Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 - 29 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 2008

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

EC Number:

248-813-1

EC Name:

Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Cas Number:

28065-97-6

Molecular formula:

C34H69NO2 C36H67NO2 C36H69NO2 C36H71NO2 C36H73NO2

IUPAC Name:

oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Method

Target gene:

his operon, trp operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone.

Test concentrations with justification for top dose:

The range-finding study served as Experiment I.

Range-finding study/Experiment I:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for all strains

Experiment I(a) (repeated):
0.3, 1, 3, 10, 33, 100, 333 and 1000 µg/plate with and without metabolic activation for TA100

Main study/Experiment II:
0.3, 1, 3, 10, 33, 100, 333 and 1000 µg/plate with and without metabolic activation for TA100
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for WP2uvrA
1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate with and without metabolic activation for TA98, TA1535 and TA1537

Vehicle / solvent:

- Solvent used: ethanol
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine: -S9: 10 and 50 µg/plate in TA98 and TA1537, respectively; 2-aminoanthracene: + S9: 2.5 or 10 µg/plate for all strains

Remarks:

Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants, clearing of the bacterial background lawn

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: ≥ 2500 and ≥ 1000 µg/plate with and without metabolic activation, respectively; Experiment II: ≥ 2500 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: ≥ 2500 µg/plate with and without metabolic activation and in Experiment II: ≥ 2500 µg/plate without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: ≥ 1000 and ≥ 333 µg/plate with and without metabolic activation, respectively; Experiment II: ≥ 1000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I and Ia: ≥ 333 and ≥ 33 µg/plate with and without metabolic activation, respectively; Experiment II: ≥ 1000 and ≥ 10 µg/plate µg/plate with and without metabolic activation, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: ≥ 2500 µg/plate without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the overlay agar in the test tubes at 333 μg/plate and above. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed in Experiment I at 1000 μg/plate and above, in Experiment Ia at 333 μg/plate and above, in Experiment II without S9 mix at 333 μg/plate and above and with S9 mix at 1000 μg/plate and above. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The range-finding study served as Experiment I. Since in Experiment I the acceptance criteria were not met in strain TA100 (without S9 mix) this part of the experiment was repeated and reported as Experiment I(a).

HISTORICAL CONTROL DATA
- Positive historical control data: The spontaneous reversion rates in the positive control are in the range of the historical control data.
- Negative (solvent/vehicle) historical control data: The spontaneous reversion rates in the negative and solvent control are in the range of the historical control data.

Any other information on results incl. tables

Table 2: Results of Experiment I

			Revertant Colony Counts (Mean ±SD)
		Dose level (per plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without metabolic activation	Ethanol		13 ± 4	10 ± 1	33 ± 8	184 ± 6	48 ± 6
	Untreated		8 ± 3	13 ± 2	34 ± 6	200 ± 16	47 ± 5
	Test substance	3 µg	15 ± 5	9 ± 3	31 ± 2	146 ± 15	47 ± 6
		10 µg	11 ± 2	8 ± 2	38 ± 4	121 ± 17	51 ± 3
		33 µg	15 ± 6	14 ± 2	31 ± 1	54 ± 12	47 ± 8
		100 µg	13 ± 3	11 ± 4^R	16 ± 4^R	22 ± 2^R	51 ± 5
		333 µg	10 ± 2^R	11 ± 3^R	14 ± 5^R	6 ± 2^R	51 ± 3^R
		1000 µg	5 ± 3^P R	11 ± 1^R P	5 ± 7^P R	2 ± 1^R P	41 ± 9^P R
		2500 µg	4 ± 1^P R	1 ± 1^P R	1 ± 1^P R	0 ± 0^R P	60 ± 3^P R
		5000 µg	4 ± 1^P R	1 ± 2^P R	1 ± 1^P R	0 ± 0^R P	63 ± 8^P R
	NaN3	10 µg	1309 ± 29	-	-	2114 ± 104	-
	4-NOPD	10 µg	-	-	395 ± 25	-	-
	4-NOPD	50 µg	-	86 ± 6	-	-	-
	MMS	2.0 µL	-	-	-	-	1034 ± 66
With metabolic activation	Ethanol		18 ± 3	13 ± 3	47 ± 14	187 ± 18	62 ± 7
	Untreated		16 ± 7	12 ± 3	39 ± 5	189 ± 10	56 ± 3
	Test substance	3 µg	13 ± 2	11 ± 1	40 ± 5	183 ± 23	61 ± 4
		10 µg	20 ± 7	12 ± 4	36 ± 4	175 ± 15	57 ± 3
		33 µg	22 ± 2	14 ± 5	40 ± 7	170 ± 19	48 ± 3
		100 µg	12 ± 3	13 ± 2	40 ± 6	152 ± 7	50 ± 3
		333 µg	19 ± 7	16 ± 3^R	43 ± 6	45 ± 9^R	62 ± 4
		1000 µg	13 ± 2^P R	11 ± 3^P R	10 ± 5^P R	5 ± 2^R P	56 ± 9^P R
		2500 µg	5 ± 2^P R M	3 ± 1^P R M	3 ± 0^P R M	1 ± 1^R P M	24 ± 6^P R M
		5000 µg	2 ± 1^P R M	2 ± 1^P R M	3 ± 1^P R M	0 ± 0^R P M	16 ± 4^P R M
	2-AA	2.5 µg	439 ± 19	144 ± 16	3843 ± 832	4759 ± 526	-
	2-AA	10.0 µg	-	-	-	-	387 ± 24

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

R: Reduced background growth

P: Precipitate

M: Manual count

Table 3: Results of Experiment I(a) (repeated experiment for TA100)

			Revertant Colony Counts (Mean   ±SD)
		Dose Level(per plate)	TA 100
Without metaolic activation	Ethanol		192 ± 21
	Untreated		188 ± 13
	Test substance	0.3 µg	176 ± 13
		1 µg	178 ± 13
		3 µg	173 ± 19
		10 µg	156 ± 18
		33 µg	58 ± 6
		100 µg	15 ± 2^R M
		333 µg	0 ± 1^P R
		1000 µg	0 ± 0^P R
	NaN3	10 µg	2409 ± 101

NaN3: sodium azide

R: Reduced background growth

P: Precipitate

M: Manual count

Table 4: Results of Experiment II

		Dose Level(per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without metabolic activation	Ethanol		12 ± 5	10 ± 1	33 ± 7	193 ± 15	42 ± 6
	Untreated		11 ± 4	14 ± 3	25 ± 10	204 ± 29	44 ± 4
	Test substance	0.3 µg	-	-	-	182 ± 38	-
		1 µg	11 ± 5	11 ± 4	37 ± 10	203 ± 21	-
		3 µg	10 ± 3	9 ± 4	32 ± 6	112 ± 9	48 ± 7
		10 µg	11 ± 1	10 ± 4	30 ± 3	76 ± 5	39 ± 7
		33 µg	11 ± 4	12 ± 1	26 ± 4	48 ± 15	44 ± 4
		100 µg	10 ± 4	8 ± 2	18 ± 3	30 ± 3	46 ± 14
		333 µg	8 ± 2^M P	6 ± 1^P	15 ± 4^P M	9 ± 2^P M	39 ± 6^P
		1000 µg	6 ± 1^M P	5 ± 1^P M	7 ± 2^P M	0 ± 1^P M	60 ± 18^P
		2500 µg	3 ± 1^P M	4 ± 1^P M	5 ± 2^P M	-	31 ± 3^P M
		5000 µg	-	-	-	-	29 ± 3^P M
	NaN3	10 µg	1404 ± 50	-	-	2228 ± 68	-
	4-NOPD	10 µg	-	-	269 ± 16	-	-
	4-NOPD	50 µg	-	98 ± 5	-	-	-
	MMS	2.0 µL	-	-	-	-	675 ± 31
With metabolic activation	Ethanol		17 ± 4	11 ± 5	41 ± 7	183 ± 6	57 ± 4
	Untreated		10 ± 1	18 ± 3	47 ± 14	215 ± 19	60 ± 3
	Test substance	0.3 µg	-	-	-	163 ± 5	-
		1 µg	17 ± 2	9 ± 4	53 ± 9	181 ± 7	-
		3 µg	15 ± 3	13 ± 4	51 ± 3	197 ± 7	54 ± 7
		10 µg	13 ± 4	17 ± 6	47 ± 11	185 ± 14	50 ± 7
		33 µg	14 ± 2	19 ± 2	43 ± 4	190 ± 10	58 ± 5
		100 µg	13 ± 3	17 ± 3	52 ± 13	170 ± 20	65 ± 3
		333 µg	16 ± 5	18 ± 3	57 ± 5	138 ± 6	63 ± 6
		1000 µg	8 ± 3^P M	16 ± 4^P M	11 ± 3^P M	22 ± 4^P M	64 ± 18^P
		2500 µg	6 ± 2^P M	16 ± 3^P M	8 ± 2^P M	-	74 ± 17^P
		5000 µg	-	-	-	-	50 ± 5^P M
	2-AA	2.5 µg	356 ± 31	168 ± 5	3792 ± 372	4303 ± 250	-
	2-AA	10.0 µg	-	-	-	-	329 ± 26

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

R: Reduced background growth

P: Precipitate

M: Manual count

Applicant's summary and conclusion

Conclusions:

Based on the results of the Ames test, the test substance did not reveal mutagenic properties in TA100, TA98, TA1535, TA1537 and WP2uvrA with and without metabolic activation.