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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
2-methylbut-2-ene (2M2B) has been examined for mutagenicity both in vitro and in vivo in a range of recognised core assay types.  It has shown negative results for mutagenicity in vitro but positive results in a number of studies in vivo in the bone marrow micronucleus assay.  It is concluded that the available data indicates that 2M2B is genotoxic in vivo.
Link to relevant study records
Reference
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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay
Test material information:
Composition 1
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
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
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
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.
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.

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

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vivo:

Non-human information

In vitro data

The key studies are considered to be a bacterial mutation assay and a mammalian cell cytogenetic assay (SRC, 1981).  These are two recognised core assay types for investigating mutation in vitro and were conducted with technically pure 2M2B.

2-Methyl-2-butene (2M2B) was tested using sealed containers for a pre-incubation phase in an Ames assay in S. typhimurium strains TA1535, TA1537, TA1538, TA100, and TA98 and E. coli strains WP2 and WP2uvrA) in both the presence and absence of rat liver S9.  A range of doses up to 4000 µg/plate was used.  2M2B was negative in this assay.  In the mammalian cell cytogenetic assay, 2M2B was examined in cultured rat liver cells (RL4) at a range of doses up to 50 µg/mL in the absence of auxiliary metabolic activation. Cells were arrested in metaphase after 24 hours exposure and evaluated for chromosomal damage. 2M2B was not clastogenic in this assay. 

A negative result was also obtained for the endpoint of gene conversion in S. cerevisiae (SRC, 1981).

In vivo data

The key studies are considered to be cytogenetic studies (bone marrow micronucleus) in the mouse (EBSI, 1991a, BASF 2009) and rat (EBSI, 1991b). This is a recognised core assay type for investigating mutation in vivo.

Male mice or rats were exposed by the inhalation route to doses of 2M2B of 1005, 3207 or 9956 ppm for 6 hours per day for 2 days (EBSI, 1991a,b).  Statistically significant increases in the incidence of micronucleated polychromatic erythrocytes (MPEs) over controls were observed at the two highest dose levels in both the mouse and the rat.  The dose levels resulted in clinical signs of laboured breathing and decreased activity in a number of the animals exposed. The magnitude of the increase in MPEs was greater in the mouse, with the maximum fold-increase over controls being to 10.6x in the mouse but only 2.2x in the rat.  A recent mouse bone marrow micronucleus assay in which two strains of mouse were exposed by the inhalation route to doses of 2M2B up to 11608 ppm for 6 hours per day for 2 days confirmed these findings with increases in the incidence of MPEs to 10x control or greater in both strains (BASF, 2009).  An assessment of the size of the micronuclei concluded that they were mainly small in size, and thus indicated that the micronuclei were most likely chromosomal fragments rather than whole chromosomes. Appropriate positive controls of an aneugen and a clastogen were used for reference with the sizing.  This would be consistent with a clastogenic effect of 2M2B in the mouse rather than an aneugenic effect.

There are also further reports of micronucleus studies in mice and rats, with results generally consistent with the studies discussed above (EBSI 1990, 1991c,d).

Human information

There is no information indicating any adverse effects of 2M2B.


Justification for selection of genetic toxicity endpoint
Available information for 2M2B indicate genotoxic potential in vivo

Justification for classification or non-classification

2-methylbut-2-ene (2M2B) warrants classification under CLP as Category 2 (H341): Suspected of causing genetic defects (positive in mammalian somatic cell mutagenicity tests in vivo).