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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1985-12-23 to 1986-02-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it was conducted according to or similar to guideline study OECD TG 476 using the L5178Y cell line.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Method: other: API procedure
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
64741-50-0
Cas Number:
64741-50-0
IUPAC Name:
64741-50-0
Constituent 2
Reference substance name:
Unrefined light paraffinic distillate
IUPAC Name:
Unrefined light paraffinic distillate
Test material form:
other: oily liquid
Details on test material:
- Light paraffinic distillate, API 84-01, CAS No. 64741-50-0
- Test substance: Light paraffinic distillate
- Physical description: Clear yellow liquid
- Gravity API: 31.5
- Wt. % Sulphur: 0.2
- Nitrogen, ppm: 256
- Olefins: 39.7
- Naphthenes: 21.7
- Viscosity at 100°C, cSt: 2.67
- Flash Pt. (°F): 372
- Molecular Wt.: 296
- Distillation ASTM D 86 equivalent (°F) range: 601-803 (10-95%)
- Initial Boiling Point (°F): 579
- PONA 37% by MS

Method

Target gene:
Thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium supplemented with pluronic solution, L-glutamine, sodium pyruvate, antibiotic, and horse serum (10% by volume)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
50 to 1000 nL/mL; visibly insoluble above 100 nL/mL
Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: ethylmethansulphonate (EMS) for nonactivation conditions and 3-methylcholanthrene (MCA)
Details on test system and experimental conditions:
Mouse lymphoma forward mutation assay

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 14 days


SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)


NUMBER OF REPLICATIONS: not reported


NUMBER OF CELLS EVALUATED: 3 x 10^6 cells were suspended in selection medium to select for mutants


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment will be a mutant frequency that is at least 150% of the concurrent background frequency plus 10X10-6. The background frequency is defined as the average mutant frequency of the solvent negative controls. A dose-related or toxicity-related increase in mutant frequency should be observed. If an increase of two times the minimum criterion or greater is observed for a single dose near the highest testable toxicity, the test material will be considered mutagenic. Treatments that induce less than 10% relative growth are included in the assay but are not used as primary evidence for mutagenicity. A test article is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 10 to 20% relative growth or, in the case of relatively nontoxic materials, a range of applied concentrations routinely extending to the maximum of 5 mg/ml or 5 ul/ml unless limited by solubility.
Statistics:
The mutant frequency is calculated by dividing the total number of colonies in each set of three mutant selection dishes by the total count in the sent of three viable count dishes and multiplying by 2X10^-4.

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data reported.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Without metabolic activation, the test material was assayed from 400 to 1000 nL/mL, and little or no toxicity was observed well into the insoluble range. No significant increases in the mutant frequency were observed. With metabolic activation, treatments from 50 to 1000 nl/ml induced significant increases in the mutant frequency at the thymidine kinase (TK+/-) locus.  Increases ranged from 2.1-fold to 7.3-fold above background. It should be noted, however, that survival was 73% of control at 50 nl/ml, 33% of control at 200 nL/mL, and less than 20% of control at all higher levels.  The mutation frequency did not increase with concentration at levels above 300 nL/mL.

Without S9 Activation
Test material dose (nl/ml) Relative Growth (%) Mutant Frequency (10E-6 units)
Solvent control 100 34.4
EMS 0.25 μl/ml 81.2 452.5
EMS 0.40 μl/ml 47.2 694.5
400 109.4 26.3
500 99.5 37.1
600 114 30.9
800 110.4 28.1
1000 95.4 42.8
With S9 Activation 
 Test material dose (nl/ml) Relative Growth (%) Mutant Frequency (10E-6 units)
Solvent control 100 34.9
MCA 2.5 μg/ml 58.6 257.2
MCA 4.0 μg/ml 35.8 331.4
50 72.6 73.4
200 32.8 142.6
300 15 230.1
500 15.6 215.5
600 14.7 164.2
800 7.3 155.4
1000 12.3 254.8

Note: Solvent control mutant frequency is the average of three solvent control plates.

EMS = ethylmethanesulphonate, positive control substance for nonactivated conditions

MCA = 3 -methylcholanthrene, positive control substance for activated conditions

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation

The test material API 84-01 was active in the mouse lymphoma forward mutation assay only in the presence of metabolic activation.
Executive summary:

Under nonactivation conditions, the test material API 84 -01 was analyzed for mutant induction from 400 nL/mL to 1000 nL/mL, and little or no toxicity was observed (percent relative growths, 114.0% to 95.4%).  None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 61.7 x 10-6. Since there was no evidence for mutagenic activity well into the insoluble range, the test material was considered nonmutagenic without activation in this assay.  In the presence of metabolic activation, the test material was analyzed for mutant induction from 50 nl/ml to 1000 nl/ml and a wide range of toxicities was induced (percent relative growths, 72.6% to 7.3%).  The test material appeared to interact with the activation mix to convert the test material to a mutagenic form or forms.  In order for a treatment to be considered mutagenic in this assay, a mutant frequency exceeding 62.3 x 10-6 was required.  All of the assayed treatments induced mutant frequencies that exceeded the minimum criterion and the increases ranged from 2.1-fold to 7.3-fold above the background mutant frequency (average of solvent controls).  The test material was therefore considered mutagenic with activation in this assay.  In the assays used in this evaluation, the average cloning efficiencies for the solvent controls varied from 86.6% without activation to 87.4% with activation which demonstrated good cloning conditions for the assays.  The negative control mutant frequencies were all in the expected range and the positive control compounds yielded mutant frequencies that were greatly in excess of the background.

This study received a Klimisch score of 1 and is classified as reliable without restriction because it was conducted according to or similar to guideline study OECD TG 476 using the L5178Y cell line.