4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Five in-vitro studies are available on the substance:

- 3 in-vitro gene mutation studies in bacteria 'Ames Tests'

- Chromosome aberration test in human lymphocytes

- Mouse lymphoma assay

All studies gave negative results.

in vitro gene mutation study in bacteria - Key study:

This report describes a study designed to assess the mutagenic potential of the test substance in a bacterial system.

In this in vitro assessment of the mutagenic potential of the test substance , histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to the test

substance diluted in purified water containing 0.15% agar. Purified water containing 0.15% agar was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). The first (range-finding) was a standard plate incorporation assay; the second involved a pre-incubation stage.

Concentrations of test substance up to 5000µg/plate were tested in the mutation tests. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log₁₀dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test.

No evidence of mutagenic activity was seen at any concentration of test substance in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions employed, the test substance showed no evidence of mutagenic activity in this bacterial system.

in vitro gene mutation study in bacteria - supporting studies:

In two supporting studies, testing Salmonella typhimurium strains TA98 and TA100 only, the test substances were judged to be non-mutagenic in the test systems.

Chromosome Aberration Study:

A study was performed to assess the ability of the test substance to induce chromosomal aberrations in human lymphocytes cultured in vitro.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

In order to assess the toxicity of ST463 to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis:

First test:

Without and with S9 mix - 3 hours treatment, 17 hours recovery: 312.5, 625 and 1250 µg/ml.

Second test:

Without S9 mix - 20 hours continuous treatment: 156.25, 312.5 and 625 µg/ml.

With S9 mix - 3 hours treatment, 17 hours recovery: 312.5, 625 and 1250 µg/ml.

In both the absence and presence of S9 mix, the test substance caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control , in either test.

A quantitative analysis for polyploidy was made in cultures treated with the negative control and highest dose level. No increases in the proportion of polyploid cells were seen in either test.

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It is concluded that the test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

in vitro gene mutation study in mammalian cells (Mouse Lymphoma Assay):

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. 

Methods:

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test:

Group	Concentration of test item (µg/mL) plated for viability and mutant frequency
4-hour without S9	0.04, 0.08, 0.16, 0.31, 0.63 ,1.25
4-hour with S9 (2%)
24-hour without S9

Results:

The maximum dose level used in the Mutagenicity Test was limited by the onset of test item precipitate. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

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:

Experimental start date: 16 January 2001. Experimental completion date: 6 February 2001.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 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:

Identity: ST 463
Chemical name: 2-Naphthalenecarboxamide, 3-hydroxy-4[[2-
methoxy-5-[(phenylamino)carbonyl] phenyl]
azo]-
Appearance: Magenta powder
Storage conditions: Room temperature
Lot number: 6611
Expiry date: 1 August 2001
Purity: 99.9%
Date received: 30 November 2000

Target gene:

histidine locus

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, rat livers stimulated by Aroclor 1254, was used as the metabolic activation system

Test concentrations with justification for top dose:

First test (range finding): 5, 15, 50, 150, 500, 1500, 5000 µg/plate. (This is the standard limit concentration recommended in the regulatory guidelines this assay follows).
Second test: 50, 150, 500, 1500, 5000 µg/plate.

No visible thinning of the background lawn of non-revertant cells was obtained following exposure to ST 463 in the range-finding test. A maximum exposure concentration of 5000 pg/plate was, therefore, selected for use in the second test.

Vehicle / solvent:

The test substance was reported to be insoluble in solvents compatible with the test system.
Suspensions of the test substance were, therefore, prepared in purified water (obtained by reverse osmosis) containing 0.15% agar.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strains TA1535 and TA100

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

30 µg/plate for strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for strain TA98

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.05 µg/plate for strain WP2uvrA/pKM101 (CM891)

Positive control substance:

other: 2-2(-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for strain WP2uvrA/pKM101 (CM891). 2 µg/plate for strain TA1535.

Positive control substance:

other: 2-Aminioanthracene

Remarks:

with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate for strains TA1537, TA98 and TA100

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

MUTATION TEST PROCEDURE:
First test (range-finding):
The test substance was added to cultures of the five tester strains at seven concentrations separated by ca half-log10 intervals. The highest concentration of ST 463 tested was 50 mg/ml in the chosen solvent, which provided a final concentration of 5000 µg/plate. This is the standard limit concentration recommended in the regulatory guidelines this assay follows. The negative control was the chosen solvent, purified water containing 0.15% agar. The appropriate positive controls were also included.

An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S9 mix or 0.5 ml 0.1 Msodium phosphate buffer (pH 7.4) were placed in glass tubes. An aliquot of 0.1 ml of the test dilution, positive or negative control was added, followed immediately by 2 ml of molten agar containing 0.5mM histidine/biotin/tryptophan. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Each petri dish was individually labelled with a unique code corresponding to a sheet, identifying the dish’s contents. Three petri dishes were used for each concentration. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at 37°C for ca 72 hours. After this period the appearance of the background bacterial lawn was examined and revertant colonies counted using a Domino automated colony counter.

Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration normally used in the second test would be the same as that used in the first. If toxic effects were observed a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at the top concentration. Ideally a minimum of three non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating dose levels should be obtained, unless otherwise justified by the Study Director.

Second test:
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. 5000 µg/plate was again chosen as the top concentration, but only five concentrations were used.

Evaluation criteria:

Refer to any other information on materials and methods 'assessment of results'.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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:

The results obtained with ST 463 and positive control compounds are presented in Tables 1-10 (attached background material).

The absence of colonies on sterility check plates confirmed the absence of microbial contamination.

The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.

The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory (except TA98 and TA100, test 2, and TA1537, where slightly higher counts were obtained; this was not considered to affect the integrity of the study). Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

FIRST TEST (RANGE-FINDING):
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to ST 463 at any concentration in either the presence or absence of S9 mix.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to ST 463. A maximum exposure concentration of 5000 pg/plate was, therefore, selected for use in the second test.

SECOND TEST
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to ST 463 at any concentration in either the presence or absence of S9 mix.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to ST 463

Conclusions:

It is concluded that, under the test conditions employed, ST 463 showed no evidence of mutagenic activity in this bacterial system.

Executive summary:

This report describes a study designed to assess the mutagenic potential of ST 463 in a bacterial system.

In this in vitro assessment of the mutagenic potential of ST 463, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to the test

substance diluted in purified water containing 0.15% agar. Purified water containing 0.15% agar was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). The first (range-finding) was a standard plate incorporation assay; the second involved a pre-incubation stage.

Concentrations of ST 463 up to 5000 µg/plate were tested in the mutation tests. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log₁₀ dilutions of the highest concentration. No signs of

toxicity were observed towards the tester strains in either mutation test.

No evidence of mutagenic activity was seen at any concentration of ST 463 in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions employed, ST 463 showed no evidence of mutagenic activity in this bacterial system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start date: 31 March 2000. Experimental completion date: 19 April 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test substance: ST383
Appearance at room temperature: Solid
Storage conditions: At room temperature in the dark
Stability: Stable under storage conditions

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital(PB) and 5,6-Benzoflavon induced rat livers were used as the metabolic activation system

Test concentrations with justification for top dose:

First Experiment:
Without and with S9 mix: 19.53, 78.13, 312.5, 1250, 5000 µg/plate.
The test was carried out at 5000 µg/plate, the highest dose, and 4 lower concentrations diluted with a geometric progression of 4 in order to determine the highest dose in the following experiment.

Second Experiment:
Without S9 mix (TA 100 only): 0.02, 0.08, 0.31, 1.22, 4.88, 19.53, 78.13, 312.5 µg/plate.
Because the growth inhibition was observed in the first experiment in TA100 without S9 Mix, the test was carried out at 312.5 µg/plate

Third Experiment
Without S9 mix (TA 100): 0.61, 1.22, 2.44, 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5 µg/plate.
Without S9 mix (TA 98): 312.5, 625, 1250, 2550, 5000 µg/plate.
With S9 mix: 312.5 (TA 98 only), 625, 1250, 2500, 5000, 10000 µg/plate.
Based on the result of the first and second experiments, a slight increase at 5000 µg/plate in TA100 with S9 Mix and the growth inhibition at 78.13 µg/plate and higher dose in TA100 without S9 Mix, 5000 /ig per plate, 10,000 Ug per plate and 312.5 µg/plate were selected as the highest dose in TA98 with and without S9, Mix, TA100 with S9 Mix and TA100 without S9.

Vehicle / solvent:

Solvent used for the mutagenicity test was dimethylsulfoxide (DMSO).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.01 µg/plate for TA100. 0.1 µg/plate TA98

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

1µg/plate for TA100. 5 µg/plate for TA98

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Preparation of test substance solution
Just before experiment, test substance was ultrasonicated with DMSO for ten minutes. After ultrasonication, the suspension and its sequent dilutions with DMSO were used as test solutions.

Test method
Test solution (0.1 ml) was mixed with 0.1 M sodium phosphate buffer (pH 7.4, 0.5 ml) and the overnight culture of tester strain (0.1 ml) and then the mixture was pre-incubated at 37 °C
for 20 minutes with gentle shaking. For metabolic activation, S9 Mix (0.5 ml) was mixed instead of 0.1 M sodium phosphate buffer. After pre-incubation, melted soft agar (2 ml) was added
and the resulting mixture was poured onto minimal glucose agar plate. The soft agar contained 0.5 mM D-biotin and 0.5 mM L-histidine.
After incubation for 2 days, revertant colonies were counted and average number of colonies in each dose and control were obtained.

Observations
Inhibition of bacterial growth was observed using a microscope after incubation for 2 days.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The first experiment
Though a slight increase in the number of revertant colonies was found in TA100 with S9 Mix at 5000 fig per plate, the twofold increase beyond spontaneous reversion was not
shown in all test strains with and without S9 Mix. The growth inhibition was observed in TA100 without S9 Mix at every concentration tested.

The second experiment
Because the strong growth inhibition was observed in the first experiment in TA100 without S9 mix, the second experiment was carried out at a lower concentration. An increase in the number
of revertant colonies was not found.

The third experiment
An increase in the number of revertant colonies was not found in all test strains with and without S9 mix. Growth inhibition was observed in TA100 without S9 mix.

Conclusions:

Mutagenicity of ST383 was judged negative because the number of revertant colonies for all strains was less than twice that of
the negative control with and without S9 Mix. It was confirmed that the test was appropriately performed, since, the positive
control substances induced significant increases in the number of revertant colonies.
Based on the results of this study, it is concluded that "ST383" was non-mutagenic in this test system.

Executive summary:

"ST383" was tested for mutagenicity using Salmonella typhimurium (TA98 and TA100) as indicator strains. The liver microsome fraction of rats or S9 was used for metabolic activation system. The test was carried out according to "Standards for Mutagenicity Test using Microorganisms" of Japanese Occupational Safety and Health Law.

The test substance was judged non-mutagenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start date: 20 september 2000. Experimental completion date: 26 October 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test substance: ST463
Appearance at room temperature: Solid
Storage conditions: At room temperature in the dark
Stability: Stable under storage conditions

Target gene:

histidine locust

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and 5,6-Benzoflavon induced rat liver S9 were used as the metabolic activation system

Test concentrations with justification for top dose:

first experiment:
Without S9 mix: 0.001 (TA98 only), 0.003, 0.01, 0.06, 0.32, 1.6, 8, 40, 200, 1000, 5000 µg/plate.
With S9 mix: 1.6, 8, 40, 200, 1000, 5000 µg/plate.

Second Experiment
Without S9 mix: 4.9 (TA100 only), 9.8, 19.5, 39.0, 78, 156.3, 312.5, 625, 1250, 2500 µg/plate.
With S9 mix: 39, 78, 156.25, 312.5, 625, 1250, 2500, 5000 µg/plate.


Third experiment (only carried out on TA98)
Without S9 mix: 9.8, 19.5, 39.0, 78, 156.3, 312.5, 625, 1250, 2500, 5000 µg/plate.
With S9 mix: 312.5, 625, 1250, 2500, 5000, 10000 µg/plate.

Vehicle / solvent:

Solvent used for the mutagenicity test was dimethylsulfoxide.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.01 µg/plate for TA100. 0.1 µg/plate for TA98.

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for TA100. 0.5 µg/plate for TA98

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Preparation of test substance solution:
Just before experiment, test substance was ultrasonicated
with DMSO for ten minutes. After ultrasonication, the suspension and its sequent dilutions with DMSO were used as test solutions.

Test method:
Test solution (0.1 ml) was mixed with 0.1 M sodium phosphate buffer (pH 7.4, 0.5 ml) and the overnight culture of tester strain (0.1 ml) and then the mixture was pre-incubated at 37'C
for 20 minutes with gentle shaking. For metabolic activation S9 Mix (0.5 ml) was mixed instead of 0.1 M sodium phosphate buffer. After pre-incubation, melted soft agar (2 ml) was added
and the resulting mixture was poured onto minimal glucose agar plate. The soft agar contained 0.5 mM D-biotin and 0.5 mM L- histidine.

After incubation for 2 days, revertant colonies were counted and average number of colonies in each dose and control were obtained.

Observations:
Inhibition of bacterial growth was observed using a microscope after incubation for 2 days.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The first experiment:
An increase in the number of revertant colonies was not found in all test strains with and without S9 Mix.
Growth inhibition was observed in both test strains.

The second experiment:
Though a slight increase in the number of revertant colonies was found in TA98 with S9 Mix at 5000 µg/plate the two fold increase beyond spontaneous reversion was not shown in all test strains with and without S9 Mix. The growth inhibition was not observed in TA98 without S9 Mix at the highest concentration 2500 µg/plate..

The third experiment:
An increase in the number of revertant colonies was not found in TA98 with and without S9 Mix.

It was confirmed that the test was appropriately performed, since the positive control substances induced significant increases in the number of

revertant colonies.

Conclusions:

Mutagenicity of ST463 was judged negative because the number of revertant, colonies for all strains was less than twice that of the negative control with and without S9 Mix.
Based on the results of this study, it is concluded that "ST463" was non-mutagenic in this test system.

Executive summary:

"ST463” was tested for mutagenicity using Salmonella typhimurium (TA98 and TA100) as indicator strains,

The liver microsome fraction of rats, or S9 was used for metabolic activation system. The test was carried out according to "Standards for Mutagenicity Test using Microorganisms" of Japanese Occupational Safety and Health Law.

The test substance was judged non-mutagenic in this test system.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 21 August 2000. Experimental completion date: 13 October 2000.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (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

Specific details on test material used for the study:

Identity: ST463
Chemical Name: 4-[[5-(Anilino) carbonyl-2-methoxyphenyl] azo]-
3-hydroxynaphthalene-2-carboxamide
Appearance: Magenta powder
Storage conditions: Room temperature
Batch number: S/No, 6553
Expiry: 14 January 2000
Purity: > 98 %
Date received: 14 July 2000

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human preripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Human blood was collected aseptically from healthy male, non smoking donors, pooled and diluted with RPMI 1640 tissue culture medium (Sigma) supplemented with 10% foetal calf serum (Globepharm ), 1 unit/ml Heparin (CP pharmaceuticals), 20 LU./ml penicillin/20 µg/ml streptomycin ( Imperial ) and 2.0 mM glutamine (Imperial ). Aliquots (0.4 ml blood : 4.5 ml medium : 0.1 ml phytohaemagglutinin (Gibco)) of the cell suspension were placed in sterile universal containers and incubated at 37°C for approximately 48 hours. The cultures were gently shaken daily to resuspend the cells.

Cytokinesis block (if used):

Two hours before the cells were harvested , mitotic activity was arrested by addition of Colcemid

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

First test: 39.06, 78.13 , 156.25, 312.5 , 625, 1250, 2500 and 5000 µg/ml.
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al 1987 ). Therefore, concentrations greater than 5000 /xg/ml -or 10 mM are not used in this test system. In this case, the highest final concentration used for subsequent testing was 5000 µg/ml.

Second test: 39.06, 78.13, 156.25, 312.5, 625 and 1250 µg/ml.

Vehicle / solvent:

Prior to commencing testing, the solubility of the test substance in solvents compatible with the test system was assessed. Information supplied by the Sponsor stated that ST463 was insoluble in water,
and was also found to be insoluble in dimethyl sulphoxide, ethanol and acetone. However, ST463 was found to form a doseable suspension in culture medium at 10 mg/ml after being ultra-turraxed in culture
medium until complete dissociation of main particles. A red coloured medium was observed in all cultures treated with ST463 throughout the study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Sterile purified water

True negative controls:

no

Positive controls:

yes

Remarks:

0.1 µg/ml

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Sterile purified water

True negative controls:

no

Positive controls:

yes

Remarks:

6 µg/ml

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

TREATMENT OF CELLS WITH TEST SUBSTANCE - FIRST TEST:
After approximately 48 hours , 2.5 ml aliquots of ST463 were added to one set of duplicate cultures to give final concentrations of 39.06, 78.13 , 156.25, 312.5 , 625, 1250, 2500 and 5000 µg/ml. Culture
medium, the solvent control, in 2.5 ml aliquots , was added to two cultures. Mitomycin C, at a final concentration of 0.1 µg/ml, was added to duplicate cultures.

Immediately before treatment of the second set of cultures, 1 ml of medium was removed from each culture and discarded . This was replaced with 1 ml of S9 mix , followed by 2.5 ml aliquots of the
various dilutions of ST463, giving the same series of final concentrations as above. Culture medium (2.5 ml) was added to two cultures. Cyclophosphamide was added to duplicate cultures at a final
concentration of 6 µg/ml .

Three hours after dosing , the cultures were centrifuged at 1700 rpm (500 g) for 5 minutes. The cell pellets were rinsed and resuspended in fresh medium. They were then incubated for a further 17 hours.

HARVESTING AND FIXATION
Two hours before the cells were harvested , mitotic activity was arrested by addition of Colcemid ® (Sigma) to each culture at a final concentration of 0.1 pg/ml. After 2 hours incubation , each cell
suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 1700 r.p.m. (500 g ). The cell pellets were treated with a hypotonic solution (0.075M KC1 prewarmed at 37°C). After a 10 minutes period of hypotonic incubation at 37°C, the suspensions were centrifuged at 1700 r.p.m. (500 g ) for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative ( 3 parts methanol : 1 part glacial acetic acid ). The fixative was replaced three times.

SLIDE PREPARATION
The pellets were resuspended, then centrifuged at 1700 r.p.m. (500 g) for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto
pre-cleaned microscope slides which were then allowed to air-dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to airdry and then mounted in DPX. pellets were rinsed and resuspended in fresh medium. They were then incubated for a further 17 hours.

MICROSCOPIC EXAMINATION
The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures.
From these results the dose level causing a decrease in mitotic index of approximately 50% of the solvent control value or, if there was no decrease, the maximum achievable concentration was used as
the highest dose level for the metaphase analysis. The intermediate and low dose levels were also selected.

The concentration of each positive control compound selected for analysis was the lowest concentration dosed unless a preliminary scan of metaphase figures indicated an insufficient level of aberrant cells. The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x 1000 using an oil immersion objective. One hundred metaphase figures were examined, where possible, from each culture. Chromosome aberrations were scored according to the classification of the ISCN (1985). Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. The vernier readings of all aberrant metaphase figures were recorded.

The incidence of polyploid metaphase cells, out of 500 metaphase cells , was determined quantitatively for negative control cultures and cultures treated with the highest dose level of the test substance used in the analysis for chromosomal aberrations.
The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973).

SECOND TEST:
Cultures were initiated and maintained as previously described. In this second test a continous treatment was used in the absence of S9 mix. In the presence of S9 mix, a three hour treatment was
used, as in the first test. The harvest time was at 20 hours for both parts of the test. Concentrations of ST463 were as follows:

Without and with S 9 mix: 39.06, 78.13, 156.25, 312.5, 625 and 1250 µg/ml.

Duplicate cultures were used for each treatment and two cultures were treated with the solvent control.
Positive control cultures were treated as in the first test.

Three hours after dosing, the cultures containing S9 mix were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 17 hours. Cultures treated in the
absence of S9 mix were incubated for 20 hours.

All cultures were treated with Colcemid, at a final concentration of 0.1 µg/ml, two hours before the end of the incubation period. They were then harvested, fixed and the slides prepared as previously
described. The slides were then examined microscopically as previously described.

Evaluation criteria:

An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.

The test substance is considered to cause a positive response if the following conditions are met:
- Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
- The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.
- The increases are reproducible between replicate cultures.
- The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity.
- Evidence of a dose-relationship is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level.

A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

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:

FIRST TEST
Toxicity data:
In both the absence and the presence of S9 mix, ST463 caused no significant reduction in the mitotic index at the six first dose levels (from 39.06 to 1250 µg/ml) when compared to the solvent control value.
Due to high levels of red precipitates observed in treated cultures at 2500 and 5000 µg/ml, mitotic indices could not be scored. Therefore, the dose levels selected for the metaphase analysis were 312.5,
625 and 1250 µg/ml.
The quantitative analysis for polyploidy showed no increases in the number of polyploid metaphase figures when compared to the solvent control.

Metaphase analysis:
In both the absence and the presence of S9 mix, ST463 caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent
control.
Both positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the
S9 mix and the sensitivity of the test system.

SECOND TEST
Toxicity data:
In the absence of S9 mix, ST463 caused no significant reduction in the mitotic index at the five first dose levels (from 39.06 to 625 µg/ml) when compared to the solvent control value. Due to high levels of
red precipitates observed in treated cultures at 1250 µg/ml, mitotic indices could not be scored. Therefore, the dose levels selected for the metaphase analysis were 156.25, 312.5 and 625 µg/ml.

In the presence of S9 mix, ST463 caused no significant reduction in the mitotic index at any dose level when compared to the solvent control value. Therefore, the dose levels selected for the metaphase
analysis were 312.5, 625 and 1250 µg/ml.

The quantitative analysis for polyploidy showed no increases in the number of polyploid metaphase cells when compared to the solvent control.

Metaphase analysis:
In both the absence and the presence of S9 mix, ST463 caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent
control.

Both positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P<0.001 ) in the proportion of aberrant cells.

Conclusions:

It is concluded that ST463 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Executive summary:

A study was performed to assess the ability of ST463 to induce chromosomal aberrations in human lymphocytes cultured in vitro.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

In order to assess the toxicity of ST463 to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis:

First test

Without and with S9 mix - 3 hours treatment, 17 hours recovery: 312.5, 625 and 1250 µg/ml.

Second test

Without S9 mix - 20 hours continuous treatment: 156.25, 312.5 and 625 µg/ml.

With S9 mix - 3 hours treatment, 17 hours recovery: 312.5, 625 and 1250 µg/ml.

In both the absence and presence of S9 mix, ST463 caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control , in either test.

A quantitative analysis for polyploidy was made in cultures treated with the negative control and highest dose level. No increases in the proportion of polyploid cells were seen in either test.

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It is concluded that ST463 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 March 2017 and 18 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene

Specific details on test material used for the study:

Identification: ST1031KH (MB002 Pigment#1)
Physical state/Appearance: Red powder
Batch: 20160212
Purity: 100%
Expiry Date: 01 February 2018
Storage Conditions: Room temperature, in the dark

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell line:
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell culture:
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Mutagenicity Test: 0.02, 0.04, 0.08, 0.16, 0.31, 0.63 ,1.25, 2.5 µg/mL

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.

Vehicle / solvent:

DMSO

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:

CELL CLEANSING:
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

TEST ITEM PREPARATION:
Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The molecular weight of the test item was 440.45 therefore the maximum proposed dose level in the solubility test was set at 2000 µg/mL initially, the maximum recommended dose level, and no correction for the purity of the test item was applied. The test item formed a suspension unsuitable for dosing at 200 mg/mL. Therefore, the test item was formulated at 50 mg/mL and dosed at 1% to give a maximum achievable dose level of 500 µg/mL. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991).
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system.

TEST PROCEDURE:
PRELIMINARY TOXICITY TEST:
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. Due to the very high levels of test item precipitate observed in the solubility test, the dose range used in the preliminary toxicity test was 0.16 to 40 µg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.

The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

MUTAGENICITY TEST:
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation.

The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (0.02 to 2.5 µg/mL for all three of the exposure groups), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).

The exposure vessels were incubated at 37°C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.


ASSESSMENTS:
MEASUREMENTS OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY:
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.

On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data, a Relative Total Growth (RTG) value.

PLATE SCORING:
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37°C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.


Calculation of Percentage Relative Suspension Growth (%RSG):
The cell counts obtained immediately post exposure and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.

4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

Calculation of Day 2 Viability (%V):
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.

P(0) = number of negative wells / total wells plated
%V = -ln P(0) x 100 / number of cells/well

Calculation of Relative Total Growth (RTG):
For each culture, the relative cloning efficiency, RCE, was calculated:

RCE = %V / Mean Solvent Control %V

Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100

Calculation of Mutation Frequency (MF):
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

The experimental data was analysed using a dedicated computer program, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear trend events.

Evaluation criteria:

Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Statistics:

See assessments section.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY TEST:
The dose range of the test item used in the preliminary toxicity test was 0.16 to 40 µg/mL.
There was no evidence of any marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in any of the three exposure groups when compared to the concurrent vehicle control groups. Precipitate of the test item was observed at and above 1.25 µg/mL in all three of the exposure groups at the end of the exposure periods. Therefore, based on the precipitate observations, the maximum dose level in the subsequent Mutagenicity Test was limited by the onset of precipitate.

MUTAGENICTY TEST:
A summary of the results from the test is presented in 'any other information on results incl. tables'.

As was seen previously, there was no evidence of any marked dose related toxicity following exposure to the test item in any of the three exposure groups, as indicated by the %RSG and RTG values. There was also no evidence of any significant reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with the positive control substances.

Precipitate of the test item was observed at 1.25 and 2.5 at the end of the exposure periods. Therefore, as the onset of precipitate was observed at 1.25 µg/mL, following the recommendations of the OECD 490 guideline, it was considered unnecessary to plate the 2.5 µg/mL dose level for viability or 5-TFT resistance.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

The test item did not induce any toxicologically significant or dose related increases in the mutant frequency x 10-6 per viable cell at any of the dose levels including the dose levels at the onset of precipitate.

Preliminary Cytotoxicity Test:

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
0.16	95	118	99
0.31	85	Culture lost	96
0.63	86	123	95
1.25	89	110	89
2.5	99	109	91
5	107	118	81
10	107	111	97
20	87	115	90
40	100	108	81

Mutagenicity Test:

Table: Summary of Results

Main Experiment

Concentration (µg/mL)			4-Hours-S9					Concentration (µg/mL)			4-Hours+S9
			%RSG	RTG	MF§						%RSG	RTG	MF§
0			100	1.00	118.82			0			100	1.00	174.72
0.02		Ø	93					0.02		Ø	101
0.04			90	1.00	134.86			0.04			89	0.93	150.35
0.08			95	0.96	119.73			0.08			102	0.93	202.97
0.16			88	0.89	134.07			0.16			101	1.00	177.23
0.31			83	0.76	135.24			0.31			94	0.87	175.39
0.63			95	1.01	118.40			0.63			93	0.88	142.19
1.25			91	0.86	158.82			1.25			105	0.85	196.57
2.5		Ø	83					2.5		Ø	88
MF threshold for a positive response = 244.82								MF threshold for a positive response = 300.72
Positive control								Positive control
EMS								CP
400			69	0.50	1341.85			1.5			75	0.45	1081.10

               

Concentration (µg/mL)		24-Hours-S9
		%RSG	RTG	MF§
0		100	1.00	146.17
0.02	Ø	92
0.04		99	0.99	117.32
0.08		84	0.77	171.83
0.16		86	0.93	123.05
0.31		75	0.79	148.44
0.63		80	0.82	156.37
1.25		84	0.86	143.30
2.5	Ø	85
MF threshold for a positive response = 272.17
Positive control
EMS
150		46	0.45	1193.20

 

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

Introduction:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

Methods:

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test:

Group	Concentration of test item (µg/mL) plated for viability and mutant frequency
4-hour without S9	0.04, 0.08, 0.16, 0.31, 0.63 ,1.25
4-hour with S9 (2%)
24-hour without S9

Results:

The maximum dose level used in the Mutagenicity Test was limited by the onset of test item precipitate. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on negative results in in-vitro studies conducted on the substance, the substance does not need to be classified for germ cell mutagenicity.