N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well-documented study performed according to GLP. Testing protocol is described in detail, but only the following general reference to internationally accepted testing guidelines is made: "This study will be conducted in general accordance with provisions specified for a 90-day subchonic oral study in US EPA and TSCA, OECD and EEC testing guidelines." No clear description on how the mean chemical consumption data were calculated from the daily feed intake data; unclear whether body weight gain is taken into account.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Principles of method if other than guideline:

This study is conducted in general accordance with provisions specified for a 90-day subchronic oral study in US EPA and TSCA, OECD and EEC testing guidelines.
Experimental design: CP 22595 was administered to groups of 20 Sprague-Dawley rats/sex at targeted levels of 0, 2500, 7500 and 1500 parts per million (ppm) in the feed. Ten male and 10 female rats/group were bled for clinicopathologic determinations, sacrificed and necropsied at approximately 7 weeks. At the end of the three months, the remaining animals were bled, sacrificed and necropsied. Tissues examined microscopically included all tissues retained from the control and highest dietary levels at the terminal sacrifice, kidneys and livers from animals of all levels sacrificed at week 7, and lungs, kidneys and livers from all animals of all levels sacrificed at termination.

A pilot study was conducted to define the test material dose corresponding to a diet concentration of 15.000 ppm. Stability of the testing substance as such and mixed in the diet, and homogeneity of the prepared diet were examined. Determination of the degree of bioabsorption of the test material is not necessary to meet the objectives of the study and as a consequence are not performed.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide

EC Number:

407-430-1

EC Name:

N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide

Cas Number:

3741-80-8

Molecular formula:

C18H17N3S4

IUPAC Name:

bis(1,3-benzothiazol-2-ylsulfanyl)(tert-butyl)amine

Details on test material:

TEST MATERIAL IDENTIFICATION
- Name of test material (as cited in study report): CP 22595
- Physical state: off-white powder
- Analytical purity: 92.8%
- Lot/batch No.: 19APP01

STABILITY OF THE TEST MATERIAL
- Stability under test conditions: Results of analyses for test material stability, conducted prior to study start and again after study termination, indicated that the test material was stable.
- Stability testing: liquid chromatography with UV/visible variable wavelength detector

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
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: males ca. 188 g, females ca. 132 g.
- Housing: individual stainless steel cages (suspended, open-mesh)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Quarantine period: 10 days
- Identification: individual tags and bar coded cage cards.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified -- mentioned: in accordance with the provisions of 'Guide to the Care and Use of Laboratory Animals'
- Humidity (%): not specified -- mentioned: in accordance with the provisions of 'Guide to the Care and Use of Laboratory Animals'
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light..

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: feed

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): approximately weekly
- Mixing appropriate amounts with Purina Mills Certified Rodent Chow #5002) neat test material mixed thoroughly with the diet using high speed mixers:
- Storage temperature of food: ambient

ANALYSIS OF DIETS:
- The homogeneity and stability of the test material in diets was determined to be adequate for this study.
- Verification of dietary concentration:
Test group 1: Target concentration: 2500 ppm; Study Mean Concentration: 2500 ppm; Standard deviation 76 ppm.
Test group 2: Target concentration: 7500 ppm; Study Mean Concentration: 7500 ppm; Standard deviation: 110 ppm.
Test group 3: Target concentration: 15000 ppm; Study Mean Concentration: 15000 ppm; Standard deviation: 350 ppm.
- Diet mixture stability: analysis of T-1 and T-3 test group samples kept at ambient temperature (open container, 7 and 14 days)/
- Homogeneity of diet mixtures: analysis of duplicate samples from top, middle and bottom of mixer of T-1 and T-3 levels.
- Dietary level verification: Extraction of test material with acetonitrile, analysis by liquid chromatography with a UV detector; all dietary levels throughout the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The feed route is a convenient and acceptable route of administration to determine the potential toxicity of materials.
- Concentration in vehicle: 0 ppm, 2.500 ppm, 7.500 ppm and 15.000 ppm.
- Amount of vehicle (if gavage): not applicable
- Lot/batch no. (if required): not specified
- Purity: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary level verification: Extraction of test material with acetonitrile, analysis by liquid chromatography with a UV detector. Analyses performed for all dietary levels throughout the study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, at libitum

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2500 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
7500 ppm
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
15000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

20 females + 20 males per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on a 30-day pilot study, the 15000 ppm level should give a test material dose of at least 1000 mg/kg bw/d for each animal.
- Rationale for animal assignment (if not random): computer generated randomization scheme that assigns animals on a weight basis giving groups of uniform body weight distribution.

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality and moribundity)
- Cage side observations avalaible in test report p. 47.

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):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Mean chemical consumption data (mg/kg bw/d) available in test report. No clear description on how these mean chemical consumption data were calculated.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once at pretest and once just prior to terminal sacrifice
- Dose groups that were examined: all at pretest, all survivors just prior to terminal sacrifice

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at weeks 7 and 14 (termination)
- Blood collection method: Posterior vena cava
- Anaesthetic used for blood collection: CO2 anesthetized
- Animals fasted: yes
- How many animals: per dose group: 10 at week 7, 10 at week 14
- Whole blood was treated with anticoagulant (EDTA pretreated commercial tubes) and processed on a COULTER S Plus II blood cell counter using the manufacturer's methods.
- Parameters checked: total erythrocyte count (RBC), total leukocyte count (WBC), platelet count (PLT), hematocrit (HCT), hemogobin (HGB) and red blood cell indices (mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC)).

LEUKOCYTE DIFFERENTIAL: Yes
- Thin blood smears on labeled glass slides were prepared, stained with Wright's stain, and examined microscopically.

RETICULOCYTE COUNT: Yes
- A portion of the EDTA-treated sample was mixed with a vital stain (methylene blue), and a slide was prepared and examined microscopically.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at weeks 7 and 14 (termination)
- Blood collection method: Posterior vena cava
- Anaesthetic used for blood collection: CO2 anesthetized
- Animals fasted: yes
- How many animals: per dose group: 10 at week 7, 10 at week 14
- Serum was harvested by centrifugation of samples sublitted in commercial clot tubes and assayed using the manufacturer's methods. Globulin was determined by subtraction of the albumin value from the total protein value. These determinations were performed on a Hitatchi 717 clinical analyzer.
- Parameters checked: albumin, total protein, blood urea nitrogen (BUN) total bilirubin, dierct bilirubin, glucose, creatine phosphokinase (CPK), glutamic pyruvic transaminase (SGPT/ALT), gama glutamyl transpeptidase (GGT), alkaline phosphatase, glutamic oxaloacetic transaminase (SGOT/AST), creatinine, cholesterol, calcium, phosphorus (inorganic phosphate), chloride, sodium and potassium.

URINALYSIS: Yes
- Time schedule for collection of urine: week 14 (termination)
- Urine collection method: via metabolism trays over an approximately 18-hour period from fasted rats removed from their water source during collection.
- Animals fasted: Yes
- Analyses were carried out using Multistix reagent strips and a Clinitek urinalysis strip reader according to the manufacturer's methods. Urine specific gravity was determined using an optical total solids meter. Urine appearance was determined by inspection and reported by exception. If a urine sample had blood and/or protein in excess of control urine samples, sediment derived from centrifuging the sample was examined microscopically to determine the presence of bacteria, epithelial cells, erythrocytes, leukocytes, casts or abnormal crystals.
- Parameters checked: total volume, bilirubin, blood, glucose, ketone, pH, protein and urobilinogen, urine specific gravity,.

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Animals examined: all
- Scheduled sacrifice: 7 weeks (10 randomly selected animals/sex/level) and 14 weeks (all survivors).
- Extent of examination: external and internal. Internal cavities were opened and organs were examined in sity and then removed. Hollow organs were opened and examined.
- Organs weighed: liver and kidneys at the interim sacrifice; adrenals, brain, kidneys, heart, liver, spleen, testes with epididymides and ovaries at terminal sacrifice.
- Tissues retained: liver and kidneys at the interim sacrifice. The following tissues were retained from all other animals: aorta, adrenals, bone (sternum), bone marrow, brain, cecum, epididymis, esophagus, exorbital lacrimal gland, eyes, heart, kidneys, colon, gross lesions, lung, liver, lymph nodes (mesenteric, submandibular), mammary gland, muscle, nasal turbinates, nerve (sciatic), pancreas, prostate, pituitary, rectum, salivary gland (submaxillary), seminal vesicle, skin, small intestine (duodenum, jejunum and ileum), spinal cord, spleen, stomacht, testes, ovaries, thymus, thyroid/parathyroid, trachea, uterus, urinary bladder.
- Fixatives: eyes: 5% buffered neutral formalin / 0.5% glutaraldehyde - remaining tissues: 10% buffered neutral formalin

HISTOPATHOLOGY: Yes
- Tissue preparation: fixed tissues washed, dehydrated, embedded in paraffin, sectioned at approximately 5 microns and stained with hematoxylin and eosin
- Tissues examined: all retained tissues from animals that diet during the study and from those in the high level and control groups that were sacrificed at the termination of the study; lungs, livers and kidneys from the intermediate dietary levels groups that were sacrificed at the termination of the study and livers and kidneys from the intermediate dietary levels at the interim sacrifice.

Statistics:

The following statistical procedures were used to detect statistically significant differences between treated animals and their respective controls:

- Dunnett's Multiple Comparison Test (two-tailed) (3): inlife body weights, cumulative body weight changes, food consumption, urine pH and urine specific gravity.

- EHL decision-tree analysis: hematology data, clinical chemistry data, terminal body weights, absolute organ weights and organ/body weight ratios were evaluated by decision-tree statistical analyses which, depending on the results of tests for normality (1) and homogeneity of variances (Bartlett's Test (2)), utilized either parametric (Dunnett's Test (3) and Linear Regression (4)) or nonparametric (Kruskal-Wallis (5), Jonckheere's (6) and/or Mann-Whitney (7) Tests) routines to detect differences and analyze for trend.

- Fisher's Exact Test (one-tailed) (8); Cochran-Armitage Test for linear trend (9,10): incidence of microscopic lesions

- Other statistical routines used for some data included Grubbs Test (11,12) to detect outliers.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
There were 2 unscheduled deaths, one low level male and one mid level female, during this study. Neither death was considered test related.

BODY WEIGHT
There were no statistically significant differences in mean body weights of males. However, statistically significant decreases in body weights occurred frequently in females at all levels throughout the study. The frequency was the greatest in the mid level. The decreases did not occur in a dose related manner and exceeded 10% only on one occasion: day 84, mid level. At study termination there were no significant body weight differences. Therefore, the changes noted were not considered toxicologically significant.

FOOD CONSUMPTION
Animals administered the test material consumed similar amounts of food as did their respective control groups, with the exception of reduced food consumption in high level females during week 5.

CHEMICAL CONSUMPTION DATA
Overall study averages for consumption of test material, based on the target concentrations, were approximately 178, 533 and 1093 mg/kg bw/d in males and 222, 673 and 1334 mg/kg bw/d in females for the low, mid and high levels, respectively.

CLINICAL SIGNS
There were few clinical signs observed; mostly scabs, loss of hair and periorbital wetness. None of these signs were considered related to test material administration.

OPHTHALMIC EXAMINATIONS
Ophthalmic examination of the control and high level animals at the end of the study showed no ocular changes attributable to test material exposure. Only one lesion was observed in a control male.

CLINICAL PATHOLOGY
There were no changes in hematologic or urinalysis parameters that were associated with treament. Cholesterol was significantly elevated in high level males at 7 weeks. This was considered to be unrelated to treatment since a change of similar magnitude was not present at 14 weeks and there was a reverse trend in treated males at 14 weeks, indicating that these changes represented spontaneous variation. Total protein, albumin and calcium were also elevated in high level males at 7 weeks; however, the elevations were slight (7%) and were not considered biologically or toxicologically significant and were not present at 14 weeks. SGPT/ALT levels were decreased in males from all three levels at both 7 and 14 weeks and in females from the 2 highest levels at 7 weeks and from all 3 levels at 14 weeks. These changes were not considered to be significant since the direction of change was opposite that expected and may have represented interference by the test material with the analytical procedure.

GROSS AND MICROSCOPIC PATHOLOGY
Terminal body weights were decreased slightly in females from all dosage groups at 14 weeks, however, since pre-fasting weights were not significantly different from controls and there was no dose response these changes were not considered related to treatment.
Absolute renal weights and liver to body weight ratio were slightly elevated in high level males at 14 weeks. A slight increase in kidney to body weight ratio in mid level females at termination was not considered related to administration of the test material as there were no differences in absolute kidney weights in treated females and there was no dose-response relationship. Although absolute and relative braing weights were slightly higherin females from the two highest levels at 14 weeks, these were not considered biologically or toxicologically significant, since no other gross or microscopic findings were associated with them, and they were probabely related to the decrease in body weight observed after an overnight fast.
There were no treatment-related gross or microscopic lesions observed in animals from this study. The slight increase in renal mineralization in females at 14 weeks was not considered toxicologically significant since the changes were extremely mild, and such changes are common in females rats of this strain.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: dietary concentration

2

Dose descriptor:

NOAEL

Effect level:

ca. 1 093 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: acutal ingested dose

3

Dose descriptor:

NOAEL

Effect level:

ca. 1 334 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: actual ingested dose

4

Dose descriptor:

NOEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: dietary concentration

5

Dose descriptor:

NOEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: dietary concentration -- effect level based upon slight non-significant decreases in body weigths.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL for males and females in this study was considered to be 15000 ppm after 90days of feeding. The NOEL in this study was considered to be 15000 ppm for males and 2500 for females, based upon slight, non-significant decreases in body weights.

Executive summary:

The test substance was administered to 20 Sprague-Dawley rats/sex/group at levels of 0, 2500, 7500, and 15000 ppm in the feed. Ten male and 10 female rats/group were bled for clinicopathologic determinations, sacrificed and necropsied at approximately 7 weeks. At the end of the three months, the remaining animals were bled, sacrificed and necropsied. Tissues examined microscopically included all tissues retained from the control and highest dietary levels at the terminal sacrifice, kidneys and livers from animals of all levels sacrificed at week 7 and lungs, kidneys and livers from animals of all levels sacrificed at termination.

Analyses to verify the stability of the test material both neat and when mixed with the diet, the diet homogeneity and concentrations of the test material in the diet were performed with satisfactory results. Overall study averages for consumption of test material were approximately 178, 533 and 1093 mg/kg bw/d in males and 222, 673 and 1334 mg/kg bw/d in females for the low, mid and high levels respectively.

There were no changes in survival, body weight, food consumption, or clinical observations considered to be treatment related. There were no changes in hematology, blood chemistry, gross or microscopic pathology considered to be treatment related. In the absence of any correlative findings, slight increases in absolute renal weights and liver/body weight ratios in high level males, and increases in brain weights in mid and high level females were not considered to be related to treatment.

The NOAEL for males and females in this study was considered to be 15000 ppm after 90days of feeding. The NOEL in this study was considered to be 15000 ppm for males and 2500 for females, based upon slight, non-significant decreases in body weights.