Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented study according to international accepted guidelines and GLP compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
Test item: Difluoro-bromo-benzene
CAS No.: 348-57-2
Batch No.: L39084N
Physical state: liquid
Colour: colourless
Active Ingredient Content (GC): 96.4 %
Storage: 2-8 °C, in a tightly closed container

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
Species and strain: Crl:(WI)Br rats
Source: TOXI COOP ZRT.
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Number of animals: 3 animals/group
Sex: Female, nulliparous and non pregnant animals
Age of animals: Young adult rat, 8 weeks old in first, second, third and fourth step
Body weight range at starting (first step): 182 - 187 g
Body weight range at starting (second step): 175 - 192 g
Body weight range at starting (third step): 170 - 190 g
Body weight range at starting (fourth step): 186 - 194 g
Acclimatization time: 5 days in first step, 6 days in second step, 7 days in third step and 8 days in fourth step
Animal health: Only healthy animals were used for the study. Health status was certified by the study director.

Housing: Group caging (3 animals/cage)
Light: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 10-15 air exchanges/hour by central air-condition system

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Helianthi annui oleum raffinatum
Details on oral exposure:
A single oral administration - followed by a fourteen-day observation period - was performed by gavage.
Starting dose was selected on the basis of the available information about the test item DIFLUORO-BROMO-BENZENE (CAS 348-57-2).
The acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg/kg bw as the starting dose (step 1; group 1) in three female rats. Since no animal died, the test was continued at 2000 mg/kg bw dose level (step 2; group 2) on further three female rats. Since two animals of group 2 died, the test was continued at 300 mg/kg bw dose level (step 3; group 3) on further three female rats. There was no death in third step, so three further female rats were treated with the same (300 mg/kg bw) dose (step 4; group 4). No animal died in the fourth step, too, so the test was finished.

All doses were formulated in the vehicle. Concentration of formulations was adjusted to maintain a treatment volume of 10 mL/kg bw. The test item was applied in a concentration of 200 and 30 mg/mL. Formulations were prepared just before the administration and stirred continuously during the treatment.

The day before treatment the animals were fasted. The food but not water was withheld overnight. The food was given back 3 hours after the treatment.
Doses:
2000, 300 mg/kg bw
No. of animals per sex per dose:
3 female/dose
Control animals:
no
Details on study design:
The observation period was 14 days. Animals were observed individually after dosing at least once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h, 5 h and 6 h after the treatment and twice each day for 14 days thereafter.
The body weights were recorded on day 0 (just before the treatment), on day 7 and on day 15 with a precision of 1 g.
At the end of the observation period rats were sacrificed and necropsy were performed.

Results and discussion

Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
300 - 2 000 mg/kg bw
Based on:
test mat.
Mortality:
Altogether two rats dosed at 2000 mg/kg DIFLUORO-BROMO-BENZENE (CAS 348-57-2) died during the study. No death occurred in group 1 (2000 mg/kg bw) single oral dose of the test item. All female rats in step 1 survived until the end of the 14-day observation period.
Two females of group 2 (step 2) died on Day 1. Both deaths seemed to be consequences of systemic toxic effect of the test item.
No death occurred at 300 mg/kg single oral dose of the test item. All female rats in step 3 and step 4 survived until the end of the 14-day observation period.
Clinical signs:
In group 1 treated with 2000 mg/kg bw dose:
Decreased activity (score -1; -2), abnormal gait (score +1; +2), incoordination (score +2) and piloerection (score +1; +2) were observed in all animals. Tremor (score +1) was detected in one animal. These symptoms were detected between Day 1 and Day 3.
In group 2 treated with 2000 mg/kg bw dose:
Abnormal gait (score +1; +2) and incoordination (score +1; +2) was observed in all animals. Decreased activity (score -1; -2) was recorded in two animals. Piloerection (score +1) was observed in two animals (No.: 9385, 9387). Blood around the nose (score +1), decreased body tone (score -1) and increased respiration rate (score +2; +3) were detected in one animal. These symptoms were detected between the treatment day and Day 2.
In group 3 treated with 300 mg/kg bw dose:
Clinical sign of reaction comprised of piloerection (1 cases of 63 observations) only in one animal. This symptom (score +1) was observed on Day 1. The other animals were free of symptoms during the study.
In group 4 treated with 300 mg/kg bw dose:
Clinical sign of reaction comprised of piloerection (1 cases of 63 observations) and decreased activity (1/63) only in one animal. Score of piloerection was +1 and score of decreased activity was -1, respectively. These symptoms were detected on the treatment day 6 hours after the treatment. The other animals were free of symptoms during the study.
Body weight:
In group 1 (2000 mg/kg bw) the mean body weight of the animals corresponded to their species and age throughout the study.
In group 2 (2000 mg/kg bw) the body weight of the survivor animal corresponded to its species and age throughout the study.
In group 3 and 4 (300 mg/kg bw) the mean body weight of the animals corresponded to their species and age throughout the study.
Gross pathology:
Two rats out of 6 animals treated with 2000 mg/kg bw dose of the test item spontaneously died during the study.
Internal necropsy finding as pale kidneys was observed in all animals of group 1. This alteration could not be related to the test item toxic effect, but was regarded an individual variation. Most likely the observation is a congenital anomaly. Moderate hydrometra was observed in one female of the group 1 is physiological finding and connected to the cycle of the animal. An internal necropsy finding as autolysis was observed in two animals of group 2, but it is normal physiological process after death. Pale kidneys were observed in one animal of group 2 and slight hydrometra was found in same female.
All animals treated with 300 mg/kg bw dose survived until the scheduled necropsy on Day 15. Pale kidneys were observed in two animals treated with 300 mg/kg bw dose (group 3) and in two animals treated with the same dose (group 4).

Any other information on results incl. tables

Groups

Treatment

Lethality

Test item

Dose (mg/kgbw)

Females

1

“DIFLUORO-BROMO-BENZENE”
Step1

2000

0/3

2

“DIFLUORO-BROMO-BENZENE”
Step2

2000

2/3

3

“DIFLUORO-BROMO-BENZENE”
Step3

300

0/3

4

“DIFLUORO-BROMO-BENZENE”
Step4

300

0/3

 

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Remarks:
Migrated information
Conclusions:
The method used is not intended to allow the calculation of a precise LD50 value.
The test item was ranked into classes of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423.
Hazard Category: Acute Tox. 4
Executive summary:

Dose (mg/kg bw) Mortality (dead/treated)  LD50 (mg/kg bw)  GHS Category
300 0/6 between 300 and 2000 4