Lithium isooctadecanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 01 February 2010 and 22 February 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

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:

Fatty acids C18-(unsaturated) lithium salts

Cas Number:

502962-81-4

IUPAC Name:

Fatty acids C18-(unsaturated) lithium salts

Test material form:

solid

Details on test material:

- Batch number: Not reported
- Expiry date: Not reported

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

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
Experiment one: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone.

- Justification for choice of solvent/vehicle: The test material was insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in–house. Sterile distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested that the test material was insoluble in water. The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

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:

Acetone

True negative controls:

not specified

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:

Acetone

True negative controls:

not specified

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:

Acetone

True negative controls:

not specified

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:

Acetone

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:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix. 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

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:

Acetone

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:

Acetone

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:

Acetone

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:

METHOD OF APPLICATION: in agar (plate incorporation) - Experiment 1

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 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 and examined for effects on the growth of the bacterial background lawn.
Example:
METHOD OF APPLICATION: in agar (pre-incubation) - Experiment 2

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): 20 minutes at 37 degrees C

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

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 9.9 x 10(+09) bacteria per mL.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
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
Dunnetts Linear Regression Analysis

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:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number.

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:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Example:
TEST-SPECIFIC CONFOUNDING FACTORS
- Vehicle solubility: The test material was insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in–house. Sterile distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested that the test material was insoluble in water. The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle.
- Precipitation: A greasy precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was toxic at and above 1500 µg/plate to TA100 without S9 and WP2uvrA- with S9 and was non-toxic to TA100 with S9 and WP2uvrA- without S9. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

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: The test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number. However, the toxicity of the test material to the tester strains was of insufficient severity to prevent testing up to the maximum recommended dose level of 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

RESULTS

Preliminary Toxicity Test

The test material was toxic at and above 1500 µg/plate to TA100 without S9 and WP2uvrA^-with S9 and was non-toxic to TA100 with S9 and WP2uvrA^-without S9. The test material 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	95	106	100	101	104	103	107	112	99	67 *	0 P *
+	TA100	101	86	97	92	102	93	107	93	95	70	75P
-	WP2uvrA^-	21	25	23	20	20	19	20	21	20	21	13P
+	WP2uvrA^-	32	26	20	29	20	30	21	31	19	56 *	60 P *

P         Precipitate

*          Partial or complete absence of 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 inTable1and 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 material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 1 (see attached document).

A history profile of vehicle and positive control values is presented in Appendix 4 (see attached document).

The test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number. However, the toxicity of the test material to the tester strains was of insufficient severity to prevent testing up to the maximum recommended dose level of 5000 µg/plate. A greasy precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

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

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)

Range-finding Test

Number of revertants (mean number of colonies per plate) Base-pair substitution type Frameshift type TA100 TA1535 WP2uvrA^- TA98 TA1537 109   25   20   18   11   97 (99) 26 (25) 20 (19) 14 (18) 11 (12) 91   25   16   22   14

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
94		21		20		17		4
107	(98)	16	(18)	22	(21)	21	(19)	13	(8)
94		17		21		20		8

 

Table 2               Test Results: Range-Finding Test– Without Metabolic Activation

Test period		From: 14 February 2010					To: 17 February 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^-			TA98			TA1537
-	0	103 110 90	(101) 10.1#	25 24 19	(23) 3.2	27 27 30		(28) 1.7	20 18 15		(18) 2.5	16 14 13		(14) 1.5
-	5	102 128 128	(119) 15.0	26 24 24	(25) 1.2	19 25 31		(25) 6.0	14 19 19		(17) 2.9	14 12 14		(13) 1.2
-	15	111 81 124	(105) 22.1	22 19 20	(20) 1.5	21 21 23		(22) 1.2	16 15 18		(16) 1.5	8 19 16		(14) 5.7
-	50	107 130 128	(122) 12.7	19 24 14	(19) 5.0	31 23 18		(24) 6.6	18 13 13		(15) 2.9	15 13 20		(16) 3.6
-	150	123 109 129	(120) 10.3	12 14 22	(16) 5.3	16 29 29		(25) 7.5	12 15 16		(14) 2.1	10 13 10		(11) 1.7
-	500	100 95 108	(101) 6.6	12 15 23	(17) 5.7	23 25 30		(26) 3.6	19 15 15		(16) 2.3	8 12 11		(10) 2.1
-	1500	85 P 79 P 91 P	(85) 6.0	15 P 15 P 14 P	(15) 0.6	18 P 19 P 22 P		(20) 2.1	15 P 18 P 19 P		(17) 2.1	4 P 3 P 5 P		(4) 1.0
-	5000	81 P * 95 P * 91 P *	(89) 7.2	16 P * 11 P * 14 P *	(14) 2.5	22 P 21 P 24 P		(22) 1.5	22 P * 11 P * 15 P *		(16) 5.6	12 P * 8 P * 5 P *		(8) 3.5
Positive controls   S9-Mix   -	Name Concentration (µg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO			9AA
		3		5		2			0.2			80
		411 408 420	(413) 6.2	2183 1544 2281	(2003) 400.2	866 872 883		(874) 8.6	124 122 134	(127) 6.4		618 1336 1015	(990) 359.7

P        Precipitate

*         Partial absence of bacterial background lawn

#        Standard deviation

Table 3               Test Results: Range-Finding Test– With Metabolic Activation

Test period		From: 14 February 2010					To: 17 February 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^-			TA98		TA1537
+	0	98 96 96	(97) 1.2#	9 13 12	(11) 2.1	20 24 22		(22) 2.0	19 32 21	(24) 7.0	12 11 14	(12) 1.5
+	5	89 81 91	(87) 5.3	8 9 9	(9) 0.6	19 26 31		(25) 6.0	15 19 18	(17) 2.1	10 11 9	(10) 1.0
+	15	99 97 89	(95) 5.3	8 8 15	(10) 4.0	20 20 24		(21) 2.3	23 24 21	(23) 1.5	13 9 12	(11) 2.1
+	50	95 106 117	(106) 11.0	10 8 12	(10) 2.0	24 20 32		(25) 6.1	19 18 18	(18) 0.6	15 9 15	(13) 3.5
+	150	98 96 89	(94) 4.7	8 9 11	(9) 1.5	15 30 29		(25) 8.4	16 25 25	(22) 5.2	8 9 7	(8) 1.0
+	500	90 73 85	(83) 8.7	11 11 13	(12) 1.2	21 27 20		(23) 3.8	19 15 16	(17) 2.1	8 12 10	(10) 2.0
+	1500	82 P 81 P 84 P	(82) 1.5	8 P 13 P 8 P	(10) 2.9	19 P 31 P 23 P		(24) 6.1	12 P 10 P 19 P	(14) 4.7	9 P 15 P 11 P	(12) 3.1
+	5000	86 P * 97 P * 89 P *	(91) 5.7	12 P 12 P 11 P	(12) 0.6	20 P 22 P 23 P		(22) 1.5	15 P * 10 P * 19 P *	(15) 4.5	14 P 10 P 9 P	(11) 2.6
Positive controls   S9-Mix   +	Name Concentration (µg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		370 409 403	(394) 21.0	219 236 242	(232) 11.9	172 200 201		(191) 16.5	66 90 95	(84) 15.5	176 195 207	(193) 15.6

P        Precipitate

*         Partial absence of bacterial background lawn

#        Standard deviation

PLEASE SEE ATTACHED IN OVERALL REMARKS, ATTACHMENTS 1) Tables 4 & 5 (Main Test)

2) Report of Results in Mutagenicity Test using Micro-organisms

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA^-were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels (5 and 15 µg/plate) and an expanded dose range were selected in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

Results.

The vehicle (acetone) 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 test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number. However, the toxicity of the test material to the tester strains was of insufficient severity to prevent testing up to the maximum recommended dose level of 5000 µg/plate. A greasy precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

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

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.