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Repeated dose toxicity: oral

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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

Referenceopen allclose all

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 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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity 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

Reference
Name:
Unnamed
Type:
Constituent
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 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 arange-findinf 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)

na

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.