Dilithium azelate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

From the results of the in vitro experimental studies of dilithium adipate (C6), azelate (C9) and sebacate (C10) and references to fatty acids in REACH Annex V, it can be concluded that there is no evidence of genotoxicity associated with the substances in the category. There is no evidence of a relevant intrinsic genotoxic properties requiring classification or substance specific risk mitigation measures (RMMs).

Link to relevant study records

Reference

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other: GLP compliant, guideline study, available as an unpublished report.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

For read across justification, see Section 13 of IUCLID

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

Principles of method if other than guideline:

Each S9 batch is characterized with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively Not the correct amount of 2-aminoanthracene was mentioned in the protocol. This writing error in the protocol had no effect on the results of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Salmonella: +Histidine
- E.Coli: Tryptophan

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 / 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:

Aroclor 1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment one: 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment two: 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli Q water for test substance and DMSO for positive controls (except sodium azelate which used saline)
- Preparation: Test substance concentrations were used within 2 hours after preparation.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2.5 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2.5 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate in direct plate assay and 5 µg/plate in preincubation assay

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 15 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Sodium azide: 5 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR 191: 2.5 µg/plate and 2-nitrofluorene: 10 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene: 10 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Methylmethanesulfonate: 650 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline N-oxide: 10 µg/plate

Remarks:

Without S9 mix

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: 30 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
To determine the toxicity, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Data evaluation and statistical procedures
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 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 control.
b) In case a repeat 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. Standard deviation was determined.

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

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:

Results: All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Precipitate: Precipitation of the substance on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity: In the direct plate test and the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
Negative controls: The negative control values were within the laboratory historical control data ranges.
Positive controls: The 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, except the response for TA1537 in the absence of S9-mix, second experiment. 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.

Remarks on result:

other: all strains/cell types tested

Table 1: Dose range finding test - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain

	Without S9		With S9
Dose	TA100	WP2uvrA	TA100	WP2uvrA
Positive control	862 ± 37	1574 ± 37	1395 ± 94	311 ± 6
Solvent control	102 ± 11	28 ± 6	82 ± 11	41 ± 10
1.7	88 ± 6	33 ± 7	93 ± 20	35 ± 8
5.4	81 ± 10	26 ± 6	105 ± 7	36 ± 12
17	89 ± 15	27 ± 9	102 ± 11	33 ± 10
52	104 ± 18	31 ± 4	104 ± 9	38 ± 8
164	95 ± 3	26 ± 7	105 ± 10	37 ± 7
512	99 ± 20	31 ± 1	95 ± 8	42 ± 10
1600	98 ± 16	33 ± 13	103 ± 18	39 ± 6
5000	87 ± 14 *	28 ± 9 *	93 ± 0 *	39 ± 7 *

* No precipitate and normal bacterial background lawn

Table 2: Experiment 1 - Mutagenic responnse in Salmonella typhimurium reverse mutation assay - Direct plate assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium

	Without S9			With S9
Dose	TA 1535	TA 1537	TA 98	TA 1535	TA 1537	TA 98
Positive control	851 ± 29	421 ± 62	949 ± 12	284 ± 16	454 ± 42	1121 ± 42
Solvent control	20 ± 6	16 ± 5	18 ± 2	15 ± 5	5 ± 0	28 ± 5
52	20 ± 10	8 ± 3	16 ± 2	15 ± 6	14 ± 6	39 ± 3
164	23 ± 5	8 ± 2	17 ± 8	12 ± 2	11 ± 1	31 ± 9
512	17± 2	6 ± 6	12 ± 3	17 ± 5	10 ± 6	32 ± 7
1600	18 ± 2	9 ± 2	19 ± 4	25 ± 2	10 ± 5	29 ± 8
5000	12 ± 0 *	11 ± 3 *	17 ± 4 *	18 ± 4 *	9 ± 4 *	30 ± 1 *

* No precipitate and normal bacterial background lawn

Table 3: Experiment 2 - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay - Pre-incubation assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

	Without S9					With S9
Dose	TA 1535	TA 1537	TA 98	TA 100	WP2uvra	TA 1535	TA 1537	TA 98	TA 100	WP2uvra
Positive control	766 ± 52	56 ± 5	1003 ± 56	682 ± 64	169 ± 22	126 ± 9	160 ± 8	432 ± 26	2266 ± 183	399 ± 21
Solvent control	17 ± 3	10 ± 6	16 ± 4	103 ± 13	33 ± 3	8 ± 2	12 ± 4	28 ± 3	115 ± 17	35 ± 5
52	17 ± 2	7 ± 3	19 ± 2	119 ± 19	26 ± 8	10 ± 5	13 ± 5	29 ± 3	126 ± 10	43 ± 16
164	16 ± 4	8 ± 4	19 ± 1	107 ± 10	27 ± 7	10 ± 5	12 ± 6	28 ± 3	110 ± 7	35 ± 5
512	11 ± 6	13 ± 2	15 ± 7	103 ± 11	30 ± 7	10 ± 5	9 ± 6	23 ± 2	109 ± 13	28 ± 5
1600	15 ± 4	9 ± 2	19 ± 7	103 ± 7	23 ± 1	10 ± 5	13 ± 11	33 ± 9	103 ± 5	42 ± 6
5000	17 ± 2 *	9 ± 6 *	18 ± 5 *	107 ± 11 *	29 ± 6 *	10 ± 5 *	15 ± 5 *	25 ± 4 *	112 ± 15 *	28 ± 12 *

* No precipitate and normal bacterial background lawn

 

Conclusions:

Based on the results of this study, it is concluded that dilithium adipate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The in vitro mutagenicity of dilithium adipate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (WIL 2015). S. typhimurium and E. coli strains were treated with suspensions of dilithium adipate 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 and positive controls confirmed the sensitivity of the assay and the efficacy of the S9 -mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The substances in the category are considered to be similar on the basis that they have common structures of a lithium ion varying only by the length of the fatty acid chain. As a result, it is expected that the substances will have similar, predictable properties. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Since published reviews do not distinguish between the properties of monocarboxylic or dicarboxylic acids as a category, then the same interpretation can be applied to the dicarboxylic acids. Due to the close structural similarity and the narrow range of carbon chain numbers covered in this category, the genotoxicity potential is expected to be similar across the category.  

  

Key bacterial reversion assays (Ames test) have been conducted on dilithium adipate (C6), azelate (C9) and sebacate (C10). In addition, a chromosomal aberration assay and a mouse lymphoma assay have been performed on fatty acids C18 (unsaturated) lithium salts and a mouse lymphoma assay has been conducted on lithium myristate. In all cases the results were negative. On the basis of the read across justification, it is appropriate to read across the negative genotoxicity data (chromosomal aberration and mammalian cell gene mutation) from the fatty acids C18 (unsaturated) lithium salts to the dilithium dicarboxylates category members. 

 

Further evidence of the lack of genotoxicity of the aliphatic acid category is provided by the following information: Lauric acid (C12) has been considered as an inhibitor of mutagenicity produced by positive control substances such as N-nitrosopyrrolidine and sodium azide (CIR 1987 review). Additional reporting of fatty acid genetic toxicity is presented in the HERA review (HERA 2002). In this review, it is stated that fatty acids are negative in in vitro bacterial systems used in the Ames test. These included capric acid (C10) and lauric acid (C12), with each substance subjected to reversion tests in Escherichia coli or Salmonella typhimurium strains with and without metabolic activation.  

 

Overall, the cation, and there is a significant volume of published data to indicate that there is no evidence of genotoxicity associated with the substances in the lithium salts of dicarboxylic acids C6-C10 category and the substances are not genotoxic. Further testing (chromosome aberration and gene mutation in mammalian cells) is being conducted on dilithium adipate to support this conclusion.

Justification for classification or non-classification

Not classified for genetic toxicity. Negative results in all studies conducted.