Benzimidazole-2-thiol

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

Based on the data available, the No Observed Adverse Effect Level (NOAEL) for the test chemical was considered to be less than 2 mg/Kg bw using male and female rats. Hence the test chemical is likely to be toxic upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: inhalation

Based on the data available, the Low Observed Adverse Effect Concentration (LOAEC) for the test chemical was considered to be 3.1 mg/m³ using male and female rats. Hence the test chemical is likely to be toxic upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: dermal

The OECD study result for acute toxicity by the dermal route indicates the LD50 value to be greater than 2000 mg/kg body weight. Also considering the use of the chemical as anti-oxidant for natural rubber and latex and considering the volatility absorption by the dermal route is not considered to be significant. Thus, given the above considerations, it is assumed that test chemical shall not exhibit repeated dose toxicity by the dermal route.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

The present 28-day repeated dose oral toxicity study of test substance followed by 2-week recovery examination in Wistar rats was conducted to evaluate adverse effects and their reversibility

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SLC Co. (Shizuoka, Japan)
- Age at study initiation: 5 weeks old
- Weight at study initiation: male 92 g, females 80g
- Fasting period before study: 16 hours
- Housing: Rats were housed in plastic cages (5 rats/cage) using chip bedding.
- Diet (e.g. ad libitum): The basal pellet diet (F-2); ad libitum
- Water (e.g. ad libitum): Tap water; ad libitum
- Acclimation period: One week prior to the initiation of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±1°C
- Humidity (%):55±5%
- Photoperiod (hrs dark / hrs light): 12 hr light/dark cycle

Route of administration:

oral: gavage

Details on route of administration:

No data

Vehicle:

corn oil

Remarks:

2%

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: test chemical was dissolved or suspended in corn oil and administered to rats by the i.g route using disposable gavage tube.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 0, 2.0, 10.0 or 50.0 mg/Kg
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of test substance in corn oil was examined by HPLC after extracting the test compound with methanol. The test chemical was confirmed to be stable for atleast 1 week at room temperature

Duration of treatment / exposure:

28 days (14 days recovery period)

Frequency of treatment:

Daily for two consecutive weeks

Dose / conc.:

0 other: mg/kg

Remarks:

Treatment period

Dose / conc.:

2 other: mg/kg

Remarks:

Treatment period

Dose / conc.:

10 other: mg/kg

Remarks:

Treatment period

Dose / conc.:

50 other: mg/kg

Remarks:

Treatment group

Dose / conc.:

0 other: mg/kg

Remarks:

Recovery period

Dose / conc.:

50 other: mg/kg

Remarks:

Recovery period

No. of animals per sex per dose:

Total: 80
0 mg/Kg: 10 males and 10 females
2.0 mg/Kg: 10 males and 10 females
10.0 mg/Kg: 10 males and 10 females
50.0 mg/Kg: 10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: For the dose-determining study, male and female rats (5 rats/group) were orally administered 80 mg, 40, 20, 10 and 5 mg/kg of 2-MBI for a consecutive 2 weeks. Taking into account the observed reduction in body weight gain in groups receiving more than 40 mg/kg and the 2 weeks longer treatment period for the 28-day repeated oral toxicity study, doses of 0 (control, corn oil alone), 2, 10 and 50 mg/kg of 2-MBI were administered by gavage to groups of 10 male and 10 female rats for 28 consecutive days.
.
- Rationale for animal assignment (if not random): No data
- Rationale for selecting satellite groups: Yes
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. The animals were observed for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Clinical signs were monitored throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Three days prior to the initiation of the treatment. On the first day of treatment, and then twice weekly throughout the study.

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;once a week

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 animals/sex
- Parameters checked : Red blood cells (REC), hemoglobin (Hb), hematocrict (HCT), mean corpuscular volume (MCV), mean hemo-globin concentration (MHC), mean corpuscular homoglobin concentration (MCHC), platelets (PLT) and white blood cells {WBC), blood clotting time

CLINICAL CHEMISTRY: Yes
- Animals fasted: No data
- How many animals: 5 animals/sex
- Parameters checked : Total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), glucose (GLC),non esterified fatty acid (NEFA), phospholipid (PL),triglyceride (TG), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE). aspartate aminotransferase (AST), alanine aminotransferase (ALT). gamma -glutamyl transpeptidase ( y -GTP), leucine aminopeptidase CLAP ), lactate dehydrogenase (LDH). calcium (Ca),magnesium (Mg2+), inorganic posphorus (Pi), sodium (Na+), potassium (K+). and chlorine (CI-) were analyzed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The weights of the brain, heart, lungs,liver, kidneys, spleen, adrenals, testes, ovaries, pituitary, thymus, submaxillary glands and thyroid glands of each animals were measured.

HISTOPATHOLOGY: Yes
The brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary, thymus, submaxillary glands and thyroid glands and the esophagus, stomach, small and large intestine, pancreas, ischiatic nerve, urinary bladder, seminal vesicles, uterus, prostate and, mysenteric lymph nodes as well as samples of spinal cord, skeletal muscle and bone marrow were fixed in 10% buffered formalin solution for routine histological processing. Paraffin sections were stained with hematoxylin and eosin for histopatbological examination.

Statistics:

All quantitative data, except for the histopathological findings were statistically analyzed by one-way analysis of variance (ANOVA) techniques with Dunnett's or Scheff ’s multiple comparison procedures. Significance was established at the p<0.05 level.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

Clinical signs and mortality:
Mortlaity: No mortality was noted in male and female rats
Clinical signs: No signs of toxicity were observed in the tested animals

Body weight and weight gain: Decrease in body weight gain was observed. The rats of both sexes receiving 50 mg/kg showed emaciation and severe suppression in body weight gain one week after treatment started. No significant differences in body weight gain were observed in the groups that received 10 mg/kg 2-MBI or less.

Food consumption and compound intake: Decreased food consumption one week after treatment started was observed.

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology: At the termination of the treatment, significant decreases in WBC in the 10 and 50 mg/kg female rats were observed. At the end of the recovery period, RBC, Hb and HCT were significantly decreased in both the male and female 50 mg/kg groups showing delayed onset of anemia. Increased active partial thromboplastin time was observed for both the males and females receiving 50 mg/kg.

Clinical chemistry: Serum levels of TP, BUN, PL, T-CHO, F-CHO,CHE and ɣ -GTP were significantly increased in both males and females given 50 mg/kg. ALP,K+ and Pi were decreased significantly in male rats receiving more than 10 mg/kg , and Na+ was increased significantly in the 50 mg/kg trated animals. CHO and PL levels remained significantly high 2 weeks after termination.

Urinanalysis: No data

Neurobehaviour: No data

Organ weights: Dose related inncreases in absolute and relative weights of thyroid, liver and kidney were observed. At 10 mg/kg, the mean absolute and relative thyroid weights in both sexes were approximately 3 times those of the control rats and in the 50mg/kg dose group the increase in relative thyroid weight was more than 10-fold. Dose related decreases in absolute and relative thymus weights in all the treatment groups of male and female rats and a derease in relative spleen weight in both males and females receiving 50 mg/kg were also observed. Significant increases in brain, lung, adrenal and pituitary gland weights and decreases in heart and sabmaxilIary gland and of relative organ weights were also observed with 50 mg/kg. Reduction in thymus weight was also observed in a dose-dependent manner without significant histopathological alteration.

Gross pathology: Marked enlargement of thyroid glands and thymus involution were evident. Diffuse hyperplasia of tall and columnar epithelial cells of follicles, and decrease in colloid and thickening of fibrous capsule appeared.

Histopathology:Hypertrophic cells in the anterior pituitary glands were found, Calcification of the collecting tubules in kidneay and fatty changes in adrenal cortex was observed

Dose descriptor:

NOEL

Effect level:

< 2 other: mg/kg/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on the significant decrease in thymus weight in the 2 mg/kg 2-MBI treatment group.

Remarks on result:

other: No toxic effects were observed

Critical effects observed:

not specified

Table: Hematological findings for rats after 28 days and 2 week recovery period

Group	Treatment				Recovery
No. of animals	Control 5	2 mg/Kg 5	10 mg/Kg 5	50 mg/Kg 5	Control 5	50 mg/Kg 5
Male
RBC	9.00±0.10	9.07±0.37	8.60±0.19	8.66±0.25	9.86±0.19	7.49±0.13**
Hb	15.8±0.4	15.9±0.4	15.4±0.4	15.7±0.4	16.3±0.3	13.7±0.2**
HCT	47.1±0.4	46.7±1.6	45.8±1.6	44.0±1.5**	48.0±0.6	39.8±0.9**
PLT	0.81±0.08	0. 84±0.09	0.79±0.18	0.63±0.05**	0.84±0.13	1.06±0.08*
WBC	7.36±0.83	6.66±0.36	6.58±0.35	6.46±0.83	8.22±0.68	6.30±0.40
PT	14.6±0.6	14.3±0.8	14.8±0.8	14.9±0.9	16.3±0.5	15.2±0.3**
APTT	24.7±2.1	24.2±4.1	25.6±2.7	35.6 v0.8*	30.0±6.7	25.2±2.6
Female
RBC	9.01±0.14	9.02±0.12	8.77±0.58	9.00±0.51	8.58±0.20	6.64±0.08
Hb	16.2±0.2	16.2±0.3	15.9±1.0	16.3 v0.7	15.6±0.3	12.7 v0.5**
HCT	46.2±1.0	45.8±1.0	45.1±31	45.1±2.6	44.6±0.9	34.8± 0.6**
PLT	0.72±0.08	0.71±0.21	0.78±0.16	0.67±0.15	1.04±0.06	1.22±0.08**
WBC	8.78±1.05	7.62±1.33	5.98±1.06**	5.24±0.31**	5.84±0.65	6.24±1.2

*, **: Significantly different from the relevant control at p<0.05, p<0.01, respectively

Table no 2: Biochemical findings for male rats after 28 days of treatment with test substance & 2-week recovery Period

Groups	Treatment				Recovery
	control	2mg/Kg	10 mg/Kg	50 mg/Kg	Control 5	50 mg/Kg
TP	5.98±0.16	6.01 ±0.15	6.20 ±0.22	6.99 ±0.08	6.32 ±0.16	5.79 ±0.10
ALB	4.25 ±0.07	4.24 ±0.12	4.33± 0.10	4.86 ±0.06	4.38 ±0.05	3.94 ±0.09
BUN	8.89±1.26	8.09±0.89	6.65±0.31	12.77±0.49	12.1±1.40	11.1±0.4
CRN	0.28±0.03	0.27±0.04	0.24±0.02	0.28±0.02	0.34±0.07	0.24±0.02
GLC	118±4	120±13	124±6	124±6	130±7	108±8
PL	103±3	108± 9	107± 9	226± 16	121± 5	160± 12
TG	76± 15	82 ±14	74 ±23	65 ±10	157± 23	85± 12
T-CHO	52± 4	54± 6	65± 7	180 ±11	59± 2	105 ±11
F-CHO	7.6± 1.6	7.1± 2.3	10.0 ±1.7	46.4± 3.5	11.4 ±1.6	23.1 ±3.7
ALP	323± 29	312 ±9	206 ±24	187± 26	198± 11	262 ±28
AST	82± 8	81± 4	69± 8	54± 3	62± 16	64± 6
CHE	174 ±15	186± 21	536 ±53	2013± 289	173 ±32	480± 42
-GTP	1.29± 0.40	1.33 ±0.22	1.42 ±0.37	1.98 ±0.09	0.01± 0	0.01± 0
LAP	47± 2	45± 1	47± 2	65± 4	42± 2	48± 2
Ca	9.9± 0.1	9.9± 0.2	9.9 ±0.2	9.8± 0.3	10.5± 0.1	10.2± 0.2
Pi	7.9 ±0.3	7.7 ±0.3	7.1± 0.3	5.4± 0.2	7.3± 0.3	7.6± 0.3
Na	137± 1	137± 1	138 ±1	142 ±1	136± 1	136± 1
K	5.2± 0.2	4.9± 0.3	4.7± 0.2	3.5 ±0.1	4.3± 0.3	5.0± 0.2
Cl	100± 0	99± 1	98 ±2	98± 1	99± 1	103± 1

Table no : Biochemical findings for female rats after 28 days of treatment with test substance & 2-week recovery Period

Groups	Treatment				Recovery
	control	2mg/Kg	10 mg/Kg	50 mg/Kg	Control 5	50 mg/Kg
TP	5.92 0.16	5.81 0.15	5.88 0.06	6.70 0.23	6.29 0.23	6.31 1.05
ALP	4.30± 0.12	4.29± 0.10	4.29± 0.05	4.60± 0.14	4.44± 0.19	3.78± 0.08
A/G	2.67± 0.18	2.83± 0.11	2.71± 0.10	2.19± 0.13	2.41± 0.15	1.66± 0.5
CRN	0.29 ±0.04	0.29 ±0.04	0.27± 0.03	0.41 ±0.07	0.32± 0.01	0.30± 0.02
PL	154 ±8	139 ±9	123± 7	265 ±22	173± 14	187 ±14
TG	52± 2	49± 12	41± 4	65 ±12	55± 11	64 ±15
T-CHO	85± 7	74± 5	73± 6	208 ±22	95± 4	121± 13
F-CHO	19.0± 2.0	15.8± 1.4	15.2± 1.7	59.1± 4.0	23.3± 1.5	30.0± 3.1
ALP	200± 29	200± 28	138± 7	183± 51	131± 12	153± 18
AST	74± 7	75± 6	68± 4	62± 4	63± 4	61± 5
CHE	1230 ±235	1350± 146	1380± 152	2096± 41	1566± 293	1421± 147
-GTP	0.70 ±0.18	0.57± 0.21	0.69± 0.24	1.25± 0.31	0.03 ±0.02	0.10± 0.21
LAP	46± 3	44 1	45 1	68 3	43 3	48 3
Pi	6.4 0.3	6.1± 0.2	6.1± 0.3	5.5± 0.2	5.1± 0.3	6.8 ±0.3
Na	138± 0	138± 1	140± 1	143± 2	137± 0	135± 1
K	4.5± 0.2	4.4± 0.2	3.9± 0.1	3.1± 0.1	4.4± 0.2	4.6 ±0.1

 

 

Conclusions:

The no-observed adverse effect level (NOAEL) of test substance in oral gavage study was considered to be less than 2 mg/kg/day based on the significant decrease in thymus weight in the 2 mg/kg test substance treatment group.

Executive summary:

In order to determine the oral toxicity of test substance, a 28-day repeated dose toxicity study in Wistar rats followed by observation over a 14-day recovery period was conducted at dose levels of 0, 2, 10 and 50 mg/kg administered by gavage. No toxic deaths occurred due to test substance treatment. Decreases of body weight gain and food consumption in the 50 mg/kg dose group were observed during the second half of the treatment period. In addition, hematological examination and serum biochemical tests revealed decreased white blood cells and hemoglobin and increased serum urea nitrogen, cholesterol, phospholipid, ɣ-glutamyl transpeptidase and the Na+/K+ ratio in the 50 mg/kg dose group. Marked thyroid enlargement (to 10 fold the control weight), histopathologically associated with diffuse hyperplasia of follicles with decreased colloid and thickening of the fibrous capsule, was found. Reduction in thymus weight was also observed in a dose-dependent manner without significant histopathological alteration. The no-observed adverse effect level (NOAEL) of test substance in oral gavage study was found to be less than 2 mg/kg/day based on the significant decrease in thymus weight in the 2 mg/kg test substance treatment group.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The data is K2 level as the data has been obtained from the experimental study from the reliable journal

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Peer reviewed journal research article

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

A 13 weeks subchronic inhalation study was conducted to investigate the toxicologic effects of test substance aerosols and to identify target organs, differences in sensitivity between sexes, and dose-response relationships with repeated exposures at various concentrations.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY).
- Age at study initiation: 6 to 7 weeks of age
- Housing: Animals were maintained in stainless steel wire-mesh cages in 2-m3 inhalation chambers.
- Diet (e.g. ad libitum): NIH-07 open formula diet; ad libitum except during exposures.
- Water (e.g. ad libitum): Filtered City of Chicago drinking water was supplied ad libitum via an automatic watering system.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 75 +/- 3°F
- Humidity (%):55 ± 15%
- Air changes (per hr): 15 ± 2 changes per hour.
- Photoperiod (hrs dark / hrs light): A 12-hr light/dark cycle (6 AM to 6 PM light)

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

< 3 µm

Remarks on MMAD:

MMAD / GSD: The mass median aerodynamic diameter (MMAD) was less than 3.0 µm and the geometric standard deviation (σg) was in the range of 1.9 to 3.0.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A wet dispersion technique using a pneumatic spray nozzle was devised to generate aerosols of test chemical from its 10% aqueous suspension.
- Source and rate of air: 0.03-in.diameter orifice at a pressure of 250 psig.
- System of generating particulates/aerosols: aerosol spray was collected with a cyclone mist collector for use in the aerosol generators.
- Temperature, humidity, pressure in air chamber: 150°C
- Method of particle size determination: The cyclone was designed to remove any droplets or natomized liquid greater than 15 pm diameter.

TEST ATMOSPHERE
- Brief description of analytical method used: RAMS real-time aerosol monitors, Quartz Crystal Microbalance (QCM)-based cascade impactor
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

-6 hr/day, 5 day/week, for 13 weeks.
-A control group of 10 rats/sex was exposed to filtered air. At least two consecutive exposures were given immediately prior to the scheduled termination.

Dose / conc.:

0 mg/m³ air

Dose / conc.:

3.1 mg/m³ air

Dose / conc.:

6.2 mg/m³ air

Dose / conc.:

12.5 mg/m³ air

Dose / conc.:

25 mg/m³ air

Dose / conc.:

50 mg/m³ air

No. of animals per sex per dose:

Groups of 10 rats/sex

Control animals:

yes

Details on study design:

Nineteen additional male rats were included in each of the control and the 3.1, 12.5, and 50 mg/m’ groups (special study) for scheduled collection of serum samples for thyroid hormone radioimmunoassay (RIA).

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations performed weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured at exposure initiation, weekly thereafter, and at necropsy.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 14 days
- Anaesthetic used for blood collection: Yes (with 70% CO2)
- Animals fasted: No
- How many animals: 5
- Parameters checked: Erythrocyte count and indices; leucocyte count and differentials; hemoglobin, hematocrit,reticulocyte, and platelet counts; and prothrombin and activated partial thromboblastin times (Hematologic determinations were performed with a Baker 9000 hematology analyzer).

CLINICAL CHEMISTRY: Yes
- Animals fasted: No
- How many animals: 5
- Parameters checked: albumin, total protein, blood urea nitrogen,creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatatase, glucose, total cholesterol,serum cholinesterase, sorbitol dehydrogenase, lactic dehydrogenase, total bihrubin, and free fatty acids.(clinical chemistry tests were performed with a Baker Centrifichem 500 automated analyzer (Serono-Baker, Allentown, PA).)

OTHER:
MORTALITY: YES
Animals were observed twice each day for mortality/moribundity

Sacrifice and pathology:

Rats necropsied at 2, 4, or 8 weeks of exposure.
All animals (excluding special study groups in the subchronic study) received a complete necropsy and were examined for gross lesions.

GROSS PATHOLOGY: Yes
Liver, thymus, thyroid, right kidney, right testis, heart, brain, and lung weights were measured.

HISTOPATHOLOGY: Yes
The following tissues were collected for histopathologic examination: gross lesions and tissue masses, lymph nodes (bronchial, mediastinal, mandibular, and mesenteric),
mammary gland with adjacent skin, thigh muscle, salivary gland, femur including marrow, rib (costochondral junction), nasal cavity and turbinates, tongue, larynx, pharnyx
and trachea, lung and mainstem bronchi, heart and aorta,thymus, thyroids, parathyroids, esophagus, stomach, large and small intestines, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, preputial or clitoral glands, prostate, testes, epididymides, seminal vesicles, scrotal sac, vagina, ovaries, uterus, brain and pituitary, spinal cord, sciatic nerve, eyes, and Zymbal’s glands. Tissues were fixed in 10% neutral buffered formalin, trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and
eosin.

Statistics:

Organ weights and organ weight/body weight ratios were analyzed by one-way (by sex) analysis of variance (ANOVA) followed by a Dunnett’s test when a significant F ratio was obtained. Thyroid hormone data were analyzed by ANOVA followed by either a Dunnett’s test, a one sample t test, or a Mann-Whitney test (RS/Explore software, version 1.1. Serial No. V-658, BBN RS/Expert Limited Partnership, BBN Software Products Corp., Cambridge, MA). TSH results were analyzed following exclusion of outliers, as determined by the method of Dixon ( 1953). Clinical chemistry results were analyzed by ANOVA and a Dunnett’s test using LABCAT software (Innovative Programming Associates, Inc., Princeton, NJ).The level of significance was p ≤ 0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The principle clinical signs of toxicity seen in rats exposed at 25 or 50 mg/m3 included hunched posture, emaciation, and hypoactivity.

Mortality:

mortality observed, treatment-related

Description (incidence):

Ten of the 19 male rats and all 10 females exposed to 50 mg/m3 died. One female control rat also died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A dose-related decrease in body weight gain occurred in both sexes exposed to 25 mg/m3 or greater, with adverse changes generally apparent after 3-4 weeks of exposure. Comparison of initial and final body weight group means indicated essentially no weight gain during the exposure period in either sex exposed at the 50 mg/m3 level or in females exposed at 25 mg/m’. No significant adverse body weight effects were seen in the rats exposed at concentrations of 12.5 mg/m3, or less.

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 specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Concentration-dependent anemia with decreased RBC counts and hemoglobin and hematocrit values was seen at levels of 12.5 mg/m3 and above. In the male rats of the 50 mg/m3 group and the female rats of the 25 mg/m3 group, prothrombin and activated partial thromboplastin times were increased. The leukocyte counts were depressed for the males of all concentration groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The 25 mg/m3 males exhibited elevated levels of aspartate aminotransferase, alinine aminotransferase, alkaline phosphatase and sorbitol dehydrogenase activity, while both sexes of the 25 mg/m3 group had increased levels of blood urea nitrogen and cholesterol as well as decreased blood levels of free fatty acids.
With regard to the thyroid hormones, the male rats of the 12.5 g/m3 group showed a drop in T3 levels at the 2, 4, and 8 week ,which however, was reversible after 13 weeks. In the 50 mg/m3 goup, T3 level were depressed at 2 weeks, but found to have recovered to control levels by the end of the study.Test chemical also produced a marked dose-related reduction in T4 levels. At the highest test chemical exposure level no serum thyroxine was detected after 2 weeks of exposure, with levels remaining below the limit of detection for the remainder of the exposure. Rats exposed to 12.5 mg/m3 had decreased T4 at 2, 4,or 8 weeks, with recovery by termination of the study, while exposure at 3.1 mg/m3 did not significantly reduce T4 levels.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Dose-related increases in absolute and relative thyroid weights occurred in both sexes of rats. At 6.2 mg/m3, the mean absolute and relative thyroid weights of either sex were approximately twice the values of the respective controls,with exposure at 25 or 50 mg/m3 resulting in increased relative weights of these organs of approximately 5 to 10 times the control weights. Increased relative liver weights were seen in males exposed at 6.2 mg/m3, or greater, and in females exposed at 3.1 mg/m3, or greater. Absolute and relative thymus weights were significantly reduced in both sexes exposed to test chemical.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy observations included dark/red adrenal glands, discolored skin of the toes, and enlarged thyroid glands in exposed rats.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological changes was seen in several organs associated with the most notable being organs of the endocrine system.
Histopathologically, both sexes of the groups receiving 3.13 g/m3 or higher doses exhibited thyroid follicular cell hyperplasia, the extent of which was concentration-dependent.
Adrenal cortical necrosis occurred in rats at the highest exposure level. This same lesion occurred at the 25 mg/m3 level, mixed with degeneration of the
zona reticularis of the adrenal cortex.
Thymic atrophy was noted with high incidence in rats exposed at 25 or 50 mg/m3. Hepatocyte hypertrophy occurred in the animals exposed at 50 mg/m3.
Histopathological lesions were noted in the kidneys (tubular atrophy and mineralisation from 25 mg/m3), the adrenal gands (necrosis and degeneration from 25 mg/m3), the pancreas (hyperplasia of islet cells from 25 mg/m3), the mysentric lymp nodes (hyperplasia from 25 mg/m3), the pituitary gland (cytoplasmic vacuolization from 12.5 mg/m3, proliferation of the thyrotrophic hormone-producing basophilic cells of the anterior adenohypophysis), the bone marrow (hypocellularity from 25 mg/m3) and the nasal cavity (cystic degeneration of the respiratory epithelium from 25 mg/3). Thyroid hyperplasia and decrease in thymus weight thus proved to be most sensitive parameters.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Dose descriptor:

LOAEC

Effect level:

3.1 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Subchronic inhalation study of test chemical : aerosol exposure conditions

 

Experimentally determined aerosol mass concentrations				Aerosol particle size distribution
Aerosol target concn (m/m3)	Mean (mg/m3)	%RSD	N	MMAD	σg
3.1	3.1	12.1	66	2.0	2.9
6.2	6.2	8.0	66	2.3	2.9
12.5	12.5	11.5	66	2.2	2.8
25.0	25.1	11.2	66	2.0	2.1
50.0	51.1	7.5	66	2.3	2.4

 

6 hr/day, 5 days/week

Determined from six daily gravimetric filter-collected aerosol samples over 66 exposure days.

The values are means of four determinations.

Relative standard deviation.

Mass median aerodynamic diameter and geometric standard deviation.

 

Conclusions:

Thus, on the basis of the study the LOAEC (lowest observed adversed effect concentration) for test chemical in male and female rats was considered to be 3.13 mg/m3.

Executive summary:

The inhalation toxicity of test chemical was evaluated in male and female F344/N rats exposed 6 hr/day, 5 days/week to respirable aerosols generated by spray atomization of aqueous suspensions of the test chemicalpowder and subsequent drying of the resulting aerosols. Subchronic exposures were conducted at target concentrations of 0, 3.1, 6.2, 12.5, 25.0, and 50.0 mg/m3 of test chemical. Rats at ≥25 mg/m3 displayed hunched posture, hypoactivity, and reduced body weight gain, with compound related mortality at the highest exposure level. Anemia; increased SGPT, SGOT, alkaline phosphatase, sorbitol dehydrogenase, BUN, and cholesterol; and reduced free fatty acid were seen in rats at ≥25 mg/m3. Increased thyroid weight and thyroid follicular cell hyperplasia were noted in both sexes at ≥6.2 mg/m3, with reduced triicdothyronine and thyroxine levels in both sexes at ≥12.5 mg/m3. Thyroid follicular cell hyperplasia was also seen in rats at 3.1 mg/m3. Thymus weights were significantly reduced in both sexes at all exposure levels with liver weight increases at ≥6.2 mg/m3. Exposure-related histopathologic changes included pituitary cytoplasmic vacuolization, adrenal cortex necrosis, lymphoid depletion, thymic atrophy, liver cell hypertrophy, renal mineralization and tubular atrophy, and hypocellularity of the bone marrow. Effects observed even at lower concentration of 3.13 mg/m3 so that it was not possible to determine a no observable effet level in this study. Thus, on the basis of the study the LOAEC (lowest observed adversed effect concentration) was considered to be 3.1 mg/m3.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

3.1 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The data is K2 level as the data has been obtained from the experimental study from the reliable journal '

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: Oral

Data available form various studies for the test chemicals was reviewed to determine the toxic nature of test chemical .The studies are as mentioned below:

Study 1

In order to determine the oral toxicity of test substance, a 28-day repeated dose toxicity study in Wistar rats followed by observation over a 14-day recovery period was conducted at dose levels of 0, 2, 10 and 50 mg/kg administered by gavage. No toxic deaths occurred due to test substance treatment. Decreases of body weight gain and food consumption in the 50 mg/kg dose group were observed during the second half of the treatment period. In addition, hematological examination and serum biochemical tests revealed decreased white blood cells and hemoglobin and increased serum urea nitrogen, cholesterol, phospholipid, ɣ-glutamyl transpeptidase and the Na+/K+ ratio in the 50 mg/kg dose group. Marked thyroid enlargement (to 10 fold the control weight), histopathologically associated with diffuse hyperplasia of follicles with decreased colloid and thickening of the fibrous capsule, was found. Reduction in thymus weight was also observed in a dose-dependent manner without significant histopathological alteration. The no-observed adverse effect level (NOAEL) of test substance in oral gavage study was found to be less than 2 mg/kg/day based on the significant decrease in thymus weight in the 2 mg/kg test substance treatment group.

Study 2

 

In order to evaluate the toxicological properties of existing chemical substances, test substance widely used in the rubber industry as a vulcanization accelerator or an antioxidant for rubber was administered orally to male and female rats once a day for 28 days, its toxicity was examined. For some animals, a recovery period of 14 days was established. The dosage was 1.2, 4, 12 and 40 mg / kg, and the vehicle (0.5% CMC) administration group was provided as a control. In females in the 40 mg / kg group, poor coat gloss was found in a few cases from day 18 after administration to recovery 8 days. In our group, one patient died on the 24th day of administration. Significant inhibition of body weight gain was observed in male of 12 mg / kg group from 18th day of administration, males in 40 mg / kg group from 11th day of administration, females from 15th day of administration, until the final administration day. During the recovery period, a significant low value of body weight was continuously observed in both males and females in the 40 mg / kg group. Significant lower values ​​of food intake were observed from males in the 12 mg / kg group from 2 weeks of administration, and males and females in the 40 mg / kg group from 1 week of administration to 4 weeks of administration. During the recovery period, a significant low value of food intake was continuously observed in both males and females in the 40 mg / kg group. The urine volume showed a significant high value in males in the 40 mg / kg group, and the urine specific gravity in the males in the group of 12 mg / kg or more showed a significant low value. The fluctuation disappeared before the end of the recovery period. The low value of platelet count and reticulocyte count and the high value of MCHC in males in the group of 12 mg / kg or more and females in 40 mg / kg group were males in the group with the low value of MCV of 12 mg / kg or more, the red blood cells In females with a low number of 12 mg / kg or more, the low value of HCT, the prolongation of PT in males and females in the 40 mg / kg group, the APTT extended in 40 mg / kg group, the white blood cell count Low values ​​were observed in females in the 40 mg / kg group. At the end of the recovery period, low numbers of erythrocyte counts were continuously observed in HCT and females in males and females of 40 mg / kg group, besides low numbers of red blood cells in males, low values ​​of HGB in males and females, white blood cells A low number of numbers was newly recognized. In males and females with a low value of K of 4 mg / kg or more, males in the group with low Ca of 4 mg / kg or more, males in the group with low values ​​of Cl and GOT of 12 mg / kg or more, Total protein, urea nitrogen, creatinine and total cholesterol were high in 12 mg / kg or higher males and 40 mg / kg females in females, γ-GTP, high value of albumin 40 mg / kg group in males and females, The males of the group with the α 2 -globulin ratio, the β-globulin ratio, the triglyceride, the low value of inorganic phosphorus not less than 12 mg / kg, the albumin ratio, the A / G ratio, the high bilirubin high in the 40 mg / kg group males , High glucose levels were observed in females in the 40 mg / kg group, and females with high Na levels above 12 mg / kg were found. At the end of the recovery period, a low value of GOT, β-globulin ratio was continuously observed in males of the 40 mg / kg group, and a high value of Na was newly observed in males. In males and females of 4 mg / kg or more, the thyroid gland was enlarged. Similar findings were obtained in males and females in the 40 mg / kg group at the end of the recovery period. Both absolute and relative weights of thyroid gland showed significant high values ​​in male groups of 4 mg / kg or more and females in groups of 12 mg / kg or more. At the end of the recovery period the degree of fluctuation became slightly weaker but a significant high thyroid weight continued to be observed in males and females in the 40 mg / kg group. In the thyroid gland, follicular cell hyperplasia and hypertrophy were found in males and females with a group of 1.2 mg / kg or more. In the adrenal gland, vacuolation of cortical cells was observed in males and females of the 40 mg / kg group. At the end of the recovery period, the same changes continued although the degree of tissue change in the thyroid gland and adrenal glands weakened in both males and females in the 40 mg / kg group. As a result, it was inferred that test substance affects hematopoietic function, thyroid, liver function, renal function and the like. The toxicologically no effect amount by repetitive oral administration for 28 days under this test condition was considered to be less than 1.2 mg / kg.

Study 3

The chronic repeated dose toxicity study of test substance in rabbits were conducted to evaluate adverse effects by the oral (gavage) route. Dosage of 20 mg/kg bw /day was administered for four months. Treatment was administered every 2 days during the first two months and every day during the third and fourth month. Haematological and clinical chemistry tests were carried out prior to study initiation and at study termination. The test chemical produced effects on serum proteins (altered albumin-globulin ratio) and the formation of prothrombin in association with haemorrhages. Whereas aldolase activity was elevated, there was no significant change in alkaline phosphatase or aminotransferase activity. Anaemia developed and there was a leftward shift in white blood cell counts. Histopathological examination revealed focal fatty dystrophy of the liver, pulmonary emphysema as well as myocardial haemorrhages. No further details available. Insufficient documentation of the conduct and the results of the study render it unsuitable for the assessment of the systemic toxicity of test substance  following repeated oral administration. The lowest observed adversed effect level (LOAEL) of test substance in this study was cosidered to be 20 mg/kg bw/day.

 

Repeated dose toxicity: inhalation

Data available form various studies for the test chemicals was reviewed to determine the toxic nature of test chemical .The studies are as mentioned below:

Study 1

The inhalation toxicity of test chemical was evaluated in male and female F344/N rats exposed 6 hr/day, 5 days/week to respirable aerosols generated by spray atomization of aqueous suspensions of the test chemicalpowder and subsequent drying of the resulting aerosols. Subchronic exposures were conducted at target concentrations of 0, 3.1, 6.2, 12.5, 25.0, and 50.0 mg/m3 of test chemical. Rats at ≥25 mg/m3 displayed hunched posture, hypoactivity, and reduced body weight gain, with compound related mortality at the highest exposure level. Anemia; increased SGPT, SGOT, alkaline phosphatase, sorbitol dehydrogenase, BUN, and cholesterol; and reduced free fatty acid were seen in rats at ≥25 mg/m3. Increased thyroid weight and thyroid follicular cell hyperplasia were noted in both sexes at ≥6.2 mg/m3, with reduced triicdothyronine and thyroxine levels in both sexes at ≥12.5 mg/m3. Thyroid follicular cell hyperplasia was also seen in rats at 3.1 mg/m3. Thymus weights were significantly reduced in both sexes at all exposure levels with liver weight increases at ≥6.2 mg/m3. Exposure-related histopathologic changes included pituitary cytoplasmic vacuolization, adrenal cortex necrosis, lymphoid depletion, thymic atrophy, liver cell hypertrophy, renal mineralization and tubular atrophy, and hypocellularity of the bone marrow. Effects observed even at lower concentration of 3.13 mg/m3 so that it was not possible to determine a no observable effet level in this study. Thus, on the basis of the study the LOAEC (lowest observed adversed effect concentration) was considered to be 3.1 mg/m3.

Study 2

A subchronic inhalation study in mice was conducted to investigate the toxic effects of test chemical with repeated exposures at various concentrations. Mice were exposed to test chemical concentrations of 0 (controls), 3.13, 6.25, 12.5, 25 and 50 mg/m3 air for 6 hours per day, 5 days per week over a period of 90 days. Mortality and clinical signs were not observed.The males which were exposed to test chemical concentrations of 12.5 mg/m3 or less, and the females which were exposed to test chemical concentrations of 25 mg/m3 or less gained slightly more body weight than did the controls. Body weight gain in the mice from the other dose groups was comparable to control mice. Male and female mice of all exposure groups were found to have enlarged thyroid glands. Relative thyroid weights were significantly increased in the male mice of the 25 and 50 mg/m3 groups and in the females of the 50 mg/m3 group. In the lower exposure groups, the relative thyroid weights were also increased, but the changes were not statistically significant. The relative liver weights from the two highest groups were significantly higher than the controls. Thyroid follicular cell hyperplasia in the male mice exposed to a concentration of 6.25 mg/m3 and in the female mice at and above an exposure level of 3.13 mg/m3, the incidence increasing in a dose-dependent manner. At the two highest concentrations, all mice exhibited these ultra structural tissue changes. In addition, 2 out of 10 males, and one out of 10 females of the 50 mg/m3 group were found to have focal hyperplasia of the thyroid gland. Hyperplasia of the epithelium of the bronchioles centrilobular hypertrophy of the liver with intranuclear inclusion bodies were diagnosed in both sexes of 12.5 mg/m3 or above.Degeneration of the zona reticularis of the adrenal gland was seen only in the female mice, with 9 out of 10 females being afflicted in the 3.13 mg/m3 group and all 10 females in the groups exposed to test chemical concentrations of 6.25 mg/m3 or above. Based upon these histopathological changes, the no observable effect level (NOEL) of test chemical for male mice was 3.13 mg/m3. For female mice, a NOEL was not determined, as even the lowest exposure group (3.13 mg/m3) was found to have ultrastructural changes in the thyroid and adrenal glands.

Study 3

Groups of five rats/sex were exposed at target concentrations of 6.3, 12.5, 25.0, 50.0, or 100.0 mg/m3 ,6 hr/day, 5 day/week, for a total of 12 exposures. No mortalities resulted from exposure to test chemical . Clinical signs of toxicity were limited to the higher exposure levels and included mild transient lethargy and hunch ed posture. Reduced body weights occurred in rats exposed at 50 or 100 mg/m3. Organ weight changes attributed to test chemical exposure included increased liver weight and decreased heart, lung,and thymus weights. Enlarged thyroids were noted in exposed rats at necropsy which corresponded with microscopically observed thyroid follicular cell hyperplasia in rats at the four highest concentrations. Additional changes included adrenal cortex fatty accumulation in males at > 12.5 mg/m3 and in females at 250 mg/m3 and pituitary atrophy in both sexes at 225 mg/m3. At the lowest test concentration, which was 6.3 mg/m3, no substance related changes were noted. Thus, the no observed adversed effct level (NOAEL) for test chemical in male and female rats was considered to be 6.3 mg/m3.

Study 4

In a dose finding study for a subchronic inhalation study rats were exposed to test chemical concentrations of 0 (controls), 5, 15, 45, 135 and 400 mg/m3 for 6 hours per day and a total of 12 exposures. Mortality at the highest concentration level (400 mg/m3) was 100%. While at the lowest concentration (5 mg/m3 and 15 mg/m3) and in the control group there were no deaths. Concentration-dependent retardation of body weight gain and decreased body weights were observed at the end of the study. As a clinical sign of toxicity, tremor occurred at the highest concentration level (3 animals). In the 15, 45 and 135 mg/m3 groups, the organ weights of the brain, thymus, heart, lungs, kidneys and testes were significantly lower than those of the controls. Liver weights were increased in the male rats of the 5 mg/m3 group and the female rats of the 5, 15 and 135 mg/m3 groups. Hematologically, there was impairment of haematopoiesis associated with mild anaemia at the higher concentration levels. Enlarged thyroid glands were observed. Histopathological changes comprised diffuse follicular cell hyperplasia of the thyroid gland, pituitary hyperplasia, hypertrophy of centrilobular hepatocytes, renal calculi, depletion of thymic lymphocytes, bone marrow hypoplasia, splenic congestion and reduced prostatic secretory activity. Thus, the lowest observed adverse effct level (LOAEL) of test chemical in male and female rat in a subacute study was considered to be 5.0 mg/m3 air.

Study 5

The test chemical is a member of the thioureylene compound family known for their potent antithyroid activity. Male Fischer-344 rats were exposed to 0.0, 3.13, 12.5, and 50.0 mg/m3 of test chemical via inhalation for 13 wk. Follicular cell hyperplasia occurred in the thyroid glands of rats from the 3 test chemical treatment groups. Thyrotrophs in the pituitary glands from rats in these 3 groups were hyperplastic, and they had varying numbers of hypertrophic cells with either eosinophilic stippled cytoplasm or with eosinophilic globules within 1 or more large vacuoles that displaced the nucleus. These cells were compared by immunohistochemistry and ultrastructural examination to "thyroidectomy cells" within the anterior pituitary glands of thyroparathyroidectomized rats and were determined to be identical to them. Immunohistochemical staining for the &beta;-chain of thyroid-stimulating hormone confirmed that the hyperplastic and hypertrophic cells were thyrotrophs. Ultrastructurally, hypertrophic cells in test chemical treated rats and thyroidectomy cells in thyroparathyroidectomized rats had expanded cytoplasm containing either increased profiles of rough endoplasmic reticulum (RER) or one or more widely dilated cisternae of RER, which displaced other cellular organelles. The test chemical appears to act comparably to other thioureylene compounds that have been shown to produce low serum concentrations of triiodothyronine and thyroxine, which results in thyrotroph hypertrophy and hyperplasia, resultant thyroid hyperplasia, and subsequent goiter. Thus, on the basis of the study the LOAEL (lowest observed adversed effect level) of test chemical in male rat was considered to be 3.13 mg/m3.

Study 6

The 14 days subacute repeated dose study was conducted,in which male and female mice were exposed to test chemical concentrations of 0 (controls), 5, 15, 45, 135 and 400 mg/m3 for 6 hours per day and a total of 12 exposures. Mortality at the highest concentration level (400 mg/m3) was 100%. While at the lowest concentration (5 mg/m3) and in the control group there were no deaths. Clinical signs of toxicity included reduced activity. Exposure to concentration levels of 135 and 400 mg/m3 was observed to be associated with tremors. Loss of body weight was observed. Final body weight changes noted in comparision with the controls were a depression in the 5 and 15 mg/m3 dose groups and an elevation in the 45 and 135 mg/m3. Macroscopically, the animals surviving the exposure regimen predominantly had an enlarged liver. The absolute liver weights in all groups except the females of the 5 and 15 mg/3 groups were significantly elevated relative to the controls. The relative liver weights also showed alterations. Histopathological observations included findings of cytoplasmic swelling of hepatocytes, thyroid and pituitary hyperplasia, lymphocytolysis and lymphocyte depletion within the thymus and to a lesser extent in a spleen. In one of the survivors from the 135 mg/m3 group, necroscopy revealed renal calculi. Thus, the lowest observed adverse effect level (LOAEL) for test chemical in male and female mice in a 14 days study was considered to be 5.0 mg/m3 air.

Repeated dose toxicity: dermal

The OECD study result for acute toxicity by the dermal route indicates the LD50 value to be greater than 2000 mg/kg body weight. Also considering the use of the chemical as anti-oxidant for natural rubber and latex and considering the volatility absorption by the dermal route is not considered to be significant. Thus, given the above considerations, it is assumed that test chemical shall not exhibit repeated dose toxicity by the dermal route.

Based on the available data for the assessment of repeated dose toxicity by oral, inhalation route and following Regulation (EC) 1272/2008 the test chemical is classified in STOT RE – 2 (oral) category.

Justification for classification or non-classification

Based on the available data for the assessment of repeated dose toxicity by oral, inhalation route and following Regulation (EC) 1272/2008 the test chemical is classified in STOT RE – 2 (oral) category.