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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registered substance and/or close structural analogues did not cause any geneotoxic or clastogenic effects in in vitro tests (Ames, HPRT, Chromosomal Aberration, micronucleus Assay)

 

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:

From 24 AUG 2012 to 13 SEP 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 471) and according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 and non-induced hamster liver S9

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: The solvent was chosen because of its solubility properties 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, 4-Nitro-o-phenylene-diamine, methyl methane sulfonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: rat liver S9 mix: 2-aminoanthracene, hamster liver S9 mix: 2-aminoanthracene, congo red

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar, plate incorporation, preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major draw¬back an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

8.4.1 Rat Liver S9 (Preparation by Harlan CCR)

Phenobarbital/b-naphthoflavone induced rat liver S9 will be used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of b-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 39.5 mg/mL (lot no. R 260412).

8.4.2 Hamster Liver S9 (Preparation by Harlan CCR)
The S9 liver microsomal fraction was prepared from the liver of 7 - 8 weeks old male Syrian golden hamsters.
After decapitation of the anaesthetised animals the livers of the animals was removed, washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in deionised water and homogenised. The homogenate, diluted 1+3 in sodium phosphate buffer was centrifuged at 9,000 g for 25 minutes at 4 °C. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as congo red.
The protein concentration in the S9 preparation was 24.0 mg/mL (lot no. H 020712).

8.4.3 Rat S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

8.4.4 Hamster S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 30% v/v. The concentrated cofactor solution yields the following concentrations in the S9 mix:
8.0 mM MgCl2
33.0 mM KCl
20.0 mM Glucose-6-phosphate
2.8 units/ml Glucose-6-phosphate-dehydrogenase
4.0 mM NADP
2.0 mM NADH
2.0 mM FMN
in 100 mM Sodium-Ortho-Phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. and Prival and Mitchell .

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.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
The test item precipitated in the overlay agar in the test tubes and on the incubated agar plates from 1000 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

 

 

 

 

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.

Executive summary:

This study was performed to investigate the potential of a 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 theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100 and theEscherichia colistrain WP2uvrA.

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:

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

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance 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. They 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 item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

From 22 AUG 1995 to 31 AUG 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

As compared to OECD guideline 471 the study design has some methodological deviations (pre-incubation assay only with but not without metabolic activation (Prival modification for azo-dyes); investigations without metabolic activation were only tested in the plate incorporation assay; not all strains tested). But considering the characteristics of the test item the study design used completely covers the most sensitive parameters relevant for azo-dyes.

Justification for type of information:

See chapter 13.2 :Read across justification document

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

not tested in E. coli WP2uvr A or TA 102

Qualifier:

according to guideline

Guideline:

other: EEC directive 92/69, L 383 A, Annex B14

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

not specified

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

Metabolic activation:

with

Metabolic activation system:

hamster liver S9 (pre-incubation test)

Test concentrations with justification for top dose:

0, 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 100, TA1535), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98)

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 (TA 100, TA 1535, TA 1537), congo red (TA 98)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- plate incorporation assay: without metabolic activation
- preincubation assay: with metabolic activation with uninduced hamster liver S9 mix (30% (v/v)

DURATION
- Preincubation period: ca. 30 minutes at 30 °C
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including controls; two independent experiments for each of the two protocols (plate incorporation assay and pre-incubation assay)

Evaluation criteria:

A test compound is classified as mutagenic if it has either of the following effects:
- it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
- it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn

Statistics:

Arithmetic means and standard deviation of the counted colonies were calculated, as well as the respective dose/control ratio.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible precipitation of the test item at 500 µg/plate and above

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay without metabolic activation and preincubation assay with metabolic activation).

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 without metabolic activation at concentrations of 0, 4, 20, 100, 500, 2500, 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 with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 0, 4, 20, 100, 500, 2500, 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.

Reference 3

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 07 SEP 2011 to 13 DEC 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 473) and in compliance with GLP.

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: 1.5, 3.0, 6.0, 12.0, 24.1, 48.1, 96.3, 192.5, 385.0 µg/mL
Experiment IIB: 1.3, 2.5, 10.0, 20.0, 30.0, 40.0, 50.0, 75.0, 100.0 µg/mL

Without metabolic activation:
Experiment I: 1.5, 3.0, 6.0, 12.0, 24.1, 48.1, 96.3, 192.5, 385.0 µg/mL
Experiment IIA: 1.5, 3.0, 6.0, 12.0, 24.1, 48.1, 96.3, 192.5, 385.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

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:

Three independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment IIA the exposure period was 18 hours without S9 mix. In Experiment IIB the exposure period was 4 hours with S9 mix. The chromosomes were prepared 18 hours (Exp. I, IIA & IIB) after the start of treatment with the test item.
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: 100 per culture, except for the positive control in Experiment IIA without metabolic activation, where 50 metaphases were evaluated.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers

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. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment IIA without metabolic activation, where 50 metaphases were evaluated.
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.

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:

cytotoxicity

Remarks:

details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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 the absence and presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment IIA the exposure period was 18 hours without S9 mix. In Experiment IIB the exposure period was 4 hours with S9 mix. The chromosomes were prepared 18 hours (Exp. I, IIA & IIB) after the start of treatment with the test item.
In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment IIA without metabolic activation, where 50 metaphases were evaluated.
The highest treatment concentration in this study, 385.0 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 3.0 µg/mL and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment IIA, in the absence of S9 mix, at 24.1 µg/mL and above and in Experiment IIB in the presence of S9 mix at 40.0 µg/mL and above. No relevant influence on osmolarity or pH value was observed.
In Experiment I in the absence of S9 mix no cytotoxicity was observed up to the highest applied concentration. In Experiment I in the presence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage due to the absence of metaphases and severe test item precipitation on the slides. In Experiment IIA in the absence of S9 mix no cytotoxicity was observed up to the highest evaluable concentration. However, higher concentrations were not evaluable due to severe test item precipitation on the slides. In Experiment IIB in the presence of S9 mix cytotoxicity indicated as reduced cell numbers was observed at the highest evaluated concentration (52.5 % of control).
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 - 4.0 % aberrant cells, excluding gaps) were slightly above the range of the solvent control values (1.0 - 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (600.0 or 1000.0 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro, when tested up to precipitating and cytotoxic concentrations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of results of the chromosome aberration study

Exp.	Preparation		Test item	Cell numbers	Mitotic indices	Aberrant cells
	interval		concentration	in %	in %	in %
			in µg/mL	of control	of control	incl. gaps*	excl. gaps*	with exchanges
		Exposure period 4 hrs without S9 mix
I	18 hrs		Solvent control^1	100.0	100.0	2.5	2.0	0.5
			Positive control^2	n.d.	92.2	24.0	24.0^S	11.0
			1.5	105.2	84.9	4.0	3.0	0.0
			3.0^P	105.9	116.1	4.0	4.0	0.0
			6.0^P	99.7	117.9	2.0	1.5	0.0
		Exposure period 18 hrs without S9 mix
IIA	18 hrs		Solvent control^1	100.0	100.0	1.0	1.0	0.0
			Positive control^#^3	n.d.	69.6	44.0	43.0^S	18.0
			12.0	87.5	100.7	2.0	1.5	0.0
			24.1^P	102.0	74.4	4.0	2.5	0.0
			48.1^P	76.1	78.5	1.5	1.5	0.0
		Exposure period 4 hrs with S9 mix
I	18 hrs		Solvent control^1	100.0	100.0	2.0	2.0	0.0
			Positive control^4	n.d.	56.7	23.0	22.0^S	4.5
			1.5	94.2	95.0	4.0	4.0	1.5
			3.0^P	101.9	94.3	2.0	2.0	1.0
			24.1^P	95.8	74.1	3.0	3.0	1.0
IIB	18 hrs		Solvent control^1	100	100	2.5	2.0	0.5
			Positive control^4	n.d.	85.3	22.5	21.5^S	7.0
			30.0	63.1	109.8	1.0	0.5	0.5
			40.0^P	87.2	102.5	1.0	1.0	0.0
			50.0^P	52.5	103.9	1.0	1.0	0.0

*     Including cells carrying exchanges

^#     Evaluation of 50 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 in the absence and presence of metabolic activation, when tested up to precipitating and cytotoxic 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 three independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. IIA	Exp. I	Exp. IIB
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. 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without metabolic activation, where 50 metaphases were evaluated.

The highest applied concentration (385.0 µg/mL) 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.

In Experiment I in the absence of S9 mix no cytotoxicity was observed up to the highest applied concentration. In Experiment I in the presence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage due to the absence of metaphases and severe test item precipitation on the slides. In Experiment IIA in the absence of S9 mix no cytotoxicity was observed up to the highest evaluable concentration. However, higher concentrations were not evaluable due to severe test item precipitation on the slides. In Experiment IIB in the presence of S9 mix cytotoxicity indicated as reduced cell numberswas observed at the highest evaluated concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to 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 4

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:

From 22 SEP 2011 to 12 DEC 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 476) and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I
4 hours treatment without S9 mix: 2.7, 5.3, 10.6, 21.3 (P), 42.5 (P), 680.0 (P) µg/mL
4 hours treatment with S9 mix: 2.7, 5.3, 10.6, 21.3 (P), 42.5 (P), 680.0 (P) µg/mL

Experiment II
24 hours treatment: 2.7, 5.3, 10.6, 21.3 (P), 42.5 (P), 680.0 (P) µg/mL
4 hours treatment: 2.7, 5.3, 10.6, 21.3 (P), 42.5 (P), 680.0 (P) µg/mL

P = precipitation
In experiment I and II the concentration of 42.5 µg/mL with and without metabolic activation was not continued to avoid analysis of too many precipitating concentrations.

Vehicle / solvent:

DMSO

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:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp. 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected
- Effects of osmolality: The osmolarity was generally above physiological values but that effect is based on a final concentration of 1% DMSO
interfering with the freezing point depression technique used to measure osmolarity.
- Evaporation from medium: Not examined
- Water solubility: Not indicated by the sponsor
- Precipitation:
Pre-experiment: Precipitation occurred at 21.3 µg/mL and above in the presence (4 hours treatment) and absence of metabolic activation (4 and 24 hours treatment).
Main experiments: Precipitation at the end of treatment, visible to the unaided eye occurred in the first and the second experiment at 21.3 µg/mL and above with and without metabolic activation.
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 680 µg/mL limited by the solubility of the test item in DMSO and aqueous medium. Test item concentrations between 5.3 µg/mL and 680 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant cytotoxic effects indicated by a relative suspension growth below 50 % were observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 21.3 µg/mL and above in the presence (4 hours treatment) and absence of metabolic activation (4 and 24 hours treatment).
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The osmolarity was generally above physiological values but that effect is based on a final concentration of 1% DMSO interfering with the freezing point depression technique used to measure osmolarity.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were generally spaced by a factor of 2 except between the two highest concentrations. A larger spacing was used to go from the lower precipitating range to the maximum possible concentration showing heavy preciptitation.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary Table

				relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	P	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL		mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12	13
Experiment I / 4 h treatment				culture I					culture II
Solvent control DMSO			-	100.0	100.0	100.0	10.8	1.0	100.0	100.0	100.0	16.2	1.0
Positive control (EMS)	150.0		-	77.9	81.2	95.2	142.4	13.2	91.0	96.6	95.5	97.8	6.0
Test item	2.7		-	102.0	81.3	107.5	13.1	1.2	101.9	103.2	107.7	17.4	1.1
Test item	5.3		-	100.6	69.6	96.1	16.3	1.5	93.7	110.7	105.3	13.5	0.8
Test item	10.6		-	96.8	81.5	96.4	19.2	1.8	96.6	117.3	104.1	19.9	1.2
Test item	21.3	P	-	99.9	101.7	98.7	13.4	1.2	93.9	116.2	101.4	22.3	1.4
Test item	42.5	P	-	93.8	culture was not continued^#				87.0	culture was not continued^#
Test item	680.0	P	-	87.2	84.2	95.9	19.3	1.8	95.1	113.2	103.4	11.4	0.7
Solvent control DMSO			+	100.0	100.0	100.0	12.3	1.0	100.0	100.0	100.0	18.5	1.0
Positive control (DMBA)	1.1		+	65.1	60.5	58.0	648.5	52.8	88.1	75.0	77.9	335.3	18.1
Test item	2.7		+	85.2	82.7	86.1	11.6	0.9	101.9	88.6	95.4	18.5	1.0
Test item	5.3		+	99.2	95.0	85.5	23.4	1.9	108.9	110.3	98.0	12.9	0.7
Test item	10.6		+	98.2	83.0	100.0	17.4	1.4	97.4	86.6	90.7	7.7	0.4
Test item	21.3	P	+	106.4	84.4	82.7	20.8	1.7	97.1	75.9	85.8	13.0	0.7
Test item	42.5	P	+	104.0	culture was not continued^#				97.2	culture was not continued^#
Test item	680.0	P	+	105.1	80.3	73.8	24.5	2.0	105.3	90.5	92.3	13.1	0.7
Experiment II / 24 h treatment				culture I					culture II
Solvent control DMSO			-	100.0	100.0	100.0	11.4	1.0	100.0	100.0	100.0	10.7	1.0
Positive control (EMS)	150.0		-	100.1	124.7	86.4	390.5	34.2	98.4	70.4	91.9	462.9	43.3
Test item	2.7		-	96.4	118.1	125.5	10.2	0.9	97.8	85.7	93.2	20.8	1.9
Test item	5.3		-	86.1	113.6	91.1	19.6	1.7	95.0	86.3	114.2	13.7	1.3
Test item	10.6		-	85.7	106.0	122.5	10.2	0.9	97.0	89.8	115.3	22.3	2.1
Test item	21.3	P	-	74.7	106.2	116.4	14.3	1.3	86.3	89.0	93.3	7.9	0.7
Test item	42.5	P	-	culture was not continued^#					86.8	culture was not continued^#
Test item	680.0	P	-	86.5	120.5	74.3	17.7	1.6	89.7	99.7	88.8	18.3	1.7
Experiment II / 4 h treatment
Solvent control DMSO			+	100.0	100.0	100.0	13.9	1.0	100.0	100.0	100.0	3.4	1.0
Positive control (DMBA)	1.1		+	60.8	88.8	102.4	328.4	23.6	66.5	84.9	80.0	414.5	121.6
Test item	2.7		+	99.3	87.5	101.8	9.4	0.7	96.7	110.7	84.9	11.1	3.2
Test item	5.3		+	91.2	97.1	110.9	6.2	0.4	95.3	106.3	90.6	13.2	3.9
Test item	10.6		+	93.7	98.0	111.4	9.0	0.6	102.0	92.0	91.7	6.3	1.8
Test item	21.3	P	+	95.7	116.4	103.3	10.2	0.7	99.4	112.5	91.9	4.3	1.3
Test item	42.5	P	+	93.5	culture was not continued^#				90.0	culture was not continued^#
Test item	680.0	P	+	82.5	112.4	107.6	6.6	0.5	93.0	113.4	90.3	4.4	1.3

^#     culture not continued to avoid evaluation of too many precipitating concentrations

P    precipitation

Conclusions:

Interpretation of results (migrated information):
negative

Conclusion:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the main experiments was limited by the solubility of the test item. Precipitation at the end of treatment, visible to the unaided eye occurred in the first and the second experiment at 21.3 µg/mL and above with and without metabolic activation.

No relevant cytotoxic effects, indicated by a relative cloning efficiency I (survival) of less than 50% compared to the corresponding solvent control occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls. An increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the second experiment with metabolic activation at the lowest concentrations of 2.7 and 5.3 µg/mL. However, this increase was based on a rather low mutation frequency of the solvent control of just 3.4 colonies per 10⁶cells. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions. Therefore, the increase of the induction factor was judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.4 up to 18.5 mutants per 10⁶cells; the range of the groups treated with the test item was from 4.3 up to 23.4 mutants per 10⁶cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

From 22 SEP 2011 to 12 DEC 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 476) and in compliance with GLP.

Justification for type of information:

see chapter 13.2 : Read across justification document

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I
4 hours treatment without S9 mix: 2.7, 5.3, 10.6, 21.3 (P), 42.5 (P), 680.0 (P) µg/mL
4 hours treatment with S9 mix: 2.7, 5.3, 10.6, 21.3 (P), 42.5 (P), 680.0 (P) µg/mL

Experiment II
24 hours treatment: 2.7, 5.3, 10.6, 21.3 (P), 42.5 (P), 680.0 (P) µg/mL
4 hours treatment: 2.7, 5.3, 10.6, 21.3 (P), 42.5 (P), 680.0 (P) µg/mL

P = precipitation
In experiment I and II the concentration of 42.5 µg/mL with and without metabolic activation was not continued to avoid analysis of too many precipitating concentrations.

Vehicle / solvent:

DMSO

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:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp. 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected
- Effects of osmolality: The osmolarity was generally above physiological values but that effect is based on a final concentration of 1% DMSO
interfering with the freezing point depression technique used to measure osmolarity.
- Evaporation from medium: Not examined
- Water solubility: Not indicated by the sponsor
- Precipitation:
Pre-experiment: Precipitation occurred at 21.3 µg/mL and above in the presence (4 hours treatment) and absence of metabolic activation (4 and 24 hours treatment).
Main experiments: Precipitation at the end of treatment, visible to the unaided eye occurred in the first and the second experiment at 21.3 µg/mL and above with and without metabolic activation.
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 680 µg/mL limited by the solubility of the test item in DMSO and aqueous medium. Test item concentrations between 5.3 µg/mL and 680 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant cytotoxic effects indicated by a relative suspension growth below 50 % were observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 21.3 µg/mL and above in the presence (4 hours treatment) and absence of metabolic activation (4 and 24 hours treatment).
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The osmolarity was generally above physiological values but that effect is based on a final concentration of 1% DMSO interfering with the freezing point depression technique used to measure osmolarity.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were generally spaced by a factor of 2 except between the two highest concentrations. A larger spacing was used to go from the lower precipitating range to the maximum possible concentration showing heavy preciptitation.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary Table

				relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	P	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL		mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12	13
Experiment I / 4 h treatment				culture I					culture II
Solvent control DMSO			-	100.0	100.0	100.0	10.8	1.0	100.0	100.0	100.0	16.2	1.0
Positive control (EMS)	150.0		-	77.9	81.2	95.2	142.4	13.2	91.0	96.6	95.5	97.8	6.0
Test item	2.7		-	102.0	81.3	107.5	13.1	1.2	101.9	103.2	107.7	17.4	1.1
Test item	5.3		-	100.6	69.6	96.1	16.3	1.5	93.7	110.7	105.3	13.5	0.8
Test item	10.6		-	96.8	81.5	96.4	19.2	1.8	96.6	117.3	104.1	19.9	1.2
Test item	21.3	P	-	99.9	101.7	98.7	13.4	1.2	93.9	116.2	101.4	22.3	1.4
Test item	42.5	P	-	93.8	culture was not continued^#				87.0	culture was not continued^#
Test item	680.0	P	-	87.2	84.2	95.9	19.3	1.8	95.1	113.2	103.4	11.4	0.7
Solvent control DMSO			+	100.0	100.0	100.0	12.3	1.0	100.0	100.0	100.0	18.5	1.0
Positive control (DMBA)	1.1		+	65.1	60.5	58.0	648.5	52.8	88.1	75.0	77.9	335.3	18.1
Test item	2.7		+	85.2	82.7	86.1	11.6	0.9	101.9	88.6	95.4	18.5	1.0
Test item	5.3		+	99.2	95.0	85.5	23.4	1.9	108.9	110.3	98.0	12.9	0.7
Test item	10.6		+	98.2	83.0	100.0	17.4	1.4	97.4	86.6	90.7	7.7	0.4
Test item	21.3	P	+	106.4	84.4	82.7	20.8	1.7	97.1	75.9	85.8	13.0	0.7
Test item	42.5	P	+	104.0	culture was not continued^#				97.2	culture was not continued^#
Test item	680.0	P	+	105.1	80.3	73.8	24.5	2.0	105.3	90.5	92.3	13.1	0.7
Experiment II / 24 h treatment				culture I					culture II
Solvent control DMSO			-	100.0	100.0	100.0	11.4	1.0	100.0	100.0	100.0	10.7	1.0
Positive control (EMS)	150.0		-	100.1	124.7	86.4	390.5	34.2	98.4	70.4	91.9	462.9	43.3
Test item	2.7		-	96.4	118.1	125.5	10.2	0.9	97.8	85.7	93.2	20.8	1.9
Test item	5.3		-	86.1	113.6	91.1	19.6	1.7	95.0	86.3	114.2	13.7	1.3
Test item	10.6		-	85.7	106.0	122.5	10.2	0.9	97.0	89.8	115.3	22.3	2.1
Test item	21.3	P	-	74.7	106.2	116.4	14.3	1.3	86.3	89.0	93.3	7.9	0.7
Test item	42.5	P	-	culture was not continued^#					86.8	culture was not continued^#
Test item	680.0	P	-	86.5	120.5	74.3	17.7	1.6	89.7	99.7	88.8	18.3	1.7
Experiment II / 4 h treatment
Solvent control DMSO			+	100.0	100.0	100.0	13.9	1.0	100.0	100.0	100.0	3.4	1.0
Positive control (DMBA)	1.1		+	60.8	88.8	102.4	328.4	23.6	66.5	84.9	80.0	414.5	121.6
Test item	2.7		+	99.3	87.5	101.8	9.4	0.7	96.7	110.7	84.9	11.1	3.2
Test item	5.3		+	91.2	97.1	110.9	6.2	0.4	95.3	106.3	90.6	13.2	3.9
Test item	10.6		+	93.7	98.0	111.4	9.0	0.6	102.0	92.0	91.7	6.3	1.8
Test item	21.3	P	+	95.7	116.4	103.3	10.2	0.7	99.4	112.5	91.9	4.3	1.3
Test item	42.5	P	+	93.5	culture was not continued^#				90.0	culture was not continued^#
Test item	680.0	P	+	82.5	112.4	107.6	6.6	0.5	93.0	113.4	90.3	4.4	1.3

^#     culture not continued to avoid evaluation of too many precipitating concentrations

P    precipitation

Conclusions:

Interpretation of results (migrated information):
negative

Conclusion:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the main experiments was limited by the solubility of the test item. Precipitation at the end of treatment, visible to the unaided eye occurred in the first and the second experiment at 21.3 µg/mL and above with and without metabolic activation.

No relevant cytotoxic effects, indicated by a relative cloning efficiency I (survival) of less than 50% compared to the corresponding solvent control occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls. An increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the second experiment with metabolic activation at the lowest concentrations of 2.7 and 5.3 µg/mL. However, this increase was based on a rather low mutation frequency of the solvent control of just 3.4 colonies per 10⁶cells. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions. Therefore, the increase of the induction factor was judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.4 up to 18.5 mutants per 10⁶cells; the range of the groups treated with the test item was from 4.3 up to 23.4 mutants per 10⁶cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

From 07 SEP 2011 to 13 DEC 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 473) and in compliance with GLP.

Justification for type of information:

See chapter 13.2 : Read across Justification document

Reason / purpose for cross-reference:

read-across source

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: 1.5, 3.0, 6.0, 12.0, 24.1, 48.1, 96.3, 192.5, 385.0 µg/mL
Experiment IIB: 1.3, 2.5, 10.0, 20.0, 30.0, 40.0, 50.0, 75.0, 100.0 µg/mL

Without metabolic activation:
Experiment I: 1.5, 3.0, 6.0, 12.0, 24.1, 48.1, 96.3, 192.5, 385.0 µg/mL
Experiment IIA: 1.5, 3.0, 6.0, 12.0, 24.1, 48.1, 96.3, 192.5, 385.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

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:

Three independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment IIA the exposure period was 18 hours without S9 mix. In Experiment IIB the exposure period was 4 hours with S9 mix. The chromosomes were prepared 18 hours (Exp. I, IIA & IIB) after the start of treatment with the test item.
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: 100 per culture, except for the positive control in Experiment IIA without metabolic activation, where 50 metaphases were evaluated.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers

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. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment IIA without metabolic activation, where 50 metaphases were evaluated.
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.

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:

cytotoxicity

Remarks:

details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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 the absence and presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment IIA the exposure period was 18 hours without S9 mix. In Experiment IIB the exposure period was 4 hours with S9 mix. The chromosomes were prepared 18 hours (Exp. I, IIA & IIB) after the start of treatment with the test item.
In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment IIA without metabolic activation, where 50 metaphases were evaluated.
The highest treatment concentration in this study, 385.0 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 3.0 µg/mL and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment IIA, in the absence of S9 mix, at 24.1 µg/mL and above and in Experiment IIB in the presence of S9 mix at 40.0 µg/mL and above. No relevant influence on osmolarity or pH value was observed.
In Experiment I in the absence of S9 mix no cytotoxicity was observed up to the highest applied concentration. In Experiment I in the presence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage due to the absence of metaphases and severe test item precipitation on the slides. In Experiment IIA in the absence of S9 mix no cytotoxicity was observed up to the highest evaluable concentration. However, higher concentrations were not evaluable due to severe test item precipitation on the slides. In Experiment IIB in the presence of S9 mix cytotoxicity indicated as reduced cell numbers was observed at the highest evaluated concentration (52.5 % of control).
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 - 4.0 % aberrant cells, excluding gaps) were slightly above the range of the solvent control values (1.0 - 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (600.0 or 1000.0 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro, when tested up to precipitating and cytotoxic concentrations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of results of the chromosome aberration study

Exp.	Preparation		Test item	Cell numbers	Mitotic indices	Aberrant cells
	interval		concentration	in %	in %	in %
			in µg/mL	of control	of control	incl. gaps*	excl. gaps*	with exchanges
		Exposure period 4 hrs without S9 mix
I	18 hrs		Solvent control^1	100.0	100.0	2.5	2.0	0.5
			Positive control^2	n.d.	92.2	24.0	24.0^S	11.0
			1.5	105.2	84.9	4.0	3.0	0.0
			3.0^P	105.9	116.1	4.0	4.0	0.0
			6.0^P	99.7	117.9	2.0	1.5	0.0
		Exposure period 18 hrs without S9 mix
IIA	18 hrs		Solvent control^1	100.0	100.0	1.0	1.0	0.0
			Positive control^#^3	n.d.	69.6	44.0	43.0^S	18.0
			12.0	87.5	100.7	2.0	1.5	0.0
			24.1^P	102.0	74.4	4.0	2.5	0.0
			48.1^P	76.1	78.5	1.5	1.5	0.0
		Exposure period 4 hrs with S9 mix
I	18 hrs		Solvent control^1	100.0	100.0	2.0	2.0	0.0
			Positive control^4	n.d.	56.7	23.0	22.0^S	4.5
			1.5	94.2	95.0	4.0	4.0	1.5
			3.0^P	101.9	94.3	2.0	2.0	1.0
			24.1^P	95.8	74.1	3.0	3.0	1.0
IIB	18 hrs		Solvent control^1	100	100	2.5	2.0	0.5
			Positive control^4	n.d.	85.3	22.5	21.5^S	7.0
			30.0	63.1	109.8	1.0	0.5	0.5
			40.0^P	87.2	102.5	1.0	1.0	0.0
			50.0^P	52.5	103.9	1.0	1.0	0.0

*     Including cells carrying exchanges

^#     Evaluation of 50 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 in the absence and presence of metabolic activation, when tested up to precipitating and cytotoxic 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 three independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. IIA	Exp. I	Exp. IIB
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. 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without metabolic activation, where 50 metaphases were evaluated.

The highest applied concentration (385.0 µg/mL) 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.

In Experiment I in the absence of S9 mix no cytotoxicity was observed up to the highest applied concentration. In Experiment I in the presence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage due to the absence of metaphases and severe test item precipitation on the slides. In Experiment IIA in the absence of S9 mix no cytotoxicity was observed up to the highest evaluable concentration. However, higher concentrations were not evaluable due to severe test item precipitation on the slides. In Experiment IIB in the presence of S9 mix cytotoxicity indicated as reduced cell numberswas observed at the highest evaluated concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to 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 7

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.

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

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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 (migrated information):
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 8

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

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

Justification for type of information:

See Chapter 13.2: Read across justification document

Reason / purpose for cross-reference:

read-across source

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.

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

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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 (migrated information):
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 9

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.

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: V79

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 (migrated information):
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 10

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: gene mutation

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:

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

Justification for type of information:

See chapter 13.2: Read across justification document

Reason / purpose for cross-reference:

read-across source

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.

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: V79

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 (migrated information):
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 11

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%.

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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 (migrated information):
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.

Reference 12

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

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.

Justification for type of information:

See chapter 13.2: Read across justification document

Reason / purpose for cross-reference:

read-across source

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%.

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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 (migrated information):
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.

Reference 13

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 22 AUG 1995 to 31 AUG 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

As compared to OECD guideline 471 the study design has some methodological deviations (pre-incubation assay only with but not without metabolic activation (Prival modification for azo-dyes); investigations without metabolic activation were only tested in the plate incorporation assay; not all strains tested). But considering the characteristics of the test item the study design used completely covers the most sensitive parameters relevant for azo-dyes.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

not tested in E. coli WP2uvr A or TA 102

Qualifier:

according to guideline

Guideline:

other: EEC directive 92/69, L 383 A, Annex B14

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

not specified

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

Metabolic activation:

with

Metabolic activation system:

hamster liver S9 (pre-incubation test)

Test concentrations with justification for top dose:

0, 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 100, TA1535), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98)

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 (TA 100, TA 1535, TA 1537), congo red (TA 98)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- plate incorporation assay: without metabolic activation
- preincubation assay: with metabolic activation with uninduced hamster liver S9 mix (30% (v/v)

DURATION
- Preincubation period: ca. 30 minutes at 30 °C
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including controls; two independent experiments for each of the two protocols (plate incorporation assay and pre-incubation assay)

Evaluation criteria:

A test compound is classified as mutagenic if it has either of the following effects:
- it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
- it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn

Statistics:

Arithmetic means and standard deviation of the counted colonies were calculated, as well as the respective dose/control ratio.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible precipitation of the test item at 500 µg/plate and above

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay without metabolic activation and preincubation assay with metabolic activation).

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 without metabolic activation at concentrations of 0, 4, 20, 100, 500, 2500, 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 with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 0, 4, 20, 100, 500, 2500, 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 registered substance and/or close structural analogues did not cause any geneotoxic or clastogenic effects in in vitro tests (Ames, HPRT, Chromosomal Aberration, micronucleus Assay)