Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All three in vitro tests (Ames-Test, Chromosome aberration assay and HPRT gene mutation assay) were negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
uvrB
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment IIA: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/plate
Pre-ExperimenUExperiment I and II: 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and relatively non-toxic to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA 1535 and TA 100 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-NOPD
Remarks:
for TA 1537 and TA 98 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
for TA 102 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
not specified
Positive control substance:
other: 2-aminoanthracene
Remarks:
for all strains with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets
the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of
our historical data
- the positive control substances should produce a significant increase in mutant colony
frequencies

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 TA 102) 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:
S. typhimurium TA 1535
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
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
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
Species / strain:
S. typhimurium TA 100
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
Species / strain:
S. typhimurium TA 102
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

The plates incubated with the test item showed reduced background growth with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the 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 and showed a distinct increase of induced revertant colonies.

Conclusions:
Based on the results of a study according to OECD Test Guideline 471 under GLP, the test substance is considered not to be mutagenic.
Executive summary:

The registered substance has been tested in a GLP-study according to OECD test guideline 471. The strain salmonella typhimurium TA 98, 100, 102, 1535 and 1537 were used without and with metabolic activation by S9. Appropriate, strain-specific controls were used giving valid results. The test substance was applied in suspension.

As the test substance did not give any positive result, it is considered not to be mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
N/A
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Without S9-mix: 0.01 to 3000 μg/ml
With S9-mix: 0.2 to 3000 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: solubility properties and relative non-toxicity to cell cultures
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
EMS without S9 and CPA with S9
Details on test system and experimental conditions:
Experiment 1:
- with and without S9
- exposure: 4h
- recovery: 14h
- preparation interval: 18h

Experiment 2:
- without S9
- exposure: 18h
- preparation interval: 18h
Evaluation criteria:
The chromosome aberration test performed in our laboratory is considered acceptable, if it meets the following criteria:
a) The number of structural aberrations found in the solvent controls falls within the range of the laboratory's historical control data.
b) The positive control substances produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory's historical control data.

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory's historical control data.
and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory's historical control data.
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).
However, both biological and statistical significance should be considered together. If the criteria mentioned above 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.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:
A test item can be classified as aneugenic if:
the number of induced numerical aberrations is not in the range of the laboratory's historical control data).
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

The test item, dissolved in tetrahydrofuran (THF), 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.

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 18 hours without 89 mix. The chromosomes were prepared 18 hours after start of treatment with the test item.

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

Neither precipitation nor relevant influence of the test item on pH value or osmolarity was observed (solvent control 380 mOsm, pH 7.3 versus 383 mOsm and pH 7.3 at 1.9 μg/mL).

In Experiment I in the absence of S9 mix clear cytotoxicity indicated by reduced cell numbers (27.4 % of control) and reduced mitotic indices (43.8 % of control) was observed at the highest evaluable concentration (0.9 μg/mL). In the presence of S9 mix no cytotoxicity indicated by reduced cell numbers and/or mitotic indices was observed up to the highest evaluable concentration of 15.0 μg/mL. However, the next dose level of 30.0 μg/mL was not evaluable. In Experiment II in the absence of S9 mix mitotic index was reduced to 46.5 % at the highest evaluable concentration (7.5 μg/mL).

In Experiment I in the absence of S9 mix the number of aberrant cells, excluding gaps (4.5 %) at 0.9 μg/mL slightly exceeded the laboratory's historical solvent control data range (0.0 - 4.0 % aberrant cells excluding gaps). However, this value was not statistically significant, when compared to the responsible solvent control value (3.5 % aberrant cells excluding gaps). In the presence of S9 mix no statistically significant or biologically relevant increase in the number of aberrant cells was observed up to the highest

evaluated concentration. The aberration rates of the cells after treatment with the test item (0.5 - 2.5 % aberrant cells, excluding gaps) were within the range of the laboratory's historical control data (0.0 - 4.0 % aberrant cells, excluding gaps).

In Experiment II in the absence of 89 mix the number of aberrant cells, excluding gaps (4.0 %) at 3.8 μg/mL slightly exceeded the laboratory's historical solvent control data range (0.0 - 3.5 % aberrant cells excluding gaps). Compared to the respective solvent control this value was statistically significantly increased. This effect however, was judged to be based upon the rather low solvent control of 1.0 % aberrant cells, excluding gaps.

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

In both experiments, either EMS (500 or 900 μg/ml) or CPA (1.0 μg/ml) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce relevant structural chromosome aberrations in V79 cells (Chinese hamster cell line), when tested up to cytotoxic and/or the highest evaluable

concentrations.

Conclusions:
In a chromomal aberration test according to OECD Test Guideline 473 under GLP, the test substance did not induce relevant structural chromosome aberrations in V79 cells.
Executive summary:

The test substancedissolved in acetone was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in-vitro in two independent experiments according to OECD guideline 473. The following study design was performed:

 

without S9-mix with S9-mix
  exp. I exp. II exp. I
Exposure period 4 h 18 h 4 h
Recovery 14 h - 14 h
Preparation interval 18 h 18 h 18 h

 

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase cells were scored for structural chromosome aberrations, except for the positive control in experiment II where only 50 metaphase cells were scored. Test item concentrations between 11.7 and 3500 µg/ml were applied in the absence and presence of S9 mix only. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. Clear toxicity was pbserved in both experiments in the absence of S9 mix. In the presence of S9, no cytotoxicity was observed. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment. No increase in the frequencies of polyploid metaphases was found after treatment as compared to the frequencies of the controls.In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in-vitro. Therefore, the test substance is considered to be non-clastogenic.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01/12/2009-06/05/2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment I (4h)
without S9: 0.1, 0.2, 0.4, 0.8, 1.6, 2.3, 3.1 microg/mL
with S9: 1.6,, 3.2, 6.3, 12.5, 18.8, 25.0, 37.5 microg/mL
Experiment II:
without S9: 0.8, 1.5, 3.0, 6.0, 8.0, 10.0, 12.0 microg/mL
with S9: 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0, 10.0, 15.0, 20.0 microg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: THF was a suitable vehicle
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
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
- Preincubation period: 24 h
- Exposure duration: 4 and 24 h

SPINDLE INHIBITOR (cytogenetic assays): 6-TG
STAIN (for cytogenetic assays): methylene blue

NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
the numbers of mutant colonies per 10^6 cells found in the solvent controls fall within the laboratory historical control data range.
the positive control substances must produce a significant increase in mutant colony frequencies.
the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria.

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
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 non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations 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.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item, were 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 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

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

The study was performed in two independent experiments, using the following experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 24 hours without metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix: 0.1, 0.2, 0.4 and 0.8 μg/ml

with S9 mix: 1.6, 3.1, 6.3 and 12.5 μg/ml; 18.8 μg/ml in culture II

Experiment II:

without S9 mix: 1.3, 2.5, 5.0, 7.5 and 10.0 μg/ml

The maximum evaluated concentrations of the test item in the main experiments were limited by relevant test item induced cytotoxicity. Al the highest evaluated test item concentration of 0.8 μg/ml following 4 hours treatment in the absence of metabolic activation the relative cloning efficiency I was reduced to 0.3 and 28.5 % of the solvent control. After 4 hours treatment with 89 mix the relative cloning efficiency I was reduced to 22.8 % ( 12.5 μg/ml) or 11.3 % (18.8 μg/ml) of the solvent control group. Following 24 hours treatment the relative cloning efficiency I was reduced to 39.1 and 54.4 % of the solvent control at 10.0

μg/ml. Al the same test item concentration the cell densities at the first subculturing step were 15.5 and 23.5 % of the solvent control.

No relevant and reproducible increase in mutant frequencies compared to the historical range of solvent controls was observed in the main experiments up to the highest evaluable concentration. However, following 4 hours incubation the levels of mutant frequencies

(105.5 mutant colonies per 10^6 cells) after treatment with a test item concentration of 0.8 μg/ml exceeded the historical data range (3.4 to 31. 7 mutant colonies per 10^6 cells) in the absence of S9 mix. Therefore, the induction factor was 15.0 and far higher than 3.0, which implies a more than threefold increase in mutation frequency.

As well, the threshold of a threefold increase in mutation frequencies was exceeded at 0.8 μg/ml in the absence of S9 mix and at 6.3 μg/ml in the presence of metabolic activation, both following 4 hours treatment in culture II.

These effects are not considered to be biologically relevant as they were either not reproducible or dependent on a rather low mutation rate in the solvent control. In all other evaluated dose groups the mutation frequency (5.1 to 29.8 % mutant colonies per 10^6 cells) was within the corresponding historical data range.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency using 8Y8TA T®11 statistics software. Two significant dose dependent trend of the mutation frequency indicated by a probability value of

<0.05 was determined in the first experiment at culture I and II without metabolic activation.

However, due to the reasons mentioned above these trends were judged as biologically irrelevant.

EMS (75 and 150 μg/ml) and DMBA (1.1 μg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

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.

Conclusions:
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, Benzaldehyde, 2-hydroxy-5-nonyl- is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of Benzaldehyde, 2-hydroxy-5-nonyl- to induce gene mutations at the HPRT locus in 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 24 hours without metabolic activation.

The highest concentration (3000.0 µg/mL, approx. 10 mM) used in the range finding pre-experiment was chosen with respect to the purity of the test item and the current OECD guideline 476. The dose range of the main experiments was limited by test item induced cytotoxicity.

The dose ranges of the first main experiment (with and without S9 mix) and the second main experiment (without S9 mix) were limited by test item induced cytotoxicity.

The tested concentrations are described in above. The experimental points evaluated for mutation rate are the following:

Experiment I (4h)

without S9: 0.1, 0.2, 0.4, 0.8, microg/mL

with S9: 1.6, 3.2, 6.3, 12.5, microg/mL

Experiment II:

without S9: 0.8, 1.5, 3.0, 6.0, 8.0, 10.0, 12.0 microg/mL

with S9: 2.5, 5.0, 10.0, 15.0 microg/mL

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames-Test

This study was performed to investigate the potential of Benzaldehyde, 2-hydroxy-5-nonyl-, oxime, branched to induce gene

mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. Due to strong toxic effects in experiment II without S9 mix in strains TA 1535, TA 1537, TA 98 and TA 100 this part was

repeated with reduced concentrations (reported as experiment Ila). The test item was tested at the following concentrations:

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

Experiment II a: 0.03; 0. 1; 0.3; 1; 3; 10; 33; and 100 μg/plate

The plates incubated with the test item showed reduced background growth with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was

observed following treatment with Benzaldehyde, 2-hydroxy-5-nonyl-, oxime, branched 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.

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.

Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

HPRT-Test

The study was performed to investigate the potential of Benzaldehyde, 2-hydroxy-5-nonyl-, oxime, branched to induce gene

mutations at the HPRT locus in 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 24 hours without metabolic activation.

The highest concentration (3000.0 μg/ml, approx. 10 mM) used in the range finding preexperiment was chosen with respect to the purity of the test item and the current OECD guideline 476. The dose range of the main experiments was limited by test item induced

cytotoxicity.

The dose ranges of the first main experiment (with and without S9 mix) and the second main experiment (without 89 mix) were limited by test item induced cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

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 material is considered to be non-mutagenic in this HPRT assay.

Chromosome aberration assay

The test item, dissolved in tetrahydrofuran (THF), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments.

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

The highest applied concentration of 3000.0 μg/ml (approx. 10 mM) was chosen with regard to the molecular weight 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.

Clear cytotoxicity was observed in Experiments I and II in the absence of S9 mix at the highest evaluable concentration. In the presence of S9 mix no cytotoxicity was observed up to the highest evaluable concentrations.

In Experiment I in the absence of S9 mix the number of aberrant cells, excluding gaps slightly exceeded the laboratory's historical solvent control data range and increased in a dose-related manner but were not statistically significant. In the presence of S9 mix no

clastogenicity was observed. In Experiment II in the absence of S9 mix a single statistically significant increase in the number of aberrant cells slightly exceeding the laboratory's historical solvent control data range was observed. This effect however, is judged to be based upon the rather low solvent control.

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.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce relevant 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 cytotoxic and/or the highest evaluable concentrations.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and the in vitro chromosome aberration assay (OECD 473, GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.