Registration Dossier

Administrative data

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diisobutylene
- Sample ID Number ID# GCRD10401R
- Physical state: Clear light brown liquid
- Chemical composition of sample of diisobutylene: approximately 70% 2,4,4-trimethylpentene (detailed chemical composition information provided in Table 1).
- Storage condition of test material: Refrigerated


Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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)

Examinations

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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.