Naphtha (petroleum), hydrotreated heavy

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

Nov. 2000 - Jan. 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report similar or equivalent to OECD TG 475.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

86290-81-5

Cas Number:

86290-81-5

IUPAC Name:

86290-81-5

Constituent 2

Reference substance name:

Baseline gasoline vapor condensate

IUPAC Name:

Baseline gasoline vapor condensate

Constituent 3

Reference substance name:

API 99-01

IUPAC Name:

API 99-01

Test material form:

other: low viscosity liquid hydrocarbon

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

inhalation

Vehicle:

- Vehicle(s)/solvent(s) used: Air control

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Air flow rate: minimum flow rate 200 Liters per minute.
- Air change rate: 12 air changes per hour.
- Method of particle size determination: TSI Aerodynamic Particle Sizer. Samples were drawn for 20 seconds at a flow rate of 5.00 Lpm.
- Treatment of exhaust air: In house filtering system, consisting of a coarse filter, a HEPA filter, activated charcoal and then through a fume incinerator.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

5 days per week for 4 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 2000, 10 000 & 20 000 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Positive control(s):

Cyclophosphamide
- Route of administration: Intraperitoneal injection
- Doses / concentrations: 40 mg/kg bodyweight

Examinations

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals from each of the dose groups were killed 24 hours after the final exposure period by CO2 asphyxiation. Both femurs were exposed, cut just above the knee and bone marrow was aspirated into a syringe containing a small volume (about 0.5 ml) of serum. The cells were then flushed into a centrifuge tube of cold serum.

DETAILS OF SLIDE PREPARATION:
The bone marrow cells were pelleted by centrifugation at about 150xg for about 5 minutes and the supernatant was drawn off, leaving a small amount of serum with the cell pellet. The cells were resuspended by aspiration with a pasteur pipette and a small drop of cells was spread onto a clean glass slide. Four slides were prepared from each animal. The slides were allowed to air dry, fixed by dipping for about 3 minutes in methanol, and aged overnight or longer prior to staining.

METHOD OF ANALYSIS:
One slide from each animal was stained according to a modified Feulgen method, which specifically stains DNA-containing bodies deep purple while leaving mast cell granules unstained. This method also allows reasonable differentiation of mature and immature erythrocytes and produces permanent preparations. The remaining slides and cell pellet were held in reserve.

The stained smears were examined under code by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal.

Evaluation criteria:

Micronuclei were identified by the following criteria:
-Large enough to discern morphological characteristics.
-Should possess a generally rounded shape with a clearly defined outline.
-Should be deeply stained and similar in colour to the nuclei of other cells - not black
- Should lie in the same focal plane as the cell
- Lack internal structure, ie, they are pyknotic
- There should be no micronucleus-like debris in the area surrounding the cell

The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes.

A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the control group (P<0.01); individual and/or group mean values should exceed the laboratory historical control range.

A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the control group and where these values fall within the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (P<0.01).

Statistics:

The results for each treatment group were compared with the results for the negative control group using non-parametric statistics.

For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels is made with the control using the Linear by Linear Association test for trend, in a step-down fashion if significance is detected; for individual inter-group comparisons (ie the positive control group only) this procedure smplifies to a straightforward permutation test. For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

No statistically significant increases in the frequency of micronucleated immature erythrocytes were observed in rats treated with baseline gasoline vapor condensate compared to negative control values.

Toxicity:

no effects

Remarks:

No substantial decrease in the proportion of immature erythrocytes was observed in rats treated with baseline gasoline vapor condensate compared to negative control values.

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The in vivo micronucleus assay of baseline gasoline vapor condensate was negative. This finding does not warrant classification of baseline gasoline vapor condensate as a genotoxin under Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Executive summary:

Baseline gasoline vapor condensate was examined for its potential to induce chromosomal damage in rat bone marrow erythrocytes from rats dosed via inhalation with the test material at 2000, 10 000, or 20 000 mg/m³ for 6 hours per day, 5 days a week for a duration of 4 weeks. Vehicle control animals were dosed with clean air alone, and positive control animals were dosed intraperitoneally with 40 mg/kg dose of cyclophosphamide in sterile water. Bone marrow was collected from 10 animals (5/sex) from each treatment and control group at 24 hours after the final administration. No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature eyrthrocytes were observed in the dose groups treated with the test material compared to the negative control values (p>0.01 in each case). The positive control material caused both large, significant increases in the frequency of micronucleated immature erythrocytes and statistically significant decreases in the proportion of immature erythrocytes (p<0.001 in each case). Baseline gasoline vapor condensate was considered to be non-genotoxic and non-clastogenic under the conditions of this test. This finding does not warrant classification of baseline gasoline vapor condensate as a genotoxin under Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.