Dioleamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies for bacterial genotoxicity in the Ames test with and without metabolic activation, for genotoxicity in CHO cells with and without metablic activation and for chromosomal aberrations in rat primary lymphocytes with and without metabolic activatin were conducted with the source substance according to the respective OECD guidelines and GLP. All results were negative indicating no genotoxic or clastogenic potential of the substance. QSAR predictions for the target substance for bacterial gene mutation and chromosomal aberration all predict that the substance is likely not genotoxic and does not induce chromosomal aberrations. Taken together these results suggest that the substance is not genotoxic and does not cause chromosomal aberrations in in vitro test systems.

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:

other: GLP guideline study with acceptable restrictions. Although 5 strains were tested no strain for detection of cross-linking or oxidising mutagens was included, but in accordance with guideline at time of testing.

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 mix

Test concentrations with justification for top dose:

100, 333, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Very low water solubility of test substance in common solvents. Appropriate solvents not suitable for bacterial culture; therefore, suspension of test substance in DMSO added to cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 without S-9 mix Migrated to IUCLID6: 1 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 100 without S-9 mix Migrated to IUCLID6: 0.5 µL/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4 NPD), 10 µg/plate

Remarks:

TA 98 and TA 1538 without S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S-9 mix Migrated to IUCLID6: 60 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA), 0.5 µg/plate

Remarks:

All strains with S-9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction of background growth

Evaluation criteria:

Acceptability of Assay:
1) Negative control data (number of spontaneous revertants per plate) should reasonably fall within the laboratory background historical range for each tester strain.
2) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory historical range documented for each positive control substance.
3) The selected dose range should include a clearly toxic concentration as demonstrated by a preliminary toxicity range-finding test with strain TA 100.

Statistics:

Mean and standard deviation of revertant numbers.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occurred up to limit concentrations.

RANGE-FINDING/SCREENING STUDIES:
Eleven serial dilutions of the test substance (0.1, 0.3, 1.0, 3.3, 10.0, 33.3, 100, 333, 1000, 3330, 5000 µg/plate) were plated with a diluted TA 100 culture on non-selective agar for viability counting. For viability counting equal numbers of bacterial cells were plated in the presence of test substance. The percentage of survival of an appropriately diluted TA 100 culture on non-selective agar was determined by comparing the number of colonies on the solvent control plate with those on the test substance plates. Even at the highest test substance concentration used (5000 µg/plate) there was no reduction in survival of TA 100. Therefore, the test substance was tested up to a concentration of 5000 µg/plate in the main test, which is the maximum test concentration to be used according to the OECD guideline.

COMPARISON WITH HISTORICAL CONTROL DATA:
The observed numbers of revertants are well within the ranges of the historical controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Maximum mean numbers of revertants (± SD):

Strain	-S9		+S9
	Control	Test substance (µg/plate)	Control	Test substance (µg/plate)
TA 1535	9 ± 2	9 ± 1 (100)	12 ± 4	14 ± 2 (333)
TA 1537	12 ± 4	14 ± 3 (1000)	9 ± 2	20 ± 19 (5000)
TA 1538	14 ± 2	19 ± 6 (100)	26 ± 6	28 ± 3 (100)
TA 98	36 ± 10	38 ± 9 (333)	30 ± 14	36 ± 8 (333)
TA 100	121 ± 6	119 ± 7 (5000)	98 ± 19	138 ± 9 (5000)

 

The test substance did not induce a statistically significant dose-related increase in the numbers of revertants in any of the tester strains.

Conclusion:

The test substance does not have to be considered as mutagenic to bacteria.

Conclusions:

The source substance was negative (not genotoxic) in an in vitro bacterial gene mutation test in S. typhimurium 1537, 1538, 98, 100 performed according to OECD TG 471 and GLP with and without metabolic acitivation. The result is considered relevant for the target substance (see read accross rational chapter 13 of IUCLID and appendix to the CSR) and are consistent with the QSAR predictions for the target substance all predicting non-genotoxicity in the Ames test with and without metabolic activation.

Executive summary:

The source substance was negative (not genotoxic) in an in vitro bacterial gene mutation test in S. typhimurium 1537, 1538, 98, 100  performed according to OECD TG 471 and GLP with and without metabolic acitivation. The result is considered relevant for the target substance (see read accross rational chapter 13 of IUCLID and appendix to the CSR) and are consistent with the QSAR predictions for the target substance all predicting non-genotoxicity in the Ames test with and without metabolic activation.

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

ISBN 92-64-12221-4, Paris, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA 40 CFR Part 798, 27 September 1985

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 rat blood

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 0.25 µg/mL Fungizone, 100 U/mL penicillin G, 0.1 mg/mL streptomycin sulfate, 2 mM L-glutamine and 20 µg/mL PHA. Treatment was done in medium without serum and PHA with or without 2% (v/v) S-9 mix. Thereafter, the old medium was applied to the cultures again.
- Properly maintained: yes, freshly prepared whole blood was cultured at 37 °C in T25 plastic tissue culture flasks
- Periodically checked for karyotype stability: not required, karyotype of cultured rat lymphocytes is stable in contrast to the relative karyotype instability of cell lines.
- Periodically "cleansed" against high spontaneous background: not applicable, freshly prepared cultures

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254-induced rat liver S-9 mix

Test concentrations with justification for top dose:

40, 133, 400 µg/mL (additionally 4 and 13 µg/mL for initial determination of mitotic indices)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: isopropyl alcohol
- Justification for choice of solvent/vehicle: limited solubility of test substance in water and various other solvents

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% (v/v) isopropyl alcohol (final concentration)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S-9 mix Migrated to IUCLID6: 1000 µg/mL (8 mM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% (v/v) isopropyl alcohol (final concentration)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S-9 mix Migrated to IUCLID6: 4.2 µg/mL (15 µM)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 and 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 28 and 52 hours

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 200 metaphases (100 of each replicate culture) (positive control: 50 metaphases/replicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Only those metaphases that contained 42 chromosomes were scored with the exception of severely damaged cells, in which case accurate counts of the chromosomes were not possible. Those cells having 10 or more aberrations/cell were classified as severely damaged cells. Gaps were not included in calculations of total cytogenetic aberrations.

Statistics:

The frequencies of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled and analysed by constructing two-dimensional contingency tables. The total Chi-square was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no difference in average scores (average number of aberrations per cell) among the dose groups and (2) no linear trend of increasing scores with increasing dose. An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If either statistic was found to be significant at alpha=0.01 versus a two-sided alternative, pairwise tests (i.e. control vs treatment) were performed at each dose level and evaluated again at alpha=0.01 versus a two-sided alternative.

The statistical procedure is designed to accommodate scoring of individual cells where the score is equal to the number of aberrations observed in the cell. Scoring is then averaged within each dose group in a method similar to one used by Bhapkar, 1968. The mean scores are then analysed using a chi-square statistic that is partitioned to provide a test of trend (Landis et al., 1978; Mantel and Haenszel, 1959).

Species / strain:

lymphocytes: primary cells from peripheral rat blood

Metabolic activation:

with and 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 examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: Precipitation occurred in the cultures treated with 133 and 400 µg/mL test substance in the culture medium; the choice of doses for the chromosomal aberration test was made due to solubility.

RANGE-FINDING/SCREENING STUDIES:
The determination of the mitotic indices after exposure to test substance concentrations of 4 to 400 µg/mL and 24 and 48 hours expression time served as kind of a range finder; precipitation was observed at test substance concentrations of 133 and 400 µg/mL in the absence and presence of metabolic activation. The average mitotic index of cultures treated with 400 µg/mL in the absence of S-9 was 6.1% (24 hours) and 4.1% (48 hours) compared to negative control values of 6.0 and 6.5%, respectively. In the presence of S-9, the average mitotic index for cultures treated with 400 µg/mL was 3.8 (24 hours) and 4.0% (48 hours) compared to negative control values of 4.8 and 3.9%, respectively.

Since no evidence of toxicity was observed (with and without S-9) in cultures treated with concentrations up to and exceeding the limit of solubility, the three top concentrations (40, 133, 400 µg/mL) were selected for determining chromosomal aberration frequencies at the 24-hour harvest with and without S-9.
At the 48-hour harvest, cultures treated with the three top doses (40, 133, 400 µg/mL) were scored in the absence of S-9; in the presence of S-9 only the 400 µg/mL dose level was scored. No positive controls were included at the 48-hour harvest.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in the chromosomal aberration assay after exposure up to 400 µg/mL and expression times of 24 and 48 hours.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results of the chromosomal aberration assay in cultured rat lymphocytes:

Concentration (µg/mL)	Mitotic index (%)	Chromatid Gaps	Total Aberrations (excluding gaps)		No. of cells with Aberrations (excluding gaps)
			absolute	(%)	absolute	(%)
24 hours after treatment, without S-9
Solvent	6.0	7	4	2.0	4	2.0
40	6.2	15	8	4.0	6	3.0
133	5.5	7	0	0	0	0
400	6.1	14	5	2.5	5	2.5
EMS(1000)	4.3	25	32	32.0	25*	25.0
24 hours after treatment, with S-9
Solvent	4.8	7	1	0.5	1	0.5
40	5.7	11	1	0.5	1	0.5
133	4.8	5	1	0.5	1	0.5
400	3.8	10	2	1.0	2	1.0
CP (4.2)	2.5	18	44	44.0	19*	19.0
48 hours after treatment, without S-9
Solvent	6.5	16	3	1.5	3	1.5
40	3.9	11	1	0.5	1	0.5
133	6.0	8	3	1.5	3	1.5
400	4.1	16	0	0	0	0
48 hours after treatment, with S-9
Solvent	3.9	9	0	0	0	0
400	4.0	7	1	0.5	1	0.5

* alpha≤0.01

In the assays conducted without S-9 (both 24- and 48-hour harvest), the frequencies of cells with aberrations in the test chemical treated cultures ranged from 0 to 3.0%. In the assays with S-9, the highest percentage of cells with aberrations was 1.0%. These values were neither significantly different from the concurrent negative (solvent) controls nor outside the reasonable range of the laboratory historical background aberration frequencies. Significant increases in aberration rates were observed in cultures treated with the positive control chemicals.

Conclusion:

The test substance did not exhibit clastogenic activity in cultured rat lymphocytes when tested up to or exceeding the limits of solubility under the conditions of this study.

Conclusions:

The source substance was negative (not clastogenic) in a chromosomal aberration test in rat primary lymphocytes according to OECD TG 473 and GLP with and without metabolic acitivation. The result is considered relevant for the target substance and is supported by the QSAR results predicting negative results for clastogenicity as well.

Executive summary:

The source substance was negative (not clastogenic) in a chromosomal aberration test in rat primary lymphocytes according to OECD TG 473 and GLP with and without metabolic acitivation. The result is considered relevant for the target substance and is supported by the QSAR results predicting negative results for clastogenicity as well.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA 40 CFR Part 798

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT-locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12 nutrient mix supplemented with 5% (v/v) heat-inactivated (56 °C, 30 min) dialysed fetal bovine serum, 25 mM HEPES, 0.25 µg/mL Fungizone, 100 U/mL penicillin and 0.1 mg/mL streptomycin sulfate. The selection medium used for the detection of HGPRT-deficient mutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 µM 6-thioguanine, 5% serum, 25 mM HEPES, 2 mM L-glutamine and the above-mentioned antibiotics-antimycotics. Treatment was conducted in medium without serum with or without 2% (v/v) S-9 mix.
- Properly maintained: yes, stocks stored at approx. -100 °C or below; cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of 5% CO2 in air at 37 °C in a humidified incubator.
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9 mix

Test concentrations with justification for top dose:

Survival test: 6.3, 12.5, 25.0, 50.0, 100.0, 200.0, 400.0 µg/mL
Assay 1+2: 50, 100, 200, 400 µg/mL
Assay 3: 10, 20, 30, 40, 50, 60 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: isopropyl alcohol
- Justification for choice of solvent/vehicle: limited solubility of test substance in water and various other solvents

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% (v/v) isopropyl alcohol (final concentration)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S-9 mix Migrated to IUCLID6: 621 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% (v/v) isopropyl alcohol (final concentration)

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with S-9 mix Migrated to IUCLID6: 4 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 8 days (replating on day 1, 3, 6)
- Selection time (if incubation with a selection agent): 7-9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-17 days

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN: crystal violet after methanol fixation

NUMBER OF REPLICATIONS: 5 replicates/treatment

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
Determination of HGPRT-deficient mutants per 10e6 clonable cells:

Mutants per 10e6 clonable cells = (No. of mutant colonies per 10e6 plated cells/Cloning efficiency)

where Cloning efficiency = (Number of colonies/Number of cells plated)

Evaluation criteria:

Acceptability: The mutation frequency in positive controls should be significantly higher than the background frequency, and the mutation frequency in the negative controls should be within reasonable limits of the historical control values of this laboratory and the literature values.

Positive: Statistically significant and reproducible increase in mutation frequency at more than one of the dose levels tested. The final interpretation also considers such factors as mutation frequency and cloning efficiencies in the negative controls and dose-response relationships.

Statistics:

The frequencies of mutants per 10e6 clonable cells observed in the treated cultures were compared to the negative control with an appropriate chi-square statistic at an alpha level of 0.01, one-sided (Steel and Torrie, 1980). Examination of trends was conducted with least squares regression using an alpha level of 0.025 (Winer, 1971).

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed in 1/3 assays in the absence of metabolic activation only without dose-response. Not reproducible in an identical assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no appreciable change in the pH of the culture medium following addition of the test material as determined by means of an Accumet pH meter in a solution containing 420 µg/mL of the test substance.
- Effects of osmolality: There was no appreciable change in the osmolality of the culture medium following addition of the test material as determined by means of an Accumet pH meter in a solution containing 420 µg/mL of the test substance.
- Water solubility: The test substance has only a very limited water solubility; therefore isopropyl alcohol was chosen as solvent.
- Precipitation: Starting at 40 µg/mL in the absence and presence of metabolic activation in assay #3. A remarkable variability in precipitation was observed from assay to assay; therefore, in assay #3 the maximum concentration was lowered to 60 µg/mL. In the preliminary toxicity assay, no precipitate was observed at 100 µg/mL or lower concentrations. In assay #1 precipitate was observed at 100 µg/mL, in assay #2 there was precipitation at the 50 µg/mL dose level, as well. This variability was considered likely to the poor solubility properties of the test material in aqueous and organic solvents.

RANGE-FINDING/SCREENING STUDIES:
In order to establish an appropriate dose range, survival of CHO cells treated with the test substance was determined in a preliminary toxicity test. The cells were exposed to 6.3, 12.5, 25.0, 50.0, 100.0, 200.0, 400.0 µg/mL in triplicate cultures in the absence and presence of metabolic activation, and the relative cell survival was calculated as percentage of the ratio of mean number of colonies/dish in the treated cultures and the mean number of colonies/dish in the negative control. The highest concentration tested was based upon limitations imposed by solubility of the test material in the solvent. Precipitation was observed at 400 and 200 µg/mL in this assay. No cytotoxicity was observed at any of the concentrations tested. Based upon these results a dose level of 400 µg/mL was selected as highest test concentration for assays 1 and 2.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies observed from the control cultures are well comparable to the respective values of the historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In assay #1 the relative cell survival (RCS) was not affected in cultures treated with the test material in the presence of S-9. However, in the assay conducted without S-9, the RCS was affected, albeit without a dose response, in the treated cultures. In assay #2 the test substance was not toxic at any dose level either in the presence or in the absence of S-9.

Results of the HGPRT Assays #1 and 2 in CHO cells:

Treatment (µg/mL)	Toxicity assay: RCS (%)	Mutation assay: TG-resistant colonies	Cloning Efficiency CE (%)	TG-resistant colonies/10e6 clonable cells
Assay #1 without S-9
neg. control	100.0	0	44.0	0
50	37.7	1	62.2	1.6
100 P	80.8	2	53.2	3.8
200 P	7.2	1	63.0	1.6
400 P	12.5	1	55.9	1.8
pos. control	72.4	120	44.4	270.3*
Assay #1 with S-9
neg. control	100.0	0	53.0	0
50	95.2	3	56.6	5.3
100 P	99.3	4	63.5	6.3
200 P	106.1	7	62.0	11.3
400 P	100.0	0	73.1	0
pos. control	107.5	81	58.3	138.9*
Assay #2 without S-9
neg. control	100.0	3	71.6	4.2
50 P	115.6	11	68.8	16.0*
100 P	113.6	5	65.9	7.6
200 P	115.6	3	67.1	4.5
400 P	118.3	10	73.6	13.6
pos. control	70.9	110	51.4	214.0*
Assay #2 with S-9
neg. control	100.0	5	68.7	7.3
50 P	90.7	24	53.6	44.8*
100 P	104.1	9	76.9	11.7
200 P	96.3	8	62.8	12.7
400 P	114.5	10	77.6	12.9
pos. control	101.3	123	71.5	172.0*

* alpha≤0.01

P: precipitation

 

Results of HGPRT Assay #3 in CHO cells:

Treatment (µg/mL)	Toxicity assay: RCS (%)	Mutation assay: TG-resistant colonies	Cloning Efficiency CE (%)	TG-resistant colonies/10e6 clonable cells
Assay #3 without S-9
neg. control	100.0	7	83.2	8.4
10	105.8	7	81.8	8.6
20	108.4	1	76.8	1.3
30	96.1	4	81.3	4.9
40 P	99.0	7	77.3	9.1
50 P	102.2	4	83.2	4.8
60 P	103.1	4	77.8	5.1
pos. control	80.0	99	45.9	215.7*
Assay #3 with S-9
neg. control	100.0	14	70.9	19.7
10	103.9	16	66.6	24.0
20	101.4	15	59.4	25.3
30	93.1	3	60.8	4.9
40 P	103.9	4	62.8	6.4
50 P	101.6	6	63.6	9.4
60 P	97.2	3	62.8	4.8
pos. control	92.0	93	66.9	139.0*

* alpha≤0.01

P: precipitation

In Assay #1 the relative cell survival was affected in the absence of S-9; however, this effect could not be reproduced in any of the other assays, neither in absence nor in presence of metabolic activation. The test material did not significantly increase the mutation frequency either in the presence or absence of S-9 in this assay.

In Assay #2 the test material was not toxic at any dose level tested either in the presence or absence of S-9. Statistical analysis of the mutation frequency data from this assay indicated a significant increase at the lowest test concentration of 50 µg/mL, which was also outside the historical background range.

In order to ascertain the biological significance of the increased mutation frequency observed at the 50 µg/mL dose level in Assay #2, a third assay with concentrations from 10 to 60 µg/mL was conducted. In this assay precipitation was observed in treatment medium at 40 µg/mL or higher. There were no significant increases in mutation frequency in cultures treated with the test material. Hence, the isolated increase in mutation frequency in Assay #2 at 50 µg/mL has to be interpreted as chance occurrence unrelated to the treatment.

The positive control chemicals induced significant increases in mutation frequency in all assays and therefore demonstrated the adequacy of the experimental conditions for detecting induced mutations.

Conclusion:

The test substance did not induce gene mutations in mammalian cells in the CHO/HGPRT gene mutation assay under the experimental conditions chosen.

Conclusions:

The source substance was negative (not genotoxic) in an in vitro mammalian gene mutation test in Chinese Hamster Ovary (CHO) Cells performed according to OECD TG 476 and GLP with and without metabolic acitivation. The result is considered relevant for the target substance (see read accross rational chapter 13 of IUCLID and appendix to the CSR).

Executive summary:

The source substance was negative (not genotoxic) in an in vitro mammalian gene mutation test in Chinese Hamster Ovary (CHO) Cells  performed according to OECD TG 476 and GLP with and without metabolic acitivation. The result is considered relevant for the target substance (see read accross rational chapter 13 of IUCLID and appendix to the CSR).

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain:

not specified

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

The predicted Ames mutagenicity (T.E.S.T. v.4.2.1) is negative.

Executive summary:

The predicted Ames mutagenicity (T.E.S.T. v.4.2.1) is negative.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain:

S. typhimurium, other:

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

The OECD QSAR Toolbox v4.2 prediction for gene mutation (no metabolic activation) is negative (p=1.42E-15).

Executive summary:

The OECD QSAR Toolbox v4.2 prediction for gene  mutation (no metabolic activation) is negative (p=1.42E-15).

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

GLP compliance:

no

Species / strain:

not specified

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Predicted endpoint: Chromosome aberration; No effect specified; No species specified; No duration
specified; No guideline specified
Predicted value: Negative
Unit/scale: Gene mutation I
Data gap filling method: Read-across analysis

Executive summary:

OECD Toolbox v4.2 predicted endpoint: Chromosome aberration; No effect specified; No species specified; No duration

specified; No guideline specified

Predicted value: Negative

Unit/scale: Gene mutation I

Data gap filling method: Read-across analysis

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

GLP compliance:

no

Species / strain:

not specified

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Predicted endpoint: Chromosome aberration with metabolism; No effect specified; No species specified; No duration
specified; No guideline specified
Predicted value: Negative
Unit/scale: Gene mutation I
Data gap filling method: Read-across analysis

Executive summary:

Predicted endpoint: Chromosome aberration with metabolism; No effect specified; No species specified; No duration

specified; No guideline specified

Predicted value: Negative

Unit/scale: Gene mutation I

Data gap filling method: Read-across analysis

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain:

S. typhimurium, other:

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

The OECD QSAR Toolbox v4.2 prediction for gene mutation (with metabolic activation) is negative (p=2E-17).

Executive summary:

The OECD QSAR Toolbox v4.2 prediction for gene mutation (with metabolic activation) is negative (p=2E-17).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

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 selection of genetic toxicity endpoint 
No study was selected, since all available in vitro genetic toxicity studies addressed different endpoints and were negative. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Justification for classification or non-classification

The available data on genetic toxicity of (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) do not meet the criteria for classification according to Regulation (EC) No 1272/2008 and are therefore conclusive but not sufficient for classification. This information is used as a read-across prediction togehter with negative QSAR predictions for oleyl oleamide (CAS 72901 -31 -6), that should consequently not be classified for this endpoint.