Thiram

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:

10 Nov - 10 Dec 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Principles of method if other than guideline:

No guideline followed. Range-finding study with mice as test organism, used for a 78 week carcinogenicity study

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Thiram

EC Number:

205-286-2

EC Name:

Thiram

Cas Number:

137-26-8

Molecular formula:

C6H12N2S4

IUPAC Name:

thiram

Test animals

Species:

mouse

Strain:

CD-1

Remarks:

Crl:CD-1(ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc, Portage, Michigan, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: group mean was 24.3 - 24.8 g for males and 19.0 - 19.3 g for females
- Fasting period before study: not applicable
- Housing: individually in stainless steel, screen-bottomed cages
- Diet: Certified rodent chow #5002 (Purina Mills, Inc), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 Nov 1988 To: 10 Dec 1988

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: the appropriate amount of Certified Rodent Chow #5002 was weighed in to a Hobart mixing bowl. 250 g of food was taken from this bowl and added with the appropriate amount of the test material to a Waring blender. This premix was mixed thoroughly and then trasnferred to the Hobart mixing bowl. Then 100 g of additional food was blended to remove residual test material from the blender.
- Storage temperature of food: frozen until in use

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Data for stability and homogeneity of similar diets were available from a previous toxicity study.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

continuously via the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 ppm

Remarks:

corresponding to approx. 51 – 58 mg/kg bw/day for males and 59 – 66 mg/kg bw/day for females

Doses / concentrations 2

Dose / conc.:

600 ppm

Remarks:

corresponding to approx. 101 – 115 mg/kg bw/day for males and 111 – 127 mg/kg bw/day for females

Doses / concentrations 3

Dose / conc.:

1 200 ppm

Remarks:

corresponding to approx. 177 – 226 mg/kg bw/day for males and 221 – 281 mg/kg bw/day for females

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: yes (overnight)

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for moribundity and mortality and once daily for signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: immediately before start of treatment, on Day 0, weekly thereafter and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks of treatment
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes (overnight)
- How many animals: all animals of all groups (treatment and control)
- Parameters checked: red blood cell (RBC) count, haemoglobin (Hb), haematocrit (Hc), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), white blood cell count (WBC), differential leucocyte count, including nucleated red blood cell count (NRBC), corrected white blood cell count (COR WBC), segmented neutrophil count (N-SEG), band neutrophil count (N-BAND), lymphocyte count (LYMPH), monocyte count (MONO), eosinophil count (EOSIN), basophil count (BASO), morphology.

CLINICAL CHEMISTRY: Yes
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes (overnight)
- How many animals: all animals of all groups (treatment and control)
- Parameters checked: Glucose (GLU), urea nitrogen (UN), total protein (T PRO), albumin (ALB), globulin (GLOB), alanine aminotransferase (ALT)

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals were anesthetized with methoxyflurane, weighed, exsanguinated and necropsied.
Organs weighed: Brain, kidneys, liver with gallbladder, ovaries, testes

HISTOPATHOLOGY: Yes
- Tissues sampled in 10% buffered formalin: Adrenals, aorta, bone, bone marrow, brain, caecum, cervix, colon, duodenum, epididymides, eyes, femur, oesophagus, gallbladder, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, mammary gland, muscle, ovaries, pancreas, parathyroid, prostate, sciatic nerve, pituitary, rectum, salivary glands, seminal vesicles, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina.
- embedding media: Paraffin
- Staining: haematoxylin and eosin
- Examined for: all animals of the control and high-dose group. Macroscopic lesions, lungs, liver with gallbladder and kidneys were also examined microscopically from all animals of the low and mid dose group.

Statistics:

Levene's test was applied to test for heterogeneity of variance between treatments. Where significant heterogeneity was found, a logarithmic, square, square root, reciprocal, angular or rank transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance (ANOVA) was carried out. If significance was found, a Dunnett's t-test was used for pairwise comparison.

If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis H-test ANOVA was used. If this was significant, the Nemenyi-Kruskal-Wallis test for multiple comparisons or the Wilcoxon-Mann-Whitney two-sample rank test was used.

Statistical significance was defined at the 5% two tailed probability level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Control: alopecia (1/10 males)
- 300 ppm: hunched posture (1/10 males), animal was thin (1/10 males), alopecia (1/10 males), squinting of both eyes (1/10 males), bloody crust on the tail (1/10 females), red tail (1/10 females), necrotic tail (1/10 females).
- 600 ppm: animal was thin (1/10 females), alopecia (1/10 females), opaque red eye (1/females)
- 1200 ppm: hunched posture (1/10 males, 2/10 females), animal was thin (1/10 males, 2/10 females), alopecia (1/10 females), lacrimation in the left eye (1/10 males)

The hunched posture and the occurrence of thin animals was considered treatment related. All other signs were considered incidental.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 300 ppm: body weight and body weight gain were statistically significantly lower in males compared to the control group in Week 3 and 4.
- 600 ppm: body weight and body weight gain were statistically significantly lower in males compared to the control group in Week 4.
- 1200 ppm: body weight was statistically significantly lower in males compared to the control group in Week 3 and 4. Body weight gain was statistically significantly lower compared to the control in Weeks 2 - 4 for males and for Weeks 3 and 4 for females.

Summarized results can be found in Attachment 1 in the attached background material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 300 ppm: food consumption was statistically significantly decreased during the complete treatment period.
- 600 ppm: food consumption was statistically significantly decreased during the complete treatment period.
- 1200 ppm: food consumption was statistically significantly decreased during the complete treatment period.

Summarized results can be found in Attachment 1 in the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 300 ppm: decreased RBC (males -6.5%), slightly decreased Hb (males -6.2%), slightly decreased Hct (males -5.1%), all changes were statistically significantly different from the control group.
- 600 ppm: decreased RBC (males -7%, females -6.2%), slightly decreased Hb (males -5%), slightly decreased Hct (males -5.1%), increased platelet count (females +18.8%), all changes were statistically significantly different from the control group.
- 1200 ppm: decreased RBC (males -7.3%), slightly decreased Hb (males -8.6%), slightly decreased Hct (males -7.7%), increased platelet count (females +27.6%), all changes were statistically significantly different from the control group.

Although these changes were statistically significant and most were treatment-related, they were relatively small and not considered indicative of severe primary pathologic effects.

Summarized results can be found in Attachment 1 in the attached background material.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The only statistically significant difference was decreased glucose in females of the 1200 ppm group. This was not considered treatment-related but incidental. Apart from that, no difference was observed in clinical chemistry between the treatment and control groups.

Summarized results can be found in Attachment 1 in the attached background material.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: histopathology of cervix, epididymis, mammary gland, ovary, prostate, seminal vesicles, testis, thyroid, uterus, vagina and adrenals, organ weights were recorded for epididymis, liver, prostate, testis and brain. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 300 ppm: the relative brain weight was statistically significantly higher in males compared to the control group.
- 600 ppm: increased relative liver weight (males +12.7%), and kidney weight (males +15.1%), decreased absolute brain weight was observed in males (-6.2%), all differences were statistically significant compared to the control group.
- 1200 ppm: increased relative liver weight (females +11%, males +13.8%), all differences were statistically significant compared to the control group.

These changes are considered secondary to decreased terminal body weight.

Summarized results can be found in Attachment 1 in the attached background material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related difference was observed between control and treatment groups up to and including the highest dose level.

Summarized results can be found in Attachment 1 in the attached background material.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment related difference was observed between control and treatment groups up to and including the highest dose level.

Summarized results can be found in Attachment 1 in the attached background material.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Effect levels

1

Key result

Dose descriptor:

LOAEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 51 mg/kg bw/day.

2

Key result

Dose descriptor:

NOAEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects were observed at this dose level.

Remarks on result:

other: corresponding to 59 mg/kg bw/day.

3

Key result

Dose descriptor:

LOAEL

Effect level:

600 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 127 mg/kg bw/day.

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The present study was conducted to assess the sub-acute toxicity of test substance on mice when given for a time frame of 4 weeks. The study was not conducted according to any guideline as it served as a dose-range finder. It was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 10 male and female mice at dose levels of 300, 600 and 1200 ppm corresponding to approximately 51-58, 101-115 and 177-226 mg/kg bw/day for males and 59-66, 111-127 and 221-281 mg/kg bw/day for females.

There were no mortalities during the study. Body weights were significantly lower for males in the 300 and 1200 ppm groups during Week 3 and 4 and for males in the 600 ppm group for Week 4. Females showed no differences. Food consumptions were significantly lower for all treated animals throughout the study period. RBC count, Hb and Hc were slightly lower in all treated males. In females RBC count was decreased in the 600 ppm group while platelet count was increased at 600 and 1200 ppm. The only statistically significant alteration in clinical chemistry was the decreased glucose value in females dosed at 1200 ppm. Absolute organ weights alterations were not dose-response-related and regarded as a result of the lower terminal body weight. There were no treatment-related macroscopic or microscopic observations noted.

Based on reduced food consumption and enhanced platelet count, the no-observable-adverse-effect level (NOAEL) for the test substance when fed to female mice for 4 weeks was 300 ppm (corresponding to 59-66 mg/kg bw for females). Due to the effects observed in the low dosed male group, a NOAEL for males could not be determined.