1,2,4-trichlorobenzene

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Trichlorobenzenes: results of a thirteen week feeding study in the rat

Author:

Cote M, Chu I, Villeneuve DC, Secours VE, and Valli VE

Year:

1988

Bibliographic source:

Drug ChemToxicol 11: 11 - 28

Reference 2

Reference Type:

secondary source

Title:

European Union Risk Assessment Report - 1,2,4-Trichlorobenzene

Author:

European Commission - European Chemicals Bureau

Year:

2003

Bibliographic source:

Office for Official Publications of the European Communities

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

No observational period of 90 days (after exposure). No ophthalmological examinations. No sensory reactivity analysis and motory assessment were conducted. Animals were not fasted before blood analysis. Some organs were not subjected to gross necropsy.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

1,2,4-trichlorobenzene

EC Number:

204-428-0

EC Name:

1,2,4-trichlorobenzene

Cas Number:

120-82-1

Molecular formula:

C6H3Cl3

IUPAC Name:

1,2,4-trichlorobenzene

Details on test material:

- Name of test material (as cited in study report): 1,2,4-trichlorobenzene
- Analytical purity: > 99%

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, Montreal, Canada
- Age at study initiation: In this study weanling rats were used
- Housing: the animals were used individually in stainless-steel mesh cages
- Acclimation period: 1week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24°C
- Humidity (%): 40-60%
- Photoperiod (hrs dark / hrs light): 12 hr alternated light/dark cycle

Administration / exposure

Route of administration:

oral: feed

Vehicle:

corn oil

Details on oral exposure:

DIET PREPARATION
The diet consumed by the control groups was prepared by blending thoroughly ground cubes (Ralston Purina) with corn oil (Mazola, 4% w/w). The test diets were made by mixing ground cubes with corn oil solutions containing appropriate amounts of test chemicals to give dietary levels of 1, 10, 100 or 1000 ppm.
Fresh diets were made every fourth week throughout the study, and kept in air-tight steel containers.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
1, 10, 100 or 1000 ppm (males: 0.07, 0.78, 7.8 or 82 mg/kg bw/day, females: 0.13, 1.5, 17 or 146 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none
- Dose selection rationale: Selection of the dose levels was based on a range-finding study in which the LD50 of 1,2,4-trichlorobenzene in rats was found to be : 0.88 g/kg

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on a daily basis


BODY WEIGHT: Yes
- Time schedule for examinations: body weight was determined weekly on all animals


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat
- Compound intake calculated as the ratio between the food consumption expressed in grams X dietary concentration (ppm) and the average body weight.

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

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood samples were collected at necropsy
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: all animals
- The following parameters were examined: haemoglobin, packed cell volume, erythrocyte counts, total and differential leukocyte counts, platelet count, and prothrombin time. Mean corpuscular haemoglobin concentration and mean corpuscular haemoglobin values were calculated.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood samples were collected at necropsy
- Animals fasted: No
- How many animals: all animals
- The following parameters were examined: sodium, potassium, inorganic phosphate, total bilirubin, alkaline phosphatase, aspartate aminotransferase, total protein, calcium, cholesterol, glucose, uric acid, and lactic dehydrogenase.


URINALYSIS: Yes
- Time schedule for collection of urine: at weeks 4, 8, and 12 od the study
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- The following parameters were examined: pH, protein and nitrite

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were examined grossly at the time of necropsy. The brain, heart, liver, spleen, and kidneys, were excised and weighed.
HISTOPATHOLOGY: Yes. The following tissues were taken and fixed in 10% buffered formalin (pH 7.4) for routine histological examination: eye, optic nerve, spinal cord, skin, tongue, brain, pituitary, liver, adrenal, thyroid, parathyroid, thymus, lungs, trachea, bronchi, thoracic aorta, esophagus, gastric cardia,, fundus and pylorus, duodenum, jejunum, ileum, pancreas, colon, cecum, kidneys, spleen, bone marrow, mesenteric and mediastinal lymph nodes, skeletal muscle, ovaries, uterus, vagina or testes, prostate, epididymis, sciatic nerve, urinary bladder, salivary gland, mammary gland, and heart. Potential fatty change in the liver was determined in frozen sections.Sections of liver and perirenal fat were excised and kept at -70°C pending residue analysis using a gas chromatographic method.

Other examinations:

Hepatic microsomal aniline hydroxylase (AH) and aminopyrine demethylase (APDM) activities were determined based on the previously reported methods adapted to automated instruments. Liver protein content was determined by biuret method. A section of femoral bone marrow was aspirated, spread on the slide from which thin films were made, und with May-Grünwald-Giemsa stain for cytological evaluation.
Potential fatty change in the liver was determined in frozen sections.Sections of liver and perirenal fat were excised and kept at -70°C pending residue analysis using a gas chromatographic method.

Statistics:

Data were analyzed by one-way analysis of variance followed by Duncan´s multiple range test to indicate the groups which are significanlty different the the control (p <= 0.05)

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 examined

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

not examined

Ophthalmological findings:

not examined

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:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY


ORGAN WEIGHTS
The liver/body weight ratios of males receiving the highest dose of 1,2,4-trichlorobenzene were significantly greater than those of the control group. The wet kidneys weights as well as kidney/body weight ratios of males receiving 1000 ppm 1,2,4 ¿trichlorobenzene were higher compared to control values.

GROSS PATHOLOGY
One male rat receiving 1000 ppm 1,2,4-trichlorobenzene diet had nephrosis

HISTOPATHOLOGY: NON-NEOPLASTIC
The liver, thyroid and kidney were the target organs which had treatment-related changes.
The changes were significant only at the highest dose level. In general, morphological alterations in males were more severe than those of females.

The livers had marked changes characterised by aggregated basophilia as well as midzonal vacuolation due to fatty infiltration. Histopathological examination of the kidney failed to reveal any abnormal changes, neither did the urinalysis. Changes in the thyroid were characterised by reduction in follicular size, increased epithelial height from flattened cuboidal cells to columnar shape, and reduced colloid density.


OTHER FINDINGS
Measurements of hepatic mixed function oxidase activities revealed that 1,2,4-trichlorobenzene at the 1000 ppm level caused a significant elevation in AH and APDM activities in males, and APDM activity in females.
There was a dose-dependent accumulation in both fat and liver in the following order:
1,3,5-trichlorobenzene>1,2,4-trichlorobenzene>1,2,3-trichlorobenzene.
The levels of trichlorobenzenes in fat were one order of magnitude higher than those in liver.

Effect levels

Dose descriptor:

NOAEL

Effect level:

7.8 mg/kg bw/day (nominal)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1.Food consumption and weight gain of male rats fed with 1,2,4-trichlorobenzene

Treatment (ppmindiet)	Foodconsumption(g/rat)	Initialweight(g)	Weightgain(g)	Amount of chemical ingested (mg/kgb.w./day)
Control	25 ± 1.1 (5)	85 ± 8 (9)	471 ± 54 (9)	0
1,2,4-trichlorobenzene
1	23 ± 0.7 (5)	85 ± 5	453 ± 27	0.07
10	25 ± 1.2 (5)	80 ± 5	474 ± 47	0.78
100	25 ± 1.1 (5)	85 ± 10	472 ± 42	7.8
1000	25 ± 2.2 (5)	84 ± 10	444 ± 33	82

 

 

Table 2.Organ weights of male rats fed diet containing 1,2,4-trichlorobenzene (mean±S.D)

 

		Liver		Kidney
Treatment (ppmin the diet)	n^a	Wet weight	Liver/b.w. ratio (% ofb.w.)	Wet weight (g)	Kidney/b.w. ratio (% ofb.w.)
Control	9		3.5 ± 0.30	1.56 ± 0.20	0.28 ± 0.02
1,2,4-trichlorobenzene
1	10	17.8 ± 2.5	3.3 ± 0.43	1.57 ± 0.11	0.29 ± 0.02
10	10	20.5 ± 2.3	3.7 ± 0.29	1.68 ± 0.18	0.30 ± 0.04
100	10	20.8 ± 2.0	3.7 ± 0.20	1.72 ± 0.10	0.31 ± 0.02
1000	10	22.2 ± 1.5	4.2 ± 0.22*	2.04 ± 0.44*	0.38 ± 0.06*

a number of animals

* Significantly different from the control (p< 0.05)

 

 

Table 3.Measurements of AHabdAPDM activitiesin males, and APDMactivitesin females

AH	Male	µmole/PAP/hr/mg protein
	Control	8.8± 3.4
	Treated	15.6 ± 6.2
APDM	Male	µmole HCHO/hr/mg protein
	Control	23 ± 3.6
	Treated	53 ± 19
	Female	µmole HCHO/hr/mg protein
	Control	24 ± 3.1
	Treated	41 ± 7.4

 

Applicant's summary and conclusion

Executive summary:

Cote et al. (1988):

 

In a study similar to OECD TG 408 with deviations (No observational period of 90 days (after exposure). No ophthalmological examinations. No sensory reactivity analysis and motory assessment were conducted. Animals were not fasted before blood analysis. Some organs were not subjected to gross necropsy) groups of ten male and ten female weanling Sprague Dawley rats were fed diets containing 1, 10, 100 or 1,000 ppm 99% pure 1,2,4-TCB for 13 weeks (Côté et al., 1988).

The dose of 1,2,4-TCB ingested was calculated to be 0.07, 0.78, 7.8, and 82 mg/kg bw/day for

males and 0.11, 1.4, 15, and 101 mg/kg bw/day for females.

During the dosing no signs of toxicity were observed. At the terminal sacrifice body weights of male rats were similar in all groups, and relative liver weights, kidney weights and relative

kidney weights showed increases which were statistically significant at the high dose level. No specific data for female body and organ weights were given. This might be taken to indicate that no statistically significant effects were observed. The activities of aniline hydroxylase and aminopyrine demethylase of the liver were significantly increased in males at the dose of 1,000 ppm, and the latter enzyme was also increased in females at 1,000 ppm. At the histopathological examination, treatment-related changes were seen in the livers, thyroids, and kidneys but significantly only at the highest dose level. In general the changes were more severe in males than in females.

The livers had marked changes characterised by aggregated basophilia as well as midzonal

vacuolation due to fatty infiltration. Histopathological examination of the kidney failed to reveal

any abnormal changes, neither did the urinalysis. Changes in the thyroid were characterised by reduction in follicular size, increased epithelial height from flattened cuboidal cells to columnar shape, and reduced colloid density.

The target organs were liver, kidneys, and thyroid. A NOAEL of 100 ppm for both sexes

(7.8 mg/kg bw/d for males and 15 mg/kg bw/d for females) can be derived from the study.