[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-N-octadecyldocos-13-enamide

EC Number:

233-226-5

EC Name:

(Z)-N-octadecyldocos-13-enamide

Cas Number:

10094-45-8

Molecular formula:

C40H79NO

IUPAC Name:

N-octadecyldocos-13-enamide

Constituent 2

Reference substance name:

stearyl erucamide

IUPAC Name:

stearyl erucamide

Test material form:

solid: pellets

Details on test material:

- Name of test material (as cited in study report): Stearyl Erucamide bead
- Physical state: solid, pale yellow beads
- Analytical purity: not reported
- Lot/batch No.: 3751D
- Expiration date of the lot/batch: 12 months from manufacture
- Stability under test conditions: not reported
- Storage condition of test material: in the fridge in the dark

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: before and after treatment RPMI 1640 with 2 mM glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin and 10% horse serum (referred to as R10); for cloning for survival and mutation cells were grown in the same base medium containing 20% horse serum instead and additionally 200 µg/mL sodium pyruvate (referred to as R20). In the range finder and during treatment the same base medium with 5% horse serum was used (referred to as R5).
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

beta-naphthoflavone and sodium phenobarbitone-induced rat liver S-9

Test concentrations with justification for top dose:

10, 25, 50, 75 and 100 µg/mL ± S-9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: positive control chemicals in DMSO, test substance in ethanol (2% v/v final concentration in culture)
- Justification for choice of solvent/vehicle: low solubility of substances in water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S-9 Migrated to IUCLID6: 750 µg/mL

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S-9 Migrated to IUCLID6: 25µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10-12 days (5% Co2, 37 °C)
- Fixation time (start of exposure up to fixation or harvest of cells): recording of colony formation on days 13-15

SELECTION AGENT (mutation assays): TFT (trifluorothymidine)

NUMBER OF REPLICATIONS: 384 wells per concentration analysed; 2 independent experiments performed

NUMBER OF CELLS EVALUATED: 2000 cells/well (10e4 cells/mL) initially plated in 4 96-well plates for assessment of mutations at the TK locus

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency: cultures were diluted to 8 cells/mL in R20, and 0.2mL aliquots of this suspension were dispensed into 2 96-well microtitre plates for each dose point (average of 1.6 cells/well).

Evaluation criteria:

Positive: A dose response curve with at least one point showing a statistically significant increase in mutation frequency (mutations per 10e6 survivors) compared to the solvent control, which is reproducible in both experiments.

Negative: Neither a dose response curve nor any statistically significant increases in mutation frequency are observed.

Statistics:

The distribution of colony forming units amongst the wells of a microtitre plate follows the Poisson Distribution, so that the plating efficiency (PE) is calculated using the zero form of the Poisson Distribution:
P(0) = number of empty wells/total wells plated

PE = -ln P(0)/n
(n = number of cells plated per well)

The mutation frequency (MF) per survivor is given by:
MF = PE(s)/PE(m) = Plating efficiency in selective medium/Plating efficiency in non selective medium

All individual mutation assessment points are compared to the controls, using the comparison of multiple treatments with control described in "Statistical Evaluation of Mutagenicity Test data" (Ed. D.J. Kirkland published by Cambridge University Press 1989).

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test substance dissolved in ethanol due to very low water solubility
- Other: There was a statistically significant difference in the negative (solvent) control's mutation frequency compared to the untreated controls in the abence of S-9 only in the first experiment. No comparable effect was observed in the second experiment.

RANGE-FINDING/SCREENING STUDIES:
Logarithmically growing L5178Y cells were suspended in R5 at 5x10e5 cells/mL and treated with the test substance for 3 hours at 37 °C in both the absence and presence of S-9 mix. At the end of the treatment period, the cells were washed with phosphate buffered saline to remove the test substance, resuspended and counted. Aliquots of each culture were diluted to 8 cells/mL and plated in R20 to measure survival. From the resuls of the range finder, doses of 10, 25, 50, 75 and 100 µg/mL in the absence and presence of metabolic activation were chosen for the main experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
the limiting factor for dose administration was the solubility limit for the test article. There was no toxicity demonstrated in the range finder.

Any other information on results incl. tables

Mutant frequencies mouse lymphoma assay:

Dose (µg/mL)	-S9		+S9
	Mutation frequency	t	Mutation frequency	t
Summary of Mutant frequencies for Experiment 1
25	1.05e-3	10.10*	4.74e-4	5,97*
50	6.83e-4	2.93	4.19e-4	3.39
75	3.04e-4	2.15	1.87e-4	2.74
100	5.77e-4	1.18	1.67e-4	4.49
Solvent control	4.42e-4	16.1*(vs. medium)	2.83e-4	0.6 (vs. medium)
Medium	2.41e-4	-	3.37e-4	-
EMS	1.09e-3	36.4*	-	-
B(a)P	-	-	2.24e-3	203.8*
Summary of Mutant Frequencies for Experiment 2
25	2.52e-4	1.24	1.53e-4	0.92
50	2.34e-4	1.84	1.40e-4	1.78
75	1.79e-4	5.86	1.47e-4	1.29
100	3.45e-4	0.03	1.39e-4	1.89
Solvent control	3.32e-4	1.3 (vs. medium)	1.94e-4	0.0 (vs. medium)
Medium	2.78e-4	-	1.95e-4	-
EMS	1.21e-3	80.1*	-	-
B(a)P	-	-	4.22e-4	29.0

* = p<0.05

Compared to the relevant negative controls the cells treated with the positive control chemicals had significantly increased mutation frequencies (p<0.05).

In experiment 1 there was a statistically signifcant difference in the negative (solvent) control's mutation frequency compared to the untreated controls in the absence of metabolic activation only. There were no statistically significant differences in the mutation frequencies of negative (solvent) controls compared to the untreated cultures in experiment 2. As that observation was not reproducible in the second experiment it was not considered to be of biological significance.

In experiment 1 the only statistically significant increase in mutation frequency was observed after dosing with the test substance at 25 µg/mL with and without metabolic activation. There was no evidence of a positive dose response. In experiment 2 there were no statistically significant increases in mutation frequency observed after dosing with the test substance.

Conclusion:

The test substance did not show mutagenic activity under the conditions of this test.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material is not mutagenic to mouse Lymphoma cells.

Executive summary:

In an OECD 476 study, conducted according to GLP, the structurally similar read across substance is non-mutagenic to mouse lymphoma.