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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The substance is not considered to be mutagenic in bacterial cells, based on read across.

HPRT-Test:

The read across-substance CAS 39322-78-6 was not mutagenic in vitro in mammalian cells.

Chromosome aberration study:

The read across-substance CAS 39322-78-6 was not clastogenic in vitro in mammalian cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: other: clastogenicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
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:
EPA OPPTS 870.5375 - 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
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
It can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the read across-substance (CAS39322-78-6) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.
Executive summary:

To investigate the potential of read across-substance Afilan V5756 (CAS39322-78-6) to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation.

Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations, except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 200 cells for the 1st culture and 100 cells for the 2nd culture.

The test item ( read across-substance) was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test item was well suspended.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 15.6, 250, 500 and 1000 µg/mL

with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL

Experiment II:

without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL

with metabolic activation: 900, 1600 and 1800 µg/mL

Precipitation of the test item was observed with and without metabolic activation in both experiments.

Toxic effects of the test item were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure) toxic effects of the test item were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

The positive controls induced the appropriate responses.

There was no evidence of Afilan V5756 induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data. 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
yes (Experiment I without S9: ≥ 1500 μg/mL; experiment I with S9: ≥ 1000 μg/mL; Experiment II without S9: ≥ 75 μg/mL; Experiment II with S9:≥ 1200 μg/mL
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item Afilan V5756 is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

In a mammalian cell gene mutation assay (HPRT locus],V79 cells culturedin vitro were exposed to the read across substance suspended in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of

- 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL (without metabolic activation, Experiment I)

- 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL (with metabolic activation, Experiment I)

- 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL (without metabolic activation, Experiment II)

- 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL (with metabolic activation, Experiment II).

The read across substance was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation the relative growth was 24.6% for the highest concentration evaluated (2000 µg/mL). The highest biologically relevant concentration evaluated with metabolic activation was 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation the relative growth was 15.7% for the highest concentration (175 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 1400 µg/mL with a relative growth of 20.0%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.00 was found at a concentration of 500 µg/mL with a relative growth of 96.5%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.75 was found at a concentration of 1500 µg/mL with a relative growth of 23.9%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.43 was found at a concentration of 175 µg/mL with a relative growth of 15.7%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.21 was found at a concentration of 150 µg/mL with a relative growth of 101.3%.

The positive controls did induce the appropriate response. 

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

 

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:
weight of evidence
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Key result
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
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.
Executive summary:

The test item (read across-substance, CAS 39322 -78 -6) was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were 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:

without S9 mix                                  1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

with S9 mix 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background growth (for details and concentrations please refer to "Any other infromation on results").

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. They showed a distinct increase of induced revertant colonies.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Key result
Species / strain:
other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the conditions of the test, the read across substance didn't show mutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 of S. typhimurium were exposed to the read across substance (CAS 68987 -29 -1), at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation, (S9 mix; Aroclor induced rat liver).

The initial Mutation Test was performed as plate incorporation method; the confirmatory and complementary assays were performed according to the pre-incubation method.

No signs of toxicity towards the bacteria could be observed. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The substance did not show any mutagenic effects in both experiments.

The read across-substance is considered as not mutagenic.

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012-06-20 to 2012-06-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Name of test material (as cited in study report): Silastol H 200-TS
- CAS No.: 68987-29-1
Target gene:
TA97a: hisD6610, uvrB, pKM 101, rfa
TA 98: hisD3052, uvrB, pKM 101, rfa
TA 100: hisG46, uvrB, pKM 101, rfa
TA102: hisG428, pKM 101, rfa
TA1535: hisG46, uvrB, rfa.
Species / strain / cell type:
other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix, Aroclor induced
Test concentrations with justification for top dose:
first experiment: 5.009, 1.507, 0.500, 0.151, 0.051 mg/plate.
second experiment: 5.001, 2.502, 1.255, 0.625, 0.314 mg/plate.
As no complete dissolution was possible, undissolved particles were visible on the plates.
Vehicle / solvent:
vehicle: sterile water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) first experiment; preincubation 2nd experiment;
DURATION
- Preincubation period: 20 min.
- Exposure duration: 48 hours

SELECTION AGENT: histidin

NUMBER OF REPLICATIONS: 4 plates/ concentration

Toxicity Control
Performed in experiment 1 only analogously to the titre control with the maximum dose of test item with and without S9 on maximal-soft agar.
Evaluation criteria:
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Key result
Species / strain:
other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
First Experiment
Confirmation of the Criteria and Validity
The treatments for the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.
Solubility and Toxicity
The test item was suspended in deionised water. All concentrations were weighed directly. As no complete dissolution was possible, undissolved particles were visible on the plates. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test item is stated as not mutagenic under the test conditions.
To verify this result, a second experiment was performed using the pre-incubation method.
Second Experiment
Confirmation of the Criteria and Validity
The treatments for the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls showed mutagenic effects with and without metabolic activation.
Solubility and Toxicity
The test item was suspended in deionised Water. All concentrations were weighed directly. As no complete dissolution was possible, undissolved particles were visible on the plates. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not significantly reduced.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions.
Conclusions:
Under the conditions of the test, the test item didn't show mutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 of S. typhimurium were exposed to Silastol H 200 TS, at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation, (S9 mix; Aroclor induced rat liver).

The initial Mutation Test was performed as plate incorporation method; the confirmatory and complementary assays were performed according to the pre-incubation method.

No signs of toxicity towards the bacteria could be observed. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item didn't show any mutagenic effects in both experiments.

The test item Silastol H 200-TS is considered as "not mutagenic under the conditions of the test".

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: other: clastogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-14 to 2012-02-23
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - 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 (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
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: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment:
with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL

Experiment I:
without metabolic activation: 15.6, 250, 500 and 1000 µg/mL
with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL

Experiment II:
without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL
with metabolic activation: 900, 1600 and 1800 µg/mL
Vehicle / solvent:
-Vehicle (s)/solvent(s) used: cell culture medium (MEM)
-Justification for choice of solvent/vehicle: The test item was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test item was well suspended. The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation Migrated to IUCLID6: 400 and 900 µg/mL
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 0.83 µg/mL
Details on test system and experimental conditions:
TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10^4 - 5 x 10^4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 300 cells
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell density
Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).


Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Results of chromosome analysis
without metabolic activation
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%) rel. Cell density (%)  Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other incl. Gaps excl. Gaps
Experiment I                              
negative control 200 - 5 4 0 0 0 0 1 1 100 100 2 4.5 2.5
15.6 µg/mL 200 no 5 0 0 0 0 0 0 0 103 96 3 2.5 0.0
250 µg/mL 200 no 6 2 0 1 0 0 0 0 81 78 3 3.5 1.5
500 µg/mL 200 yes 4 3 2 0 0 0 2 0 53 55 1 5.0 3.0
1000 µg/mL 200 yes 4 1 0 0 1 0 0 0 51 44 0 3.0 0.5
EMS (900 µg/mL) 200 - 7 10 8 2 0 1 2 0 85 94 3 12.5 9.5
Experiment II                                  
negative control 200 - 1 4 0 0 0 0 0 0 100 100 2 2.5 2.0
15.6 µg/mL 200 no 6 3 0 1 0 0 0 0 76 102 3 4.5 2.0
31.3 µg/mL 300 no 5 7 1 1 0 0 0 0 74 94 1 4.7 3.0
62.5 µg/mL 200 yes 2 2 0 0 0 0 1 0 51 89 1 2.5 1.5
125.0 µg/mL 200 yes 1 2 0 0 0 0 1 0 30 57 1 2.0 1.5
EMS (400 µg/mL) 200 - 7 17 3 3 3 0 0 0 72 103 0 13.0 10.5
Results of chromosome analysis
with metabolic activation
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%) rel. Cell density (%)  Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other incl. Gaps excl. Gaps
Experiment I                              
negative control 200 - 6 2 0 0 0 0 0 0 100 100 0 4.0 1.0
15.6 µg/mL 300 no 4 3 3 1 0 0 2 0 96 102 1 4.0 3.0
500 µg/mL 200 no 2 0 2 0 0 0 0 0 90 86 0 2.0 1.0
1000 µg/mL 200 yes 3 0 0 0 0 0 0 0 62 85 1 1.5 0.0
1500 µg/mL 200 yes 2 1 0 0 1 0 0 0 54 63 1 2.0 0.5
CPA (0.83 µg/mL) 200 - 3 12 11 1 0 1 1 0 104 85 3 12.0 10.5
Experiment II                                  
negative control 200 - 4 5 0 1 1 0 1 0 100 100 2 5.5 3.5
900 µg/mL 200 no 1 3 0 0 0 0 0 0 71 88 0 2.0 1.5
1600 µg/mL 200 yes 3 3 0 0 0 0 0 0 54 75 0 2.5 1.5
1800 µg/mL 200 yes 0 2 0 0 0 0 0 1 45 56 0 1.5 1.5
CPA (0.83 µg/mL) 200 - 3 9 3 3 1 1 2 1 96 100 0 10.0 9.0
Conclusions:
In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item (CAS39322-78-6) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.
Executive summary:

To investigate the potential of Afilan V5756 to induce structural chromosome aberrations in Chinese hamster V79 cells, anin vitrochromosome aberration assay was carried out.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation.

Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations, except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 200 cells for the 1st culture and 100 cells for the 2nd culture.

The test item was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test item was well suspended.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 15.6, 250, 500 and 1000 µg/mL

with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL

Experiment II:

without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL

with metabolic activation: 900, 1600 and 1800 µg/mL

Precipitation of the test item was observed with and without metabolic activation in both experiments.

Toxic effects of the test item were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure) toxic effects of the test item were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

The positive controls induced the appropriate responses.

There was no evidence of Afilan V5756 induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data. 

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:
2011-09-26 to 2012-02-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment for experiment I (without metabolic activation):
15.6, 31.3, 62.5, 125, 500, 1000 and 2500 µg/mL
Pre-experiment for experiment I (with metabolic activation):
31.3, 62.5, 125, 250, 500, 1000, 1750 and 2500 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
125, 250, 500, 750, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600 and 2800 µg/mL
Experiment I
without metabolic activation: 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL
and with metabolic activation: 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL
Experiment II
without metabolic activation: 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL
and with metabolic activation: 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL

Vehicle / solvent:
Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test item was supended in cell culture medium and processed by ultrasound for 5 min.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation Migrated to IUCLID6: 300 µg/mL
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation Migrated to IUCLID6: 1 µg/mL and 1.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth; cloning efficiency
Evaluation criteria:
A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
yes (Experiment I without S9: ≥ 1500 μg/mL; experiment I with S9: ≥ 1000 μg/mL; Experiment II without S9: ≥ 75 μg/mL; Experiment II with S9:≥ 1200 μg/mL
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item Afilan V5756 is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

In a mammalian cell gene mutation assay (HPRT locus],V79 cells culturedin vitro were exposed to Afilan V5756 suspended in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of

- 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL (without metabolic activation, Experiment I)

- 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL (with metabolic activation, Experiment I)

- 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL (without metabolic activation, Experiment II)

- 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL (with metabolic activation, Experiment II).

Afilan V5756 was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation the relative growth was 24.6% for the highest concentration evaluated (2000 µg/mL). The highest biologically relevant concentration evaluated with metabolic activation was 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation the relative growth was 15.7% for the highest concentration (175 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 1400 µg/mL with a relative growth of 20.0%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.00 was found at a concentration of 500 µg/mL with a relative growth of 96.5%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.75 was found at a concentration of 1500 µg/mL with a relative growth of 23.9%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.43 was found at a concentration of 175 µg/mL with a relative growth of 15.7%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.21 was found at a concentration of 150 µg/mL with a relative growth of 101.3%.

The positive controls did induce the appropriate response. 

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

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

 

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16. July - 06. Aug 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform GLP study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine and tryptophan reversion
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
1; 3; 10;, 33; 100; 333; 1000; and 2500 µg/plate / experiment II without S9 mix
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II with S 9mix
Vehicle / solvent:
Deionised Water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Deionised Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 48 hours


NUMBER OF REPLICATIONS: 3 plates
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.
Key result
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:
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'.

Summary Tables
The assay was performed in two independent experiments. Each concentration, including the controls, was tested in triplicate.

Summary of Results Pre-Experiment and Experiment I

Metabolic
Activation

Test
Group

Dose Level
(µg/plate)

Revertant Colony Counts (Mean±SD)

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

 

 

 

 

 

 

 

 

Without

Deionised water

 

26±3

8±1

24±2

149±18

47±3

Activation

Untreated

 

26±4

10±4

25±5

135±17

60±6

 

Leomin PN pa

3

25±3

10±6

28±8

140±15

58±11

 

 

10

23±4

16±1

29±8

105±8

45±7

 

 

33

25±4

11±3

28±5

124±13

61±5

 

 

100

20±4

8±1

29±6

129±6

57±3

 

 

333

14±4R

2±1R

9±4R

53±4R

54±8

 

 

1000

2±1R

4±2R

7±2R

16±3R

57±10

 

 

2500

0±0R,P

0±0R,P

1±1R,P

2±1R,P

29±7R,P

 

 

5000

0±0R,P

0±0R,P

0±0R,P

0±0R,P

15±1R,P

 

NaN3

10

2702±63

 

 

2609±118

 

 

4-NOPD

10

 

 

384±6

 

 

 

4-NOPD

50

 

105±6

 

 

 

 

MMS

3.0 µL

 

 

 

 

1234±35

R     Reduced background growth
P       Precipitate

Metabolic
Activation

Test
Group

Dose Level
(µg/plate)

Revertant Colony Counts (Mean±SD)

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

 

 

 

 

 

 

 

 

With

Deionised water

 

19±3

12±3

31±2

134±13

58±8

Activation

Untreated

 

19±4

20±4

39±3

129±19

61±3

 

Leomin PN pa

3

24±8

10±2

38±10

157±6

58±6

 

 

10

21±4

13±2

41±3

132±19

57±13

 

 

33

18±3

13±1

37±2

138±4

61±5

 

 

100

14±7

12±4

38±5

124±10

69±7

 

 

333

11±5

14±1

30±5

91±11

65±13

 

 

1000

5±2R

5±1R

9±1R

45±3R

60±8

 

 

2500

0±1R,P

3±2R,P

5±2R,P

20±3R,P

38±2R,P

 

 

5000

0±1R,P

0±0R,P

0±0R,P

0±1R,P

28±2R,P

 

2-AA

2.5

276±18

196±22

1276±153

1977±146

 

 

2-AA

10

 

 

 

 

320±19

R     Reduced background growth
P       Precipitate

Summary of Results Experiment II

Metabolic
Activation

Test
Group

Dose Level
(µg/plate)

Revertant Colony Counts (Mean±SD)

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

 

 

 

 

 

 

 

 

Without

Deionised water

 

29±5

15±3

32±4

138±11

51±2

Activation

Untreated

 

34±1

14±3

31±11

139±12

52±6

 

Leomin PN pa

1

27±2

12±4

32±9

138±13

52±10

 

 

3

31±7

12±5

29±1

134±12

53±3

 

 

10

29±5

14±5

28±9

137±10

50±3

 

 

33

31±7

12±6

26±1

136±12

48±1

 

 

100

25±9R

6±4R

19±7R

71±2R

50±6

 

 

333

12±5R,P

0±0R,P

7±3R,P

32±3R,P

34±7R,P

 

 

1000

0±1R,P

0±0R,P

2±1R,P

4±2R,P

29±12R,P

 

 

2500

0±0R,P

0±0R,P

0±0R,P

0±0R,P

12±5R,P

 

NaN3

10

2655±112

 

 

2614±196

 

 

4-NOPD

10

 

 

628±6

 

 

 

4-NOPD

50

 

135±13

 

 

 

 

MMS

3.0 µL

 

 

 

 

240±27

R     Reduced background growth
P       Precipitate

Metabolic
Activation

Test
Group

Dose Level
(µg/plate)

Revertant Colony Counts (Mean±SD)

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

 

 

 

 

 

 

 

 

With

Deionised water

 

32±6

21±7

38±5

171±17

64±8

Activation

Untreated

 

37±1

18±4

32±5

164±20

54±2

 

Leomin PN pa

3

30±2

18±3

37±11

163±13

70±3

 

 

10

29±1

17±4

32±1

159±9

62±9

 

 

33

31±0

19±2

37±4

162±12

63±6

 

 

100

30±3

16±4

36±2

156±4

56±5

 

 

333

34±4P

15±5R,P

35±2P

127±3R,P

64±10P

 

 

1000

9±4R,P

8±4R,P

14±3R,P

54±3R,P

33±3R,P

 

 

2500

1±2R,P

1±2R,P

4±1R,P

18±4R,P

35±3R,P

 

 

5000

2±1R,P

0±0R,P

2±3R,P

1±1R,P

21±5R,P

 

2-AA

2.5

249±29

191±35

1275±69

1920±73

 

 

2-AA

10

 

 

 

 

273±18

R     Reduced background growth
P       Precipitate

Conclusions:
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.
Executive summary:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were 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:

without S9 mix                                  1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

with S9 mix 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background growth (for details and concentrations please refer to "Any other infromation on results").

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. They showed a distinct increase of induced revertant colonies.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

The test item (read across-substance, CAS 39322-78-6) was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were 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:

without S9 mix: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

with S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 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. 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.

 

In the second available Ames test another read across-substance (CAS 68987-29-1) was tested.

The study was also conducted according to OECD guideline 471. The strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 of S. typhimurium were exposed to the read across substance (CAS 68987 -29 -1), at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation, (S9 mix; Aroclor induced rat liver). The initial Mutation Test was performed as plate incorporation method; the confirmatory and complementary assays were performed according to the pre-incubation method. No signs of toxicity towards the bacteria could be observed. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The substance did not show any mutagenic effects in both experiments. The read across-substance is considered as not mutagenic.

 

HPRT Test:

In a mammalian cell gene mutation assay (HPRT locus), V79 cells culturedin vitro were exposed to the read across substance(CAS 39322 -78 -6) suspended in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of

- 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL (without metabolic activation, Experiment I)

- 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL (with metabolic activation, Experiment I)

- 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL (without metabolic activation, Experiment II)

- 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL (with metabolic activation, Experiment II).

The read across substance was tested up to cytotoxic concentrations.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.00 was found at a concentration of 500 µg/mL with a relative growth of 96.5%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.75 was found at a concentration of 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.43 was found at a concentration of 175 µg/mL with a relative growth of 15.7%. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.21 was found at a concentration of 150 µg/mL with a relative growth of 101.3%. The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background. In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item Afilan V5756 is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

 

Chromosome Aberration Study:

To investigate the potential of read across-substance (CAS39322-78-6) to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation.

Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations, except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 200 cells for the 1st culture and 100 cells for the 2nd culture.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 15.6, 250, 500 and 1000 µg/mL

with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL

Experiment II:

without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL

with metabolic activation: 900, 1600 and 1800 µg/mL

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

It can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the read across-substance (CAS39322-78-6) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.

Therefore, it is considered to be non-clastogenic in this chromosome aberration test.

 

Based on the data published on the ECHA homepage the second read across-substance, CAS 39322-78-6, is also not classified for genetic mutagenicity.

 

Conclusion: 3 different genetic toxicity studies were performed with appropriate read across substances according to OECD guidelines 471, 473 and 476 to determine the genetic toxicity potential. No genetic toxicity was observed. Therefore, the substance to be registered is not considered to have a genotoxic property.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.