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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 lithium cation or the fatty acid anion. 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:
migrated 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other: GLP compliant, guideline study, available as an unpublished report.
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
Remarks:
Migrated from field 'Test system'.

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:
Interpretation of results (migrated information):
negative

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

Additional information from genetic toxicity in vitro:

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. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances. As the fatty acid substances listed in Annex V are exempt, it can reasonably be interpreted that they are not considered to be hazardous to human health (with the noted potential exceptions of skin and eye irritation) or the environment. 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. Thus, the fatty acid components of the category ‘dilithium salts of dicarboxylic acids C6-C10’ are not expected to be hazardous. As all category members are lithium salts, any toxicity is expected to be driven by the lithium ion. 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. 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: Castor oil and magnesium stearate have been tested in the Ames test with negative results, as reported in the API Robust Summaries (2008, referencing NTP 1992 and Litton Bionetics 1976, respectively). Stearic acid was tested for mutagenicity with and without S9 in the Ames test (reported in CIR 1987) and no mutagenic activity was observed. A sister chromatid exchange study on oleic acid was reported in the same review, using Chinese hamster V79 lung fibroblasts. The mean number of SCE’s per metaphase was similar to the control, but higher incidences of aneuploidy were apparently seen with the oleic acid cultures. However, oleic and lauric acids have been considered as inhibitors 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), lauric acid (C12), stearic acid (C18), oleic acid (C18 unsaturated), and fatty acids C18-22. Each of these substances has been subjected to reversion tests in Escherichia coli or Salmonella typhimurium strains with and without metabolic activation.

Overall, there is no evidence of genotoxicity associated with the lithium cation, and there is a significant volume of published data to indicate that the C6-C10 fatty acids within the category are not genotoxic.


Justification for selection of genetic toxicity endpoint
This substance is representative of the lithium salts of C6-C10 fatty acids and can be read across to other lithium dicarboxylate category members.

Justification for classification or non-classification

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

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