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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

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.

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.

Link to relevant study records

Referenceopen allclose all

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 FEB 2005 to 29 MAR 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 471) with Prival modifications for azo-dyes, GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9; hamster liver S9
Test concentrations with justification for top dose:
0, 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate (plate incorporation test)
0, 33, 100, 333, 1000, 2500, 5000 µg/plate (pre-incubation test)
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), 4-nitro-o-phenylene-diamine (TA 1537, TA 98), methyl methane sulfonate (WP2uvrA)
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 (all strains)
Remarks:
with metabolic activation (rat liver S9 mix)
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, WP2uvrA), congo red (TA 98)
Remarks:
with metabolic activation (hamster liver S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- plate incorporation assay without and with induced rat liver S9 mix (15% (v/v); induction with Phenobarbital/beta-Naphthoflavone)
- preincubation assay without and 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

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
- an increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent secnd experiment
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in strain TA1537 and TA100 in the pre-incubation test at 5000 µg/plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible at 333 to 5000 µg/plate (plate incorporation assay with and without metabolic activation), at 1000 to 5000 µg/plate (pre-incubation assay without metabolic activation), at 33 to 5000 µg/plate (pre-incubation assay with metabolic activation); in the plate incorporation assay the highest test concentration of 5000 µg/plate could not be analysed in TA100 (without metabolic activation) and in TA 98, TA 100 and WP2uvrA (with metabolic activation), due to densely coloured plates


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- in strain TA1537 and TA100 minor reductions in the number of revertants were observed in the pre-incubation test at 5000 µg/plate without metabolic activation
- the reduction in the number of revertants in strain TA 1535 at 333 µg/plate with S9 mix in the pre-incubation test was judged by the authors of the study to be based on biological fluctuations


In the plate incorporation assay with metabolic activation TA 98 showed a minor increase in revertant colony numbers at 2500 µg/plate. To verify the results of this experiment an independent repeat experiment was performed under indentical conditions with strain TA 98 in the presence of metabolic activation. No increase in the number of revertant colonies occurred in the repeat experiment and the effect observed in the first experiment was judged by authors of the study as biologically irrelevant.

Revertant colony counts in TA 98 (plate incorporation assay with metabolic activation)

Control group

Test item concentration (µg/plate)

First experiment (mean +/- SD)

Repeat experiment (mean +/- SD)

DMSO

 

34 +/- 9

40 +/- 13

Untrated

 

44 +/- 7

46 +/- 4

 

3

30 +/- 4

37 +/- 1

 

10

36 +/- 7

37 +/- 7

 

33

35 +/- 4

45 +/- 4

 

100

47 +/- 4

38 +/- 5

 

333

37 +/- 12

42 +/- 3

 

1000

50 +/- 2

53 +/- 5

 

2500

70 +/- 5

50 +/- 3

 

5000

Analysis not possible

49 +/- 3

Conclusions:
Interpretation of results:
negative

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

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and E. coli WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay. Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

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

Endpoint:
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).
Key result
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.

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 control1

100.0

100.0

2.5

2.0

0.5

 

 

 

Positive control2

n.d.

92.2

24.0

24.0S

11.0

 

 

 

1.5

105.2

84.9

4.0

3.0

0.0

 

 

 

3.0P

105.9

116.1

4.0

4.0

0.0

 

 

 

6.0P

99.7

117.9

2.0

1.5

0.0

 

 

Exposure period 18 hrs without S9 mix

IIA

18 hrs

Solvent control1

100.0

100.0

1.0

1.0

0.0

 

 

 

Positive control#3

n.d.

69.6

44.0

43.0S

18.0

 

 

 

12.0

87.5

100.7

2.0

1.5

0.0

 

 

 

24.1P

102.0

74.4

4.0

2.5

0.0

 

 

 

48.1P

76.1

78.5

1.5

1.5

0.0

 

 

Exposure period 4 hrs with S9 mix

I

18 hrs

Solvent control1

100.0

100.0

2.0

2.0

0.0

 

 

 

Positive control4

n.d.

56.7

23.0

22.0S

4.5

 

 

 

1.5

94.2

95.0

4.0

4.0

1.5

 

 

 

3.0P

101.9

94.3

2.0

2.0

1.0

 

 

 

24.1P

95.8

74.1

3.0

3.0

1.0

 

IIB

18 hrs

Solvent control1

100

100

2.5

2.0

0.5

 

 

 

Positive control4

n.d.

85.3

22.5

21.5S

7.0

 

 

 

30.0

63.1

109.8

1.0

0.5

0.5

 

 

 

40.0P

87.2

102.5

1.0

1.0

0.0

 

 

 

50.0P

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:
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.

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.
Key result
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
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 106cells factor efficiency I density efficiency II 106cells 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:
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.

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
Justification for type of information:
For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
assessment report
Key result
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
Conclusions:
Interpretation of results:
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 study used as source investigated C.I. Pigment Yellow 1. The study results of the source compound were considered applicable to the target compound and were used for classification and labelling acc. to REGULATION (EC) No 1272/2008. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

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
Justification for type of information:
For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
assessment report
Key result
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
Conclusions:
Interpretation of results:
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 study used as source investigated C.I. Pigment Yellow 1. The study results of the source compound were considered applicable to the target compound and were used for classification and labelling acc. to REGULATION (EC) No 1272/2008. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

No classification

The test item and closs structural analogues did not show genotoxic effects in bacterial and mammalian cells.