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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with GLP and appropriate OECD Guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
denomination:
- protocol: Iso-PROPYL BROMIDE
- labelling: Iso-PROPYL BROMIDE
batch number:
- protocol: 0-302-1
- labelling: 0-302-1
description: colourless liquid
container: one glass flask
date of receipt: 4.10.94
storage conditions: at room temperature, protected from light
purity: 99.9%

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
Animals

Number -- three male and three female mice for the preliminary toxicity test
-- 56 mice: 28 males and 28 females for the first cytogenetic study (unvalidated)
-- 36 mice: 18 males and 18 females for the second cytogenetic study.
Strain: Swiss OF1/ICO:OF1 (lOPS Caw).
Reason for this choice: rodent species commonly requested by the international regulations for this type of study.
Breeder: lffa Credo, L' Arbresle, France.
Age: on the day of treatment the animals were approximately seven weeks old.
Veterinary care at C.LT.: upon their arrival at C.LT., the animals were given a complete examination to ensure that the animals are in good clinical condition.
Acclimatization: at least five days before the day of treatment.
Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex. Subsequently, each group was assigned to a different treatment group.
Identification: individual tail identification upon treatment.

Environmental conditions

The animal room
conditions were set as follows:
· temperature: 21 ± 2°C
· relative humidity: 50 ± 20%
· light/dark cycle: 12 h / 12 h (07:00 - 19:00)
· ventilation: about 12 cycles/hour of filtered non-recycled fresh air.
The housing conditions (temperature, relative humidity) are checked regularly.
The animals were housed in polycarbonate cages and each cage contained five animals of the same sex and group (three for the supplementary animals). Each cage contained autoclaved sawdust (SICSA, Alfortville, France).
Bacteriological analysis and detection of possible contaminants (pesticides, heavy metals) of the sawdust are conducted periodically by approved laboratories.

Food and water

All animals had free access to A04 C pelleted sustenance diet (U.A.R., Villemoisson-sur-Orge, France) and tap water (filtered using a 0.22 micron filter).
Each batch of food was analysed (composition and contaminants) by the suplier.
Bacteriological and chemical analyses of diet and water and detection of possible contaminants (pesticides, heavy metals and nitrosamines) are performed regularly by approved laboratory.
There are no known contaminants in the diet, water or sawdust at levels likely to influence the outcome of the study.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Corn oil
Details on exposure:
Rationale for dose selection

The choice of doses was performed according to the following criteria specified in international regulations.
A limit test using one dose, i.e.: the top dose recommended by international regulations:
2000 mg/kg/day was performed if no observable toxic effects are produced top dose:
- if observable toxic effects are produced, the top dose is defined as the dose producing signs of toxicity, such that a higher dose would be expected to produce lethality.
middle and low doses:
- the two other doses will be separated by no more than a factor between 2 and square root of 10.
Three doses (400, 800 and 2000 mg/kg/day) were used in the first study (unvalidated) and thereafter only 2000 mg/kg/day was used.

Preliminary toxicity test

In order to determine the top dose and depending upon the amount of information supplied by the Sponsor, several preliminary assays were performed on groups of six animals (three males and three females). Clinical signs and any mortality were recorded for a period of 48 hours. At the end of this period, the animals were sacrificed after CO2 inhalation in excess.

Administration
The test substance was administered by the intraperitoneal route using a dose volume of 10 ml/kg.
Each animal was given the test substance twice.
The quantity of the test substance administered to each animal was adjusted according to the body weight recorded at the time of dosing.
The vehicle control animals received the vehicle alone, under the same conditions.
The positive control animals received cyclophosphamide alone, by oral route.

Preparation of smears

At the time of sacrifice, all the animals were killed after CO2 inhalation in excess. The femurs of the mice were removed and the bone marrow eluted-out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. All the slides were coded for scoring.

Doses / concentrations
Remarks:
Doses / Concentrations:
40, 80 and 200 mg/ml
Basis:
nominal conc.
No. of animals per sex per dose:
First cytogenic study (sacrificed 24 hr post-exposure)
Vehicle -- 5 male/5 female
400 mg/kg/day -- 5 male/5 female
800 mg/kg/day -- 5 male/5 female
2000 mg/kg/day -- 5 male/5 female

Second cytogenetic study (sacrificed 48 hr post-exposure)
Vehicle -- 5 male/5 female
2000 mg/kg/day -- 5 male/5 female
treated supplementary -- 2000 mg/kg/day -- 5 male/5 female
Cyclophosphamide -- 50 mg/kg/day -- 5 male/5 female
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide 5 mg/ml dissolved in distilled water

Results and discussion

Any other information on results incl. tables

Preliminary Toxicity Test (see Table 1 below)

In order to select the top dose of the test substance to be administered in the cytogenetic study: 1500 and 2000 mg/kg/day were administered by intraperitoneal route for two consecutive days to six male and six female mice. The administration of 1500 and 2000 mg/kg/day induced neither clinical signs nor mortality. Consequently, the dose of 2000 mg/kg/day, being the top dose requested by international regulations, was selected for the cytogenetic study. The two other doses were 800 or 400 mg/kg/day.

Cytogenetic Test

First Study (unvalidated) (see Table 2 below)

No clinical signs and no mortality were observed after treatment with the test substance. Since the PE/NE ratio in the vehicle control group was lower than usually obtained in our laboratory, the results of this first test were not taken into account. Indeed the biological significance of the results obtained in the conditions of an insufficient erythropoiesis is difficult to assess.

Second Study (see Tables 3 and 4 below)

Therefore, a second cytogenetic study was performed only at the top dose of 2000 mg/kg/day. In the vehicle control group, the mean value of micronucleated polychromatic erythrocytes (MPE) was within the range of laboratory historical data. Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under the experimental conditions. In addition, the PE/NE ratio decreased significantly (p < 0.01) showing the toxic effect of this substance to bone marrow cells. In the group treated with iso-propyl bromide, the mean value of MPE was slightly higher to the vehicle group, and statistically significant differences were observed (p < 0.05). However, the value obtained (2.3% vs 1.3%) was in the range of our historical data. The PE/NE ratio was statistically lower than that of the vehicle control group in the study (p < 0.001), showing that bone marrow was effectively affected by the test substance.

Table 1 -- Results of the Preliminary Toxicity Test
Test substance diluted in corn oil
Doses Time Animals Clinical
mg/kg/day   Males Females Signs
1500 2-6 hr      
  24-48 hr 01-02-03 01-02-03 None
2000 2-6 hr      
  24-48 hr 01-02-03 01-02-03 None
Table 2 -- Data Summary of the First Study (unvalidated)
Group Doses (1) MPE/1000PE PE/NE ratio
  mg/kg/day mean (sd)   mean (sd)  
Vehicle - - 0.8 (0.5)   0.5 (0.1)  
Test Substance 400 1.7 (1.1) ** 0.6 (0.2)  
  800 2.2 (1.2) *** 0.4 (0.1) *
  2000 2.6 (1.3) *** 0.4 (0.2)  
Cyclophosphamide 50 24.5 (6.3) *** 0.4 0.2)  
10 animals (5 males/5 females) per group.
Time of sacrifice after the last administration = 24 hr
Doses (1) frequency -- vehicle and test substance: 2 administrations at 24 hr intervals
-- cyclophosphamide:1administration (oral route)
* p<0.05
** p<0.01
*** p<0.001
Table 3 -- Results of the Second Study
Doses Time Animals Clinical
mg/kg/day   Males Females Signs
2000 2-6 hr      
  24-48 hr 01-02-03 01-02-03 None
Table 4 -- Data Summary of the Second Study
Group Doses (1) MPE/1000PE PE/NE ratio
  mg/kg/day mean (sd)   mean (sd)  
Vehicle - - 1.3 (0.7)   0.8 (0.2)  
Test Substance 2000 2.3 (1.5) * 0.4 (0.2) ***
Cyclophosphamide 50 33.6 (8.5) *** 0.4 (0.2) ***
10 animals (5 males/5 females) per group.
Time of sacrifice after the last administration = 24 hr
Doses (1) frequency -- vehicle and test substance: 2 administrations at 24 hr intervals
-- cyclophosphamide:1administration (oral route)
* p<0.05
*** p<0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions described, the test substance Iso-PROPYL BROMIDE did not induce cytogenetic damage to the bone marrow cells of mice treated by intraperitoneal route at 2000 mg/kg/day in the micronucleus test.
Executive summary:

In a CoR 1 Swiss mouse bone marrow micronucleus assay (OECD Guideline 474 Mammalian Erythrocyte Micronucleus Test), 5 male & 5 female mice per dose were treated intraperitoneally with 2-bromopropane at a dose of 2,000 mg/kg bw.  Bone marrow cells were harvested at 48 hours post-treatment.  The vehicle was corn oil.

 

There were no signs of toxicity during the study. The positive control (cyclophosphamide) induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow of mice exposed to 2 -bromopropane.