Trisodium 5-[[5-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-sulphonatophenyl]azo]-1-ethyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-3-methanesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the Ames tests performed with the source substance according to OECD TG 471, the source substance did not induce mutations with or without metabolic activation.

Under the conditions of the in vitro mammalian chromosome aberration test performed with the source substance according to OECD TG 473, the source substance did not induce chromosome aberrations in the S9-activated test systems using V79 cells. However, in the absence of S9 mix the test article increased the structural aberration rate after treatment with 400 µg/mL at fixation interval 28 hours.

Under the conditions of the in vitro mammalian cell gene mutation test (HPRT) performed with the source substance according to OECD TG 476, the source substance did not induce mutagenic effects in the non-activated and S9-activated test systems using V79 cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and the target substance have very similar physicochemical and (eco)toxicological properties because their chemical structures are nearly identical. An analogue approach has thus been employed. The target substance is the meta-isomer of the dye Reactive Yellow 095, where the sulphonate group is bound at the meta-position of the amino benzene moiety. The source chemical is the reaction mass of both the meta-isomer and the para-isomer of Reactive Yellow 095.
The presence of sulphonate groups make both dyes highly water soluble and therefore less critical for human health and environmental issues. Based on their chemical similarity, similar properties are expected in both humans and the environment.
2. SOURCE AND TARGET CHEMICAL(S)
Source: Reactive Yellow 095 meta/para (CAS# --- / EC# 944-218-2)
Target: Reactive Yellow 095 meta (CAS# 84045-63-6 / EC# 281-865-3)
3. ANALOGUE APPROACH JUSTIFICATION
see attachment under 4.12 Auto flammability

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The substance was not mutagenic in the bacterial reverse mutation assay.

Executive summary:

A key study was performed to investigate the potential of the source substance to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations : 10.0, 33.3, 100.0, 333.3, 1000.0; and 5000.0 ug/plate A slight toxic effect, evidenced by a reduction in the number of revertants, occurred only in strain TA 98 at 5000.0 ug/plate in the presence of S9 mix in experiment I. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the source substance at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the source substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

The structurally related target substance will show the same behaviour and is therefore also considered non-mutagenic in the Ames test.

Reference 2

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:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and the target substance have very similar physicochemical and (eco)toxicological properties because their chemical structures are nearly identical. An analogue approach has thus been employed. The target substance is the meta-isomer of the dye Reactive Yellow 095, where the sulphonate group is bound at the meta-position of the amino benzene moiety. The source chemical for most endpoints is the reaction mass of both the meta-isomer and the para-isomer of Reactive Yellow 095. The presence of sulphonate groups make both dyes highly water soluble and therefore less critical for human health and environmental issues. Based on their chemical similarity, similar properties are expected in both humans and the environment.
For this endpoint, the source chemical is Reactive Yellow 175 instead. This source chemical is used as it is also used by the registrants of the reaction mass of the meta-isomer and para-isomer of Reactive Yellow 095 to cover this endpoint.
2. SOURCE AND TARGET CHEMICAL(S)
Source: Reactive Yellow 175 (CAS# 111850-27-2 / EC# 402-740-3)
Target: Reactive Yellow 095 meta (CAS# 84045-63-6 / EC# 281-865-3)
3. ANALOGUE APPROACH JUSTIFICATION
see attachment under 4.12 Auto flammability

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON GENOTOXICITY:
In the presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.00 % - 3.25 %) are in or very near to the range of the control values: 0.50 % - 2.50 %. In the absence of S9 mix the test article increased the structural aberration rate after treatment with 400 µg/mL at fixation interval 28 h. Additionally, 3.75 % of the cells were carrying exchanges as compared to 0.00 % in the corresponding solvent control.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix with concentration of 400 µg/mL the colony forming ability was clearly reduced as compared to the solvent controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main experiment the mitotic index was reduced after treatment with the highest dose level in the absence and presence of S9 mix, indicating that FAT 40'277/B had cytotoxic properties.

Conclusions:

The test substance is considered to be clastogenic in this chromosomal aberration test.

Executive summary:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, chinese hamster lung fibroblast (V79) were exposed to the source substance with and without metabolic activation. Different preparation intervals (up to 28 hours) and dosages (up to 400 µg/mL) were chosen in this study. The mitotic index was reduced after treatment with the highest concentrations in the presence and absence of S9 mix indicating that the test substance had cytotoxic properties. In the presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval, but in the absence of S9 mix the test article increased the structural aberration rate after treatment with 400 µg/mL at fixation interval 28 hours. Based on the results the test substance is considered to be clastogenic in this chromosomal aberration test.

It is anticipated that the structurally related target substance will show similar behaviour.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and the target substance have very similar physicochemical and (eco)toxicological properties because their chemical structures are nearly identical. An analogue approach has thus been employed. The target substance is the meta-isomer of the dye Reactive Yellow 095, where the sulphonate group is bound at the meta-position of the amino benzene moiety. The source chemical for most endpoints is the reaction mass of both the meta-isomer and the para-isomer of Reactive Yellow 095. The presence of sulphonate groups make both dyes highly water soluble and therefore less critical for human health and environmental issues. Based on their chemical similarity, similar properties are expected in both humans and the environment.
For this endpoint, the source chemical is Reactive Yellow 175 instead. This source chemical is used as it is also used by the registrants of the reaction mass of the meta-isomer and para-isomer of Reactive Yellow 095 to cover this endpoint.
2. SOURCE AND TARGET CHEMICAL(S)
Source: Reactive Yellow 175 (CAS# 111850-27-2 / EC# 402-740-3)
Target: Reactive Yellow 095 meta (CAS# 84045-63-6 / EC# 281-865-3)
3. ANALOGUE APPROACH JUSTIFICATION
see attachment under 4.12 Auto flammability

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥ 400 μg/mL; experiment I with S9: ≥ 50 μg/mL; Experiment II without S9: ≥ 500 μg/mL; Experiment II with S9:≥ 40 μg/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster in an in vitro cell gene mutagenicity test.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to the source substance at concentrations of

- 10, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200 µg/mL (without metabolic activation, Experiment I)

- 1.0, 2.5, 5, 10, 25, 50, 100 and 125 µg/mL (with metabolic activation, Experiment I)

- 5, 10, 25, 50, 100, 250, 500, 1000 and 2000 µg/mL (without metabolic activation, Experiment II)

- 1.00, 3.16, 10, 20, 40, 60, 80, 100 and 120 µg/mL (with metabolic activation, Experiment II).

The source substance was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 14.0% for 1200 µg/mL evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 125 µg/mL with a relative growth of 14.3%. In experiment II without metabolic activation the relative growth was 13.9% for the highest concentration (2000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 120 µg/mL with a relative growth of 10.6%.

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.13 was found at a concentration of 1200 µg/mL with a relative growth of 14.0%.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 3.07 was found at a concentration of 100 µg/mL with a relative growth of 20.7%.
In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.14 was found at a concentration of 10 µg/mL with a relative growth of 109.2%.
In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.74 was found at a concentration of 10 µg/mL with a relative growth of 79.1%.

The observed elevated number of mutant colonies and the mutation factor of 3.07 in experiment I (with metabolic activation) at a concentration of 100 µg/mL could not be reproduced in experiment II (with metabolic activation). At the same concentration (100 µg/mL) only 32.43 mutants per 10⁶cells were observed, resulting in a mutation factor of 1.34. Due to this finding, the absence of a dose-response relationship as well as due to the overall integrity of the data the observed increased mutant values resulting in an elevated mutation factor in experiment I (with metabolic activation) is considered to be not biologically relevant.

There was no evidence of a concentration related positive response of induced mutant colonies over background.

The structurally related target substance will show similar behaviour.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

None

Qualifier:

according to guideline

Guideline:

other: Ames et al, 1973

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no tester strain to detect cross-linking mutagens has been included

Principles of method if other than guideline:

None

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

None

Target gene:

None

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

25, 7 5, 225, 675, and 2025 µg / 0 .1 ml

Vehicle / solvent:

phosphate buffer

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation - for TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation - for TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunoblastin

Remarks:

Without metabolic activation - for TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation - for TA 100 strain

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic acyivation - for TA 1535 strain

Details on test system and experimental conditions:

None

Rationale for test conditions:

None

Evaluation criteria:

When the colonies had been counted, the arithmetic mean was calculated. The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration.

Statistics:

None

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, 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:

not examined

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

None

Conclusions:

FAT 40000/B was determined to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

A supporting study was performed wherein preparation FAT 40000/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. Since considerable fluctuations in the number of back-mutants produced were observed in the experiments with Strain TA 100, the experiments were repeated, in accordance with the usual practice in the laboratory. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiments performed with and without microsomal activation, comparison of the numbers of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 40000/B revealed no marked deviations. No evidence of the induction of point mutations by FAT 40000/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

Treatment was performed with the following concentrations:
- 7 hours: 100, 200, 300, 400 µg/mL
- 18 hours: 10, 50, 100, 200, 300, 400 µg/mL
- 28 hours: 100, 100, 300, 400 µg/mL

The following dose levels were evaluated:
- 7 hours: 400 µg/mL
- 18 hours: 10, 200, 400 µg/mL
- 28 hours: 400 µg/mL

Vehicle / solvent:

culture medium without fetal calf serum

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation: Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension was prepared. The cells were seeded into Quadriperm dishes ( Heraeus, D-6450 Hanau, F.R.G. ) which contained microscopic slides (4 chambers per dish and test group). In each chamber 5E4 - 1E5 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS.
- Exposure: After 48 h (7 h, 28 h preparation interval) and 55 h (18 h preparation interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 µL/ml S9 mix. After 4h this medium was replaced with normal medium after rinsing twice. All incubations were done at 37°C in a humidified atmosphere with 5 % CO2.
- Fixation: 5, 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.0 h (7 h interval) or 2.5 h later, (18 h and 28 h interval). the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37°C. After incubation in the hypotonic solution the cells are fixed with 3:1 absolute methanol:glacial acetic acid. All four slides per group were prepared. After fixation the cells were stained with aceto-orcein.

NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED:
- At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 22 +/- 1 were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

The test article will be classified as mutagenic if it induces a significantly increased aberration rate with at least one of the concentrations tested as compared with the negative control. A test article which produce no significant positive response at any test point will be regarded as non-mutagenic in this assay.

Statistics:

nonparametric Mann-Whitney test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON GENOTOXICITY:
In the presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.00 % - 3.25 %) are in or very near to the range of the control values: 0.50 % - 2.50 %. In the absence of S9 mix the test article increased the structural aberration rate after treatment with 400 µg/mL at fixation interval 28 h. Additionally, 3.75 % of the cells were carrying exchanges as compared to 0.00 % in the corresponding solvent control.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix with concentration of 400 µg/mL the colony forming ability was clearly reduced as compared to the solvent controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main experiment the mitotic index was reduced after treatment with the highest dose level in the absence and presence of S9 mix, indicating that FAT 40'277/B had cytotoxic properties.

Remarks on result:

other: all strains/cell types tested

Conclusions:

The test substance is considered to be clastogenic in this chromosomal aberration test.

Executive summary:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, chinese hamster lung fibroblast (V79) were exposed to the test substance with and without metabolic activation. Different preparation intervals (up to 28 hours) and dosage (up to 400 µg/mL) were chosen in this study. The mitotic index was reduced after treatment with the highest concentrations in the presence and absence of S9 mix indicating that the test substance had cytotoxic properties. In the presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval, but in the absence of S9 mix the test article increased the structural aberration rate after treatment with 400 µg/mL at fixation interval 28 hours. Based on the results the test substance is considered to be clastogenic in this chromosomal aberration test.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no tester strain to detect cross-linking mutagens has been included

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

None

Target gene:

None

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

10.0, 33.3, 100.0, 333.3, 1000.0 and 5000.0 ug/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - for TA 1535, TA 100 strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Without metabolic activation - for TA 1537, TA 1538, TA 98 strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation - for TA 1535, TA 1537, TA 1538, TA 98, TA 100 strains

Details on test system and experimental conditions:

None

Rationale for test conditions:

None

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are: corresponding background growth on both negative control and test plates normal range of spontaneous reversion rates.

Statistics:

None

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, 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:

not examined

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

None

Conclusions:

FAT 40000/G was determined to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

A key study was performed to investigate the potential of FAT 40000/G to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations : 10.0, 33.3, 100.0, 333.3, 1000.0; and 5000.0 ug/plate A slight toxic effect, evidenced by a reduction in the number of revertants, occurred only in strain TA 98 at 5000.0 ug/plate in the presence of S9 mix in experiment I. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 40000/G at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 40000/G is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-12-12 to 2013-03-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix (pre-experiment) and Liver S9 of Sprague Dawley Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix (main experiments)

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation):
5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/mL
Experiment I
without metabolic activation: 10, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200 µg/mL
and with metabolic activation: 1.0, 2.5, 5, 10, 25, 50, 100 and 125 µg/mL
Experiment II
without metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000 and 2000 µg/mL
and with metabolic activation: 1.00, 3.16, 10, 20, 40, 60, 80, 100 and 120 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used:
For the pre-experiment the test item was dissolved in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment).
For the main experiments a stock solution of the test item in Aqua ad injectabilia were prepared (tenfold) and processed by sterile filtration. The dilution series was prepared in Aqua ad injectabilia. 10% of the dilution series and/or Aqua ad injectabilia were added to cell culture medium prior to treatment (resulting in the designated concentrations of the test item).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 1 and 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in Aqua ad inj. / medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥ 400 μg/mL; experiment I with S9: ≥ 50 μg/mL; Experiment II without S9: ≥ 500 μg/mL; Experiment II with S9:≥ 40 μg/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

In the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 40277/D TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus),V79 cells culturedin vitro were exposed to FAT 40277/D TE at concentrations of

- 10, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200 µg/mL (without metabolic activation, Experiment I)

- 1.0, 2.5, 5, 10, 25, 50, 100 and 125 µg/mL (with metabolic activation, Experiment I)

- 5, 10, 25, 50, 100, 250, 500, 1000 and 2000 µg/mL (without metabolic activation, Experiment II)

- 1.00, 3.16, 10, 20, 40, 60, 80, 100 and 120 µg/mL (with metabolic activation, Experiment II).

FAT 40277/D TE was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 14.0% for 1200 µg/mL evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 125 µg/mL with a relative growth of 14.3%. In experiment II without metabolic activation the relative growth was 13.9% for the highest concentration (2000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 120 µg/mL with a relative growth of 10.6%.

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.13 was found at a concentration of 1200 µg/mL with a relative growth of 14.0%.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 3.07 was found at a concentration of 100 µg/mL with a relative growth of 20.7%.
In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.14 was found at a concentration of 10 µg/mL with a relative growth of 109.2%.
In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.74 was found at a concentration of 10 µg/mL with a relative growth of 79.1%.

The observed elevated number of mutant colonies and the mutation factor of 3.07 in experiment I (with metabolic activation) at a concentration of 100 µg/mL could not be reproduced in experiment II (with metabolic activation). At the same concentration (100 µg/mL) only 32.43 mutants per 10⁶cells were observed, resulting in a mutation factor of 1.34. Due to this finding, the absence of a dose-response relationship as well as due to the overall integrity of the data the observed increased mutant values resulting in an elevated mutation factor in experiment I (with metabolic activation) is considered to be not biologically relevant.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 forin vitromutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The source substance did not induce micronuclei in the micronucleus assay, hence considered to be not clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The four in vitro studies performed with the source substances, according to OECD TG 471 (twice), OECD TG 473 and OECD TG 476, showed that the substance(s) did not induce mutations in bacteria and in mammalian cells, nor did it induce chromosomal aberrations in mammalian cells in the presence of metabolic activation. However, the source substance induced chromosomal aberrations in mammalian cells in the absence of metabolic activation, thus being clastogenic in vitro.

In an in vivo erythrocyte micronucleus assay, the source substance did not induce micronuclei, hence it can be considered not clastogenic.

The structurally related target substance will show similar behaviour and therefore it is anticipated that it will not be mutagenic and clastogenic either.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No.1272/2008).