Reaction mass of L-Glutamic acid, N-coco acyl derivs., monosodium salts and sodium hydrogen N-(1-oxooctadecyl)-L-glutamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Mammalian Cell Gene Mutation Test: MLA (read-across from structurally similar substance)

Under the conditions of this study, the test material was found not to be mutagenic in mouse lymphoma L5178Y cells in the presence and absence of exogenous metabolic activation up to precipitating and cytotoxic concentrations.

Mammalian Chromosome Aberration Test (read-across from structurally similar substance)

Under the conditions of this study, the test material was found not to be clastogenic in Chinese hamster lung fibroblasts (V79) in the presence and absence of metabolic activation up to cytotoxic and precipitating concentrations.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2016 to 22 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997, ninth ammendment

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected agent.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory Water Bath Shaker.
- The strains are stored at -80 ± 10 °C in the testing laboratory. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly.
-Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
- The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The number of viable cell of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory Water Bath Shaker.
- The strains are stored at -80 ± 10 °C in the testing laboratory. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly.
-Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
- The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The number of viable cell of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

-Preliminary Concentration Range Finding Test: 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate
-Initial Mutation Test and in the Confirmatory Mutation Test: 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate in all strains with metabolic activation 1 581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate in all strains without metabolic activation.
-Complementary Confirmatory Mutation Test: 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate in S. typhimurium TA1537 strain without metabolic activation and 5 000, 1 581, 500, 158.1, 50 and 15.81 μg/plate in E. coli WP2 uvrA strain without metabolic activation.
-Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test same concentrations were used. Furthermore, a Complementary Confirmatory Mutation Test was also performed based on the results of the Confirmatory Mutation Test using a modified concentration range.

Vehicle / solvent:

- Vehicle used: Distilled water
- Justification for choice of solvent/vehicle: The appropriate vehicle and the behaviour of the test material formulations with the solution of top agar and phosphate buffer were determined in a preliminary compatibility test.
-The solubility of the test material was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-Dimethylformamide (DMF). The test material was insoluble in DMF and DMSO at 100 mg/mL concentrations. However, the formulation at 100 mg/mL concentration using Distilled water as vehicle (solvent) was a clear white solution. Therefore, Distilled water was selected as vehicle (solvent) for the study. The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

For test material and positive controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene and 4-nitro-1,2-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: Pre-incubation

PRELIMINARY CONCENTRATION RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
-Based on the available information and the solubility and compatibility test, 100 mg/mL stock solution was prepared in Distilled water, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate of the test material. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

INITIAL MUTATION TEST AND CONFIRMATORY MUTATION TEST
-Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared in Distilled water, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5 000 μg test material/plate.
-A pre-incubation method was used in the preliminary experiment and main tests of the study as requested by the Sponsor. In the pre-incubation procedure bacteria were exposed to the test material both in the presence and absence of a metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. The test material and other components were prepared freshly.
Before the overlaying, 50 μL of test material formulations or its vehicle (or positive reference controls or their solvent), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test material. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator.
-After the incubation period, 2 mL of molten top agar was added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hour.

Evaluation criteria:

DATA EVALUATION
-The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor (Mutation factor (MF): mean number of revertants on the test material plate / mean number of revertants on the vehicle control plate) values were calculated for each concentration level of the test material and for the controls using Microsoft Excel software.

VALIDITY CRITERIA
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analysable concentrations were presented in all strains of the main tests.

CRITERIA FOR A POSITIVE RESPONSE
A test material was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than twice higher than the reversion rate of the negative (solvent) control.

CRITERIA FOR A NEGATIVE RESPONSE
The test material was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Key result

Species / strain:

bacteria, other: S typhimurium TA98, TA100, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S. typhimurium TA98, TA100 and TA1537 strains at 5 000 μg/plate (+S9 mix) and at 1 581, 500 and 158.1 μg/plate concentrations (-S9 mix); in S. typhimurium TA1535 strains at 5 000 μg/plate (+S9 mix) and at 1 581 and 500 μg/plate (-S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CONCENTRATION RANGE FINDING TEST
-Concentrations of 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test.
In the Preliminary Concentration Range Finding Test, the numbers of revertant colonies were mostly in the normal range which is the Mutation Factor between 0.8 and 1.2 (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
-Precipitate was not detected in the Preliminary Concentration Range Finding Test.
-Inhibitory, cytotoxic effect of the test material (reduced/slightly reduced background lawn development) was observed in the Preliminary Concentration Range Finding Test in Salmonella typhimurium TA98 and TA100 strains at 5 000 and 2 500 μg/plate concentration and Salmonella typhimurium TA100 strain at 1 000 μg/plate Concentration (+S9 mix) and at 5 000, 2 500, 1 000 and 316 μg/plate concentrations (-S9 mix).

INITIAL AND CONFIRMATORY MUTATION TESTS
-In the Initial Mutation Test (using the pre incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 15.81 μg/plate concentration without metabolic activation (the observed mutation factor values were: MF: 1.36). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
-In the Confirmatory Mutation Tests (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 0.5 and 0.1581 μg/plate concentration without metabolic activation (the observed mutation factor values were: MF: 1.63). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
-Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were within the historical control range in all cases, thus they were considered as biological variability of the test system.
-Precipitate was not detected in the Initial Mutation Test or Confirmatory Mutation Tests.
-Inhibitory, cytotoxic effect of the test item (reduced/slightly reduced background lawn development) was observed in the Initial Mutation Test and in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5 000 μg/plate concentration (+S9 mix) and at 1 581, 500 and 158.1 μg/plate concentrations (-S9 mix); in Salmonella typhimurium TA1535 strains at 5 000 μg/plate concentration (+S9 mix) and at 1 581 and 500 μg/plate concentrations (-S9 mix); in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1537 strain at 158.1 μg/plate concentration (-S9 mix).

Table 1: Summary Table of the Initial Mutation Test (Pre-Incubation Method)

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Untreated DMSO Distilled water 5000 1581 500 158.1 50 15.81 5 1.581 0.5	113.3 - 108.3 - 0.0 88.0 102.7 104.7 111.0 100.7 114.0 97.3	13.0 - 11.0 - 0.0 4.0 9.3 9.0 15.0 10.3 10.0 13.3	39.0 - 36.7 - 34.0 32.3 36.0 32.0 35.7 35.7 40.0 38.3	20.7 23.0 22.3 - 0.0 5.0 14.0 17.3 20.0 29.0 23.0 19.0	5.3 9.0 10.7 - 0.0 1.0 3.3 4.3 6.7 7.3 8.0 9.0
+	Untreated DMSO Distilled water 5000 1581 500 158.1 50 15.81 5 1.581 0.5	125.3 144.7 141.7 0.0 0.0 10.3 51.0 66.3 87.7 105.3 108.0 -	16.7 9.3 13.0 0.0 4.7 9.0 10.0 11.0 7.7 10.3 10.3 -	40.0 40.0 41.0 40.3 41.7 42.3 42.7 40.3 39.0 39.7 39.7 -	31.3 29.3 28.7 0.0 22.7 24.7 23.0 31.7 27.7 27.0 27.7 -	11.3 7.0 9.0 0.0 5.7 5.0 7.3 5.0 6.7 10.3 6.0 -
Positive Controls
-	Name	SAZ	SAZ	MMS	NPD	9AA
	Concentration (µg/plate)	2	2	2	4	50
	Mean no. colonies/plate	850.7	1234.7	989.3	410.7	424.7
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2	2	50	2	2
	Mean no. colonies/plate	2528.0	217.3	215.3	2309.3	205.3

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SAZ = Sodium azide

MMS = Methyl-methanesulfonate

NPD = 4-nitro-1,2-phenylenediamine

Table 2: Summary Table of the Confirmatory Mutation Test and the Complementary Confirmatory Mutation Test (Pre-Incubation Method)

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Untreated DMSO Distilled water 5000 1581 500 158.1 50 15.81 5 1.581 0.5 0.1581	107.7 - 100.3 - 0.0 26.7 53.3 54.3 75.3 87.7 98.7 98.3 -	13.7 - 12.7 - 0.0 6.7 12.0 13.3 10.3 15.0 11.0 14.7 -	26.7 - 30.3 28.3 34.7 29.3 25.3 28.0 28.7 - - - -	26.0 24.7 26.0 - 0.0 9.7 11.3 15.3 26.0 29.0 23.0 30.0 -	6.0 8.3 6.3 - - - 4.3 8.7 6.0 6.3 8.0 10.3 10.3
+	Untreated DMSO Distilled water 5000 1581 500 158.1 50 15.81 5 1.581 0.5 0.1581	120.7 117.0 114.7 2.3 35.0 63.3 73.0 78.7 73.0 90.0 118.7 - -	12.3 9.7 14.0 0.0 10.0 11.0 11.3 14.0 14.3 15.3 10.7 - -	42.3 46.7 45.3 45.3 51.3 52.0 51.7 50.0 59.0 49.3 52.3 - -	33.3 31.0 35.3 0.0 26.3 27.0 34.0 37.0 34.0 46.0 36.0 - -	14.0 11.3 14.3 0.0 8.3 10.3 14.7 8.7 14.7 11.0 10.0 - -
Positive Controls
-	Name	SAZ	SAZ	MMS	NPD	9AA
	Concentration (µg/plate)	2	2	2	4	50
	Mean no. colonies/plate	1042.7	1262.7	824.0	342.7	397.3
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2	2	50	2	2
	Mean no. colonies/plate	2372.0	350.0	238.7	2477.3	209.3

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SAZ = Sodium azide

MMS = Methyl-methanesulfonate

NPD = 4-nitro-1,2-phenylenediamine

Preliminary Compatability Test

-Based on the available information and the results of the solubility testing, distilled water was selected as vehicle (solvent) of the study.

Validity of the Tests

-Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.

-The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Conclusions:

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100 under GLP conditions using the bacterial reverse mutation assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (S. typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (E. coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

Based on the results of a solubility test, the test material was formulated in distilled water. Concentrations of 5 000; 2 500; 1 000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate in all strains with metabolic activation and 1 581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate in all strains without metabolic activation. Examined concentrations in the Complementary Confirmatory Mutation Test were 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test material/plate in Salmonella typhimurium TA1537 strain without metabolic activation and 5 000, 1 581, 500, 158.1, 50 and 15.81 μg test material/plate in Escherichia coli WP2 uvrA strain without metabolic activation.

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test material treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

Precipitate was not detected in the Initial Mutation Test or Confirmatory Mutation Tests.

Inhibitory, cytotoxic effect of the test material (reduced/slightly reduced background lawn development) was observed in the Initial Mutation Test and in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5 000 μg/plate concentration with metabolic activation and at 1 581, 500 and 158.1 μg/plate concentrations without metabolic activation; in Salmonella typhimurium TA1535 strains at 5 000 μg/plate concentration with metabolic activation and at 1 581 and 500 μg/plate concentrations without metabolic activation; in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1537 strain at 158.1 μg/plate concentration without metabolic activation.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test materialdid not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 February 2013 to 19 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Remarks:

Study conducted on read-across material

Justification for type of information:

A RAAF report will shortly be provided.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10 000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10 (v/v) % heat-inactivated foetal bovine serum (DMEM-10, culture medium). This was reduced to 5 % for the assays (DMEM-5).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

ASSAYS 1 AND 3
- Without metabolic activation: 300, 200, 150, 125, 100, 75, 50 and 25 μg/mL (3 hour treatment, harvest 20 hours from beginning of treatment)
- With metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 20 hours from beginning of treatment)

ASSAY 2
- Without metabolic activation: 100, 80, 60, 50, 40, 30, 20, 10 and 5 µg/mL (20 hour treatment, harvest 28 hours from beginning of treatment)
- With metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 28 hours from beginning of treatment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
For the cytogenetic experiments, 1 to 3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 10⁵ cells/dish concentration and incubated for approximately 24 hours at 37 °C in 10 mL of culture medium (DMEM-10). After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) for the assay without metabolic activation or with 9.4 mL treatment medium with 0.5 mL S9-mix in case of experiments with metabolic activation.
Cells were treated with different concentration test material solutions, negative (vehicle) or positive control solutions (treatment volume: 100 μL/dish in all cases) for the given period of time at 37 °C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1 v/v % antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.

DURATION
- Exposure duration: Assays 1 and 3: 3 hours in the presence and absence of metabolic activation. Assay 2: 3 hours in the presence of metabolic activation and 20 hours in the absence of metabolic activation.
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours for assays 1 and 3 and 28 hours for Assay 2

SPINDLE INHIBITOR (cytogenetic assays): 2 to 2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 μg/mL).
STAIN (for cytogenetic assays): 5 % Giemsa solution. The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (methanol:acetic acid 3:1 (v:v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried (at least three slides were prepared for each culture in the main tests). The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted.

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: At least one hundred metaphases with 22 ± 2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Where there were insufficient metaphases in one replicate, the total was made up to 200 cells examined per concentration using the other replicate.

DETERMINATION OF CYTOTOXICITY
- Method: % relative survival (determined through counting with a haemocytometer)

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:

The assay was considered positive if the following criteria were met:
- Increases in the frequency of metaphases with aberrant chromosomes were observed at one or more test concentrations (only data without gaps are considered).
- The increases were reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases were statistically significant.
- The increases were not associated with large changes in pH or osmolarity of the treated cultures.
The historical control data for the testing laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.
The test material was concluded to have given a negative response if no reproducible, statistically significant increases were observed.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Assay 1 produced a positive response in 1 concentration without metabolic activation and 2 concentrations with metabolic activation. No positive response was observed in assays 2 and 3 (assay 3 was performed using the same conditions)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Marked cytotoxicity was observed in the high dose concentrations in all assays with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant effects relating to pH were observed.
- Effects of osmolality: No significant effects relating to osmolality were observed.
- Water solubility: The test material was found to be soluble in distilled water at up to 250 mg/mL. This was selected as the vehicle for use in the study.
- Precipitation: Precipitation was observed at the end of treatment in Assay 1 from 75 to 300 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 2, precipitation was observed at 80 to 100 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 3, the test material was found to precipitate at 75 to 300 µg/mL without metabolic activation and at 100 to 400 µg/mL with metabolic activation.
- Other confounding effects: In Assay 1, there was an increase in the number of chromosome aberrations at 125 μg/mL without metabolic activation, and at 150 and 200 μg/mL with metabolic activation. However, in each case there was a substantial difference between the replicates and the general level of aberrations was higher than usual, including in the negative controls. Also, there were more polyploid and endoreduplicated cells than are normally observed, indicating some instability in the cultures. Assay 2, carried out one week later with the same cell line, demonstrated that the result was not reproduced. Therefore, an additional main test (Assay 3) was carried out with identical conditions to Assay 1 for clarification. Assay 1 was considered to be invalid and was discounted.

RANGE-FINDING/SCREENING STUDIES
Two concentration selection cytotoxicity assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 3-hour treatment with metabolic activation or 20-hour treatment without metabolic activation, 28-hour harvesting time) were performed to establish appropriate concentration ranges for the definitive tests.
A total of eight test concentrations between 5000 and 2.29 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.
In assay A, without metabolic activation 24 % relative survival was noted at 185.2 µg/mL; above this concentration (555.6, 1666.7 and 5000 µg/mL), 0 % relative survival was recorded. With metabolic activation, % relative survival was 65 % at 185.2 µg/mL; above this concentration, % relative survival was 0 % (555.6, 1666.7 and 5000 µg/mL).
In assay B without metabolic activation, relative survival was 10 % at 61.73 µg/mL and 0 % at concentrations above this level (185.2, 555.6, 1666.7 and 5000 µg/mL). With metabolic activation, relative survival at 185.2 µg/mL was 68 %; above this concentration (555.6, 1666.7 and 5000 µg/mL) relative survival was 0 %.

MAIN STUDY
In Assay 1, marked cytotoxicity was observed at 300, 200 and 150 μg/mL c without metabolic activation (relative survival values were 0, 18 and 33 %, respectively) and 400, 300 and 250 μg/mL with metabolic activation (relative survival values were 0, 0 and 26 %, respectively).Therefore, concentrations of 150, 125, 100 and 75 μg/mL were chosen for evaluation without metabolic activation and 250, 200, 150 and 100 μg/mL were chosen for evaluation with metabolic activation.
In Assay 2, cytotoxicity was also observed at 100, 80 and 60 μg/mL concentrations without metabolic activation (relative survival values were 0, 15 and 29 %, respectively) and 400, 300, 250 and 200 μg/mL with metabolic activation (relative survival values were 0, 0, 23 and 45 %, respectively). Therefore, concentrations of 60, 50 and 40 μg/mL were chosen for evaluation without metabolic activation and 200, 150 and 100 μg/mL were chosen for evaluation with metabolic activation.
In Assay 3, marked cytotoxicity was observed at 300, 200 and 150 μg/mL without metabolic activation (relative survival values were 0, 18 and 37 %, respectively) and 400, 300 and 250 μg/mL with metabolic activation (relative survival values were 0, 0 and 22 % , respectively)4. Therefore, concentrations of 150, 125, 100 and 75 μg/mL were chosen for evaluation without metabolic activation and 250, 200, 150 and 100 μg/mL were chosen for evaluation with metabolic activation.

Assay 3 did not cause an increase in the number of cells with structural chromosome aberrations at the same concentrations as seen in Assay 1. Additionally, Assay 2 showed no increase in aberration levels with or without metabolic activation at any concentration of the test material.
Polyploid metaphases (1-6) or endoreduplicated (1-4) were found in some cases in the negative (vehicle) control, positive control or test material treated cells in Assays 1 and 3. No polyploidy or endoreduplicated metaphases were detected in Assay 2.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative (vehicle) control data were within the acceptable range for spontaneous aberration frequency.

POSITIVE CONTROL DATA: The positive control substances caused a statistically significant increase in the number of structural aberrations in the experiments with or without metabolic activation, demonstrating the sensitivity of the test system.
In the 3 hour treatment without metabolic activation for Assay 1 and 3, the aberration frequency of the EMS positive control was slightly lower than anticipated. However, the increase in the number of aberrant cells compared to the negative control value was statistically highly significant and the proper status of the cells was also proven in the same assay in the experiment with metabolic activation. Furthermore, there was a good response using this positive control substance using 20-hour treatment without metabolic activation. Therefore, the observed values were considered to be acceptable.

Table 1: Results for Assay 1 (discounted)

Concentration (µg/mL)	Treatment time (hours)	Harvesting time (hours)	% Relative survival	Mean % aberrant cells		No. cells observed		Observations
				Including gaps	Excluding gaps	Polyploid	Endo.
	Without metabolic activation
Vehicle control	3	20	100	2.5	2.5	3	1	normal
300	3	20	0	NE	NE	NE	NE	ppte, disc. medium
200	3	20	18	NE	NE	NE	NE	ppte, disc. medium
150	3	20	33	2.5	2.5	6	1	ppte, disc. medium
125	3	20	63	9.5	9.0**	4	0	ppte, disc. medium
100	3	20	80	4.5	4.5	5	1	ppte, disc. medium
75	3	20	94	1.0	1.0	1	0	ppte
50	3	20	96	NE	NE	NE	NE	normal
25	3	20	77	NE	NE	NE	NE	normal
EMS 1 µL/mL	3	20	73	14.4	14.4***	5	0	normal
	With metabolic activation
Vehicle control	3	20	100	4.0	4.0	5	1	normal
400	3	20	0	NE	NE	NE	NE	ppte, disc. medium
300	3	20	0	NE	NE	NE	NE	ppte, disc. medium
250	3	20	26	6.0	6.0	2	4	ppte, disc. medium
200	3	20	56	11.0	10.5*	4	2	ppte, disc. medium
150	3	20	75	12.0	12.0**	2	2	ppte, disc. medium
100	3	20	99	3.5	3.5	4	2	ppte, disc. medium
50	3	20	91	NE	NE	NE	NE	normal
25	3	20	105	NE	NE	NE	NE	normal
CP 6 µg/mL	3	20	69	93.8	93.8***	0	1	normal

Endo. = Endoreduplicated

EMS = Ethyl methanesulfonate

CP = Cyclophosphamide

ppte = Precipitate

disc. medium = discoloured medium

NE = Not examined

*p<0.05 comparing numbers of aberrant cells excluding gaps with negative control

**p<0.01 comparing numbers of aberrant cells excluding gaps with negative control

***p<0.001 comparing numbers of aberrant cells excluding gaps with negative control

 

Table 2: Results from Assay 2

Concentration (µg/mL)	Treatment time (hours)	Harvesting time (hours)	% Relative survival	Mean % aberrant cells		No. cells observed		Observations
				Including gaps	Excluding gaps	Polyploid	Endo.
	Without metabolic activation
Vehicle control	20	28	100	4.0	2.5	0	0	normal
100	20	28	0	NE	NE	NE	NE	ppte, disc. medium
80	20	28	15	NE	NE	NE	NE	ppte
60	20	28	29	4.5	2.0	0	0	normal
50	20	28	58	6.5	3.5	0	0	normal
40	20	28	97	2.5	2.0	0	0	normal
30	20	28	95	NE	NE	NE	NE	normal
20	20	28	102	NE	NE	NE	NE	normal
10	20	28	108	NE	NE	NE	NE	normal
5	20	28	120	NE	NE	NE	NE	normal
EMS 0.4 µL/mL	20	28	79	26.9	23.1***	0	0	normal
	With metabolic activation
Vehicle control	3	28	100	5.0	2.5	0	0	normal
400	3	28	0	NE	NE	NE	NE	ppte, disc. medium
300	3	28	0	NE	NE	NE	NE	ppte, disc. medium
250	3	28	23	NE	NE	NE	NE	ppte, disc. medium
200	3	28	45	8.5	3.5	0	0	ppte, disc. medium
150	3	28	70	5.5	2.0	0	0	ppte, disc. medium
100	3	28	93	3.5	2.0	0	0	ppte, disc. medium
50	3	28	92	NE	NE	NE	NE	normal
25	3	28	108	NE	NE	NE	NE	normal
CP 6 µg/mL	3	28	75	37.8	33.3***	0	0	normal

Endo. = Endoreduplicated

EMS = Ethyl methanesulfonate

CP = Cyclophosphamide

ppte = Precipitate

disc. medium = discoloured medium

NE = Not examined

*p<0.05 comparing numbers of aberrant cells excluding gaps with negative control

**p<0.01 comparing numbers of aberrant cells excluding gaps with negative control

***p<0.001 comparing numbers of aberrant cells excluding gaps with negative control

 

Table 3: Results from Assay 3

Concentration (µg/mL)	Treatment time (hours)	Harvesting time (hours)	% Relative survival	Mean % aberrant cells		No. cells observed		Observations
				Including gaps	Excluding gaps	Polyploid	Endo.
	Without metabolic activation
Vehicle control	3	20	100	2	1.5	0	0	normal
300	3	20	0	NE	NE	NE	NE	ppte, disc. medium
200	3	20	18	NE	NE	NE	NE	ppte, disc. medium
150	3	20	37	3.5	2	1	4	ppte, disc. medium
125	3	20	64	2	1.5	1	2	ppte, disc. medium
100	3	20	69	5.5	4.5	0	1	ppte, disc. medium
75	3	20	87	3.5	3	2	0	ppte
50	3	20	85	NE	NE	NE	NE	normal
25	3	20	100	NE	NE	NE	NE	normal
EMS1 µL/mL	3	20	78	12.5	11.5***	1	0	normal
	With metabolic activation
Vehicle control	3	20	100	5	3.5	1	0	normal
400	3	20	0	NE	NE	NE	NE	ppte, disc. medium
300	3	20	0	NE	NE	NE	NE	ppte, disc. medium
250	3	20	22	4.5	3	0	2	ppte, disc. medium
200	3	20	62	7	4	0	0	ppte, disc. medium
150	3	20	75	4.5	3.5	0	0	ppte, disc. medium
100	3	20	90	4.5	3.5	0	0	ppte, disc. medium
50	3	20	90	NE	NE	NE	NE	normal
25	3	20	100	NE	NE	NE	NE	normal
CP 6 µg/mL	3	20	66	88.2	88.2***	1	0	normal

Endo. = Endoreduplicated

EMS = Ethyl methanesulfonate

CP = Cyclophosphamide

ppte = Precipitate

disc. medium = discoloured medium

NE = Not examined

*p<0.05 comparing numbers of aberrant cells excluding gaps with negative control

**p<0.01 comparing numbers of aberrant cells excluding gaps with negative control

***p<0.001 comparing numbers of aberrant cells excluding gaps with negative control

Conclusions:

Negative with and without metabolic activation.

Under the conditions of this study, the test material was found not to be clastogenic in Chinese hamster lung fibroblasts (V79) in the presence and absence of metabolic activation up to cytotoxic and precipitating concentrations.

Executive summary:

The clastogenicity of the test material was investigated in Chinese hamster lung fibroblasts (V79) in a chromosome aberration assay performed in accordance with the standardised guidelines OECD 473, EU Method B.10 and EPA OPPTS 870.5375 under GLP conditions.

Cells were seeded into culture dishes (5 x 10⁵ cells/dish concentration) and incubated for 24 hours prior to exposure to the test material. The cells were exposed to the test material in the presence and absence of metabolic activation (S9). The cells were exposed to the test material at the following concentrations:

- Assays 1 and 3

Without metabolic activation: 300, 200, 150, 125, 100, 75, 50 and 25 μg/mL and with metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 20 hours from beginning of treatment).

- Assay 2

Without metabolic activation: 100, 80, 60, 50, 40, 30, 20, 10 and 5 µg/mL (20 hour treatment, harvest 28 hours from beginning of treatment) and with metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 28 hours from beginning of treatment).

Precipitation was observed at the end of treatment in Assay 1 from 75 to 300 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 2, precipitation was observed at 80 to 100 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 3, the test material was found to precipitate at 75 to 300 µg/mL without metabolic activation and at 100 to 400 µg/mL with metabolic activation.

Marked cytotoxicity was observed at the higher doses; however a minimum of three analysable concentrations were available for evaluation for each assay.

In Assay 1, there was an increase in the number of chromosome aberrations at 125 μg/mL without metabolic activation and at 150 and 200 μg/mL with metabolic activation. However, in each case there was a substantial difference between the replicates and the general level of aberrations was higher than usual, including in the negative controls. Also, there were more polyploid and endoreduplicated cells than are normally observed, indicating some instability in the cultures. Assay 1 was considered to be invalid and was discounted.

Assay 3 did not cause an increase in the number of cells with structural chromosome aberrations at the same concentrations as seen in Assay 1. Additionally, Assay 2 showed no increase in aberration levels with or without metabolic activation at any concentration of the test material.

Under the conditions of this study, the test material was found not to be clastogenic in Chinese hamster lung fibroblasts (V79) in the presence and absence of metabolic activation up to cytotoxic and precipitating concentrations.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

A RAAF report will shortly be provided.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Assay 1 produced a positive response in 1 concentration without metabolic activation and 2 concentrations with metabolic activation. No positive response was observed in assays 2 and 3 (assay 3 was performed using the same conditions)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Marked cytotoxicity was observed in the high dose concentrations in all assays with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 October 2013 to 16 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Remarks:

Study conducted on read-across material

Justification for type of information:

A RAAF report will shortly be provided.

Reason / purpose for cross-reference:

other: Read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK+/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-10 medium. The RPMI-10 medium consisted of RPMI 1640 medium with the following components added (final concentration in medium): heat inactivated horse serum 10 % v/v, antibiotic-antimycotic solution 0.01 mL/mL (containing 10 000 IU/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin–B), Pluronic-F68 0.5 mg/mL and pyruvic avid 0.2 mg/mL. L-glutamine 0.3 mg/mL was also added freshly to the media. NaHCO₃ 2 mg/mL was already included in the ready to use liquid media.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

ASSAY 1
- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL
- Three hour treatment without metabolic activation: 5, 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL

ASSAY 2
- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL
- Twenty four hour treatment without metabolic activation: 2.5, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Based on a previous chromosome aberration study performed by the same laboratory, the test material was soluble in distilled water. The highest achievable concentration using this vehicle was 250 mg/mL. As this vehicle is also compatible with the test system, it was selected to be the vehicle (solvent) for this study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
0.2 mL of RPMI-5 medium, vehicle (solvent), test material formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or 150 mM KCl (in the case of 3 h treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, at least 10⁷ cells were placed in each of a series of 75 cm² sterile flasks. For the 24-hour treatment, at least 4 x 10⁶ cells were placed in each of a series of 25 cm² sterile flasks.
RPMI-5 medium was made in the same way as the RPMI-10, with the exception that it contained a reduced level of heat inactivated horse serum (5 % v/v).
Cultures were incubated at 37 ± 1 °C (approximately 5 % CO₂ in air). Gentle shaking was used during the 3-hour treatments. After the treatment period, cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer. Measurement of pH and osmolality was also performed after the treatment period.
Where a sufficient number of cells survived, the cell density was adjusted to a concentration of 2 x 10⁵ cells/mL. Cells were transferred to flasks for growth through the expression period (maximum 25 mL of suspension) or diluted to be plated for survival.

DURATION
- Exposure duration: Assay 1: 3 hours in the presence and absence of metabolic activation. Assay 2: 3 hours in the presence of metabolic activation, 24 hours in the absence of metabolic activation.
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): about 2 weeks

SELECTION AGENT (mutation assays): trifluorothymidine (TFT). The cultures were sub-cultured daily and the cell density adjusted to 2 x 10⁵ cells/mL.

NUMBER OF REPLICATIONS: Performed in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency and the corresponding relative survival (%) and relative total growth
Plating for survival - Cultures of cell density 2 x 10⁵ cells/mL were further diluted to 8 cells/mL. 0.2 mL of the final concentration of each culture were placed into each well of two 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C in air containing approximately 5 % (v/v) CO₂ for about two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Plating for viability - At the end of the expression period (cultures treated with TFT), the cell density in the selected cultures was determined and adjusted to 1 x 10⁴ cells/mL with RPMI-20 (20 % heat inactivated horse serum, no addition of Pluronic F-68) for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL. 0.2 mL of the final concentration of each culture was placed into each well of two 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C in air containing approximately 5 % (v/v) CO₂ for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:

The test material was considered to be mutagenic in this assay if all the following criteria were met:
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency were observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There was a significant concentration-relationship as indicated by linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 10⁶ viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.
Results which only partially satisfied the acceptance and evaluation criteria were evaluated on a case-by-case basis.

Statistics:

The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis in any assay with or without S9

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Due to excessive toxicity in the cultures, not all assays had eight analysable concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in pH
- Effects of osmolality: There were no large changes in osmolality
- Precipitation: In assay 1, minimal precipitation was observed at all concentrations in at least one replicate. In assay 2 in the presence of metabolic activation (3 hour exposure) minimal precipitation was observed at all concentrations in at least one replicate. In assay 2 in the absence of metabolic activation (24 hour exposure) minimal precipitation was observed at all concentrations in at least one replicate with the exception of the lowest dose tested in the series.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed at the following concentrations: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL for 3 hour treatments in the presence and absence of metabolic activation (toxicity tests A and B, respectively) and for 24 hours in the absence of metabolic activation (toxicity test C).
All concentrations were performed in duplicate. Due to the solubility of the test material, the test solution administration in the preliminary toxicity test was 0.4 mL. Precipitate was observed at all concentrations in all three tests in at least one replicate.

In test A, relative survival at 78.13 µg/mL was 41 % on day 0 and 92 % on day 3. All concentrations above this level demonstrated excessive toxicity (0 % relative survival).
In test B, relative survival at 78.13 µg/mL was 5 % on day 0 and 45 % on day 3. All concentrations above this level demonstrated excessive toxicity (0 % relative survival). At 36.06 µg/mL, relative survival was 61 % on day 0 and 84 % on day 3.
In test C, relative survival at 19.53 µg/mL was 21 % on day 1 and 80 % on day 3. All concentrations above this level demonstrated excessive toxicity (0 % relative survival).

COMPARISON WITH HISTORICAL CONTROL DATA:
Comparison with the historical control data confirmed that the test system was functioning correctly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Assay 1 with metabolic activation, excessive cytotoxicity of the test material was observed at 160, 140 and 120 μg/mL concentrations; cells of these samples did not survive the treatment or the expression period. Evaluation of the mutagenicity was made using data of the first surviving concentration of 100 μg/mL (relative survival value of 24 %) and the five lower concentrations (a total of six concentrations).
In Assay 1 without metabolic activation, excessive cytotoxicity of the test material was observed at the 100 μg/mL concentration; cells of this sample did not survive the treatment period. Evaluation was performed on data from the next concentration of 90 μg/mL (relative survival value of 18 %) and seven lower concentrations (a total of eight concentrations).

In Assay 2, for the 3-hour treatment with metabolic activation, cells of the 160, 140 and 120 μg/mL concentration samples did not survive the treatment or the expression period. Evaluation was performed on the data from the first surviving concentration which was 100 μg/mL (relative survival value of 46 %) and the five lower concentrations (a total of six concentrations).
In Assay 2, for the 24-hour treatment without metabolic activation, excessive cytotoxicity was observed at the 40, 35, 32.5, 30 and 27.5 μg/mL concentrations; cells of these samples died during the treatment or in the expression period. Evaluation of the data was made using the next concentration of 25 μg/mL (relative survival of 8 %) and the following six lower concentrations (a total of seven concentrations).

Table 1: Summary of Results for Assay 1

Concentration (µg/mL)	Survival Data			Viability Data	Mutation Frequency
	PE	RS (%)	RTG (%)	PE
Metabolic activation	With (+ S9-mix); 3 hour exposure
160	ND	ND	ND	ND	ND
140	0.007	0	ND	ND	ND
120	0.015	1	ND	ND	ND
100	0.374	24	1	0.860	120.7
80	0.575	51	37	0.810	116.4
60	0.748	79	59	0.706	139.1
40	0.805	89	85	0.687	109.3
20	0.787	95	108	0.727	102.2
10	0.776	97	117	0.701	123.3
Vehicle control	0.810	100	100	0.759	109.9
Vehicle control for positive control (DMSO)	0.712	94	113	0.743	99
Untreated control	0.737	98	91	0.732	111.8
Positive control CP (4 μg/mL)	0.405	45	16	0.398	1733.3*
Metabolic activation	Without (- S9-mix); 3 hour exposure
100	ND	ND	ND	ND	ND
90	0.765	18	3	0.732	100.4
80	0.732	31	13	0.754	101.6
70	0.776	41	25	0.781	104.8
60	0.712	65	54	0.712	122.6
50	0.810	89	59	0.759	108.9
40	0.707	84	78	0.754	108.6
20	0.776	98	78	0.712	106.5
10	0.816	115	108	0.816	96.5
5	0.799	105	100	0.787	110.9
Vehicle control	0.781	100	100	0.722	99.8
Vehicle control for positive control (DMSO)	0.816	108	94	0.687	103.8
Untreated control	0.754	103	99	0.793	101.3
Positive control NQO (0.15 μg/mL)	0.453	60	29	0.440	1087.9*

CP = Cyclophosphamide

RS= Relative survival values (%) corrected with the post treatment cell concentrations

ND = No data (no cells were plated for colony growing due to the observed cytotoxicity during treatment or in the expression period)

NQO = 4-Nitroquinoline-N-oxide

RTG = Relative total growth

PE = Plating efficiency

* = Statistically significant

 

Table 2: Summary of Results for Assay 2

Concentration (µg/mL)	Survival Data			Viability Data	Mutation Frequency
	PE	RS (%)	RTG (%)	PE
Metabolic activation	With (+ S9-mix); 3 hour exposure
160	ND	ND	ND	ND	ND
140	0.008	0	ND	ND	ND
120	0.008	1	ND	ND	ND
100	0.424	46	35	0.787	141.1
80	0.644	83	47	0.640	147.4
60	0.743	86	79	0.835	99.0
40	0.841	105	89	0.737	132.1
20	0.706	97	110	0.701	120.0
10	0.770	100	99	0.663	118.9
Vehicle control	0.765	100	100	0.653	94.3
Vehicle control for positive control (DMSO)	0.770	107	116	0.776	90.9
Untreated control	0.805	106	114	0.810	89.8
Positive control CP (4 μg/mL)	0.380	41	12	0.357	1935.9*
Metabolic activation	Without (- S9-mix); 24 hour exposure
40	ND	ND	ND	ND	ND
35	ND	ND	ND	ND	ND
32.5	ND	ND	ND	ND	ND
30	ND	ND	ND	ND	ND
27.5	ND	ND	ND	ND	ND
25	0.398	8	18	0.706	92.4
22.5	0.408	9	23	0.822	80.3
20	0.539	14	29	0.805	83.0
15	0.532	25	41	0.748	84.3
10	0.805	92	121	0.860	70.8
5	0.743	99	119	0.722	91.5
2.5	0.727	83	103	0.776	89.0
Vehicle control	0.754	100	100	0.717	95.2
Vehicle control for positive control (DMSO)	0.754	74	91	0.793	85.2
Untreated control	0.727	99	122	0.810	86.1
Positive control NQO (0.1 μg/mL)	0.360	17	6	0.386	1473.9*

CP = Cyclophosphamide

RS= Relative survival values (%) corrected with the post treatment cell concentrations

ND = No data (no cells were plated for colony growing due to the observed cytotoxicity during treatment or in the expression period)

NQO = 4-Nitroquinoline-N-oxide

RTG = Relative total growth

PE = Plating efficiency

* = Statistically significant

Conclusions:

Negative with and without metabolic activation

Under the conditions of this study, the test material was found not to be mutagenic in mouse lymphoma L5178Y cells in the presence and absence of exogenous metabolic activation up to precipitating and cytotoxic concentrations.

Executive summary:

The mutagenicity of the test material was assessed in an in vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay) which was performed in accordance with the standardised guidelines OECD 476 and EU Method B.17, under GLP conditions.

The test was performed using the mouse lymphoma L5178Y cell model up to cytotoxic and precipitating concentrations in the absence and presence of an exogenous metabolic activation system (S9 -mix).

The following conditions were used in the study:

ASSAY 1

- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL

- Three hour treatment without metabolic activation: 5, 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL

ASSAY 2

- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL

- Twenty four hour treatment without metabolic activation: 2.5, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL

The expression time and selection time for both assays (both in the presence and absence of metabolic activation) were 3 days and 2 weeks, respectively.

No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis was observed in any of the assays either with or without metabolic activation.

Under the conditions of this study, the test material was found not to be mutagenic in mouse lymphoma L5178Y cells in the presence and absence of exogenous metabolic activation up to precipitating and cytotoxic concentrations.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

A RAAF report will shortly be provided.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis in any assay with or without S9

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Due to excessive toxicity in the cultures, not all assays had eight analysable concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100 under GLP conditions using the bacterial reverse mutation assay. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (S. typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (E. coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

Based on the results of a solubility test, the test material was formulated in distilled water. Concentrations of 5 000; 2 500; 1 000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate in all strains with metabolic activation and 1 581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate in all strains without metabolic activation. Examined concentrations in the Complementary Confirmatory Mutation Test were 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test material/plate in Salmonella typhimurium TA1537 strain without metabolic activation and 5 000, 1 581, 500, 158.1, 50 and 15.81 μg test material/plate in Escherichia coli WP2 uvrA strain without metabolic activation.

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test material treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

Precipitate was not detected in the Initial Mutation Test or Confirmatory Mutation Tests.

Inhibitory, cytotoxic effect of the test material (reduced/slightly reduced background lawn development) was observed in the Initial Mutation Test and in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5 000 μg/plate concentration with metabolic activation and at 1 581, 500 and 158.1 μg/plate concentrations without metabolic activation; in Salmonella typhimurium TA1535 strains at 5 000 μg/plate concentration with metabolic activation and at 1 581 and 500 μg/plate concentrations without metabolic activation; in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1537 strain at 158.1 μg/plate concentration without metabolic activation.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test materialdid not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Mammalian Cell Gene Mutation Test: MLA (read-across from structurally similar substance)

In the key study (Hargitai 2014), the mutagenicity of the read-across material was assessed in an in vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay) which was performed in accordance with the standardised guidelines OECD 476 and EU Method B.17, under GLP conditions. The study was therefore assigned a reliability score of 1 in accordance with the criteria for assessing data quality as described in Klimisch et al (1997).

The test was performed using the mouse lymphoma L5178Y cell model up to cytotoxic and precipitating concentrations in the absence and presence of an exogenous metabolic activation system (S9 -mix).

The following conditions were used in the study:

ASSAY 1

- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL

- Three hour treatment without metabolic activation: 5, 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL

ASSAY 2

- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL

- Twenty four hour treatment without metabolic activation: 2.5, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL

The expression time and selection time for both assays (both in the presence and absence of metabolic activation) were 3 days and 2 weeks, respectively.

No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis was observed in any of the assays either with or without metabolic activation.

Under the conditions of this study, the read-across material was found not to be mutagenic in mouse lymphoma L5178Y cells in the presence and absence of exogenous metabolic activation up to precipitating and cytotoxic concentrations.

Mammalian Chromosome Aberration Test (read-across from structurally similar substance)

In the key study (Hargitai 2014), the clastogenicity of the read-across material was investigated in Chinese hamster lung fibroblasts (V79) in a chromosome aberration assay performed in accordance with the standardised guidelines OECD 473, EU Method B.10 and EPA OPPTS 870.5375 under GLP conditions. The study was therefore assigned a reliability score of 1 in accordance with the criteria for assessing data quality as described in Klimisch et al (1997).

Cells were seeded into culture dishes (5 x 10⁵ cells/dish concentration) and incubated for 24 hours prior to exposure to the test material. The cells were exposed to the test material in the presence and absence of metabolic activation (S9). The cells were exposed to the test material at the following concentrations:

- Assays 1 and 3

Without metabolic activation: 300, 200, 150, 125, 100, 75, 50 and 25 μg/mL and with metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 20 hours from beginning of treatment).

- Assay 2

Without metabolic activation: 100, 80, 60, 50, 40, 30, 20, 10 and 5 µg/mL (20 hour treatment, harvest 28 hours from beginning of treatment) and with metabolic activation: 400, 300, 250, 200, 150, 100, 50 and 25 μg/mL (3 hour treatment, harvest 28 hours from beginning of treatment).

Precipitation was observed at the end of treatment in Assay 1 from 75 to 300 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 2, precipitation was observed at 80 to 100 µg/mL without metabolic activation and 100 to 400 µg/mL with metabolic activation. In Assay 3, the test material was found to precipitate at 75 to 300 µg/mL without metabolic activation and at 100 to 400 µg/mL with metabolic activation.

Marked cytotoxicity was observed at the higher doses; however a minimum of three analysable concentrations were available for evaluation for each assay.

In Assay 1, there was an increase in the number of chromosome aberrations at 125 μg/mL without metabolic activation and at 150 and 200 μg/mL with metabolic activation. However, in each case there was a substantial difference between the replicates and the general level of aberrations was higher than usual, including in the negative controls. Also, there were more polyploid and endoreduplicated cells than are normally observed, indicating some instability in the cultures. Assay 1 was considered to be invalid and was discounted.

Assay 3 did not cause an increase in the number of cells with structural chromosome aberrations at the same concentrations as seen in Assay 1. Additionally, Assay 2 showed no increase in aberration levels with or without metabolic activation at any concentration of the test material.

Under the conditions of this study, the read-across material was found not to be clastogenic in Chinese hamster lung fibroblasts (V79) in the presence and absence of metabolic activation up to cytotoxic and precipitating concentrations. 

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to genetic toxicity.