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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
No data reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it was carried out according to or similar to OECD TG 474.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Oily liquid
Details on test material:
Unrefined lubricating oil (ULO) feedstock derived from a naphthenic crude oil: CAS# 64741-53-3

In addition a heavy fuel oil substance was included as part of the validation work
Catalytically cracked clarified oil (CCCO) : CAS # 64741-62-4

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada (Quebec, Canada)
- Age at study initiation: 6 to 9 weeks old
- Weight at study initiation: 17 to 35 grams
- Assigned to test groups randomly: not reported
- Fasting period before study: not reported
- Housing: singly housed in wire mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: quarantine for 17 days


ENVIRONMENTAL CONDITIONS: not reported


IN-LIFE DATES: not reported

Administration / exposure

Route of administration:
other: oral gavage or intraperitoneal injection
Vehicle:
Four separate micronucleus studies were conducted:
Experiment 1: CCCO in corn oil via oral gavage or intraperitoneal injection
Experiment 2: CCCO extracted using DMSO via oral gavage
Experiment 3: ULO neat or ULO DMSO extracted via oral gavage
Experiment 4: CCCO in corn oil via intraperitoneal injection
Details on exposure:
Four separate micronucleus studies were conducted based on a range-finding experiment.
Duration of treatment / exposure:
Experiment 1: Cells harvested 24 hours after last administration of test substance
Experiment 2: Cells harvested 24 hours and 48 hours after last administration of test substance
Experiment 3: Cells harvested 24 hours after last administration of test substance
Experiment 4: Cells harvested 24 hours after last administration of test substance
Frequency of treatment:
Experiment 1: two consecutive daily doses
Experiment 2: two consecutive daily doses
Experiment 3: two consecutive daily doses
Experiment 4: two consecutive daily doses
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.188, 0.375, 0.75, 1.50 g/kg (highest dose in gavage group only)
Basis:
other: experiment 1 (validation with heavy fuel oil)
Remarks:
Doses / Concentrations:
0, 1.25, 2.5, 5.0 g/kg
Basis:
other: experiment 2 (validation with heavy fuel oil)
Remarks:
Doses / Concentrations:
0, 1.25, 2.5, 5.0 g/kg
Basis:
other: experiment 3 (unrefined lube oil - 64741-53-3)
Remarks:
Doses / Concentrations:
0, 0.75, 1.5, 3.0 g/kg
Basis:
other: experiment 4 (validation with heavy fuel oil)
No. of animals per sex per dose:
Experiment 1: 5/sex/dose
Experiment 2: 2/sex/dose
Experiment 3: 2/sex/dose
Experiment 4: 2/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Experiment 1: cyclophosphamide (CPP) in water, 0.04 g/kg
Experiment 2: 7,12-dimethylbenzanthracene (DMBA), 0.1 g/kg
Experiment 3: 7,12-dimethylbenzanthracene (DMBA), 0.15 g/kg

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
Both femurs were removed from each treated mouse, and the proximal ends were cut to expose the bone marrow which was then aspirated with foetal bovine serum into a centrifuge tube. The cells were collected by centrifugation, and slides were prepared. After fixation in methanol, the slides were stained with acridine orange for approximately 1 to 2 minutes and evaluated at 400X magnification by fluorescence microscopy. A total of 1000 erythrocytes were counted from each animal, and the total number of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. One thousand PCEs were evaluated for the presence of micronuclei.
Evaluation criteria:
Means and standard deviations were calculated for each treatment and compared to controls to determine differences in micronucleus frequency.
Statistics:
Means and standard deviations were calculated. Test of equality of group means by standard one way analysis of variance at each time period was calculated. When ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan's Multiple Range Test. A standard regression analysis was performed to test for a dose response. Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion. The residuals were normally distributed (values were greater than 0.01 level of significance) in more than 75% of the analyses. Therefore non-parametric analysis was not performed. Sexes were analyzed separately in the experiment 1. In subsequent studies, the sexes were not separated due to the smaller group sizes.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In an initial range-finding study, CCCO was orally administered in doses of 1, 2, 3, or 4 g/kg on two consecutive days to CD-1 mice in groups of four (two males and two females). All mice in the highest dose group died as did one mouse in the 3 g/kg group, and 2 mice in the 2 g/kg group. All remaining mice in 2 g/kg and 3 g/kg groups were observed to be hypoactive or prostrate. All mice given 1 g/kg survived. Based on these findings, 1.5 g/kg was selected as the high dose for oral gavage studies. There were no notable increases in the mean number of micronucleated polychromatic erythrocytes in any of the dose groups in the range-finding study. All values were within the normal range of the corn oil control for this laboratory.

Any other information on results incl. tables

There were no signs of clastogenicity in any of the four studies, even though a lethal response was observed in mice administered DMSO-extracted CCCO where one of four mice in the 2.5 g/kg group and three of four mice in the 5 g/kg group died. The positive and negative controls of all studies induced the appropriate response. 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The sample of unrefined lubticating oil was not clastogenic following oral administration to mice, either neat or as a DMSO extract.
Executive summary:

Read across justification

IP346 data were not available for these samples.  Accordingly it was not possible to differentiate them on the basis of IP 346 levels. However neither unrefined and acid treated oils or heavy fuel oils were not active when tested in this assay when applied as neat or DMSO-extracted materials. Accordingly, if the aromatic constituents of these oils are not active when tested separately, it seems reasonable to assume that none of the oils in the lubricant base oil category would be active in bone marrow assays for chromosomal mutations.

Four separate bone marrow micronucleus assays were conducted using two types of petroleum-derived materials: catalytically cracked clarified oil (CCCO) and an unrefined lubricating oil (ULO). 

In the test (experiment 3) with neat or DMSO-extracted ULO, material was administered to CD-1 mice (2/sex/dose) in two consecutive daily doses via oral gavage at dose levels of 0, 1.25, 2.5, or 5.0 g/kg. Bone marrow cells were harvested at 24 hours following the final dose.

In the first study, CD-1 mice (5/sex/dose) were administered CCCO in corn oil in two consecutive daily doses via oral gavage or intraperitoneal injection at dose levels of 0, 0.188, 0.375, or 0.75 g/kg. An additional high dose of 1.50 g/kg was administered to the oral gavage group only. Bone marrow cells were harvested at 24 and 48 hours after the final dose. In a second micronucleus test, CD-1 mice (2/sex/dose) were administered a DMSO extract of CCCO in two consecutive daily doses via oral gavage at dose levels of 0, 1.25, 2.5, or 5.0 g/kg. Bone marrow cells were harvested at 24 hours after the final dose.  In the fourth test, CCCO in corn oil was administered to CD-1 mice (2/sex/dose) in two consecutive daily doses by IP injection at dose levels of 0, 0.75, 1.5, or 3.0 g/kg. Bone marrow cells were harvested at 24 hours after the final dose.

There were no signs of clastogenicity in any of the four studies, even though a lethal response was observed in mice administered DMSO-extracted CCCO where one of four mice in the 2.5 g/kg group and three of four mice in the 5 g/kg group died.  The positive and negative controls of all studies induced the appropriate response. 

This study received a Klimisch score of 1 and is classified as reliable without restriction because it was carried out according to or similar to OECD TG474.