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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
There is no evidence of mutagenicity in vitro or in vivo in a range of recognised core assay types. In vitro studies comprise of 2 bacterial reverse mutation assays, one with 2,4,4-trimethylpentene (purity >95%) and one with diisobutylene (purity about 85%) and a mammalian chromosome aberration test with 2,4,4-trimethylpentene (>95% pure). A negative in vivo mammalian erythrocyte micronucleus test following exposure of rats to diisobutylene (about 85% purity) by inhalation has also been reported.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
yes
Remarks:
exposed by inhalation (generally in compliance with EPA OPPTS 870.1300
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
rat
Strain:
other: Crl:CD®(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 233-271 g (males) and 174-206 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: Individually in suspended wire-mesh cages. On the day of exposure, the animals were placed in wire mesh batteries containing 24 separate cages.
- Diet: Certified Rodent Lab Diet® 5002 (PMI Nutrition International, LLC) ad libitum except during exposure
- Water: Municipal water ad libitum except during exposure
- Acclimation period: Minimum of 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21.3-21.4ºC
- Humidity: 36.6-39.2%
- Air changes (per hr): Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 13 December 2005 To: 15 December 2005
Route of administration:
inhalation: vapour
Vehicle:
Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Exposure apparatus: 1000 L whole-body inhalation chambers operated at a minimum of 10 air changes per hour. Exposure atmosphere conditions were recorded approximately every 30 minutes during the exposure. Oxygen content was measured pre-exposure. The time required to attain 99% of the equilibrium concentration (or clearance time for the concentration to decrease from the equilibrium concentration) was calculated.

A vapour atmosphere of the test article was generated and piped to the inlet of the whole body chamber where it was mixed with the chamber supply air. Exhaust atmosphere passed through an in-house exhaust system.

TEST ATMOSPHERE
- Brief description of analytical method used: Two primary compounds of the test article, DIB-1 (2,4,4-trimethyl-1-pentene) and DIB-2 (2,4,4-trimethyl-2-pentene), were analyzed independently with each sample obtained by gas chromatography.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:
4 h
Frequency of treatment:
single exposure
Post exposure period:
24/48 hours
Remarks:
Doses / Concentrations:
0, 1048, 2159, 4158 ppm diisobutylene
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 995, 2035, 4015 ppm diisobutylene
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0, 579, 1184, 2335 ppm DIB-1
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0, 181, 371, 733 ppm DIB-2
Basis:
analytical conc.
No. of animals per sex per dose:
10/sex/group for sham controls and 4015 ppm, 5/sex/group for 995, 2035 ppm and for positive controls
Control animals:
yes, sham-exposed
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses/concentrations: 10 mL/kg (analysed)
Tissues and cell types examined:
erythrocytes in rat bone marrow
Details of tissue and slide preparation:
Bone marrow smears were prepared from the femurs of each animal, fixed in methanol and air dried.
Evaluation criteria:
2000 polychromatic erythrocytes (PCEs), were scored per animal for the presence of micronuclei (micronucleated PCEs, MPCEs). The number of micronucleated normocytes in the field of 2000 polychromatic erythrocytes were enumerated, but not used to evaluate the response of the test article. The proportion of polychromatic erythrocytes to total erythrocytes was recorded per 1000 erythrocytes in test article-treated animals should not be less than 20% of the control value. The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the negative control. The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes for each animal and per 10,000 PCEs per each treatment group was determined.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Mortality: No mortalities
- Clinical Signs: At post-exposure observation period: red material around the nose in the 0 ppm, 4015 ppm and positive control groups and yellow material on the urogenital and ventral abdominal area and clear material on the upper left hind limb in the 4015 ppm group.
- Bodyweight: All animals lost weight during the post-exposure observation period. By the end of the post-exposure observation period, animals at 0, 995, 2035 and 4015 ppm groups lost up to 6, 3, 8 and 17 grams, respectively, below their initial body weight. Animals in the positive control group lost (up to 18 grams) weight similarly to the 4015 ppm group.
- MPCEs: The number of micronucleated polychromatic erythrocytes in the bone marrow for the 995, 2035 and 4015 ppm groups was not significantly increased relative to the negative control group at either the 24 hour or 48 hour bone marrow collections.

Summary of acute study test atmosphere characteristics of diisobutylene

Test atmosphere characteristics

Target concentration (ppm)

0 (control)

1000

2000

4000

40 mL/kg CP

Nominal concentration (ppm)

0

1048

2159

4158

-

Test article used (g)

0

226

477

941

-

Total volume chamber air (L)

47040

48240

49440

50640

-

Analytical concentration (mean)

0

995

2035

4015

10 mL/kg

Mean concentration of DIB-1(ppm)

0

579

1184

2335

-

Mean concentration of DIB-2 (ppm)

0

181

371

733

756 ppm (n=5)

Flowrate (mL/min)

N/A

12

29

56

-

Temperature

21°C ± 0 (n=8)

24°C ± 0.7 (n=8)

22°C ± 0.5 (n=8)

25°C ± 0.7 (n=8)

-

Humidity

46% ± 0.9 (n=8)

30% ± 1.2 (n=8)

34% ± 1.2 (n=8)

35% ± 0.7 (n=8)

-

CP = cyclophosphamide monohydrate

- not reported
Conclusions:
Interpretation of results (migrated information): negative
Diisobutylene at concentrations of 995, 2035 and 4015 ppm was negative in the mammalian erythrocyte micronucleus assay when male and female albino rats were exposed to a single, 4-hour, whole-body exposure.
Executive summary:

Based on the results of this study, Diisobutylene at concentrations of 995, 2035 and 4015 ppm did not induce a statistically significant increase in the incidence of micronucleated polychromatic erythocytes in the bone marrow when male and female albino rats were exposed to test article as a single, 4-hour, whole-body exposure.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro Genetic Toxicity

2,4,4-trimethylpentene (purity >95%) was not mutagenic to S. typhimurium or E. coli strains in the presence or absence of metabolic activation at concentrations up to 5,000 ug/plate (HLS, 1996 e). Similarly, diisobutylene (purity about 85%) was negative a Bacterial Reverse Mutation Assay, using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9 (BioReliance, 2005).

In addition, 2,4,4-trimethylpentene (purity>95%) was negative when tested in an in vitro chromosomal aberration test using human peripheral blood lymphocytes in the presence or absence of metabolic activation (HLS, 1997 d).

In Vivo Genetic Toxicity

Diisobutylene (purity about 85%) did not significantly increase the incidence of micronucleated polychromatic erythrocytes in male or female rats exposed by inhalation to concentrations of 0, 995, 2035 or 4015 ppm for 4 hours (WIL, 2006b).

Overall there is no evidence of mutagenic activity with either 2,4,4-trimethylpentene (>95% purity) or diisobutylene (about 85% purity) either in vitro or in vivo from the core assay systems reported.


Justification for selection of genetic toxicity endpoint
Available information indicates that this substance is not genotoxic in vitro or in vivo

Justification for classification or non-classification

The evidence from in vitro and in vivo studies indicate that 2,4,4 -trimethylpentene is not mutagenic and hence classification under DSD or CLP is not warranted.