(Z)-N-octadecyldocos-13-enamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:

Gene mutation in bacteria (Reverse Mutation Test, OECD 471): negative

Cytogenicity in mammalian cells (Chromosomal Aberration, OECD 473): negative

Gene mutation in mammalian cells (MLA, OECD 476): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP guideline study with acceptable restrictions. Although 5 strains were tested no strain for detection of cross-linking or oxidising mutagens was included.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Strain for detection of oxidising or cross-linking mutagens is missing, but in accordance with guideline at the time of testing.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: hexane
- Justification for choice of solvent/vehicle: solubility of test compound in vehicle

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

hexane

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S-9 Migrated to IUCLID6: 80 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

hexane

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

TA 100 and TA 1535 without S-9 Migrated to IUCLID6: 3 µg/plate (TA 100), 5 µg/plate (TA 1535)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

hexane

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 and TA 1538 without S-9 Migrated to IUCLID6: 1 µg/plate (TA 98) and 2 µg/plate (TA 1538)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

hexane

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene: 0.5 µg/plate with TA 1538 and TA 98, 1 µg/plate with TA 100, and 2 µg/plate with TA 1535 and TA 1537.

Remarks:

all strains with S-9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: triplicates; independent repetition of complete test

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: reduction of background growth

Evaluation criteria:

Positive: increase in revertant colony numbers of at least twice of those of the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in presence or absence of S9-mix. No statistical analysis is performed.
Negative: no reproducible increases in revertant colony numbers of at least 1.5 times of those of the concurrent solvent controls, at any dose level with any bacterial strain. No statistical analysis is performed.
Ambiguous: If the results fail to satisfy the criteria for a clear positive or negative response as described the following approach is taken: 1) Repeat tests with modified experimental method may be performed. These modifications include (but are not restricted to) the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies the 'positive' criteria the material is considered to be mutagenic. No statistical analysis is performed. 2) If no clear 'positive' response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedure used is normally analysis of variance followed by Student's t-test.

Statistics:

In case of ambiguous results analysis of variance followed by Student's t-test is performed. In case of unequivocally positive or negative results according to the described criteria, no statistical analysis is performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation starting at 1500 µg/plate in test 1 and at 5000 µg/plate in test 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation starting at 1500 µg/plate in test 1 and at 5000 µg/plate in test 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation starting at 1500 µg/plate in test 1 and at 5000 µg/plate in test 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation starting at 1500 µg/plate in test 1 and at 5000 µg/plate in test 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation starting at 1500 µg/plate in test 1 and at 5000 µg/plate in test 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Starting at 1500 µg/plate with and without metabolic activation in all strains in test 1 and at 5000 µg/plate with and without activation in all strains in test 2.

RANGE-FINDING/SCREENING STUDIES:
Dose range finding tests were performed with 5, 50, 500 and 5000 µg/plate. No cytotoxicity was observed, therefore 5000 µg/plate was chosen as maximum concentration for main test.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed.

Maximum mean number of revertants (Test with highest revertant numbers chosen):

	Mean revertant numbers ±SD
	without S-9		with S-9
Strain	Solvent control	Test substance (µg/plate)	Solvent control	Test substance (µg/plate)
TA 1535	10±2.6	11±3.1 (150)	13±1.5	13±3.0 (50)
TA 1537	12±2.5	11±2.6 (150)	9±1.4	11±2.0 (50)
TA 1538	11±1.5	11±1.5 (500)	10±1.7	15±1.5 (500)
TA 98	23±1.2	25±2.0 (500)	23±3.6	24±0.6 (500)
TA 100	75±5.1	87±6.1 (150)	93±5.5	90±9.1 (5000)

 

The concurrent positive controls demonstrated the validity of the test system.

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation.

Conclusion:

Under the conditions chosen the test substance was not mutagenic in bacteria.

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP guideline study with acceptable restrictions. Purity not reported.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: primary cells from peripheral human blood

Metabolic activation:

with and without

Metabolic activation system:

beta-naphthoflavone and sodium phenobarbitone-induced rat liver S9

Test concentrations with justification for top dose:

Exp 1: -S9: 0.5, 2.5, 5, 25, 50 µg/mL; +S9: 10, 50, 100 µg/mL
Exp 2: -S9: 5, 25, 50 µg/mL; +S9: 10, 50, 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (2% v/v with S9 mix and 1% v/v without S9 mix)
- Justification for choice of solvent/vehicle: low solubility of test substance in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment 1: 3 hours with S9, 20 hours (1.5 cell cycles) without S9; Experiment 2, Set 1+2: 3 hours with S9, 20 hours without S9
- Expression time (cells in growth medium): Experiment 1: 20 hours (1.5 cycle times, cycle length 12.5 hours) with/without S9; Experiment 2, set 1: as experiment 1; Experiment 2, set 2: 44 h with/without S9
- Fixation time (start of exposure up to fixation or harvest of cells): Experiment 1: 23 hours with S9, 20 hours without S9; Experiment 2, set 1: as experiment 1; Experiment 2, set 2: 47 hours with S9, 44 hours without S9

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg/mL), 2 hours before harvest
STAIN (for cytogenetic assays): 5% Gurrs Giemsa R66, pH 6.8

NUMBER OF REPLICATIONS: duplicates (2 cultures from different blood donors)

NUMBER OF CELLS EVALUATED: 200 (100 from each donor), solvent controls 400

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (minimum of 1000 cells)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells with more than 48 chromosomes were recorded as numerical aberrations.
- Determination of endoreplication: yes

OTHER: only well spread metaphases with a chromosome count of 44-48 were scored for chromosome damage.

Evaluation criteria:

Positive: A dose response with at least one point having statistically more aberrations (p<0.05) than the solvent control is required. As the complex interaction between cell cycle and induced damage can give reduced damage scores at higher dose levels, the dose response curve will not always be a strict increase in damage with dose.

Negative: Neither a dose response nor any statistically significant increases in aberrations were observed.

Statistics:

Analysis of both total aberrations and percent aberrant cells by Fisher's exact test, as recommended by UKEMS ("Statistical Analysis of Mutagenicity Test Data" (1989) ed. D.J. Kirkland, Cambridge University Press).

Key result

Species / strain:

lymphocytes: primary cells from peripheral human blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested concentrations limited by solubility in the maximum applicable solvent concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects on pH were observed: pH 7.3 in the absence of S9
- Effects of osmolality: Osmolality in the absence of S9: medium 272.270 mOsm, 2% ethanol 612.619 mOsm, 100 µg/mL test substance concentration 671.668 mOsm
- Water solubility: limited by maximum applicable solvent concentration of 2% (v/v) ethanol: 50 and 100 µg/mL test substance without and with S9, respectively

RANGE-FINDING/SCREENING STUDIES:
In order to avoid toxicity to the cultures the maximum concentration of ethanol added was 2% (v/v). This limited maximum treatment concentration to 100 µg/mL. Osmolality and pH measurements were taken of medium control and preparations of 2% ethanol and 100 µg/mL test substance in treatment medium without S9. The cells were treated either with medium only, medium containing 2% (v/v) ethanol as solvent control, or a preparation of the test substance in ethanol added to medium at 2% (v/v). The concentrations of test substance were 1, 5, 10, 50 and 100 µg/mL. Cultures were also included for treatment with 1% ethanol as an extra control.

Results demonstrated effects of 2% ethanol on the cultures (reduction of mitotic index) in the absence of S9; therefore, for the actual experiments 1% (v/v) ethanol was chosen as solvent concentration in the cultures without S9. This limits the maximum attainable test substance concentration in the absence of S9 to 50 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The control data was well comparable with that of the historic controls.

Chromosomal Aberration Experiment 1:

Dose (µg/mL)	Total cells	Aberrant cells	Mitotic Index	Gaps	% cells with aberrations
					with gaps	w/o gaps
Without metabolic activation, exposure period 20h, fixation time 20h (1% v/v ethanol)
5	200	5	6.75	0	2.5	2.5
25	200	5	7.05	3	2.5	1
50	200	6	10.60	2	3	2
solvent	400	7	6.23	4	1.8(*)	1.3
untreated	200	1	11.55	0	0.5	0.5
MMC	159	61	4.15	6	38.4(<***)	37.1(<***)
With metabolic activation, exposure period 3h, fixation time 20h (2% v/v ethanol)
10	200	9	6.20	3	4.5(*)	3(**)
50	200	3	4.85	1	1.5	1
100	200	6	5.85	1	3	2.5(*)
solvent	400	9	6.68	7	2.3	0.5
untreated	200	6	5.70	5	3	0.5
CPA	133	51	1.30	20	38.3(<***)	30.8(<***)

* = p<0.05

** = p<0.01

*** = p<0.001

<*** = p<0.00005

 

Chromosomal Aberration Experiment 2:

Dose (µg/mL)	Total cells	Aberrant cells	Mitotic Index	Gaps	% cells with aberrations
					with gaps	w/o gaps
Without metabolic activation, exposure period 20h, fixation time 20h (1% v/v ethanol)
5	200	2	10.90	1	1	0.5
25	200	3	9.65	1	1.5	1
50	200	5	11.45	1	2.5	2
solvent	400	13	9.15	4	3.3	2.3
untreated	200	4	10.40	1	2	1.5
MMC	200	62	2.95	7	31(<***)	28.5(<***)
With metabolic activation, exposure period 3h, fixation time 20h (2% v/v ethanol)
10	200	2	6.50	2	1	0.5
50	200	4	6.20	1	2	1.5
100	200	3	8.30	1	1.5	1
solvent	400	5	6.68	2	1.3	0.8
untreated	200	2	11.15	1	1	0.5
CPA	200	33	1.70	12	16.5(<***)	13(<***)
Without metabolic activation 2nd harvest, exposure period 20h, fixation time 44h (1% v/v ethanol)
50	200	1	3.85	0	0.5	0.5
solvent	400	12	5.60	7	3	1.3
untreated	200	7	7.80	4	3.5	2
With metabolic activation 2nd harvest, exposure period 3h, fixation time 44h (2% v/v ethanol)
100	200	5	3.65	3	2.5	1
solvent	400	3	6.08	1	0.8	0.5
untreated	200	3	8.70	3	1.5	0

<*** = p<0.00005

All experimental points were compared statistically by the Fisher's exact test with the relevant negative controls (solvent). Solvent control values were compared statistically by the fisher's exact test with the relevant medium controls (untreated).

The positive control chemicals have caused statistically significant increases in the number of aberrations scored in both experiments. This demonstrates that the cells were sensitive to the effects of a known clastogen.

Treatment with the test substance showed some statistically significant increases in aberrations. These were not dose related or consistent between donors or experiments and are therefore not thought to demonstrate clastogenicity.

Conclusion:

The test substance has not demonstrated a consistent clastogenic effect under the conditions of this test.

 

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP guideline study with acceptable restrictions. Purity not reported.

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

Target gene:

Thymidine Kinase locus

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

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).

Key result

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

True negative controls validity:

not examined

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.

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.

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Waiver - No in vivo testing required as none of the in vitro genetic toxicity were positive.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro

- Gene mutation in bacteria

The mutagenic potential of (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) was assessed in a GLP-compliant gene mutation assay in bacteria (Ames test) according to OECD 471 (Huntingdon Research Centre Ltd., 1990). In two independent experiments, the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to the test material diluted in hexane for 48 h at concentrations of 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). Consistent with the preliminary range finding test, no cytotoxic effects were observed in any tester strains up to the maximum concentration of 5000 µg/plate in the both experiments of the main study. In the first experiment, precipitation of the test substance was observed starting at 1500 µg/plate (with and without S9 mix) in all strains, whereas in the second experiment, precipitation was noted at 5000 µg/plate with and without activation. No increase in the mean number of revertants compared to controls was noted after treatment in either the presence or absence of S9 mix in both independent experiments. The concurrent positive and solvent controls demonstrated the expected results, and thus verified the sensitivity of the assay. Based on these results, the test substance was not mutagenic in the presence and absence of metabolic activation in the selected strains of S. typhimurium.

 

- Chromosome aberrations

An in vitro mammalian chromosome aberration test was performed with (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) in primary cells from peripheral human blood according to OECD guideline 473 and in compliance with GLP (Quintiles Preclinical Services, 1997a). Treatment of cells was performed in two independent experiments, both in the presence and absence of metabolic activation (S9 mix). Based on a preliminary solubility and cytotoxicity tests, 100 µg/mL was selected as the highest concentrations for treatment in both experiments due to the limited solubility in the maximum applicable concentration of ethanol (2% v/v). In the first experiment, cells were exposed for 3 h to the test substance concentrations of 0.5, 2.5, 5, 25 and 50 µg/mL without S9 mix, whereas concentrations of 9, 10, 50, 100 µg/mL were selected for the 20-h treatment with S9 mix. Concentrations selected for chromosome analysis were 5, 25 and 50 µg/mL in the presence and 5, 10 and 100 µg/mL in the absence of S9 mix. After treatment with the test substance, statistically significant, but not dose-dependent increases in chromosomal aberrations compared to solvent controls were observed at concentrations of 10 and 100 µg/mL in the presence of S9 mix. In addition, a statistically significant difference in the solvent control's mutation frequency compared to the negative (untreated) controls in the absence of S9 was noted. However, in an independent repeat experiment with treatment concentrations of 5, 25 and 50 µg/mL with S9 mix and 10, 50 and 100 µg/mL without S9 mix, no statistically significant increase in treated cells compared to the concurrent solvent controls as well as solvent controls compared to negative (untreated) controls was observed. Thus, the observed increase in chromosomal aberrations in the first experiment were not reproducible and thus considered to be of no toxicological relevance. No cytotoxicity was observed at any test concentration in both experiments. The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values in all experiments and the positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations. Therefore, under the conditions of this assay, the test substance did not exhibit clastogenic activity in cultured rat lymphocytes in the presence and absence of metabolic activation.

 

- Gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay with (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) was performed in mouse-lymphoma L5178Y cells according to OECD guideline 476 and GLP (Quintiles Preclinical Services, 1997b). In a preliminary cytotoxicity test with concentrations ranging from 10 to 1000 µg/mL, no cytotoxic effects were observed in the cells up to the highest concentration tested in the presence and absence of metabolic activation. Based on these results and due to the fact that the substance was only not completely soluble at 1000 µg/mL, mutations at the TK locus of mouse-lymphoma L5178Y cells were investigated in two independent experiments at test substance concentrations of 10, 25, 50, 75 and 100 µg/mL for a period of 3 h. After an expression period of 72 h in the presence of 5-trifluorothymidine (TFT) selective medium, the test substance induced a statistically significant, but isolated increase in mutation frequency at 25 µg/mL with and without metabolic activation, respectively. Furthermore, a statistically significant difference in the negative (solvent) control's mutation frequency compared to the untreated controls in the absence of S9 mix was observed. As the positive responses from the first experiment was neither dose-dependent nor reproducible in the second experiment, it was not considered to be of toxicological significance. No cytotoxic effects were seen at any test concentration in the presence and absence of S9 mix in both experiments. The positive controls significantly increased mutant frequency and thus verified the sensitivity of the assay. In conclusion, the test substance did not induce gene mutation in mouse-lymphoma L5178Y cells in the presence and absence of metabolic activation.

 

In vivo

According to Regulation (EC) No 1907/2006, Annex IX, column 2, testing for genetic toxicity in vivo is not indicated as the test substance did not demonstrate any genotoxic activity in bacteria or mammalian cells in vitro.

Justification for classification or non-classification

The available data on the genetic toxicity of (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) do not meet the criteria for classification according to Regulation (EC) No 1272/2008 and are therefore conclusive but not sufficient for classification.