N,N-dicyclohexylbenzothiazole-2-sulphenamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1988 - April 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study conducted to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no ophthalmological examinations performed

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dicyclohexylbenzothiazole-2-sulphenamide

EC Number:

225-625-8

EC Name:

N,N-dicyclohexylbenzothiazole-2-sulphenamide

Cas Number:

4979-32-2

Molecular formula:

C19H26N2S2

IUPAC Name:

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

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Portage MI
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: approx 6 weeks
- Weight at study initiation: males: 213.1-240.7g, females: 151.7-170.4
- Fasting period before study: not specified
- Housing: individual stainless steel cages
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib (St. Louis public water supply)
- Acclimation period: not specified

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70-64˚F
- Humidity (%): 30-60%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

IN-LIFE DATES: animal arrival 31 May 1988

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Basal Diet: Purina Mills Certified RODENT CHOW #5002
Frequency of Preparation: Approximately weekly
Levels Prepared: T-3 (5000 ppm), T-2 (2500 ppm), T-1 (500 ppm)
Mixing Machine: HOBART HCM-450
Mixing Time: 10 minutes
Batch size: Premix (also used as T-3), 14.6 kilograms; other levels,
9 kgs each.
Mixing Procedure Number: DP-88121-1

Vehicle:

other: DCBS contained in the diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test material stability: liquid chromotography using variable wavelength UV/VIS detector.
Homogeneity of Diet Mixtures: Analysis of duplicate samples from top, middle, and bottom of mixer of T-1 and T-3 levels.
Diet Mixture Stability: Analysis of T-1 and T-3 level samples kept frozen (closed container, days) or at ambient temperature (open container, 7 and 14 days).
Dietary Level Verification: Extraction of
SANTOCURE DCBS with 90/10 hexane/methylene chloride, analysis by liquid chromotography using variable wavelength UV/VIS detector; all dietary levels for first 7 weeks, thereafter at least once/week.

Duration of treatment / exposure:

3 months

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

control

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

500 ppm (ca. 36.9 mg/kg bw/d)

Doses / concentrations 3

Dose / conc.:

2 500 ppm

Remarks:

2500 ppm (ca. 176.7 mg/kg bw/d)

Doses / concentrations 4

Dose / conc.:

5 000 ppm

Remarks:

5000 ppm (ca. 342.6 mg/kg bw/d)

No. of animals per sex per dose:

15 per dose and sex

Control animals:

yes, plain diet

Details on study design:

Post-exposure period: no data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes, once weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes - see 'any other information on materials and methods'

CLINICAL CHEMISTRY: Yes - see 'any other information on materials and methods'

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes - see 'any other information on materials and methods'

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: see 'any other information on materials and methods'

Statistics:

Dunnett’s Multiple Comparison Test (two-tailed) (1): Body weights and food consumption.

Fisher’s Exact Test (2) with Bonferroni Inequality Procedure (4): Incidence of microscopic lesions.

Terminal body weights, absolute organ weights, organ/body weight ratios and selected noncategorical clinical pathology data were evaluated by decision-tree statistical analysis procedures which, depending on the results of tests for normality (3) and homogeneity of variances (Bartlett’s Test), chose either the parametric (Dunnett’s Test and Linear Regression) or nonparametric (Kruskal-Wallis, Jonckheere’s and/or Mann-Whitney Tests) routines to detect group differences and analyzed for trend.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

there were no clinical signs considered to be related to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male and females of the two highest dietary levels had reduced weight gains throughout the study (compared to control: males 2600 ppm:-6.4 %, 5200 ppm:-9 %, females 2600 ppm -10.0%, 5200 ppm: -15.6%. Lowest dietary level males and females were not significant different from control (males 540 ppm 0.3 %, females 540 ppm: -1.8 %). See table 'body weight'.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slight decrease in food intake at the two highest exposure levels (2600, 5200 ppm, males: -10.5%, -12.4%, females: -9.7%, -20.5%. The lowest food intake values occurred during the first week of exposure, and the animals gradually began to approach control group feeding levels during the remainder of the study. Animals from the 540 ppm group consumed similar food as compared to control (males: -3.7%, females: -0.6%). See table 'food consumption'.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

there were no relevant changes in hematologic parameters at either sampling period

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean glucose values were slightly to moderate decreased in males of the highest dose group (period 1), in males of the 2600 ppm group (period 2) and in all treated females(period 2). These changes may have been related to treatment; however the male 5200 ppm group (period 1) was largely affected by a low value in one animal. The apparent decrease in values for treated females at period 2 (compared to the control group values) was a function of the slightly higher than expected control group values, and therefore was considered to be of equivocal biological significance.
Inorganic phosphorus values were slightly increased in males of the highest dose groups (2600, 5200 ppm) and in females of the highest dose group. Total protein and albumin values were slightly decreased for females of the 540 and 2600 ppm groups (period 2), and globulin was slightly decreased in all treated females groups. The mean potassium value for the males of the highest dose group (period 1) was slightly increased. All of these changes were either of small magnitude, inconsistent between periods or sex, or not related to dose and therefore were considered equivocal in their relationship to treatment.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The only statistically significant absolute organ weight change observed was a slight decrease in absolute heart weights probably resulted from the decreased mean body weight in this group; in addition no biologically relevant change in organ/body weight ratios

Gross pathological findings:

no effects observed

Description (incidence and severity):

no significant gross necropsy changes noted at sacrifice.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

there were no microscopic changes attributable to administration of the test substance

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Slight decrease in body weight gain compared to control (males -6.4%, females -10%)

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Analysis of test material and diets: 

Results of analyses for neat compound stability conducted over the span of time approximately equal to the study length, indicated the test material was stable. The homogeneity and stability of the diet mixtures was determined to be adequate for study use. The following table shows the overall averages (uncorrected for quality control results):

Test Group:	T1	T2	T3
Target exposure (ppm):	500	2500	5000
Study mean concentration (ppm):	540	2600	5200
St. Deviation (ppm):	11	71	130
% difference from target:	8.0	4.0	4.0

 

Body weight:

Group	Study Mean	% diff. from control (study mean)	% diff. from control (final bw)	% gain from initial
MN	417.8	-	-	132.9
M1	419.0	0.3	0.1	133.4
M2	391.1	-6.4	-8.1	114.8
M3	380.2	-9.0	-9.3	111.4
FN	236.2	-	-	73.8
F1	232.0	-1.8	-3.7	67.1
F2	212.5	-10.0	-12.9	51.0
F3	199.4	-15.6	-20.1	38.7

 

Food Consumption:

Group	Study Mean	% diff. from control (study mean)
MN	26.7	-
M1	25.7	-3.7
M2	23.9	-10.5
M3	23.4	-12.4
FN	17.6	-
F1	17.5	-0.6
F2	15.9	-9.7
F3	14.0	-20.5

Applicant's summary and conclusion

Conclusions:

The authors concluded that the No Observed effect level (NOEL) for this study is 500 ppm (36.9 mg/kg bw/day) (Monsanto 1989).
The LOAEL for the study can be concluded as 176.7 mg/kg bw/d (2500ppm) based on the noted effects on body weight and food consumption in male and female animals.

Executive summary:

Study outline
In a 90 day feeding study according to OECD 408, DCBS was administered to male and female Sprague-Dawley rats at target levels of 0, 540 ppm, 2600 ppm and 5400 ppm (study mean concentrations) in the diet (corresponding to mean compound consumption of 0, 36.9, 176.7 and 342.6 mg/kg bw/day). 

 

Results

Analysis to verify the stability of the test material both neat and when mixed wiuth the diet, the diet homogeneity and concentrations of the test material in diet were undertaken with satisfactory results. Overall averages of dietary concentrations were found to be 540, 2,600 and 5,200 ppm.

Both sexes at the two highest exposure levels had reduced food consumption (males: -10.5 %, -12.4 %, and females: -9.7 %, -20.5 %) and reduced body weight gain compared to control (males:-6.4 %, -9 %, and females -10.0 %, -15.6 %). The lowest food intake values occurred during the first week of exposure, and the animals gradually began to approach control group feeding levels during the remainder of the study. The authors suggest that this pattern in food consumption after the initial exposure reveal that the animals acclimated themselves to the treated diets. However, the lower weight gain appeared to have resulted from decreased food intake, rather than a direct toxic response to the test chemical. This interpretation was supported by the absence of any significant clinical, gross or microscopic pathologic findings.

Changes seen in some serum chemistry parameters (especially decreased glucose) and organ weights or their ratios may have resulted from the lower food intake and/ or the lower body weight. 

 

Conclusions

The authors concluded that the No Observed effect level (NOEL) for this study is 500 ppm (36.9 mg/kg bw/day) (Monsanto 1989).