Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the in vitro test results, the test substance, C16-18 AMP is considered to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 21, 2017 to July 20, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/plate
The maximum concentration was 5000 µg/plate (the maximum recommended dose level)

Vehicle / solvent:

The test substance was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test substance formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Identity: Dimethyl formamide, Supplier: Fisher Scientific, Batch number (purity): 1418073 (99.99%), Expiry: 05/2021

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Test System and Supporting Information
Bacteria: The five strains of bacteria used, and their mutations, are as follows:
Salmonella typhimurium
Strains: Genotype: Type of mutations indicated
TA1537: his C 3076; rfa-; uvrB-: frame shift
TA98: his D 3052; rfa-; uvrB-;R-factor: frame shift
TA1535: his G 46; rfa-; uvrB-: base-pair substitution
TA100: his G 46; rfa-; uvrB-;R-factor: base-pair substitution

Escherichia coli
Strain: Genotype: Type of mutations indicated
WP2uvrA: trp-; uvrA-: base-pair substitution
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000). The bacteria used in the test were obtained from: University of California, Berkeley, on culture discs, on 04 August 1995 and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 h. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 6147883 03/21) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 44673 07/17 and 44674 07/17).

Study controls
The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate. The positive control substances used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.

The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix;
and The maximum dosing solution of the test substance in the absence of S9-mix only (test in singular only).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test substance activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Results

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test substance formulation was also shown to be sterile. These data are not given in the report. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test substance precipitate (particulate in appearance) was observed under a low power microscope at 1500 µg/plate and to the naked eye at 5000 µg/plate (with a creamy film also noted), these observations did not prevent the scoring of revertant colonies. A film was not observed in Experiment 2 after employing the pre-incubation modification. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). Small, statistically significant increases in revertant colony frequency were observed in Experiment 2 at 500 and 1500 µg/plate (TA98 dosed in the absence of S9-mix) and 150 µg/plate (WP2uvrA dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the mean maximum fold increase was only 1.5 times the concurrent vehicle controls.The test substance was considered to be non-mutagenic under the conditions of this test.

Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1 (Plate Incorporation)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
74		18		19		14		28
78	(81)	24	(19)	18	(21)	15	(17)	15	(18)
92		15		26		22		11

Experiment 2 (Pre-Incubation)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
98		27		14		25		14
82	(90)	27	(27)	20	(16)	16	(19)	8	(10)
89		28		15		17		9

Table 2 Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 04 July 2017					To: 07 July 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	71 81 73	(75) 5.3#	24 20 24	(23) 2.3	15 12 19		(15) 3.5	14 13 15	(14) 1.0	18 17 17	(17) 0.6
	1.5 µg	75 70 79	(75) 4.5	17 7 20	(15) 6.8	18 25 21		(21) 3.5	13 14 14	(14) 0.6	13 21 23	(19) 5.3
	5 µg	74 67 65	(69) 4.7	10 26 18	(18) 8.0	14 23 24		(20) 5.5	14 12 15	(14) 1.5	16 17 20	(18) 2.1
	15 µg	108 77 60	(82) 24.3	24 25 11	(20) 7.8	16 13 18		(16) 2.5	19 17 16	(17) 1.5	16 14 17	(16) 1.5
	50 µg	64 79 72	(72) 7.5	15 26 20	(20) 5.5	17 16 19		(17) 1.5	19 11 12	(14) 4.4	17 18 25	(20) 4.4
	150 µg	84 91 63	(79) 14.6	28 17 23	(23) 5.5	8 18 18		(15) 5.8	16 19 16	(17) 1.7	20 16 22	(19) 3.1
	500 µg	65 79 65	(70) 8.1	16 28 16	(20) 6.9	7 18 26		(17) 9.5	16 9 15	(13) 3.8	15 16 17	(16) 1.0
	1500 µg	78 78 67	(74) 6.4	29 19 22	(23) 5.1	18 16 23		(19) 3.6	10 12 10	(11) 1.2	14 18 16	(16) 2.0
	5000 µg	63 PF 61 PF 66 PF	(63) 2.5	26 PF 25 PF 20 PF	(24) 3.2	20 PF 23 PF 14 PF		(19) 4.6	10 PF 11 PF 14 PF	(12) 2.1	11 PF 14 PF 10 PF	(12) 2.1
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		618 612 641	(624) 15.3	489 560 362	(470) 100.3	729 706 709		(715) 12.5	238 242 220	(233) 11.7	302 153 199	(218) 76.3

Table 3 Test Results: Experiment 1 – With Metabolic Activation(Plate Incorporation)

Test Period		From: 04 July 2017					To: 07 July 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	78 61 70	(70) 8.5#	16 25 15	(19) 5.5	21 18 19		(19) 1.5	19 11 18	(16) 4.4	17 9 18	(15) 4.9
	1.5 µg	75 67 91	(78) 12.2	14 15 10	(13) 2.6	22 22 15		(20) 4.0	16 16 21	(18) 2.9	15 14 24	(18) 5.5
	5 µg	77 93 81	(84) 8.3	12 12 9	(11) 1.7	17 22 20		(20) 2.5	16 9 18	(14) 4.7	24 13 15	(17) 5.9
	15 µg	71 79 76	(75) 4.0	11 14 24	(16) 6.8	16 13 11		(13) 2.5	13 11 25	(16) 7.6	25 16 14	(18) 5.9
	50 µg	91 75 68	(78) 11.8	26 16 17	(20) 5.5	18 21 19		(19) 1.5	9 24 23	(19) 8.4	14 19 18	(17) 2.6
	150 µg	74 70 80	(75) 5.0	17 13 18	(16) 2.6	31 20 18		(23) 7.0	16 22 21	(20) 3.2	8 14 10	(11) 3.1
	500 µg	70 67 67	(68) 1.7	23 18 14	(18) 4.5	14 8 13		(12) 3.2	15 17 15	(16) 1.2	18 14 13	(15) 2.6
	1500 µg	71 78 88	(79) 8.5	21 23 22	(22) 1.0	17 11 23		(17) 6.0	18 14 23	(18) 4.5	6 11 18	(12) 6.0
	5000 µg	65 PF 61 PF 62 PF	(63) 2.1	19 PF 26 PF 25 PF	(23) 3.8	18 PF 17 PF 22 PF		(19) 2.6	28 PF 16 PF 11 PF	(18) 8.7	14 PF 19 PF 14 PF	(16) 2.9
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1765 1659 1657	(1694) 61.8	290 247 222	(253) 34.4	259 301 308		(289) 26.5	216 245 278	(246) 31.0	433 376 437	(415) 34.1

Table 4 Test Results: Experiment 2 – Without Metabolic Activation(Pre-Incubation)

Test Period		From: 17 July 2017					To: 20 July 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	71 66 74	(70) 4.0#	27 17 19	(21) 5.3	19 21 15		(18) 3.1	20 17 16	(18) 2.1	7 12 13	(11) 3.2
	15 µg	87 77 78	(81) 5.5	10 11 18	(13) 4.4	13 19 14		(15) 3.2	20 12 13	(15) 4.4	9 5 17	(10) 6.1
	50 µg	74 91 72	(79) 10.4	7 22 30	(20) 11.7	7 16 27		(17) 10.0	17 13 16	(15) 2.1	4 11 4	(6) 4.0
	150 µg	81 64 62	(69) 10.4	9 9 11	(10) 1.2	25 21 31		(26) 5.0	13 19 14	(15) 3.2	5 2 6	(4) 2.1
	500 µg	64 63 64	(64) 0.6	15 8 11	(11) 3.5	23 23 26		(24) 1.7	32 21 26	* (26) 5.5	10 8 9	(9) 1.0
	1500 µg	66 P 65 P 62 P	(64) 2.1	8 P 10 P 13 P	(10) 2.5	32 P 15 P 22 P		(23) 8.5	22 P 28 P 29 P	* (26) 3.8	10 P 9 P 9 P	(9) 0.6
	5000 µg	71 P 60 P 65 P	(65) 5.5	12 P 9 P 8 P	(10) 2.1	16 P 13 P 12 P		(14) 2.1	14 P 17 P 17 P	(16) 1.7	5 P 8 P 9 P	(7) 2.1
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		626 628 700	(651) 42.2	1184 1629 1573	(1462) 242.4	569 632 634		(612) 37.0	365 337 368	(357) 17.1	366 243 218	(276) 79.2

Table 5 Test Results: Experiment 2 – With Metabolic Activation(Pre-Incubation)

Test Period		From: 17 July 2017					To: 20 July 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	77 71 86	(78) 7.5#	25 25 23	(24) 1.2	16 17 19		(17) 1.5	13 19 33	(22) 10.3	11 11 10	(11) 0.6
	15 µg	98 100 91	(96) 4.7	21 24 27	(24) 3.0	13 20 18		(17) 3.6	28 23 27	(26) 2.6	10 8 10	(9) 1.2
	50 µg	85 94 77	(85) 8.5	22 23 18	(21) 2.6	16 23 27		(22) 5.6	24 33 27	(28) 4.6	8 19 7	(11) 6.7
	150 µg	66 78 75	(73) 6.2	24 27 28	(26) 2.1	26 24 26		* (25) 1.2	19 19 25	(21) 3.5	6 10 7	(8) 2.1
	500 µg	67 65 65	(66) 1.2	22 27 28	(26) 3.2	21 25 18		(21) 3.5	28 18 24	(23) 5.0	20 8 9	(12) 6.7
	1500 µg	66 P 63 P 86 P	(72) 12.5	28 P 26 P 21 P	(25) 3.6	15 P 24 P 21 P		(20) 4.6	22 P 20 P 25 P	(22) 2.5	9 P 9 P 11 P	(10) 1.2
	5000 µg	73 P 65 P 65 P	(68) 4.6	24 P 29 P 18 P	(24) 5.5	16 P 19 P 22 P		(19) 3.0	24 P 21 P 21 P	(22) 1.7	8 P 5 P 7 P	(7) 1.5
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		745 676 450	(624) 154.3	290 310 297	(299) 10.1	72 90 178		(113) 56.7	78 107 81	(89) 15.9	488 499 518	(502) 15.2

2AA - 2-Aminoanthracene

BP - Benzo(a)pyrene

ENNG - N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO - 4-Nitroquinoline-1-oxide

9AA - 9-Aminoacridine

P - Test substance precipitate

F - Test substance film

# Standard deviation

* p £0.05

Conclusions:

Under study conditions, the test substance was determined to be non-genotoxic with or without metabolic activation in the Ames test.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance, C16-18 AMP, according to OECD Guideline 471 (Reverse Mutation Assay - 'Ames Test') in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using both the Ames plate incorporation and pre-incubation methods at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was between 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended (15 to 5000 µg/plate) following the results of Experiment 1. Six test substance concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test substance following the change in test methodology. The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test substance precipitate (particulate in appearance) was observed under a low power microscope at 1500 µg/plate and to the naked eye at 5000 µg/plate (with a creamy film also noted), these observations did not prevent the scoring of revertant colonies. A film was not observed in Experiment 2 after employing the pre-incubation modification. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). Small, statistically significant increases in revertant colony frequency were observed in Experiment 2 at 500 and 1500 µg/plate (TA98 dosed in the absence of S9-mix) and 150 µg/plate (WP2uvrA dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the mean maximum fold increase was only 1.5 times the concurrent vehicle controls. Under study conditions, the test substance was considered to be non-mutagenic in the Ames test (Envigo, 2017).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 27, 2017 to September 19, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

OECD Guideline 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016,

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The test method was designed to be in alignment with the following Japanese Guidelines
1) Kanpoan No. 287 - - Environment Protection Agency
2) Eisei No. 127 - - Ministry of Health and Welfare
3) Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burrough Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix [S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%].

Test concentrations with justification for top dose:

10 dose levels (0, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250 µg/mL) in duplicate
The dose levels plated for viability and expression of mutant colonies were as follows:
4-h without S9: 2, 4, 8, 10, 12, 16 µg/mL
4-h with S9 (2%): 4, 8, 16, 20, 24, 32 µg/mL
24-h without S9: 1, 2, 4, 8, 10, 12 µg/mL

Vehicle / solvent:

Acetone
Fisher batch: 1664439
Expiry: January 19, 2021
Purity: 99.97%

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Fisher batch: 1664439, Expiry: January 19, 2021, Purity: 99.97%

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burrough Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at approximately 37 deg C with 5% CO2 in air. The cells had a generation time of approximately 12 h and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), were used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Microsomal Enzyme Fraction
Lot No. PB/NF S9 30/06/17 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test. The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Evaluation criteria:

Following evaluation for test substance were made:
-Measurement of Survival, Viability and Mutant Frequency
-Plate Scoring
-Calculation of Percentage Relative Suspension Growth (%RSG)
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-H Suspension Growth (SG) = (24-h cell count/2) x (48-h cell count/2)
24-H Suspension Growth (SG) = (0-h cell count/1.5) x (24-h cell count/2) x (48 h cell count/2)
Day 0 Factor = dose 0-h cell count/vehicle control 0-h cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100
-Calculation of Day 2 Viability (%V)
-Calculation of Relative Total Growth (RTG)
RTG = (RCE x RSG)/100%
-Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear trend events. The Delta building monitoring system was used during the course of the study.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Preliminary Cytotoxicity Test

The dose range of the test substance used in the preliminary toxicity test was 0.98 to 250 µg/mL.

Table 1: The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (mg/mL)	% RSG (-S9) 4 h Exposure	% RSG (+S9) 4 h Exposure	% RSG (-S9) 24 h Exposure
0	100	100	100
0.98	100	92	66
1.95	100	91	83
3.91	91	102	58
7.81	80	102	26
15.63	6	83	6
31.25	1	1	0
62.5p	0	0	0
125p	0	0	0
250p	0	0	0

p = precipitate of test substance at the end of exposure

In the all three exposure groups there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test substance when compared to the concurrent vehicle controls. A precipitate of the test substance was observed at and above 62.5 µg/mL in all three exposure groups. In the subsequent mutagenicity experiments the maximum dose level was limited by test substance induced toxicity.

Mutagenicity Test

Table 2: Main Experiment

Treatment (µg/mL)		4-hours-S-9				Treatment (µg/mL)		4-hours+S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	172.10		0		100	1.00	117.41
1	Ø	97				1	Ø	108
2		92	0.99	147.75		2	Ø	97
4		90	1.07	113.45		4		93	1.00	89.51
8		78	0.88	147.90		8		98	0.71	131.68
10		84	1.01	111.51		16		86	0.59	156.29
12		49	0.58	119.73		20		54	0.43	140.93
16		18	0.19	176.22		24		58	0.41	152.04
32	Ø	1				32	X	11	0.08	140.24
						40	Ø	1
						48	Ø	0
MF threshold for a positive response 298.1						MF threshold for a positive response 243.41
EMS						CP
400		74	0.72	862.33		1.5		93	0.16	802.54

      

 

Treatment (µg/mL)		24-hours-S-9
		%RSG	RTG	MF§
0		100	1.00	131.86
0.5	Ø	77
1		78	0.77	133.82
2		77	0.80	147.87
4		61	0.80	129.14
8		43	0.68	102.98
10		35	0.50	144.64
12		14	0.27	121.56
16	Ø	2
MF threshold for a positive response 257.86
EMS
150		82	0.64	1464.75

Table 3: Summary Analysis: Mutagenicity Test (-S9) 4 -h Exposure

Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
0		12.97	100	73.67	1.00	172.10
1	Ø	13.84	97
2		12.47	92	79.06	0.99	147.75
4		13.70	90	87.30	1.07	113.45
8		11.78	78	82.33	0.88	147.90
10		12.63	84	88.81	1.01	111.51
12		9.12	48	88.05	0.58	119.73
16		4.58	18	66.28	0.19	176.22
32	Ø	0.20	1
Positive Control EMS
Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
400		10.44	74	71.44	0.72	862.33

GEF = 126, therefore MF threshold for positive response = 298.10

Table 4: Summary Analysis: Mutagenicity Test (+S9) 4-Hour Exposure

Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
0		16.97	100	82.33	1.00	117.41
1	Ø	17.49	108
2	Ø	16.48	97
4		14.75	93	88.05	1.00	89.51
8		14.67	98	59.85	0.71	131.68
16		14.89	86	57.33	0.59	156.29
20		11.89	54	65.80	0.43	140.93
24		12.41	58	68.28	0.41	152.04
32	X	2.83	11	63.89	0.08	140.24
40	Ø	0.31	1
48	Ø	0.18	0
Positive Control
Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
1.5		14.02	93	54.16	0.16	802.54

GEF = 126, therefore MF threshold for a positive response = 243.41

Table 5: Summary Analysis: Mutagenicity Test (-S9) 24-Hour Exposure

Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
0		70.48	100	75.11	1.00	131.86
0.5	Ø	60.62	77
1		61.47	78	65.80	0.77	133.82
2		65.10	77	64.83	0.80	147.87
4		57.33	61	74.24	0.80	129.14
8		44.84	43	81.00	0.68	102.98
10		39.67	35	66.28	0.50	144.64
12		21.57	14	64.83	0.27	121.56
16	Ø	4.43	2
Positive Control EMS
Treatment (µg/mL)		SG	%RSG	%V	RTG	MF§
150		54.74	82	61.16	0.64	1464.75

GEF = 126, therefore MF threshold for a positive response = 257.86

There was evidence of marked toxicity following exposure to the test substance in all three exposure groups, which were indicated by the %RSG and RTG values. There was evidence of modest reductions in viability (%V) in all three exposure groups, therefore indicating that residual toxicity had occurred. Optimum levels of toxicity were achieved in the 4 and 24 h exposure in the absence of metabolic activation. Near to optimum levels if toxicity were achieved in the 4 h exposure in the presence of metabolic activation, however the RTG value was marginally too toxic and was therefore excluded from analysis. The dose levels of 32 µg/mL in the 4 h exposure in the absence of metabolic activation, 40 and 48 µg/mL in the 4 h exposure in the presence of metabolic activation and 16 µg/mL in the 24 h exposure in the absence of metabolic activation were not plated out for 5-TFT resistance and viability due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test item did not induce any toxicologically significant increases in the mutant frequency x 10^-6 per viable cell in either of the three exposure groups. The GEF value of the test item dose levels were not exceeded in any of the three exposure groups, including a dose level (32 µg/mL) beyond the acceptable level of toxicity in the 4 h exposure in the presence of metabolic activation. A precipitate of the test item was observed at 48 µg/mL in the 4 h exposure in the presence of metabolic activation, however this dose level was not analysed due to excessive toxicity.

Conclusions:

Under study conditions, the test substance is determined to be non genotoxic with or without metabolic activation in the mouse lymphoma assay.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance, C16-18 AMP, using the Thymidine Kinase Gene method,according to OECD Guideline 490 (Mammalian Cell Gene Mutation Tests)and EU Method B.17, in compliance with GLP. The dose range used in the main test was selected following the results of a preliminary toxicity study. In the main mutagenicity test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at up to 10 dose levels (0, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250 µg/mL) in duplicate, together with vehicle (Acetone), and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-h exposure group in the absence of metabolic activation. A precipitate of the test substance was observed at 48 µg/mL in the 4-h exposure in the presence of metabolic activation, however this dose level was not analysed due to excessive toxicity. There was evidence of marked toxicity following exposure to the test substance in all three exposure groups, as indicated by the %RSG (Relative suspension growth) and RTG (Relative total growth) values. There was evidence of modest reductions in viability (%V) in all three exposure groups, therefore indicating that residual toxicity had occurred. Optimum levels of toxicity were achieved in in the 4-h and 24-h exposure in the absence of metabolic activation. Near to optimum levels if toxicity were achieved in the 4-h exposure in the presence of metabolic activation, however the RTG value was marginally too toxic and was therefore excluded from analysis. The dose levels of 32 µg/mL in the 4-h exposure in the absence of metabolic activation, 40 and 48 µg/mL in the 4-h exposure in the presence of metabolic activation and 16 µg/mL in the 24-h exposure in the absence of metabolic activation were not plated out for 5-TFT resistance and viability due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test substance did not induce any toxicologically significant increases in the mutant frequency x 10^-6per viable cell in either of the three exposure groups. The GEF (Global evaluation factor) value of the test substance dose levels were not exceeded in any of the three exposure groups, including a dose level (32 µg/mL) beyond the acceptable level [GEF of 126 x 10^-6] of toxicity in the 4-h exposure in the presence of metabolic activation. Under study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation in the mouse lymphoma assay (Envigo, 2017).

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From January 01, 2013 to April, 03, 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

- Type and identity of media:The basic culture medium used was the RPMI 0 medium containing sodium bicarbonate, non-essential aminoacids, penicillin, streptomycin and HCl. The RPMI 0 medium was supplemented with 10% inactivated horse serum, pluronic acid, L-Glutamine, sodium pyruvate and Amphotericine B.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix : The S9 fraction was prepared at Institut Pasteur de Lille using the method described by Ames et al. (1975) in male rat OFA Sprague Dawley induced by Aroclor 1254 (origin - Monsanto, Saint Louis, U.S.A) .

Test concentrations with justification for top dose:

Toxicity assay :
1.25 - 0.63 - 0.31 - 0.16 - 0.08 - 0.04 - 0.02 - 0.01 - 0.005 - 0.002 mM
Genotoxicity assays
Assay with 3 h treatment and 27 h recovery period:
-Without S9-mix:0.20 – 0.15 – 0.10 – 0.075 – 0.05 mM but only the following concentration were assessed : 0.20 - 0.15 - 0.10 mM
-With S9-mix (5% S9-mix):0.40 – 0.30 – 0.20 – 0.15 – 0.10 – 0.075 mM but only the following concentration were assessed : 0.30 – 0.20 – 0.15 mM
Assay with 27 h treatment and no recovery period:
- Without S9-mix:0.075 – 0.05 – 0.025 – 0.013 – 0.0063 – 0.0031 mM but only the following concentration were assessed : 0.075 – 0.05 – 0.025 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: As indicated by the Sponsor and specified in the Final Study Plan, the test substance, N-(2-hydroxypropyl)Oleamide, was dissolved in ethanol.

The N-(2-hydroxypropyl)Oleamide was dissolved in ethanol (Merck, batch K429 09 883 148) at a maximum initial concentration of 2000 mM. Nevertheless, this concentration as well as the 2 concentrations below induced a strong precipitate when used at 1% in culture medium. The test item was thus dissolved at a maximum initial concentration of 125 mM and used at 1% in culture medium, giving a final concentration of 1.25 mM.
This concentration induced no variation in osmolality higher than 50 mOsmol when compared to the solvent control and was thus retained as top concentration to be tested in cytotoxicity assays. Successive dilutions were also performed in distilled water and used at 1%.

Furthermore, the pH was in the acceptable range of 6-8 at the highest concentrations tested from 1.25 to 0.31 mM.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Short treatment without S9-mix: mitomycin C 0.5 µg/mL and With S9-mix: cyclophosphamide 10 µg/mL Continuous treatment: mitomycin C 0.1 µg/mL and griseofulvin 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no
- Exposure duration: 3 h with 24 h recovery period both with and without metabolic activation and 27 h without recovery period
- Expression time (cells in growth medium): 27 h after the beginning of the treatment
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 27 h after the beginning of the treatment

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): Giemsa reagent (at 2% in water)

NUMBER OF REPLICATIONS: 2 cultures per assay

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable

Evaluation criteria:

The results obtained in the different treatment cultures are presented giving the mean number of micronuclei for 2000 mononucleated cells, and the RPD per concentration.
A test substance is found to demonstrate clastogenic or aneugenic properties against TK6 cells:
-if it results in a statistically significant increase in the number of micronuclei compared with the control,
-if this increase amounts to at least a doubling of the control value and
-if the genotoxicity detected shows a concentration-effect relationship.

A test substance is found to have no genotoxic effect on TK6 cells, if it does not comply with any of the 3 criteria listed above. If neither situation occurs, the results are discussed case by case.

Statistics:

Statistical analysis of the results obtained in the cells treated at each concentration level is performed using the c2 test in comparison with those in control groups.

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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
- Evaporation from medium: unknown
- Water solubility: poorly
- Precipitation: The N-(2-hydroxypropyl)Oleamide was dissolved in ethanol at a maximum initial concentration of 1000 mM. Nevertheless, this concentration as well as the 2 concentrations below induced a strong precipitate when used at 1% in culture medium. The test substance was thus dissolved at a maximum initial concentration of 125 mM and used at 1% in culture medium, giving a final concentration of 1.25 mM.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity assay using a 3 h treatment without metabolic activation, a potent toxicity was observed at the 3 highest concentrations from 1.25 to 0.31 mM. At the immediately lower concentration of 0.16 mM, a strong but acceptable toxicity was observed with a RPD of 49.1%, respectively corresponding to a percentage of cytostasis of 50.9%, when compared to the respective solvent control (Table 3). Under these conditions, the concentration of 0.20 mM was retained as the maximum concentration to be tested in the corresponding mutagenicity assay. A narrowed range of concentration was chosen in order to reach a concentration that induces a RPD of around 45 ± 5%.

In the preliminary toxicity assay using a 27 h treatment without metabolic activation, a potent toxicity was observed at the 5 highest concentrations from 1.25 to 0.08 mM. At the immediately lower concentration of 0.04 mM, a moderate toxicity was observed with a RPD of 66.2%, respectively corresponding to a percentage of cytostasis of 33.8%, when compared to the respective solvent control (Table 5). Under these conditions, the concentration of 0.075 mM was retained as the maximum concentration to be tested in the corresponding mutagenicity assay. A narrowed range of concentration was chosen in order to reach a concentration that induces a RPD of around 45 ± 5%.

In the preliminary toxicity assay using a 3 h treatment with metabolic activation, a potent toxicity was observed at the 2 highest concentrations of 1.25 and 0.63 mM. At the immediately lower concentration of 0.31 mM, a strong but acceptable toxicity was observed with a RPD of 45.2%, respectively corresponding to a percentage of cytostasis of 54.8%, when compared to the respective solvent control (Table 4). Under these conditions, the concentration of 0.40 mM was retained as the maximum concentration to be tested in the corresponding mutagenicity assay. A narrowed range of concentration was chosen in order to reach a concentration that induces a RPD of around 45 ± 5%.

COMPARISON WITH HISTORICAL CONTROL DATA:
In the solvent control group, the number of micronucleated cells per mononucleated cells were within the limits of values generally observed under our experimental conditions.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the mutagenicity assay using a 3 h treatment without metabolic activation (Table 6), the concentration of 0.20 mM induced a moderate toxicity with a RPD of 67.2%, corresponding to percentages of cytostasis of 32.8%, when compared to the respective solvent control. Under these conditions, the concentration of 0.20 mM was retained as the maximum concentration to be analyzed. Two lower concentrations were also assessed for genotoxicity.

In the mutagenicity assay using a 27 h treatment without metabolic activation (Table 8), the concentration of 0.075 mM induced a moderate toxicity with a RPD of 67.8%, corresponding to percentages of cytostasis of 32.2%, when compared to the respective solvent control. Under these conditions, the concentration of 0.075 mM was retained as the maximum concentration to be analyzed. Two lower concentrations were also assessed for genotoxicity.

In the mutagenicity assay using a 3 h treatment with metabolic activation (Table 7), the concentration of 0.40 mM induced a too strong toxicity. The immediately lower concentration of 0.30 mM induced a moderate toxicity with a RPD of 55.9%, corresponding to percentages of cytostasis of 44.1%, when compared to the respective solvent control. Under these conditions, the concentration of 0.30 mM was retained as the maximum concentration to be analyzed. Two lower concentrations were also assessed for genotoxicity.

Remarks on result:

other: no mutagenic potential

The investigation of a genotoxic activity of the test substance N-(2 -hydroxypropyl)Oleamide provided by SEPPIC has been carried out compliance with the OECD Guideline 487, using in vitro mammalian cell micronucleus test on TK6 lymphoblastoid human cells. The test was performed at the highest dose compatible with the toxic activity of the test substance in the three assays with and without S9 -mix, both with and without recovery period. Two concentrations below were also analysed. All the concentrations tested in this study are expressed as mM of N-(2-hydroxypropyl)Oleamide. In the short treatment without metabolic activation followed by a 24 h recovery period (assay S9 - 3h/+ 24h), the test substance, N-(2 -hydroxypropyl)Oleamide, induced neither biologically nor statistically significant increase in the number of micronucleated cells at all the concentrations analyzed from 0.20 to 0.10 mM. Indeed, 8 to 14 micronucleated mononucleated cells were observed per 2000 cells,vs.7 in the negative control without any dose-effect relationship. The test substance N-(2-hydroxypropyl)Oleamide, was thus considered as not genotoxic under this condition. In the short treatment with metabolic activation followed by a 24 h recovery period (assay S9+ 3h/24h), the test substance, N-(2 -hydroxypropyl)Oleamide, induced neither biologically nor statistically significant increase in the number of micronucleated cells at all the concentrations analyzed from 0.30 to 0.15 mM. Indeed, 7 and 8 micronucleated mononucleated cells were observed per 2000 cells,vs.8 in the negative control without any dose-effect relationship. The test substance, N-(2 -hydroxypropyl)Oleamide, was thus considered as not genotoxic under this condition. In the continuous treatment without metabolic activation without recovery period (assays S9 - 27h/+0h), the test substance, N-(2 -hydroxypropyl)Oleamide, induced neither biologically nor statistically significant increase in the number of micronucleated cells at all the concentrations analyzed from 0.075 to 0.025 mM. Indeed, 4 to 6 micronucleated mononucleated cells were observed per 2000 cells,vs.3 in the negative control. The test substance, N-(2 -hydroxypropyl)Oleamide was thus considered as not genotoxic under this condition.

Conclusions:

Under study conditions, the test substance was determined to be non genotoxic with or without metabolic activation in the micronucleus assay.

Executive summary:

A study was conducted to determine the genotoxic potential of the read across substance, Oleamide MIPA, using TK6 lymphoblastoid human cells, according to OECD Guideline 487 (in vitro mammalian cell micronucleus test), in compliance with GLP. Three assays (two short and one continuous exposure period) with and without metabolic activation, both with and without recovery period were performed with 0.025 to 0.40mM test substance concentrations. In a first study, 0.10 to 0.20 mM test substance with 3 h exposure, without metabolic activation followed by a 24 h recovery period, it did not induce biologically or statistically significant increase in the number of micronucleated cells. Although, 8 to 14 micronucleated mononucleated cells were observed per 2000 cells in comparison to 7 in the negative control without any dose-effect relationship. The test substance was thus considered as non-genotoxic under this condition by the study author. In a another study, 0.15 mM to 0.30 mM test substance with 3 h exposure period with metabolic activation followed by a 24 h recovery period, it did not induce biologically or statistically significant increase in the number of micronucleated cells. Although, 7 and 8 micronucleated cells were observed per 2000 cells compared to 8 in the negative control without any dose-effect relationship. The test substance was thus considered as non-genotoxic under this condition by the study author. In the continuous exposure period of 27 h with 0.025 to 0.075 mM test substance, without metabolic activation without recovery period, it did not induce biologically or statistically significant increase in the number of micronucleated cells. Although, 4 to 6 micronucleated mononucleated cells were observed per 2000 cells compared to 3 in the negative control. Overall the study author concluded that, the test substance induced no biologically or statistically significant increase in the number micronucleated cells either with or without metabolic activation, either with a short or with a continuous treatment. The acceptance criteria for the assay were fulfilled. The study is thus considered as valid. Under study conditions, the test substance was determined to be non-genotoxic with or without metabolic activation in the micronucleus assay (Nakab, 2013). Based on the results of the read across study, the test substance, C16 -18 AMP is also considered to be non-genotoxic in the micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1:

A study was conducted to determine the genotoxic potential of the test substance, C16-18 AMP, according to OECD Guideline 471 (Reverse Mutation Assay - 'Ames Test') in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using both the Ames plate incorporation and pre-incubation methods at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was between 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended (15 to 5000 µg/plate) following the results of Experiment 1. Six test substance concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test substance following the change in test methodology. The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test substance precipitate (particulate in appearance) was observed under a low power microscope at 1500 µg/plate and to the naked eye at 5000 µg/plate (with a creamy film also noted), these observations did not prevent the scoring of revertant colonies. A film was not observed in Experiment 2 after employing the pre-incubation modification. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). Small, statistically significant increases in revertant colony frequency were observed in Experiment 2 at 500 and 1500 µg/plate (TA98 dosed in the absence of S9-mix) and 150 µg/plate (WP2uvrA dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the mean maximum fold increase was only 1.5 times the concurrent vehicle controls. Under study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation in the Ames test (Envigo, 2017).

Study 2:

A study was conducted to determine the genotoxic potential of the test substance, C16-18 AMP, using the Thymidine Kinase Gene method,according to OECD Guideline 490 (Mammalian Cell Gene Mutation Tests)and EU Method B.17, in compliance with GLP. The dose range used in the main test was selected following the results of a preliminary toxicity study. In the main mutagenicity test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at up to 10 dose levels (0, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250 µg/mL) in duplicate, together with vehicle (Acetone), and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-h exposure group in the absence of metabolic activation. A precipitate of the test substance was observed at 48 µg/mL in the 4-h exposure in the presence of metabolic activation, however this dose level was not analysed due to excessive toxicity. There was evidence of marked toxicity following exposure to the test substance in all three exposure groups, as indicated by the %RSG (Relative suspension growth) and RTG (Relative total growth) values. There was evidence of modest reductions in viability (%V) in all three exposure groups, therefore indicating that residual toxicity had occurred. Optimum levels of toxicity were achieved in in the 4-h and 24-h exposure in the absence of metabolic activation. Near to optimum levels if toxicity were achieved in the 4-h exposure in the presence of metabolic activation, however the RTG value was marginally too toxic and was therefore excluded from analysis. The dose levels of 32 µg/mL in the 4-h exposure in the absence of metabolic activation, 40 and 48 µg/mL in the 4-h exposure in the presence of metabolic activation and 16 µg/mL in the 24-h exposure in the absence of metabolic activation were not plated out for 5-TFT resistance and viability due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test substance did not induce any toxicologically significant increases in the mutant frequency x 10^-6per viable cell in either of the three exposure groups. The GEF (Global evaluation factor) value of the test substance dose levels were not exceeded in any of the three exposure groups, including a dose level (32 µg/mL) beyond the acceptable level [GEF of 126 x 10^-6] of toxicity in the 4-h exposure in the presence of metabolic activation. Under study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation in the mouse lymphoma assay (Envigo, 2017).

Study 3:

A study was conducted to determine the genotoxic potential of the read across substance, Oleamide MIPA, using TK6 lymphoblastoid human cells, according to OECD Guideline 487 (in vitro mammalian cell micronucleus test), in compliance with GLP. Three assays (two short and one continuous exposure period) with and without metabolic activation, both with and without recovery period were performed with 0.025 to 0.40mM test substance concentrations. In a first study, 0.10 to 0.20 mM test substance with 3 h exposure, without metabolic activation followed by a 24 h recovery period, it did not induce biologically or statistically significant increase in the number of micronucleated cells. Although, 8 to 14 micronucleated mononucleated cells were observed per 2000 cells in comparison to 7 in the negative control without any dose-effect relationship. The test substance was thus considered as non-genotoxic under this condition by the study author. In a another study, 0.15 mM to 0.30 mM test substance with 3 h exposure period with metabolic activation followed by a 24 h recovery period, it did not induce biologically or statistically significant increase in the number of micronucleated cells. Although, 7 and 8 micronucleated cells were observed per 2000 cells compared to 8 in the negative control without any dose-effect relationship. The test substance was thus considered as non-genotoxic under this condition by the study author. In the continuous exposure period of 27 h with 0.025 to 0.075 mM test substance, without metabolic activation without recovery period, it did not induce biologically or statistically significant increase in the number of micronucleated cells. Although, 4 to 6 micronucleated mononucleated cells were observed per 2000 cells compared to 3 in the negative control. Overall the study author concluded that, the test substance induced no biologically or statistically significant increase in the number micronucleated cells either with or without metabolic activation, either with a short or with a continuous treatment. The acceptance criteria for the assay were fulfilled. The study is thus considered as valid. Under study conditions, the test substance was determined to be non-genotoxic with or without metabolic activation in the micronucleus assay (Nakab, 2013). Based on the results of the read across study, the test substance, C16 -18 AMP is also considered to be non-genotoxic in the micronucleus assay.

Justification for classification or non-classification

Based on the negative results in the in vitro Ames and mouse lymphoma assays available with the test substance and micronucleus assay with the read across substance, the test substance, C16 -18 AMP, does not warrant classification for genotoxicity, according to the EU CLP criteria (Regulation 1272/2008/EC).​​