Azelaic acid, calcium salt (1:1)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as an unpublished report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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

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

Test material

Test material information

Constituent 1

Reference substance name:

Dilithium azelate

EC Number:

254-184-4

EC Name:

Dilithium azelate

Cas Number:

38900-29-7

Molecular formula:

C9H14O4.2Li

IUPAC Name:

dilithium azelate

Test material form:

other: solid

Details on test material:

- Physical state: White powder
- Purity: 98.33%
- Batch number: FLTF G14057
- Analysis code: A058/99
- Expiration date: 2018-03-06
- Storage of test material: Room temperature in the dark

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:

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 3 hours after preparation.

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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

Controls 6

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

Controls 7

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

Controls 8

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

Controls 9

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

Controls 10

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.

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:

Results: The substance was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate. In the absence of S9-mix in tester strain TA1535, the mean plate count of the solvent control (3) was below the limit of the range (6). Therefore this part was repeated in an additional (second experiment), the dose range tested was 52, 164, 512, 1600 and 5000 μg/plate in the absence of S9-mix.
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: In the direct plate assay, no reduction of the bacterial background lawn was observed. No decrease in the number of revertants less than the minimal value of the historical control data range were observed in all tester strains tested. However a high solvent control value in tester strain TA1537 in the absence of S9-mix resulted in a doubtful toxic observation. Therefore this part of the experiment was repeated in an additional (second experiment), the dose range tested was 52, 164, 512, 1600 and 5000 μg/plate in the absence of S9-mix. In the pre-incubation assay, 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.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

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	798 ± 110	1375 ± 91	1312 ± 105	217 ± 7
Solvent control	104 ± 11	21 ± 5	107 ± 18	25 ± 7
1.7	96 ± 6	33 ± 6	111 ± 5	27 ± 4
5.4	90 ± 15	23 ± 6	102 ± 15	29 ± 16
17	100 ± 3	26 ± 6	95 ± 9	31 ± 13
52	104 ± 5	21 ± 6	124 ± 7	34 ± 8
164	91 ± 7	21 ± 3	108 ± 6	39 ± 5
512	99 ± 17	25 ± 8	97 ± 9	22 ± 10
1600	99 ± 3	24 ± 6	97 ± 3	33 ± 7
5000	99 ± 6 *	27 ± 4 *	113 ± 23 *	30 ± 6 *

* 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	750 ± 26	868 ± 40	1052 ± 46	209 ± 10	337 ± 37	943 ± 40
Solvent control	3 ± 2	15 ± 6	16 ± 5	9 ± 3	8 ± 5	23 ± 2
52	10 ± 3	3 ± 3	18 ± 6	8 ± 3	10 ± 2	24 ±8
164	10 ± 6	4 ± 2	17 ± 5	11 ± 4	8 ± 4	14 ± 3
512	13 ± 3	4 ± 4	18 ± 4	11 ± 2	6 ± 2	20 ± 8
1600	18 ± 2	5 ± 2	14 ± 5	14 ± 6	11 ± 3	22 ± 8
5000	13 ± 4 *	9 ± 3 *	14 ± 7 *	11 ± 4 *	4 ± 1 *	23 ± 6 *

* No precipitate and normal bacterial background lawn

Table 3: Experiment 2 - Mutagenic response in the 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
Dose	TA100	WP2uvrA
Positive control	773 ± 22	667 ± 58
Solvent control	13 ± 6	6 ± 4
52	11 ± 10	5 ± 4
164	16 ± 3	8 ± 4
512	21 ± 7	5 ± 2
1600	10 ± 3	6 ± 4
5000	14 ± 2 *	3 ± 4 *

* No precipitate and normal bacterial background lawn

Table 4: 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	791 ± 18	63 ± 14	974 ± 26	697 ± 91	158 ± 74	134 ± 12	169 ± 13	512 ± 17	2137 ± 204	343 ± 9
Solvent control	15 ± 1	4 ± 1	20 2) 3	97 ± 15	28 ± 9	10 ± 2	4 ± 3	35 ± 2	96 2) 16	30 ± 7
52	20 ± 6	3 ± 2	15 ± 4	106 ± 5	24 ± 4	12 ± 4	7 ± 4	27 ± 8	108 ± 14	42 ± 12
164	19 ± 7	6 ± 2	20 ± 6	87 ± 10	21 ± 8	19 2) 5	10 ± 2	28 ± 8	100 ± 15	39 ± 5
512	13 ± 4	8 ± 4	16 ± 10	102 ± 8	20 ± 6	11 ± 4	8 ± 4	26 ± 7	89 ] 11	31 ± 5
1600	15 ± 3	4 ± 3	16 ± 4	91 ± 14	24 ± 5	18 ± 3	8 ± 4	28 ± 6	80 ± 9	31 ± 3
5000	13 ± 3 *	5 ± 2 *	13 ± 3	109 ± 12 *	22 ± 3 *	11 ± 1 *	9 ± 3 *	19 ± 10 *	88 ± 9 *	29 ± 6 *

* No precipitate and normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, it is concluded that dilithium azelate 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 azelate 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 azelate 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.