3-nitro-o-xylene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-nitro-o-xylene

EC Number:

201-474-3

EC Name:

3-nitro-o-xylene

Cas Number:

83-41-0

Molecular formula:

C8H9NO2

IUPAC Name:

1,2-dimethyl-3-nitrobenzene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 23; received from the sponsor of the study; BG Chemie


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a refrigerator (at 4°C)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

(Bor:WISW; Wistar-outbred)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Breeding Centre for Laboratory Animals, F. Winkelmann GmbH, Borchen, F.R. Germany

- Age at study initiation:
ca. 5 weeks

- Weight at study initiation: 92-93g (females); 99-100g (male animals)

- Housing:
5 animals per cage, separated by sex, in suspended stainless steel cages, fitted with wire mesh floor and front

- Diet (e.g. ad libitum):
Institute's stock diet. A powdered cereal-based, open-formula diet

- Water (e.g. ad libitum):
drinking bottles were filled daily with fresh tap water, except for the weekends

- Acclimation period:
10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5 °C - 22.5 °C
- Humidity (%): 60-80 %
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): twice during the study
- Mixing appropriate amounts with (Type of food): standard lab diet
- Storage temperature of food: -20 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Immediately after preparation of the first batch of diets, three samples of each test diet were taken fron different locations in the blender. These samples were analysed in duplo for the homogenous distribution of the test substance in the diet. One sample of the control diet was analysed as well.

The diet was extracted with ethanol and analyzed using a HPLC technique.

Duration of treatment / exposure:

28 days/ 42 days (recovery groups)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

100 ppm

Doses / concentrations 3

Dose / conc.:

500 ppm

Doses / concentrations 4

Dose / conc.:

2 500 ppm

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on the results of a 5-day range finding study

- Rationale for selecting satellite groups: to examine the reversibility of the effects found

- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days; once daily during the weekend

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0; then twice weekly as well as on the day of autopsy

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: No; food consumption was recorded twice weekly

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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; The efficiency of food utilization was calculated over one-week periods and expressed as grams weight gain per gram food consumed.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At autopsy on day 28
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Not specified
- How many animals: All; except for the animals of the recovery group
- Parameters examined:
- red blood cell count; haemoglobin concentration; packed cell volume; prothrombin time; total white blood cell count; differential white blood cell count; thrombocyte count; mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day 26 for glucose measurements; on day 28 for the other parameters.
- Animals fasted: Yes (prior to blood sampling on day 26): deprivation of water for 24 hours and of food during the last 16 hours.
- How many animals: All; except for those of the recovery groups
- Parameters examined:
- alkaline phosphatase activity; total protein; aspartate aminotransferase activity; albumin/globulin ratio; alanine aminotransferase activity; urea; gamma qlutamyl transferase activity; creatinine; total bilirubin; sodium; potassium; calcium; inorganic phosphate; chloride; albumin

At autopsy of the rats of the recovery study on day 42, blood was collected and analysed for:
- alanine aminotransferase activity
- gamma glutamyl transferase activity
- total protein


URINALYSIS: Yes
- Time schedule for collection of urine: Day 25-26
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
- volume; density; appearance; protein; glucose; urobilinogen; bilirubin; occult blood; ketones; pH; microscopic examination of the sediment

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Samples of the following tissues and organs were preserved:
adrenals; ovaries, heart, testes, kidneys, spleen, liver, all gross lesions
Following organs were weighed: adrenals, testes, kidneys, spleen, liver



HISTOPATHOLOGY: Yes
liver, kidneys, heart, adrenals and spleen of all rats of the control group and the topdose
group

Statistics:

Body weights were evaluated by one-way analysis of co-variance followed by Dunnett's multiple comparisons test; or (for the two groups of the
recovery study) by the two sample t-test. Data on red blood cell variables, total white blood cell counts, clinical chemistry values, volume and density of the urine, and organ weights were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. The above data of the recovery study were analysed by the two sample t-test. Differential white blood cell counts and semi-quantitative urinary observations were analysed by the Mann-
Whitney u-test. The histopathological changes were examined by Fishers exact probability test. All data were tested two-sided.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One rat showed some alopecia and another rat inflammation of the eyes.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were slightly decreased in the high-dose group in both sexes. The differences with the controls were not always statistically significant. Body weights of the mid-dose rats tended to be lower than in controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake of both sexes was slightly lower in the high-dose group than in the controls.

Food efficiency:

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption showed slightly lower values in the high-dose females than in controls in both the main study and the recovery study. In males no dose-related differences between test groups and controls were observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Thrombocyte count was slightly increased in the high-dose group. Such an effect was not observed at the end of the recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma protein concentration was slightly decreased in the high-dose group in both sexes, and in females of the mid-dose group. Plasma alanine aminotransferase activity was slightly decreased in females of the high dose-group. Albumin/globulin ratio and gamma glutamyl transferase activity were slightly increased in the
high-dose group in males, although with respect to the latter finding the difference with the controls was not statistically significant. An increase in albumin/globulin ratio occurred also in males of the mid-dose group. A decreased plasma alanine aminotransferase activity was still present after the recovery period, whereas gamma glutamyl transferase activity and protein concentration had returned to normal.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At autopsy of the rats of the sub-acute study (killed an day 28), one male of the mid-dose group showed one kidney with a pale area. At autopsy of the rats of the recovery study (killed on day 42), gross examination was essentially negative.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Because no distinct changes of toxicological importance were observed in the low- and mid- dose groups in the present 28-day study, it is concluded

that the no-effect level of 3-nitro-1,2-dimethylbenzol was 500 ppm in the diet of rats when fed for a period of four weeks. This dietary level

provided an intake of about 50 mg/kg body weight/day.