2-[(2-methoxy-4-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pigment Yellow 74 also yielded negative results in an in vitro gene mutation study in mammalian cells (mouse lymphoma assay) in concentration up to 2000 µg/ml in the presence and absence of metabolic activation. This study was selected as key study.

Pigment Yellow 74 did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation under the experimental conditions used when test up to precipitating concentrations.

In conclusion, Pigment Yellow 74 is not mutagenic in the bacterial reverse mutation assay, in the mouse lymphoma assay and in the in vitro micronucleus assay in the presence and absence of metabolic activation up to the tested concentrations.

Mutagenic properties of Pigment Yellow 74 were investigated in several bacterial reverse mutation assays (Ames test; test strains used: S. typhimurium TA 97, TA 98, TA 100, TA 1535, TA 1537, TA 1538, E. coli WP2 uvr A), in an in vitro gene mutation study in mammalian cells (Mouse lymphoma assay) and in an in vitro micronucleus assay. Negative results were obtained in all tests with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 November 2006 to 08 December 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: performed in accordance with OECD and GLP guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetz and OECD Principles of Good Laboratory Practice

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment and Experiment I: 0 (control), 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment II: 0 (control), 33, 100, 333, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below for additional information

Details on test system and experimental conditions:

The assay was performed in two independent experiments:

experiment I: plate incorporation assay with and without induced rat liver S9 mix
experiment II: pre-incubation test with and without non-induced hamster liver S9 mix

Hamster liver S9 mix, but not rat liver S9 mix, contained the reductive agent FMN.


DURATION
- Preincubation period: 30 min, 30°C
- Exposure duration: after solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: existence of evaluable plates (> 0 colonies) at five concentrations or more

POSITIVE CONTROL SUBSTANCES:
without metabolic activation: sodium azide (TA 1535, TA 100), 4-nitro-o-phenylene-diamine ((TA 1537, TA 98), methyl methane sulfonate (WP2 uvrA);
with metabolic activation: 2-aminoanthracene (all strains with rat liver S9 mix and TA 1535, TA 100, TA 1537, WP2 uvrA with hamster liver S9 mix), cKongo red (TA 98 with hamster liver S9 mix)

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I, TA 1537, without metabolic activation: minor reduction in number of revertants at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Mean mutant number ratios treated/solvent control

Exp. I: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 3 -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 1.0 -- 0.8 -- 0.8 -- 1.1 -- 0.8 -- 0.7 -- 0.9 -- 0.7

TA1537 -- 1.5 -- 1.1 -- 0.9 -- 0.9 -- 0.9 -- 1.3 -- 0.9 -- 0.4

TA98 -- 0.9 -- 1.0 -- 1.0 -- 1.1 -- 0.8 -- 0.9 -- 0.9 -- 0.7

TA100 -- 0.8 -- 0.9 --1.0 -- 1.1 -- 0.9 -- 1.0 -- 0.9 -- 0.8

WP2uvrA -- 1.0 -- 1.0 -- 1.1 -- 1.2 -- 0.9 -- 0.9 -- 0.8 -- 0.8

Exp. I: plate incorporation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 3 -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 1.1 -- 1.1 -- 1.1 -- 1.0 -- 1.1 -- 1.2 -- 1.2 -- 0.7

TA1537 -- 1.1 -- 0.9 -- 1.1 -- 1.3 -- 1.1 -- 0.9 -- 0.9 -- 0.7

TA98 -- 1.1 -- 1.1 -- 1.1 -- 1.0 -- 0.9 -- 0.9 -- 1.0 -- 0.8

TA100 -- 1.0 -- 1.1 --1.0 -- 1.0 -- 1.0 -- 1.0 -- 0.9 -- 0.8

WP2uvrA -- 1.0 -- 1.0 -- 0.9 -- 0.9 -- 0.9 -- 0.8 -- 0.8 -- 0.8

Exp. II: pre-incubation method without S9 mix

Concentrations given in µg/plate

Strain -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 1.2 -- 0.9 -- 1.0 -- 0.9 -- 1.0 -- 0.9

TA1537 -- 1.2 -- 1.3 -- 1.4 -- 1.4 -- 1.1 -- 0.9

TA98 -- 1.0 -- 1.2 -- 0.9 -- 1.1 -- 0.8 -- 0.7

TA100 --1.2 -- 1.1 -- 1.1 -- 1.1 -- 0.9 -- 1.0

WP2uvrA -- 1.2 -- 0.9 -- 1.1 -- 0.9 -- 1.0 -- 0.9

Exp. II: pre-incubation method with hamster S9 mix

Concentrations given in µg/plate

Strain -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 0.9 -- 0.8 -- 0.7 -- 0.7 -- 0.7 -- 0.7

TA1537 -- 1.3 -- 1.0 -- 1.1 -- 0.8 -- 0.8 -- 0.8

TA98 -- 1.0 -- 1.0 -- 1.0 -- 1.0 -- 0.7 -- 0.7

TA100 --1.0 -- 0.8 -- 1.0 -- 0.9 -- 0.8 -- 0.7

WP2uvrA -- 1.2 -- 0.8 -- 0.7 -- 1.0 -- 0.9 -- 0.7

Conclusions:

Interpretation of results:
negative with and without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by
frameshifts or base-pair substitutions in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I) and the pre-incubation test with hamster liver S9 (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I : 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both experiments.

No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains. Only in experiment I in strain TA 1537 in the absence of metabolic activation a minor reduction in the number of revertants, were observed at 5000 µg/plate.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor 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 biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to Draft Proposal for a new Guideline No. 487

Qualifier:

according to guideline

Principles of method if other than guideline:

OECD Guideline Draft Proposal for a new Guideline No. 487, Version 3

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (Greiner,
72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium; Invitrogen GIBCO, 76131 Karlsruhe, Germany) supplemented with 10 % fetal calf serum (FCS; Invitrogen, 76131 Karrlsruhe, Germany). Additionally, the medium was supplemented with 1 % 100x Penicillin/ Streptomycin solution (10.000 Units/mL Penicillin, 10 mg/mL Streptomycin; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % Amphotericin B (250 µg/mL, PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide (95.5% air).

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Exp. I: with and without S9 mix: 4.7, 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, 600.0, and 1200.0 µg/mL
Exp. II: without S9 mix: 2.4, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, and 600.0 µg/mL
with S9 mix: 1.2, 2.4, 4.7, 9.4, 18.8, 37.5, 75.0, and 150.0 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol (EtOH) (E. MERCK, 64293 Darmstadt, Germany; purity 99.8 %)
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

EtOH

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

griseofulvin, cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.

METHOD OF APPLICATION: in minimal essential medium

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): May Gruenwald and Giemsa

NUMBER OF REPLICATIONS: 1.5 - 2

NUMBER OF CELLS EVALUATED: 2000

EVALUATION: Evaluation of the cultures was performed manually using NIKON microscopes with 40 x oil immersion objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 2000 cells from clones with 2 - 8 cells were scored per test group. The frequency of micronucleated cells was reported as % micronucleated cells.

DETERMINATION OF CYTOTOXICITY
- Method: Proliferation Index

OTHER EXAMINATIONS:

Evaluation criteria:

A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical control data, and
- either a concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.
A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated test groups is in the range of the historical control data, and/or
- no concentration-related increase in the number of micronucleated cells is observed.
Statistical significance can be confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria above mentioned for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Statistics:

Statistical significance can be confirmed by means of the Chi square test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item, suspended in ethanol, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and the presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed and 1000 cells per culture were scored for micronuclei.
In both experiments no cytotoxicity was observed up to the highest evaluated concentration. However, due to strong test item precipitation higher concentrations were not evaluable for cytogenetic damage. In addition, no relevant influence of the test item on the pH value or osmolarity was observed (Exp. I: solvent control 377 mOsm, pH 7.6 versus 344 mOsm and pH 7.6 at 1200 µg/mL; Exp. II: solvent control 381 mOsm, pH 7.6 versus 373 mOsm and pH 7.5 at 1200 µg/mL).
In Experiment I in the absence of metabolic activation no statistically significant and biologically relevant increase in the number of micronucleated cells was observed at the evaluated concentrations. In the presence of metabolic activation one increase in micronucleated cells (2.10 %) was observed after treatment with 4.7 µg/mL . This value slightly exceeded the laboratory´s historical control data range (0.15 – 1.70 % micronucleated cells), but was dose-independently and statistically not significant.
In Experiment II in the absence and presence of metabolic activation no statistically significant and biologically relevant increase in micronucleated cells was observed at the evaluated concentrations. The slight increase in the number of micronucleated cells obtained in Experiment I in the presence of metabolic activation was not confirmed. Therefore, this observation is regarded as biologically irrelevant.
Griseofulvin (9.0 µg/mL), Mitomycin C (0.03 and 0.1 µg/mL), and CPA (10 and 15 µg/mL) were evaluated as positive controls and showed a distinct increase in the percentage of micronucleated cells.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation.

Dose selection was performed following the current Draft Proposal for a new Guideline No. 487. In general, the test item should be tested up to a maximum concentration of 5 mg/mL, 5 µL/mL, or 10 mM if the test item is not toxic. The highest treatment concentration chosen for the evaluation of genotoxicity should reduce the cell growth to approx. 50 %, determined by a Proliferation Index (PI). The solubility of the test item and changes in the pH value and the osmolarity may influence the dose selection.

With respect to the solubility of the test item, a concentration of 1200 µg/mL of the test item (approx. 3 mM) was applied as top concentration for treatment of the cultures in Experiment I. Dose selection of Experiment II was influenced by the results obtained in Experiment I. In Experiment I precipitation of the test item in culture medium occurred at 37.5 µg/mL in the absence and presence of metabolic activation. In Experiment II concentrations between 2.4 and 600 µg/mL in the absence of metabolic activation as well as concentrations between 1.2 and 150 µg/mL in the presence of metabolic activation were applied.

Summary of results of the micronucleus test

Exp.	Preparation	Test item	Proliferation	Micronucleated
	interval	concentration	Index	cells
		in µg/mL		in %
Exposure period 4 hrs without S9 mix
I	24 hrs	Solvent control^1	2.68	0.55
		Positive control^2	2.57	10.85^S
		9.4	2.63	0.90
		18.8	2.58	1.05
		37.5^P	2.80	0.50
Exposure period 24 hrs without S9 mix
II	24 hrs	Solvent control^1	2.70	0.40
		Positive control^3	2.11	13.40^S
		Positive control^4	2.35	7.15^S
		2.4	2.69	0.40
		4.7	2.74	0.25
		9.4	2.75	0.20
Exposure period 4 hrs with S9 mix
I	24 hrs	Solvent control^1	1.95	1.35
		Positive control^5	1.71	19.05^S
		4.7	1.84	2.10
		9.4	1.96	1.50
		18.8	2.00	1.05
Exposure period 4 hrs with S9 mix
II	24 hrs	Solvent control^1	2.28	0.85
		Positive control^6	1.72	10.40^S
		1.2	1.99	0.90
		2.4	2.21	1.10
		4.7	2.23	0.65

^S      Number of micronucleated cells statistically significant higher than corresponding
control values  

^P       Precipitate

¹      EtOH           0.5  % (v/v)

²            Mitomycin C                 0.03 µg/mL

³            Mitomycin C                 0.1   µg/mL

⁴            Griseofulvin                   9.0   µg/mL

⁵            CPA                 10.0   µg/mL

⁶            CPA                 15.0   µg/mL

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test item did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation under the experimental conditions used. Therefore, the test item has to be considered as non-mutagenic in this in vitro test system when tested up to precipitating or the highest evaluated test item concentrations.

Executive summary:

The test item, suspended in ethanol, was assessed for its potential to induce micronuclei in Chinese hamster V79cells in vitro in the absence and the presence of metabolic activation by S9 mix.

In each experimental group two parallel cultures were analysed and 1000 cells per culture were scored for micronuclei.

The following test item concentrations were applied (e=evaluated):

Exp. I: with and without S9 mix: 4.7 (e), 9.4 (e), 18.8 (e), 37.5, 75.0, 150.0, 300.0, 600.0, and 1200.0 µg/mL

Exp. II: without S9 mix: 2.4 (e), 4.7 (e), 9.4 (e), 18.8, 37.5, 75.0, 150.0, 300.0, and 600.0 µg/mL

Exp. II: with S9 mix: 1.2 (e), 2.4 (e), 4.7 (e), 9.4, 18.8, 37.5, 75.0, and 150.0 µg/mL.

The highest applied concentration (1200 µg/mL; approx. 3 mM) was chosen with regard to the solubility properties of the test item in ethanol following the current Draft Proposal for a new Guideline No. 487. Test item precipitation was observed at 37.5 µg/mL and higher.

In the absence and the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. Higher concentrations were not evaluable for cytogenetic damage because strong test item precipitation occurred in any step of evaluation.

In Experiment I no clastogenicity was observed at the concentrations evaluated in the absence of S9 mix. In the presence of S9 mix a dose-independent and statistically not significant induction of micronucleated cells (2.10 %) was observed after treatment with 4.7 µg/mL. This value marginally exceeded the laboratory’s historical control data range (0.15 – 1.70 % micronucleated cells).

In Experiment II in the absence and the presence of S9 mix no clastogenicity was observed at the concentrations evaluated. The slight increase in micronucleated cells obtained in Experiment I in the presence of S9 mix was not confirmed. Therefore, this observation has to be regarded as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p<0.05) in the percentage of micronucleated cells.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and the presence of metabolic activation. Therefore, the test item has to be considered as non-mutagenic in this in vitro test system when tested up to precipitating or the highest evaluated test item concentrations.

Reference 3

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:

year of publication: 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

It is unclear from the report whether the test was performed according to OECD and GLP guidelines. Important aspects (duplicate cultures, dosing range) in line with current OECD guideline, but study design is restricted because no second experiment with 24-hour incubation was done.

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

L5178Y TK +/- mouse lymphoma assay

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's medium for leukemic cells of mice (supplemented with 10% horse serum)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0 (control), 972.0, 1231.0, 1488.0, 1744.0, 2000.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO or acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

see below for additional information

Details on test system and experimental conditions:

Number of Replications: 2

- positve control substances:
without metabolic activation: ethyl methanesulfonate
with metabolic activation: 3-methylcholanthrene


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): trifluorothymidine

Evaluation criteria:

A response was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequency, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Results without rat liver S9 mix:

Concentrations (µg/ml): 0 -- 972 -- 972 -- 1231 -- 1231 -- 1488 -- 1488 -- 1744 -- 1744 -- 2000 -- 2000

Rel. suspension growth (%): x -- 75 -- 86 -- 100 -- 93 -- 94 -- 85 -- 86 -- 54 -- 53 -- 74

Rel. cloning efficiency (%): x -- 110 -- 118 -- 92 -- 110 -- 97 -- 101 -- 108 -- 103 -- 99 -- 109

Rel. total growth (%): x -- 82 -- 102 -- 92 -- 103 -- 91 -- 85 -- 93 -- 55 -- 52 -- 80

Average number of colonies trifluorothymidine / viable: x -- 73/178 -- 53/191 -- 42/149 -- 64/179 -- 58/157 -- 70/163 -- 50/175 -- 62/167 -- 54/160 -- 85 -176

Mutant frequency (per 10E4 cells): 0.77 -- 0.82 -- 0.55 -- 0.56 -- 0.72 -- 0.74 -- 0.86 -- 0.57 -- 0.74 --0.68 -- 0.97

Results with rat liver S9 mix:

Concentrations (µg/ml): 0 -- 972 -- 972 -- 1231 -- 1231 -- 1488 -- 1488 -- 1744 -- 1744 -- 2000 -- 2000

Rel. suspension growth (%): x -- 110 -- 115 -- 109 -- 114 -- 105 -- 103 -- 108 -- 104 -- 110 -- 81

Rel. cloning efficiency (%): x -- 113 -- 119 -- 109 -- 106 -- 113 -- 99 -- 104 -- 121 -- 109 -- 99

Rel. total growth (%): x -- 124 -- 137 -- 119 -- 121 -- 118 -- 102 -- 113 -- 126 -- 120 -- 80

Average number of colonies trifluorothymidine / viable: x -- 69/181 -- 65/191 -- 74/175 -- 77/169 -- 59/180 -- 61/158 -- 73/166 -- 79/194 -- 77/174 -- 43/159

Mutant frequency (per 10E4 cells): 0.76 -- 0.68 -- 0.85 -- 0.56 -- 0.91 -- 0.66 -- 0.77 -- 0.88 -- 0.81 --0.8 -- 0.54

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Pigment Yellow 74 was not mutagenic under the conditions tested.

Executive summary:

Pigment Yellow 74 was tested in a mammalian cell gene mutation assay (mouse lymphoma L5178Y cells) in the presence and absence of metabolic activation. Relative cloning efficiency and growth as well as mutant frequency were not affected by the test substance (concentration range tested: 972.0 to 2000.0 µg/ml). Pigment Yellow 74 was not mutagenic under the conditions tested.

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

Pigment Yellow 74 does not have to be classified for mutagenicity according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC) because Pigment Yellow 74 did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation at concentrations up to 10000 µg/plate, in the mouse lymphoma assay at concentrations up to 2000 µg/ml and in the in vitro micronucleus test in V79 cells at up to a precipitating concentration of 37.5 µg/ml.