Carbamic acid, N-[(butylthio)thioxomethyl]-, butyl ester

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-11-10 - 2007-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also conducted according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1995

GLP compliance:

yes (incl. QA statement)

Remarks:

2005-11-21

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

476-230-4

EC Name:

-

Cas Number:

39142-36-4

Molecular formula:

C10H19NO3S

IUPAC Name:

n-Butyoxycarbonyl-O-n-butyl thionocarbamate

Test material form:

liquid

Specific details on test material used for the study:

- IUPAC Name: n-Butyoxycarbonyl-O-n-butyl thionocarbamate
- Synonyms: O,O-dibutyl imidothiodicarbonate, S-10036
- CAS number: 39142-36-4
- Form: Liquid
- Lot/batch No.of test material: MR1143
- Storage condition of test material: At room temperature, in the dark

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 (UK) Limited, Margate, Kent, UK
- Age at study initiation: 5-8 weeks
- Weight at study initiation: males 143-172g; females 131-152g
- Fasting period before study: no
- Housing: groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): ad libitum, Rodent 5LF2 Certified Diet provided by BCM IPS Limited, London, UK
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by the laboratory using a validated HPLC method. The formulations were shown to be stable for at least 14 days. They were therefore prepared weekly and stored at 4°C in the dark.
Samples of each test material formulation were taken and analysed for concentration of the test substance at the laboratory. The results indicate that the prepared formulations were within +/- 5% of the nominal concentrations.

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a 14-day range-finder .
- Fasting period before blood sampling for clinical biochemistry: no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before and post dosing, 1 and 5 hours afer dosing during the work week or 1h after dosing on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule for examinations: prior to the start of the treatment and on days 3, 10, 17 and 34 observation for signs of functional/behavioural toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 and at weekly intervals thereafter and prior to terminal kill

FOOD CONSUMPTION:
Food consumption was recorded for each cage group at weekly intervals

WATER CONSUMPTION: Yes
- Time schedule for examinations: measured gravimetrically throughout the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All control and test animals
- Parameters examined: haemoglobin, total erythrocyte count, haematocrit, mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration, total leucocyte count, differential leucocyte count, platelet count, reticulocyte count, prothrombin time, activated partial thromboplastin time,

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: No
- How many animals: All control and test animals
- Parameters examined: urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, ASAT, ALAT, alkaline phosphatase, creatinine, total cholesterol, total bilirubin, cholinesterase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional performance tests were also performed on all animals during week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
- Dose groups that were examined: all
- Battery of functions tested: grip strength / motor activity / sensory reactivity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination.
Organ weights: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin and, for control and 100 mg/kg bw animals, subsequently prepared for microscopic examination: adrenals, bone & marrow (sternum), brain, caecum, colon, duodenum, epididymides, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, ovaries, prostate, rectum, sciatic nerve, seminal viscles, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus.
Other organs were also collected but not systematically prepared.
The liver, spleen, kidneys, theyroid and thymus from all 15 and 40 mg/kg dose group animals were also analysed because of indications of treatment-related changes.

Statistics:

Where appropriate (with exception of food and water consumption), quantitative data were analysed by the Provantis™ Tables and Statistics Module or the SPSS statistical program (for cholinesterase only). For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

On Day 1 of the study, incidents of hunched posture were detected for animals of either sex treated with 100 or 40 mg/kg/day. For animals treated with 100 mg/kg.day, this was accompanied by increased lachrymation for two males and one female; and noisy respiration for one female.
No such observations were detected for animals of either sex treated with 15 mg/kg/day.

Staining around the mouth was detected for one male treated with 100 mg/kg/day and one male treated with 15 mg/kg/day, these were isolated observations and are associated with the oral administration of an unpalatable test material formulation and in isolation are considered unrelated to test material toxicity. Fur loss was detected for two females treated with 100 mg/kg/day from Day 19 onwards and Day 22 onwards. This observation is occasionally seen following prolonged group housing and in isolation is considered to be incidental and not indicative of test material toxicity.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A reduction in bodyweight development was detected for males treated with 100 and 40 mg/kg/day during Week 1 of the study. This reduction persisted throughout the remainder of the study for 100 mg/kg/day males, whilst subsequent recovery was observed after the first week of treatment for males treated with 40 mg/kg/day.
No such observations were detected for females treated with 100 or 40 mg/kg/day and for animals of either sex treated with 15 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A reduction in food consumption was detected for animals of either sex treated with 100 mg/kg/day during the first week of treatment. This reduction subsequently recovered for 100 mg/kg/day females, whilst the reduction continued for 100 mg/kg/day males for the remainder of the study period.
No such observations were detected for animals of either sex treated with 15 or 40 mg/kg/day.

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

effects observed, treatment-related

Description (incidence and severity):

An increase in water consumption was detected for males treated with 40 or 100 mg/kg/day during the first week of treatment, and for all treated males (15, 40 or 100 mg/kg/day) for the remainder of the study when compared to controls. Females treated with 40 or 100 mg/kg/day had an increased water consumption throughout the study period.
No such observations were detected for females treated with 15 mg/kg/day.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 100 mg/kg/day showed a statistically significant reduction in haemaglobin, haematocrit and erythrocyte count and increase of reticulocyte count. In addition males of this treatment group showed reduction in neutrophil count, whilst females showed an increase in mean cell volume. At 40 mg/kg/day reductions in haemoglobin and haematocrit were detected for animals of either sex, whilst females of this treatment group also showed a statistically significant reduction in erythrocyte count and an increase in reticulocyte count (both effects also seen in males but not statistically significant).
No such observations were detected for animals of either sex treated with 15 mg/kg/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg/day, animals of either sex showed a decrease in total protein and an increase in alkaline phosphatase levels. In addition males had elevated cholinesterase and plasma urea levels; whilst females of this treatment group showed statistically significant reductions in both glucose and cholesterol levels. The reduction in plasma cholesterol levels extended to females treated with 40 and 15 mg/kg/day. However the reduction in cholesterol values showed no dose related trend and the actual difference between high dose and control values for females was small, so the relevance of the statistical significance is questionable.
Males treated with 40 mg/kg/day had reduced total protein levels and showed elevations in plasma glucose levels.
No treatment-related changes were detected in the blood parameters measured for males treated with 15 mg/kg/day.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- There were no treatment-related changes in the behavioural parameters measured (urination, defecation and transfer arousal scores).

- There were no toxicologically significant changes in functional performance parameters measured
A statistically significant decrease in hind limb grip strength was detected for males treated with 100 and 40 mg/kg/day, and in fore limb grip strength for females treated with 100 mg/kg/day. These findings were only detected for one out of the three trials carried out, and in the absence of clinical observations to suggest a neurotoxic effect, these findings were considered not to be related to test material toxicity. In addition, a statistically significant reduction in overall activity and overall mobility was detected for females treated with 100 mg/kg/day when compared to controls. As these findings were not detected for males treated with 100 mg/kg/day; and in the absence of evidence to suggest a neurotoxic effect, these findings were considered incidental and not indicative oftest material toxicity.
No such effects were detected for females treated with 40 mg/kg/day or for animals of either sex treated with 15 mg/kg/day.

- There were no treatment-related changes in sensory reactivity.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weight
Males: significantly increased liver weight at all doses; increased absolute liver weight for 15 and 40 mg/kg/day males (no effect for 100 mg/kg/day). A statistically significant difference in male absolute liver weight was not supported by group mean values and may well be due to the distribution of individual values.
Females: increased liver weight for 40 and 1000 mg/kg/day females

Kidney weight
Males: significantly increased kidney weight at all doses; increased absolute kidney weight for 15 and 40 mg/kg/day males and decreased for 100 mg/kg/day males. This is possibly due to a lower bodyweight detected for high dose males when compared to controls, and is supported by the increase in liver and kidney weight relative to terminal bodyweight for all treated males when compared to controls.
Females: no effects

Spleen weight
Males: increased spleen weight at 100 mg/kg/day
Females: increased spleen weight at 100 mg/kg/day

Thymus weight
All treated animals had reduced thymus weight

Brain weight
Males: no effects
Females: reduced brain weight for 100 mg/kg/day group. This reduction was of minimal statistical significance, and in the absence of histopathological correlates was considered to be an incidental finding and unrelated to treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Three males treated with 100 mg/kg/day had pallor of the liver and kidneys. In addition three females treated with 100 mg/kg/day and one treated with 40 mg/kg/day had pallor of the kidneys.
Three males treated with 100 mg/kg/day had small seminal vesicles, but in the absence of supporting microscopic effects, this finding was considered unrelated to treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver:
Histopathological changes characterised by centrilobular hepatocyte enlargement, vacuolation of centrilobular hepatocytes, centrilobular hepatocyte inflammatory cell infiltrates, pigment deposits, and a higher incidence and generally higher grades of severity of generalised hepatocyte vacuolation (glycogen type in appearance), were seen in relation to treatment for animals of either sex treated with 100 mg/kg/day, and to a lesser extent for animals treated with 40 and 15 mg/kg/day. Vacuolation of centrilobular hepatopcytes was demonstrated to be a consequence of lipid accumulation by frozen sections stained with Oil Red O. Pigment deposits stained positively with Perl's stain and were thus likely to be haemosiderin.

Centrilobular hepatocyte degeneration and necrosis were observed for animals of either sex treated with 100 mg/kg/day; for one female and two males treated with 40 mg/kg/day and for three males treated with 15 mg/kg/day. This is considered to be an adverse morphological change.

Spleen:
Higher grades of severity of extramedullary haempoiesis and haemosiderin pigment deposition, positively stained with Perl's stain, were seen in relation to treatment for animals of either sex treated with 100 mg/kg/day. Higher grades of severity of extramedullary haemopoiesis were also seen for males treated with 40 and 15 mg/kg/day, and higher grades of severity of pigment deposition were seen for females treated with 40 mg/kg/day.

Kidneys:
Tubular basophilia and karyomegaly of tubular cells affecting tubules of the inner cortex were seen for animals of either sex treated with 100 mg/kg/day, but not convincingly at any other treatment level.

Thyroid:
Follicular cell hypertrophy was observed in relation to treatment for animals of either sex treated with 100 and 40 mg/kg/day.

Thymus:
Atrophy of the thymus was seen as a variable response among treated animals of either sex but there was no evidence of a dose relationship particularly for the females, where no atrophy was observed at 100 mg/kg/day. The atrophy could just be a coincidental finding but if it is associated with treatment, this would probably be only as a secondary effect.


All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Effect levels

Dose descriptor:

NOEL

Effect level:

< 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Adverse effects on the liver

Remarks on result:

not determinable

Remarks:

Adverse effects on the liver were seen even at the lowest concentration tested.

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material, to rats for a period of 28 consecutive days at dose levels of 100, 40 and 15 mg/kg/day resulted in toxicologically significant effects for animals of either sex from all dose levels. The 'No Observed Effect Level' (NOEL) was, therefore, not achieved.

Executive summary:

The systemic toxicity of the test material was investigated in a study conducted according to OECD TG 407 and GLP principles.

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley rats, for twenty-eight consecutive days, at dose levels of 15, 40 and 100 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 100, 40 and 15 mg/kg/day resulted in toxicologically significant effects for animals of either sex from all dose levels. In particular, centrilobular hepatocyte degeneration and necrosis were observed for animals of either sex treated with 100 mg/kg/day; for one female and two males treated with 40 mg/kg/day and for three males treated with 15 mg/kg/day. This is considered to be an adverse morphological change.

The 'No Observed Effect Level' (NOEL) was, therefore, not achieved.

The CLP guidance value (for a 28-day study) for classification as STOT-RE Category 1 is <= 30 mg/kg bw/day when significant toxic effects are seen to occur within this guidance range. The effects observed at 15 mg/kg bw/day support classification based on the CLP criteria as they were considered to be adverse changes (liver weight changes associated with cellular degeneration and necrosis).

The LOAEL in this study is considered to be 15 mg/kg/day, and applying the security factors of GHS classification due to the fact that this is a 28 day study, this substance is considered as STOT RE 1 for its liver effects.