Fatty acids, C16-18, lithium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as an unpublished report.

Justification for type of information:

See IUCLID section 13 for category and read across justification

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lithium myristate

EC Number:

243-743-8

EC Name:

Lithium myristate

Cas Number:

20336-96-3

Molecular formula:

C14H28O2.Li

IUPAC Name:

lithium myristate

impurity 1

Reference substance name:

Lithium hydroxide

EC Number:

215-183-4

EC Name:

Lithium hydroxide

Cas Number:

1310-65-2

Molecular formula:

HLiO

IUPAC Name:

lithium hydroxide

Test material form:

solid

Details on test material:

- Batch number: A194-99
- Expiry date: 30 June 2013

Method

Target gene:

- Salmonella: +Histidine
- E.Coli: Tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- Main test experiment one: 50, 150, 500, 1500 and 5000 µg/plate
- Main test experiment two: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
- Justification for choice of solvent/vehicle:

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix. Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Remarks:

Without S9 mix

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix. 9-Aminoacridine: 80 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix. N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix. N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix. N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHODS OF APPLICATION
- Experiment 1: In agar (plate incorporation)
- Experiment 2: Pre-incubation

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 µg/plate because of excessive test item precipitation.

Evaluation criteria:

ACCEPTANCE CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 1 to 9.0 x 10(+09) bacteria per ml.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should not be an excessive loss of plates due to contamination.

EVALUATION CRITERIA
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test item was insoluble in sterile distilled water (25 and 50 mg/mL), dimethyl sulphoxide, dimethyl formamide and acetonitrile (50 mg/mL), acetone (100 mg/mL) and tetrahydrofuran (200 mg/mL) in solubility checks performed in-house. The test item formed the best doseable suspension in sterile distilled water at 25 mg/mL; therefore, this solvent was selected as the vehicle.
- Precipitation: A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
- Preliminary Toxicity Test:The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Master strains: Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
- Negative control: Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
- Positive control: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

RESULTS

Preliminary ToxicityTest

The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile. The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	83	91	92	87	101	91	97	85	86P	67SP	45SP
+	TA100	87	102	79	101	67	75	74	71	74P	82P	39SP
-	WP2uvrA	12	11	15	13	12	17	15	15	18P	15P	10SP
+	WP2uvrA	22	16	13	18	16	21	22	15	24P	12P	5P

P: Precipitate

S: Sparse bacterial background lawn

      

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table1(see below) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1               Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
106	114	9	16	27	23	20	17	3	10
118		28		20		15		17
119		11		21		16		11

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
103	119	17	14	21	21	19	18	7	7
124		13		21		17		7
130		13		20		17		7
		25	20*					12	10*
		13						8
		23						9

* Experimental procedure repeated at a later date (without S9-mix) due to excessive toxicity in the original test

Table 2: Test Results: Experiment 1– Without Metabolic Activation

With or without S9-Mix	Dose Level Per Plate	Number of revertants (mean) ± SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
		Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)
S9-Mix (-)	Solvent Control (Acetone)	104	112 (10.8)	19	19 (2.0)	20	20 (0.6)	13	13 (6.0)	1	6 (6.4)
		107		17		20		7		3
		124		21		19		19		13
	5 μg	136	125 (11.0)	16	14 (7.8)	25	25 (8.0)	17	19 (3.5)	5	6 (1.2)
		126		5		33		23		5
		114		20		17		17		7
	15 μg	102	116 (11.8)	28	29 (3.2)	24	20 (9.6)	12	14 (1.7)	7	5 (2.0)
		123		33		27		15		5
		122		27		9		15		3
	50 μg	83	91 (10.6)	17	17 (1.5)	32	20 (10.2)	8	13 (5.7)	1	4 (3.5)
		103		19		13		11		8
		87		16		16		19		4
	150 μg	104	98 (9.8)	13	13 (0.6)	24	24 (0.6)	4	10 (9.0)	5	5 (2.0)
		87		13		24		20		7
		104		12		25		5		3
	500 μg	116P	98 (15.7)	5P	9 (3.5)	17P	18 (1.7)	13P	13 (1.5)	8P	5 (3.5)
		91P		11P		17P		15P		5P
		87P		11P		20P		12P		1P
	1500 μg	115P	107 (18.0)	13SP	20 (7.0)	28P	23 (4.4)	15SP	14 (1.2)	7SP	7 (2.0)
		86P		27SP		21P		13SP		9SP
		119P		19SP		20P		15SP		5SP
	5000 μg	144SP	97 (42.2)	11SP	9 (1.5)	24SP	17 (6.1)	7SP	6 (1.5)	0SP	0 (0.0)
		83SP		8SP		13SP		6SP		0SP
		63SP		9SP		14SP		4SP		0SP
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG		4NQO		9AA
	Dose level	3 μg		5 μg		2 μg		0.2 μg		80 μg
		Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)
	No. revertants	529	435 (181.6)	160	161 (6.1)	521	504 (20.5)	186	185 (11.0)	668	605 (82.2)
		226		156		481		196		635
		551		168		509		174		512

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

S: Sparse bacterial background lawn

P: Precipitate

Table 3: Test Results: Experiment 1– With Metabolic Activation

With or without S9-Mix	Dose Level Per Plate	Number of revertants (mean) ± SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
		Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)
S9-Mix (+)	Solvent Control (Acetone)	90	104 (17.1)	13	13 (3.5)	33	30 (2.6)	16	16 (0.6)	7	9 (2.1)
		123		16		28		17		11
		99		9		29		16		8
	5 μg	103	117 (19.5)	8	13 (6.2)	19	25 (6.0)	29	19 (8.5)	8	5 (3.5)
		108		20		24		13		5
		139		11		31		16		1
	15 μg	115	112 (2.6)	9	8 (3.1)	29	23 (6.0)	15	19 (4.0)	11	11 (0.0)
		110		11		23		19		11
		111		5		17		23		11
	50 μg	95	102 (9.1)	20	16 (4.5)	23	22 (1.7)	16	20 (6.4)	4	3 (0.6)
		98		16		23		16		3
		112		11		20		27		3
	150 μg	88	96 (10.6)	7	12 (4.6)	13	15 (3.8)	23	18 (5.0)	7	8 (3.1)
		108		15		19		13		5
		92		15		12		19		11
	500 μg	115P	108 (6.4)	15P	10 (4.2)	33P	29 (4.0)	19P	18 (6.0)	1P	4 (3.1)
		104P		7P		25P		12P		7P
		104P		9P		29P		24P		3P
	1500 μg	116P	112 (10.6)	21SP	20 (1.0)	16P	17 (0.6)	21SP	21 (2.5)	4SP	4 (2.5)
		100P		19SP		17P		24SP		6SP
		120P		20SP		17P		19SP		1SP
	5000 μg	112SP	118 (8.1)	1SP	7 (5.3)	12SP	16 (7.2)	16SP	14 (2.6)	0SP	0 (0.0)
		114SP		11SP		24SP		11SP		0SP
		127SP		9SP		11SP		15SP		0SP
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA		BP		2AA
	Dose level	1 μg		2 μg		10 μg		5 μg		2 μg
		Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)	Value	Mean (s.d.)
	No. revertants	1070	1029 (92.2)	191	190 (2.3)	255	305 (47.3)	289	302 (11.0)	406	426 (25.0)
		1093		187		311		307		454
		923		191		349		309		418

2AA 2-Aminoanthracene

BP Benzo(a)pyrene

P Precipitate

S: Sparse bacterial background lawn

Tables 4 and 5 below

Applicant's summary and conclusion

Conclusions:

Interpretation of results: Negative
Lithium myristate was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The in vitro mutagenicity of lithium myristate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (Harlan 2013). S. typhimurium and E. coli strains were treated with suspensions of lithium myristate using both the Ames plate incorporation and pre-incubation methods at five dose levels in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The vehicle, positive and negative controls confirmed the sensitivity of the assay and the efficacy of the S9 -mix.