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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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MRD-90-830
- Physical state: Colourless gas
- Analytical purity: >99.2%
- Purity test date: Responsibility of sponsor
- Lot/batch No.: I
- Expiration date of the lot/batch: July 01, 1995
- Stability under test conditions: Responsibility of sponsor
- Storage condition of test material: Room temperature

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: 6 -7 weeks
- Weight at study initiation: 24-28 g
- Assigned to test groups randomly: yes, based on body weights. Animal numbers were not sequential for each group and constitute a blind code.
- Housing: single housed in suspended steel cages.
- Diet : Purina Certified rodent chow (pellets) ad libitum during non-exposure periods.
- Water : automatic watering system, ad libitum during non-exposure periods.
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature: 68 - 76°F
- Humidity: 40- 70%
- Air changes (per hr): not reported
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: From: 7 August 1990 To: 4 September 1990

Administration / exposure

Route of administration:
inhalation
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1m3 stainless steel and glass exposure chambers
- Method of holding animals in test chamber: individually in stainless steel cages
- Source and rate of air: room air
- Method of conditioning air: not reported
- System of generating vapour: Liquid test substance was pumped into a heated glass vapour generator and the resulting vapours were mixed with room air and then delivered to the exposure chamber.
- Temperature, humidity, pressure in air chamber: recorded at approximately 30 minute intervals
- Air flow rate: 200 L/min (exhaust flow rate)
- Air change rate: 1 air change / 5 minutes
- Treatment of exhaust air: flow rate measured by calibrated orifice meter.

TEST ATMOSPHERE
- Brief description of analytical method used: on-line gas chromatography with flame ionisation detector
-Samples taken from breathing zone: not specified
Duration of treatment / exposure:
2 consecutive days
Frequency of treatment:
6 hours/day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1000, 3260 or 10,000 ppm
Basis:

Remarks:
Doses / Concentrations:
0, 1005, 3207, or 9956 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Positive control(s):
1,3-butadiene
- Route of administration: inhalation
- Doses / concentrations: 1000 ppm

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Mice were exposed to concentrations of the test substance , or the positive control or to air only (negative control ) by inhalation, 6 h per day on 2 consecutive days. Approximately 24 h after the last exposure, mice were killed by CO2 asphyxiation.

DETAILS OF SLIDE PREPARATION:
Bone marrow was removed from the femur , suspended in foetal bovine serum and centrifuged. The pellet was then resuspended and smears were prepared (2 slides per animal). Slides were stained with acridine orange prior to microscopic evaluation.

METHOD OF ANALYSIS: microscopic evaluation
1000 polychromatic erythrocytes (PCE) from each animal were examined for micronuclei. The ratio of polychromatic erythrocytes to normochromatic erythrocytes (NCE) was determined for each animal by counting 1000 erythrocytes (PCEs & NCEs).

OTHER:
Evaluation criteria:
A dose related and statistically significant increase in the number of micronuclei, compared to air controls, was taken as a positive response.
A dose related and statistically significant decrease in the percentages of polychromatic erythrocytes was taken as a measure of toxicity.
Statistics:
Means and standard deviations of micronuclei data. ANOVA to test for equality of group means followed by Duncan's Multiple Range Test if appropriate. Standard regression analysis to test for dose-related response. Wilk's Criterion for normality.

Results and discussion

Test results
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Statistically significant, dose related increase in the mean number of micronuclei at 3,260 and 10,000 ppm.
- Ratio of PCE/NCE (for Micronucleus assay): Statistically significant, dose related decrease in percentages of PCEs at 10,000 ppm.
- Appropriateness of dose levels and route: Dose levels showed adequate dose response and negative and positive controls gave expected results.
- Statistical evaluation: Statistical significance p<0.01 for increase in micronuclei and decrease in PCEs.

Any other information on results incl. tables

On Day 1, all mice appeared normal. A few (i.e., 10-30%) of the animal in the high dose group (10,000 ppm) displayed decreased activity that started the second hour of exposure and continued for the remainder of the exposure. A few animals also exhibited laboured breathing at the second hour of exposure and continued throughout the exposure. All of the mice in the positive control group (1000 ppm 1,3-butadiene) appeared normal during most of the exposure and a few animals displayed white ocular discharge during the sixth hour of exposure. On Day 2, all the mice in the air and positive control groups appeared normal. All the mice in the low and high dose group appeared normal for the first three hours of exposure. Few (i.e.,10-30%) to some (i.e., 40-60%) of the mid dose group animals displayed decreased activity at the second hour of exposure that continued for the remainder of the exposure. A few mice also exhibited laboured breathing for the last three hours of the exposure. A few to most (i.e., 70-90%) of the high dose animals displayed decreased activity during the last three hours of exposure and a few to some also exhibited laboured breathing during the last three hours of exposure.

 

The test substance induced statistically significant (p<0.01) and dose-related increases in micronucleated PCEs at 3207 and 9956 ppm (Table 1). The positive control produced a statistically significant increase in micronucleated PCEs (29.7). A statistically significant (p<0.01) decrease in the %PCEs, which is a measure of haematotoxicity, was also observed at 9956 ppm.

Table 1 - In vivo mammalian bone marrow micronucleus assay in male mice

Dose

Number

Mean PCE

STD-PCE

Mean MNE

STD-MNE

Air Control

10

57.38 R+

3.25228

3.4 R+

3.23866

1005ppm MRD-90-830

10

57.42

2.90777

4.2

1.75119

3207 ppm MRD-90-830

10

54.30

6.82837

16.6**

5.33750

9956 ppm MRD-90-830

10

37.90 **

2.97134

36.1**

8.88757

1045 ppm Butadiene

10

46.45 **

4.23668

29.7**

5.27152

N = Number of animals in dose group

MEAN PCE = Mean % PCE for dose group

STD PCE= Standard deviation of mean % PCE

MEAN MNE = Mean micronuclei per 1,000 polychromatic erythrocytes

STD MNE= Standard deviation of mean micronuclei

*  Significantly different from control at P<0.05

** Significantly different from control at P<0.01

R Significant regression coefficient at P<0.05

R+Significant regression coefficient at P'<0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive
Under the conditions of this study, inhalation exposure to 3207 and 9956 ppm of the test substance induced statistically significant increases in micronucleated polychromatic erythrocytes in male B6C3F1 mice.
Executive summary:

Under the conditions of this study, inhalation exposure to 3207 and 9956 ppm of the test substance induced statistically significant increases in micronucleated polychromatic erythrocytes in male B6C3F1 mice. The test substance is therefore considered to be clastrogenic under the conditions of the test.