Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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:
1985-10-17 to 1986-01-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it is in compliance with GLPs as published in 21 CFR 58, 40 CFR 160, and 40 CFR 792 and was conducted according to EPA OPPTS 870.5375, EPA OTS 789.5375, and OECD 473.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report date:
1987

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Deviations are minor not expected to interfere with study results
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
64742-53-6
Cas Number:
64742-53-6
IUPAC Name:
64742-53-6
Constituent 2
Reference substance name:
55/60 Pale Oil, highly refined, IP 346 < 3%
IUPAC Name:
55/60 Pale Oil, highly refined, IP 346 < 3%
Test material form:
other: Oily liquid
Details on test material:
This substance is very similar in structure to the substance being registered.

- Name of test material (as cited in study report): L-01 (55/60 Pale Oil)
CAS number - 64742-53-6
- Substance type: Lubricant base oils (sufficiently refined, IP 346 < 3 wt%)
- Physical state: Liquid (clear, colorless)
- Lot/batch No.: TA288
- Expiration date of the lot/batch: 1990-09-19
- Storage condition of test material: Room temperature, protected from light

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium supplemented with 10 percent and 2.5 percent fetal bovine serum, 100 units penicillin and 100 micrograms streptomycin per milliliter, and 2 mM L-glutamine (complete medium)
- Properly maintained: yes (kept at 37±1 degrees Celsius in 5±1 percent carbon dioxide in air)
- Periodically checked for Mycoplasma contamination: Not reported
- Periodically checked for karyotype stability: Not reported
- Periodically "cleansed" against high spontaneous background: Not reported
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Metabolic activation was prepared immediately prior to use from Aroclor-pretreated Sprague-Dawley rat livers. S9 mix consisted of 15 μL S9, 2.4 mg NADP, and 4.5 mg isocitric acid per mL Ham's F-12 medium with 2.5% serum.
Test concentrations with justification for top dose:
Preliminary toxicity test: 0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, and 1 μL/mL, ±S9
Chromosome aberration test: 0.02, 0.04, 0.08, 0.15 μL/mL, -S9; 0.05, 0.1, 0.2, 0.4 μL/mL, +S9
Triethylenemelamine (TEM, positive control, -S9): dissolved in distilled water for a working concentration of 1 μg/mL
Cyclophosphamide (CP, positive control, +S9): dissolved in distilled water for a working concentration of 100 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (lot 851283)
Controls
Untreated negative controls:
yes
Remarks:
Complete medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Triethylenemelamine (-S9) and cyclophosphamide (+S9)
Details on test system and experimental conditions:
For the chromosome aberration assay, cultures were seeded in duplicate flasks at 5.0x105 cells/25 cm2 flask for each treatment condition and incubated at 37±1ºC in 5±1% carbon dioxide in air for 16-24 hours. Test material dissolved in DMSO (50 μL), DMSO alone, TEM (-S9), or CP (+S9) was added to the flasks containing either complete (-S9) or S9-reaction mixture (+S9). In the presence of metabolic activation, cells were exposed for 2 hours at 37±1ºC in 5±1% carbon dioxide in air, washed with phosphate buffered saline, refed complete medium, and incubated again for an additional 8 hours. In the absence of metabolic activation, cells were exposed for 10 hours at 37±1ºC in 5±1% carbon dioxide in air. All treated cultures were then incubated with Colcemid (0.1 μg/mL) during the last 2-3 hours prior to harvest. Nontreated flasks contained complete medium only. All other procedures were conducted in a similar manner to treatment flasks.

Metaphase cells were harvested by mitotic shake-off. Collected cells were centrifuged; cell pellet was resuspended in 2-4 mL 0.075 M KCl and allowed to stand at room temperature for 5 minutes. Cells were centrifuged, the supernatant was aspirated, and cells were fixed with two washes with approximately 2 mL Carnoy's fixative and stored overnight (or longer) in Carnoy's fixative at approximately 4ºC.

To prepare slides, fixed cells were centrifuged, supernatant fluid was decanted, and cells were resuspended in Carnoy's fixative. Cell suspension (1-2 drops) was placed in the center of a side and dried overnight. Slides were coded, and dried slides were stained with 5% Giemsa, air dried, and permanently mounted.

Fifty metaphase cells from coded slides were scored in each duplicate flask (i.e., 100 cells per treatment group). Cells were evaluated for chromatid gaps and breaks, chromosome gaps and breaks, chromatid fragments, acentric fragments, dicentrics, rings, triradicals, quadriradials, complex rearrangements, pulverized chromosome(s) and cells, and severely damaged cells (>10 aberrations). Slides were examined microscopically, and the number of polyploid metaphases and metaphases with endoreduplication were counted in a total of 100 metaphase cells.
Evaluation criteria:
An assay was considered valid if the number of cells with chromosome aberrations in the negative and solvent controls was no greater than 12%, and the frequency of structural chromosome aberrations per cells in the positive control was statistically increased (p≤0.05, Student's t test) relative to the solvent control or untreated control if a solvent other than water was used.

A positive response was considered to be a significant increase (p≤0.05, Student's t test) in the number of structural aberrations per cell in a dose-response manner. A "suspect" response was considered to be a significant increase at the high dose only with no dose-response. An equivocal response was considered to be a significant increase at one dose level other than the high dose with no dose-response.
Statistics:
Treatment-related effects were expressed relative to controls. The number and types of aberrations were presented for each treatment condition. The percentage of damaged cells in the total population of cells examined was calculated for each treatment condition. The frequency of structural aberrations per cell was calculated for each group. Chromatid and chromosome gaps are presented but were not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell. The Student's t test was used to analyze the frequency of structural aberrations per cells. The number of aberrations per cell for each treatment group was compared to the control group using the t test. Significant differences between the number of cells with numerical aberration in the treatment and control group was evaluated by a chi-square analysis using a 2 x 2 contingency table.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results from the cytotoxicity test showed a marked decrease in relative cell growth at dose 0.3 μL/mL, -S9, and 1 μL/mL, +S9. There were no surviving cells at 1 μL/mL, -S9.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Cytogenic Analysis of CHO Cell Treated with L-01

Treatment

Cells with Structural Aberrations (%)a

Cells with Numerical Aberrations (%)b

Structural Aberrations per Cella, c

Without Metabolic Activation

Untreated cells

1

3

0.01

DMSO

0

2

0.00

L-01

 

0.15 μL/mL

2

0

0.02

0.08 μL/mL

1

2

0.01

0.04 μL/mL

0

1

0.00

0.02 μL/mL

0

1

0.00

TEM

 

1.0 μg/mL

22

1

0.26**

With Metabolic Activation

Untreated cells

0

2

0.00

DMSO

3

2

0.03

L-01

 

0.4 μL/mL

0

3

0.00

0.2 μL/mL

1

2

0.01

0.1 μL/mL

1

3

0.01

0.05 μL/mL

0

3

0.00

CP

 

100 μg/mL

14

2

0.14**

aChromatid and chromosome gaps are not included.

bIncludes endoreduplications and polypoid cells.

cSDC and pulverizations were counted as 10 aberrations.

** Significant at p≤0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The highly refined lubricant base oil did not induce any significant increases in chromosome aberrations in cultured CHO cells with or without metabolic activation under the conditions of this study. Positive, vehicle, and nontreated controls performed in an appropriate manner, indicating that the test system could detect both activation-dependent and direct-acting clastogens.
Executive summary:

In a mammalian cell chromosome aberration assay, Chinese hamster ovary cells were exposed to a highly refined lubricant base oil (batch TA288) at concentrations of 0.02, 0.04, 0.08, or 0.15 μL/mL, -S9, for 10 hours and 0.05, 0.1, 0.2, or 0.4 μL/mL, +S9, for 2 hours. This read across is considered conservative since lubricant base oils are not as severely refined as white oils, but because compositional and physico-chemical data show that white mineral oils are very similar to Lubricating base oils, it is considered appropriate to read across from the Lubricating base oils data to White mineral oils.

Results from the cytotoxicity test showed a marked decrease in relative cell growth at dose 0.3 μL/mL, -S9, and 1 μL/mL, +S9. There were no surviving cells at 1 μL/mL, -S9. Doses used in the chromosome aberration study were based from these results. Results from the chromosome aberration assay showed no significant structural or numerical aberrations in CHO cells at any dose level, with or without metabolic activation. Positive controls induced the appropriate response.

The highly refined oil substances in this category are all derived from crude petroleum and produced by sequential and increasingly severe refinement. They are all hydrocarbon liquids of varying viscosity, and non volatile. They are complex combinations ofsaturated, isoparaffinic and naphthenic hydrocarbons with carbon numbers ranging from C12- C50, and boiling range of 200oC to >600oC.

The complex and variable composition of UVCB substances means that it is not possible to define precisely their physico-chemical, toxicological and environmental properties, but they will fall into a range, defined by the properties, and amounts present, of the individual hydrocarbon constituents. To take account of the variable composition, hazard properties are determined using a ‘worst case’ approach, except where specified. Where data do not exist for highly refined base oils, a conservative read across is conducted to lubricant base oils. This read across is considered conservative since lubricant base oils are not as severely refined as highly refined base oils.

This study is rated 'reliable without restriction' because it is in compliance with GLPs as published in 21 CFR 58, 40 CFR 160, and 40 CFR 792 and was conducted according to EPA OPPTS 870.5375, EPA OTS 789.5375, and OECD 473.