (0 to 3), eschar, exfoliation
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
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):
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:
not examined
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 occuring in the control group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
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
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Acanthosis, hyperkeratosis, and dermal inflammatory cell infiltration in the treated skin of rabbits from the 2000 mg/kg
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
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 mg/kg bw/day
System:
other: skin
Organ:
skin
Treatment related:
yes
Dose response relationship:
yes

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 skin at 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.


Conclusions:
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 ant not related to the application of the test substance.
Executive summary:

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.

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 ant not related to the application of the test substance.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
6 mg/cm²
Study duration:
subacute
Species:
rabbit
Quality of whole database:
GLP and guideline study

Additional information

Repeated dose toxicity: oral

In a 30-day gavage study, male and female Sprague Dawley rats (5 per dose and sex) were administered with the test substance TBBS at concentrations of 0, 100, 300, 1000 and/or 3000 mg/kg bw and day. All female rats and one male rat treated with 3000 mg/kg bw and day were dead within six days. The remaining high dose males were sacrificed in a moribund condition on day six. All other treated animals survived for the duration of the study. Animals of the high dose (3000 mg/kg bw and day) exhibited decreased food consumption, anogenital staining, tremors, abdominal distention, and generally poor condition prior to their death. At 1000 mg/kg bw and day, anogenital staining was also noted. This dosage group and the 300 mg/kg bw and day dosage group exhibited excessive salivation during the dosing procedure. No other unusual physical observations were noted in these groups or in any of the lower dose groups. In the high dose group, males showed a 38 % decrease in body weight during the first 5 days; due to mortality, weights could not be obtained for females. Body weights of males in the 300 and 1000 mg/kg bw and day groups were slightly but not significantly reduced. Low dose males and females from all treatment groups surviving the study, had body weight gains comparable to control animals. Food consumption for high dose males was significantly reduced during the first week of the study. Significant reductions were also noted during week 1 for males and females at 1000 mg/kg bw and day, with slight reduction noted in weeks 2 and 3, for week 4 values were comparable to controls. Post-mortem findings, observed grossly, appear to occur in treated and control animals with similar frequency and severity and did not appear to be related to treatment, with the exception of the high dose animals. These animals, which died during the study, exhibited staining of fur, distention of stomach, and/or intestines, in several animals, encrustations around eyes and ocular opacities. Organ weight differences were observed for several tissues. Heart weights were lower for males in 100, 300 and 1000 mg/kg bw and day and for females in the 1000 mg/kg bw and day group. Absolute and relative kidney weights were increased in all treated females. In males the absolute and relative kidney weights were slightly increased at 100 mg/kg bw and day, whereas the findings at the higher dose levels (300 and 1000 mg/kg bw and day) were unclear due to the reduced terminal body weights and the lack of a dose response. Liver weights were elevated for females treated at 300 and 1000 mg/kg bw and day (Monsanto Co. 1982). Based on the changes in organ weights observed a LOAEL of 100 mg/kg bw was suggested.

In a subsequent subchronic toxicity study (90 days) the test substance TBBS was administered orally by gavage to four groups of 20 male and 20 female Sprague-Dawley rats at doses of 0, 100, 300 and 1000 mg/kg bw and day. All animals survived the treatment period. Incidences of salivation, resistance to dosing, and or pawing motions after dosing occurred for both sexes in the 300 and 1000 mg/kg bw and day dose groups. These observations were probably indicative of the animals aversion to dosing caused by poor palatability of the test substance suspension rather than toxic response. Furthermore, there were no gross or microscopic findings to suggest any gastrointestinal factors which may have contributed to these signs. Males and females from the highest dose group had urine stained abdomen. However, there were no renal or bladder changes indicating a treatment effect. A reduction of body weight was noted in males of the 300 and 1000 mg/kg bw and day groups, in absence of reduced food intake. Thus the authors suggested a relationship to treatment. In haematology, statistically significant changes in calculated haematology were observed, resulted from minimal increases in red cell counts or MCV values and were considered as not biologically relevant. However, the increased mean cholesterol level in females of the highest dose group (1000 mg/kg bw and day) was suggested to be related to administration of the test substance, although there were no correlative changes in other serum chemistry parameters or microscopic lesions which clarify this significance. Other changes in serum chemistries were minimal sporadic fluctuations that remained within normal limits and were of no biological relevance. Urine specific gravity was significant increased in males at 1000 mg/kg bw and day and in females at 300 and 1000 mg/kg bw and day. The solutes responsible for the increase in specific gravity were not determined. Mild ketonuria was evident in males from all treatment groups. However, no correlative changes in microscopic pathology were seen in the kidneys of high dose animals. Gross lesions found at necropsy were few and not related to treatment. Although several significant increases in relative organ weights were noted, the majority of these were due to disproportional body weight decreases in treated male rats. The organ weights of the kidneys and livers of high dose females showed absolute and/or relative weight increases which were considered to be treatment related. There were no associations between microscopic lesions and chemical administration in any tissue or organ examined. The authors concluded that the low dose level (100 mg/kg bw and day) produced no significant toxicological effects; therefore it is considered the NOAEL for this study (Monsanto Co. 1985).

In addition, the oral toxicity of the test substance TBBS was evaluated in a combined repeat dose and reproductive/developmental toxicity screening test (OECD TG 422). Sprague-Dawley rats (13 males and 13 females per group) were dosed by gavage with 0, 40, 200 and 1000 mg/kg bw and day test substance (MHWJ 1997). Temporary salivation after administration of the test substance was observed in males at 200 mg/kg bw and day or higher. In addition a suppression of food consumption and body weight gain was noted in males of the highest dose group (1000 mg/kg bw and day). At the autopsy after 42 days treatment, the number of animals with eosinophilic bodies in the kidney was increased in all test substance-treated males. However, the incidence of eosinophilic bodies was increased in treated animals, but the severity of these lesions was comparable with findings of the control group. An increase of the absolute and relative kidney weights was observed in males at 1000 mg/kg bw and day. Although slight hypertrophy of hepatocytes in the central zone was observed in males at 200 mg/kg bw and day and higher, an increase in relative liver weights related to the administration was observed in the 1000 mg/kg bw and day group. Decrease in fatty change of hepatocytes in the periportal zone was noted in male of the highest dose group. In males of the higher dose groups a slight increase in the total bilirubin concentration and a dose-dependent increase in the deposition of hemosiderin in the spleen were observed. In addition, haemoglobin and hematocrit values were slightly decreased and haemolytic anaemia was induced in the 1000 mg/kg group. A slight increase in the total cholesterol concentration was noted in males of the highest dose group. No other organs showed abnormalities suggesting effects of the test substance TBBS. In females, temporary salivation after administration of the test substance was noted at concentrations of 200 mg/kg bw and day and higher. Food consumption was decreased in the 1000 mg/kg bw and day group prior to mating and along with slight suppression of body weight gain during pregnancy. At autopsy on postpartum day 4, slight vacuolar degeneration in the proximal tubules of the kidney was observed in females of the higher dose groups, as well as slight increase in the relative kidney weights. Slight hypertrophy of hepatocytes in the central zone was noted in females at 200 and 1000 mg/kg bw and day. In addition, an increase in absolute and relative liver weights was noted at 1000 mg/kg bw and day. However, deposits of brown pigment in the spleens in the 1000 mg/kg bw and day group tended to increase, but in many cases were comparable to those in the control group. Thus, the authors suggested, that haemolysis resulting from the administration did not occur in females. No abnormalities related to the treatment were observed in other organs.

Based on the increase incidence of eosinophilic bodies in the kidney in all treated male rats the authors suggested an NOAEL less than 40 mg/kg for males. However, the incidence was increase but the severity of lesions was comparable with findings in control animals. Thus, the biological relevance of this effect is questionable and therefore a NOAEL of 40 mg/kg bw and day for males and females is suggested.

Repeated dose toxicity: inhalation

In a subacute inhalation study (Monsanto Co. 1981) male and female Sprague-Dawley rats were exposed to the dust of TBBS six hours per day, 5 days per week during a 29-day experimental period. The mean nominal exposure concentrations were 0.058, 0.17, 0.52 mg/l (corresponding to mean analytical concentrations of 0.0024, 0.029 and 0.084 mg/l). The equivalent aerodynamic diameter was 8.4 µm with a geometric standard deviation of 4.31. This distribution of TBBS particle sizes was above the recommendations in current inhalation guideline tests. Immediately after some of the 6-hours exposure periods, the rats exhibited nasal irritation in relation to the concentration. By the following morning this symptom had generally disappeared. Urogenital discharge and alopecia were observed in some rats of the highest dose group during the first half of the study. This symptom was observed after exposure and was still apparent the following morning (no more data). The biological relevance of this finding is questionable. No body weight effects were observed and no significant differences in food consumption were revealed. No compound induced gross pathologic lesions or organ weight variations were observed in any animals at necropsy. Haematology and urinalysis were all within the normal rage of variation. Of the blood chemistry parameters measured, SGOT values were significant elevated at 0.029 (males 25 %, females 15 %) and 0.084 mg/ml (males 23 %, females 23 %) as compared to controls. Microscopic evaluations were done for both control groups and the highest dose group (0.084 mg/l); histological data for the low and mid dose groups (0.0024, 0.029 mg/l) are missing. Microscopic lesions were present in lymph nodes (brown pigment in sinusoidal macrophages and liver (focal/multifocal hepatocellular necrosis with inflammation) at low incidences. However, these effects seen in a few high dose group animals were also observed in controls (at lower incidence) from this study or have been observed in control rats from other studies at this facility.

The biologically relevance of the increase of SGOT values is questionable because no relevant changes in organ weight was observed and the microscopic lesions noted were of low incidence and were also observed in control rats. In summary, the study is limited concerning the recommended particle-size distribution given in current guidelines. To allow for exposure of all relevant regions of the respiratory tract, aerosols with mass median aerodynamic diameters (MMAD) ranging from 1 to 3 µm are recommended. The MMAD used was above the recommendations given (8.4 µm) and therefore, a limited systemic exposure seems reasonable. Thus, the findings of this study are questionable, especially for systemic effects. Whereas the local effects on the respiratory tract noted should be used as supporting evidence.

Repeated dose toxicity: dermal

The test substance TBBS was administered by dermal application to three groups of 10 male and female rabbits, one-half with intact skin and one-half with abraded skin, five days per week for three consecutive weeks at dose levels of 125, 500 and 2000 mg/kg bw and day. An identical control group was treated with saline. Criteria evaluated for treatment effect included mortality, pharmacotoxic signs, body weights, dermal irritation, haematological and clinical biochemical determinations, organ weights and macroscopic and selected microscopic evaluation of tissue. No mortality occurred in this study. The majority of rabbits in both control and test groups appeared normal with the exception of a few rabbits in all groups exhibiting occasional spontaneous pharmacotoxic signs. No statistical significance difference was noted in body weights. No remarkable changes or differences in dermal irritation were observed between the control and test groups. Statistically significant changes in haematological values occurred in the 500 mg/kg bw and day males in lymphocytes and segmented neutrophils. A few statistically significant changes occurred in the biochemical values; 125 and 2000 mg/kg bw and day males showed a decrease in total bilirubin and 2000 mg/kg bw and day 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 bw and day group. All other microscopic lesions observed were incidental in nature and not related to the application of the test substance (Monsanto Co 1981).

Based on these findings a NOAEL systemic of 2000 mg/kg bw/d and a NOAEL local of 500 mg/kg bw and day are suggested.

In conclusion:

Oral

Several repeated dose gavage toxicity studies were conducted to evaluate the toxicity of the test substance TBBS. Temporary salivation after test substance administration, a reduction of food consumption and body weight gain were noted consistently in the high dose groups. Moreover, an increase in absolute and/or relative kidney and liver weights were noted in treated animals. The increase incidence of eosinophilic bodies in all treated male rats noted in the TG 422 study (MHWJ 1997) was considered as questionable because the incidence was increase but the severity was comparable with findings from the control group.

The subchronic gavage study (90 day-study) is considered to be the most relevant study for risk assessment and thus based on the findings of this study a NOAEL oral of 100 mg/kg bw and day is suggested.

Inhalation

The inhalation toxicity of TBBS was examined in a subacute inhalation study (Monsanto Co. 1981). The study is limited concerning the recommended particle-size distribution given in current guidelines and thus an exposure of all relevant regions of the respiratory tract might not have been given. The biologically relevance of sporadic findings reported, especially systemic effects are questionable. In light of these limitations the data from the oral subchronic toxicity study (NOAEL: 100 mg/kg bw and day, Monsanto 1985) will be used for calculation of the systemic DNEL; whereas local effects noted in the inhalation study will be used as supporting evidence. Based on the fact that signs of nasal irritation observed at clinical examination were reversible, only short in duration and could not be correlated to histopathological effects, no toxicological significance was attached to this finding and thus, no relevant local effects were observed up to 0.084 mg/l.

Dermal

The dermal toxicity of the test substance TBBS was evaluated in a 21-day dermal toxicity study (Monsanto Co. 1981). No death or systemic toxicity was indicated in any of the treated animals. No effects on body weight or dermal irritation were noted. Some incidental changes in haematology and biochemistry was observed. No compound related macroscopic lesions or statistically significant organ weight variations were found in this study. However, 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 dose group. Thus, a NOAEL dermal systemic of 2000 mg/kg bw and day and a NOAEL dermal local of 500 mg/kg bw and day were considered.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The most valid study is used (key study)

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Only a single study is available.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Only a single study is available.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Only a single study is available.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Only a single study is available.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Repeated dose toxicity: inhalation - systemic effects (target organ) cardiovascular / hematological: other

Justification for classification or non-classification

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.