Lithium salt of branched-aliphatic dicarboxylic acid

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:

2014-07-28 to 2014-08-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted under GLP conditions according to OECD guideline 471.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(Adopted July 21, 1997)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(May 31, 2008)

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical state: light yellow powder with lumps (determined at WIL Research Europe B.V.)
- Storage condition of test material: at room temperature in the dark

Method

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):

- Properly maintained: yes

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

- Experiment 1: 52, 164, 512, 1600, 5000 µg/plate (5% (v/v) S9-mix) with TA1535, TA1537, TA98
- Experiment 2: 52, 164, 512, 1600, 5000 µg/plate (10% (v/v) S9-mix) with TA1535, TA1537, TA98, TA100, WP2uvrA

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli-Q water

Controls

Untreated negative controls:

yes

Remarks:

Milli-Q water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoant

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +- 4 h


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

other: not examined as the vehicle control was with Milli-Q water

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: not examined as the vehicle control was with Milli-Q water

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: the tester strains TA100 and WP2uvrA were tested in triplicate, both with and without 5% (v/v) S9-mix. Concentrations: 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate. The highest concentration of test substance used in the subsequent mutation assay was 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Deviations:

1. In the second mutation experiment only two plates could be determined in tester strain WP2uvrA (absence of S9 -mix) at the dose level of 512 µg/plate

Evaluation: One of the triplicate cultures was infected. The two remaining plates showed responses well within the range and the testing of an extra plate would have given no additioinal information: therefore this deviation had no influence on the study result.

2. The positive control substance of tester strain WP2uvrA (second experiment, presence of S9 -mix) showed a response (mean plate count) which was not within the laboratory historical range.

Evaluation: The value (1357) was above the limit of the range (1271). The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

The study integrity was not affected by the deviations.

Table 1: Dose range finding test: Mutagenic response of Lithium salt of branched-aliphatic dicarboxylic acid in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain
	TA100	WP2uvrA
	Without S9-mix
Positive control	887 +-13	1354 +/-133
Solvent control	101 +-9	27 +/-4
1.7	103 +-12	33 +/-4
5.4	119 +-8	33 +/-4
17	122 +-16	37 +/-9
52	108 +-8	32 +/-5
164	105 +-7	41 +/-6
512	117 +-11	33 +/-11
1600	98 +-12	33 +/-2
5000	107 +-2 *	29 +/-2*
	With S9 -mix**
Positive control	1359 +-158	152 +/-28
Solvent control	92 +-13	44 +/-9
1.7	105 +-6	35 +/-4
5.4	92 +-18	39 +/-12
17	98 +-23	30 +/-11
52	105 +-11	42 +/-6
164	93 +-5	34 +/-4
512	98 +-4	35 +/-7
1600	98 +-11	38 +/-3
5000	102 +-9*	34 +/-8*

*No precipitate and Normal bacterial background lawn

** Plate incorporation assay (5% S9)

Table 2: Experiment 1: Mutagenic response of Lithium salt of branched-aliphatic dicarboxylic acid in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium.
	TA1535	TA2537	TA98
	Without S9-mix
Positive control	603 +/-29	610 +/-73	942 +/-11
Solvent control	18 +/-3	9 +/-4	10 +/-5
52	15 +/-2	5 +/-4	12 +/-4
164	13 +/-4	4 +/-1	15 +/-10
512	20 +/-5	8 +/-3	16 +/-2
1600	22 +/-5	3 +/-2	18 +/-2
5000	15 +/-5*	4 +/-2*	16 +/-3*
		With S9-mix**
Positive control	257 +/-17	228 +/-36	655 +/-23
Solvent control	13 +/-2	7 +/-3	19 +/-6
52	16 +/-2	7 +/-2	25 +/-7
164	17 +/-4	4 +/-2	17 +/-3
512	13 +/-3	6 +/-5	23 +/-3
1600	14 +/-6	9 +/-5	22 +/-6
5000	13 +/-2 *	8 +/-2*	21 +/-2*

*No precipitate and Normal bacterial background lawn

**Plate incorporation assay (5% S9)

Table 3: Experiment 2: Mutagenic response of Lithium salt of branched-aliphatic dicarboxylic acid in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
	Without S9 -mix
Positive control	599 +/-13	592 +-103	847 +/-32	891 +/-10	304 +/-18
Solvent control	10 +/-4	4 +/-3	29 +/-5	121 +/-8	36 +/-6
52	10 +/-2	10 +/-6	37 +/-5	123 +/-6	48 +/-6
164	14 +/-3	6 +/-2	20 +/-3	126 +/-12	48 +/-12
512	15 +/-1	6 +/-2	39 +/-13	98 +/-17	33 +/-8***
1600	16 +/-3	8 +/-1	32 +/-6	109 +/-11	43 +/-2
5000	13 +/-3*	6 +/-3*	31 +/-12*	104 +/-10*	30 +/-2*
	With S9 -mix**
Positive control	173 +/-31	454 +/-30	519 +/-23	1174 +/-80	1357 +/-8
Solvent control	14 +/-5	11 +/-2	48 +/-5	122 +/-16	38 +/-9
52	14 +/-3	11 +/-5	32 +/-7	124 +/-13	32 +/-4
164	13 +/-4	6 +/-3	41 +/-11	103 +/-13	26 +/-6
512	16 +/-5	9 +/-3	36 +/-8	107 +/-23	48 +/-3
1600	18 +/-1	12 +/-4	43 +/-4	105 +/-16	48 +/-8
5000	18 +/-4*	10 +/-6*	46 +/-4*	105 +/-14*	39 +/-8*

*No precipitate and Normal bacterial background lawn

**Plate incorporation assay (10% S9)

***one plate infected: Mean of two plates

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that Lithium salt of branched-aliphatic dicarboxylic acid is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of Lithium salt of branched-aliphatic dicarboxylic acid in theSalmonella typhimurium reverse mutation assay and the Escherichia colireverse mutation assay.

 

The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP₂uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

The test substance was a light yellow powder with lumps with a purity of>=99 %. The test substance was dissolved in Milli-Q water.

 

In the dose range finding test, the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP₂uvrA. The test substance did not precipitate on the plates at the highest dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the mutation assay.

 

Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 52 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In a follow-up experiment of the assay with additional parameters, the test substance was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

Lithium salt of branched-aliphatic dicarboxylic acid did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, with deviations that were determined not to have affected the results.

 

Based on the results of this study it is concluded that Lithium salt of branched-aliphatic dicarboxylic acid is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in the Escherichia colireverse mutation assay.