N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(2-methoxyphenyl)azo]-3-oxobutyramide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay. Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Chromosomal Aberration

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

 

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I	Exp. II
Exposure period	4 hrs	18 hrs	4 hrs	4 hrs
Recovery	14 hrs	-	14 hrs	14 hrs
Preparation interval	18 hrs	18 hrs	18 hrs	18 hrs

 

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentration (500.0 µg/mL, approx. 1.4 mM) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The evaluated experimental points and the results are summarised in Table1.

In the absence and presence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration, where test item precipitation occurred.

No clastogenicity was observed at the concentrations evaluated, either with or without metabolic activation.

No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

No relevant increase in endomitotic cells was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

HPRT

The genotoxic potential of the test item C.I.Pigment Yellow 194 induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

 

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 C.I.Pigment Yellow 194 was insoluble in sterile water and formed a free flowing suspension in Dimethyl sulfoxide (DMSO) at 200 mg/mL.

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 27 and 32 % at the
1000 µg/mL, in the presence and absence of metabolic activation, respectively. There was precipitation of the test item in the test medium at and above
1000 µg/mL, both in the presence and absence of metabolic activation. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 1500 µg/mL was tested in the gene mutation assay. 

 In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 23.44, 93.75, 375 and 1500 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The results of the forward gene mutation test at thehprtlocus with C.I.Pigment Yellow 194 indicated that the test item was non-mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian cell gene mutation test using the Hprt gene

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1,
(ATCC CCL-61, Lot 4765275)

Metabolic activation:

with and without

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air)

Rationale for test conditions:

Test approaches currently accepted under the OECD for the assessment of mammalian cell gene mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.

Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time

Evaluation criteria:

CRITERIA FOR ACCEPTABILITY OF THE TEST
The assay will be considered valid if the following criteria are met:
a) The concurrent vehicle control data is within the range of the laboratory historical control data.
b) The concurrent positive control substances should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent vehicle control.
c) Two experimental conditions are tested unless one results in positive response.
d) Adequate number of cells and analyzable concentrations are tested under each of the experimental conditions.
e) The criteria for the selection of top concentration are consistent with those described in the guideline.

EVALUATION AND INTERPRETATION OF RESULTS
When all the validity criteria are fulfilled:

1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

2 A test chemical is considered to be clearly negative if, in all experimental conditions examined:

• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

It is concluded that the test item, C.I.Pigment Yellow 194 does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.

Executive summary:

 

The genotoxic potential of the test item C.I.Pigment Yellow 194 induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

 

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

C.I.Pigment Yellow 194 was insoluble in sterile water and formed a free flowing suspension in Dimethyl sulfoxide (DMSO) at
200 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 27 and 32 % at the
1000 µg/mL, in the presence and absence of metabolic activation, respectively. There was precipitation of the test item in the test medium at and above
1000 µg/mL, both in the presence and absence of metabolic activation. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 1500 µg/mL was tested in the gene mutation assay. 

 

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 23.44, 93.75, 375 and 1500 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The results of the forward gene mutation test at thehprtlocus with C.I.Pigment Yellow 194 indicated that the test item was non-mutagenic under the conditions of this study

Reference 2

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:

From JUN 16 2011 to 26 JUL 2011

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:

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)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / beta-naphtoflavone induced rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0
Experiment II: 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0

Without metabolic activation:
Experiment I: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0
Experiment II: 0.2, 0.7, 2.1, 6.2, 18.5, 55.6, 166.7, 500.0

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of test item

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:

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 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium (minimal essential medium)

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 18 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: at least 100 per culture, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers


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

Evaluation criteria:

Evaluation of the cultures was performed according to the OECD Guideline using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) and relative cell numbers were determined.

In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases)

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Species / strain:

Chinese hamster lung fibroblasts (V79)

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 examined

Positive controls validity:

valid

Additional information on results:

The highest treatment concentration in this study, 500.0 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I, visible precipitation of the test item in the culture medium was observed at 15.6 µg/mL and above in the absence and presence of S9 mix at the end of treatment. In Experiment II precipitation was observed at 6.2 µg/mL and above in the absence of S9 mix and at 12.5 µg/mL and above in the presence of S9 mix at the end of treatment.
No relevant influence on osmolarity or pH value was observed. The osmolarity and pH-value were determined in the solvent control and the maximum concentration of Experiment I and II without metabolic activation:

Exp. Solvent control Test item 500.0 µg/mL
I Osmolarity (mOsm) 380 390
pH-value 7.3 7.3
II Osmolarity (mOsm) 395 392
pH-value 7.5 7.4

No cytotoxicity indicated by reduced cell numbers or mitotic indices were observed after treatment with the test item .
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.3 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.0 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory’s historical control data: 0.0 - 4.0 % aberrant cells, excluding gaps.
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.3 - 3.9 %) as compared to the rates of the solvent controls (1.3 - 3.6 %).
In both experiments, either EMS (600 or 1000 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line), when tested up to precipitating concentrations.

Remarks on result:

other: strain/cell type: V79 cells

Remarks:

Migrated from field 'Test system'.

Table 1: Summary of results of the chromosome aberration study with the test substance

Exp.	Preparation	Test item	Endomitotic	Polyploid	Cell numbers	Mitotic indices	Aberrant cells
	interval	concentration	cells	cells	in %	in %	in %
		in µg/mL	in %	in %	of control	of control	incl. gaps*	excl. gaps*	with exchanges
Exposure period 4 hrs without S9 mix
I	18 hrs	Solvent control^1	0.0	3.6	100.0	100.0	1.0	1.0	0.0
		Positive control^2#	n.d.	n.d.	n.d.	91.0	33.0	33.0^S	20.0
		3.9	0.0	3.4	82.0	95.3	0.5	0.5	0.5
		7.8	0.0	3.0	115.6	100.0	0.5	0.5	0.5
		15.6^P	0.0	2.7	92.7	111.4	1.5	1.0	0.0
Exposure period 18 hrs without S9 mix
II	18 hrs	Solvent control^1	0.0	2.4	100.0	100.0	3.0	2.0	0.0
		Positive control^3	n.d.	n.d.	n.d.	71.2	18.0	17.5^S	10.0
		0.7	0.0	2.7	106.6	101.8	1.0	1.0	0.0
		2.1	0.0	1.9	76.4	119.6	1.0	1.0	0.0
		6.2^P	0.0	1.9	75.7	83.2	2.5	2.0	0.0
Exposure period 4 hrs with S9 mix
I	18 hrs	Solvent control^1	0.1	3.6	100.0	100.0	1.5	1.0	0.5
		Positive control^4	n.d.	n.d.	n.d.	87.0	14.0	13.0^S	4.5
		3.9	0.1	2.4	79.3	115.1	1.5	1.5	0.0
		7.8	0.1	3.6	78.9	92.5	3.0	2.5	0.5
		15.6^P	0.3	3.9	113.1	102.5	2.5	2.0	0.5
II	18 hrs	Solvent control^1	0.0	2.3	100	100	2.5	2.5	0.0
		Positive control^4	n.d.	n.d.	n.d.	71.4	16.0	14.5^S	7.0
		3.1^##	0.0	1.9	111.9	78.3	4.0	3.3	1.3
		6.3^##	0.0	2.0	90.5	99.2	3.0	3.0	0.8
		12.5^P	0.0	1.3	76.8	112.0	2.5	2.5	2.5

*     Inclusive cells carrying exchanges

^#     Evaluation of 50 metaphases per culture

^##    Evaluation of 200 metaphases per culture

n.d. Not determined

^P     Precipitation occurred at the end of treatment

^S     Aberration frequency statistically significant higher than corresponding control values

¹     DMSO    0.5 % (v/v)

²           EMS  1000.0 µg/mL

³           EMS   600.0 µg/mL

⁴           CPA       1.4 µg/mL

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

Executive summary:

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I	Exp. II
Exposure period	4 hrs	18 hrs	4 hrs	4 hrs
Recovery	14 hrs	-	14 hrs	14 hrs
Preparation interval	18 hrs	18 hrs	18 hrs	18 hrs

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentration (500.0 µg/mL, approx. 1.4 mM) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The evaluated experimental points and the results are summarised in Table1.

In the absence and presence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration, where test item precipitation occurred.

No clastogenicity was observed at the concentrations evaluated, either with or without metabolic activation.

No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

No relevant increase in endomitotic cells was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Reference 3

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:

From 28 APR 2006 to 12 MAY 2006

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 (induced with phenobarbital/beta-naphtoflavone; experiment I); hamster liver S9 (non-induced; experiment II)

Test concentrations with justification for top dose:

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

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (for all strains)

Remarks:

with metabolic activation (rat liver S9)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (TA 1535, TA 100, TA 1537, WP2 uvrA), congo red (TA 98)

Remarks:

with metabolic activation (hamster liver S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: plate incorporation assay without and with induced rat liver S9 mix (induction with phenobarbital/beta-naphthoflavone)
Experiment II: preincubation assay without and with non-induced hamster liver S9 mix

DURATION
- Preincubation period: Experiment II: 30° C for 30 minutes
- Exposure duration: at least 48 hours at 37° C

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including the control

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, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony 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 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.

Statistics:

Arithmetic means and standard deviation of the counted colonies were calculated.

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:

strain TA1535 (exp.II, w/o S9, at 5000 µg/plate), in TA98 (exp. II, w/o S9, starting from 2500 µg/plate), and in TA1535 (exp.II, with S9 mix, atarting from 2500 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The laboratory´s historical control range was slightly exceeded in the untreated (experiment I) and solvent (experiment II) control of strain WP2 uvrA in the presence of metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and has no impact on the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation in experiment I. In experiment II, slight toxic effects were observed without metabolic activation in strains TA 1535 at 5000 µg/plate and TA 98 at 2500 and 5000 µg/plate and with metabolic activation in strain TA 1535 at 2500 and 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:

The test item precipitated in the overlay agar at the following concentrations (microgram/plate):

Strain	ExperimentI		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	333- 5000	333- 5000	10- 5000	333- 5000
TA 1537	333- 5000	333- 5000	333- 5000	333- 5000
TA 98	333- 5000	333- 5000	333- 5000	333- 5000
TA 100	333- 5000	333- 5000	333- 5000	333- 5000
WP2 uvrA	333- 5000	333- 5000	333- 5000	333- 5000

The undissolved particles had no influence on the data recording.

Conclusions:

Interpretation of results (migrated information):
negative

The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay. Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

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

No classification

The test material did not cause mutagenic effects in bacteria and mammalian cells.