Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP and OECD Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- WISKf (SPF71)
- Source: Hoechst AG, breeding colony
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 110 - 123 g (male); 99 - 125 g (female)
- Fasting period before study: none
- Housing: group housing of 5 animals per sex in Macrolon cages type 4 on soft wood granulate
- Diet (e.g. ad libitum): rat diet Altromin 1324, ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: approx. 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0 ± 3.0°C
- Humidity (%):30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/v) 0.8, 40 and 20.0 % were prepared daily immediately before treatment; the test item was suspended homogenously in the vehicle by means of magnetic stirrer. 5 ml/kg bw were administered.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance formulations in sesame oil were noted as stable for at least 4 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed mean values within the range of about 84-103 % of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.
Duration of treatment / exposure:
28 days treatment
Frequency of treatment:
28 applications within 29 days, 7 days/week; application between 7.00 and 12.00 a.m.. Animals were dosed up to the day prior to necropsy.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
40 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 (with 5 additional animals/sex in the control group)
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: in a preliminary study groups of 3 male and 3 female rats received the test item by oral gavage at dose levels of 500 or 1000 mg/kg bw per day over a period of 14 days. No symptoms occured after administration of 500 mg/kg bw per day. Male and female animals treated with 1000 mg/kg bw per day showed decreased spontaneous activity as well as impairments of motility and respiration until day 3 of the study. At day 4 of the study all symptoms were reversible
Based on these results the test substance was tested at the dose levels of 0, 40, 200 and 1000 mg/kg bw per day.
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality, viability
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study and then twice weekly
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: twice weekly
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
- Time schedule for examinations: once weekly over a period of 16 h, results given as water consumption/animal/16 h
OPHTHALMOSCOPIC EXAMINATION: opacity of the refracting media of the eyes
HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (Ketanest (R))
- Animals fasted: No
- How many animals: 40 (end of treatment)
- Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, mean cellular volume (MCV), mean cellular hemoglobin (MCH), mean cellular hemoglobin conc. (MCHC), Leucocyte count, Thrombocyte count, differential leucocyte count and red cell morphology, Reticulocyte count, Heinz bodies, coagulation time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: No
- How many animals: 40 (end of treatment)
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea nitrogen, Creatinine, Glucose, Cholesterol, Triglyderides, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphorus, Uric acid, Bilirubin total, Bilirubin direct
URINALYSIS: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: 40 (urine collected overnight from day 26 to 27)
- Parameters examined: Appearance, Volume, Colour, Specific gravity, pH, Protein, Hemoglobin, Glucose, Ketone, Bilirubin, Urobilinogen, Sediment
NEUROBEHAVIOURAL EXAMINATION: Yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were necropsied and descriptions of all macroscopic abnormalities recorded.

The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy: Heart, lung, liver, kidneys, spleen, ovaries, testes, epididymides, adrenals, brain, thymus

HISTOPATHOLOGY: the following tissues or organs (or pieces of them) were preserved in a suitable fixative and processed for histopathological investigations: heart, liver, kidneys, adrenals, spleen, lung, brain, thymus, ovaries, testes, epididymides, trachea, stomach, jejunum, colon, urinary bladder, uterus, prostata, seminal vesicles, skeletal muscle, N. ischiadicus, femur with bone marrow, spinal cord, lymph nodes, throid glands (high dose group)
Other examinations:
None

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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):
effects observed, treatment-related
Description (incidence and severity):
both sexes in high dose group
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
both sexes in high dose group
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
both sexes in high dose group
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
both sexes in high dose group
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
both sexes in high dose group
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
both sexes in high dose group

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No test item related effects were observed in male rats after repeated application of 200 mg/kg bw per day.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No test item related effects were observed in female rats after repeated application of 40 mg/kg bw per day.
Dose descriptor:
dose level:
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Slight hemolytic anemia effects develolped
Dose descriptor:
dose level:
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Distinct effects, elevated respectively were observed at a dose of 1000 mg/kg bdw for males and females

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Since for females slight effects developed at the dose level of 200 mg/kg bwd and effects were distinct at a dose level of 1000 mg/kg bwd an effect level sufficiently for classification at a dose level of 300 mg/kg bwd cannot be excluded. Therefore o-aminobenzotrifluoride is preventatively classified as:

CLP 1272/2008/EC: STOT RE Cat.2, H373
Directive 67/548/EEC: Xn, R48/22
Executive summary:

Groups of 5 male and 5 female Wistar rats received the test item by oral gavage at dose levels of 0, 40, 200 and 1000 mg/kg bw per day for 28 days and were necropsied at day 29.

Behaviour and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly, water consumption once weekly.

Hematological examinations and clinical chemistry were carried out at the termination of the study. Urine analysis was also performed at the end of the study.

No compound-related effects were observed in male and female Wistar rats after repeated application of 40 mg/kg bwd for females.

Slight hemolytic anemia also developed in females at the dose level of 200 mg/kg bwd. No compound-related effects were observed in males under these conditions.

Repeated application of 1000 mg/kg bwd caused distinct hemolytic anemia, which was macrocytic and hyperchromic, together with reactive extramedullary erythropoesis and increased medullary erythropoesis. The elevated bilirubin levels are considered to be a consequence of increased hemoglobin degradation. Futhermore, clinical symptoms and increased water consumption occured in this group. Three males showed decreased secretion in the seminal vesicles.

With regard to the present study the no observed effect level is 40 mg/kg bw per day for females and 200 mg/kg bw per day for males.