Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro tests were performed (AMES test OECD 471, Chromosome aberration test OECD 473 and HPRT assay OECD 476).

The substance was shown to be negative with and without metabolic activation in all three tests.

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:
March 09, 2010 - April 21, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1 (Pre-Experiment), all strains:
Without and with S9-mix: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: methyl methane sulfonate, 3.0 µL/plate in deionised water for TA102
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide, 10 µg/plate in deionised water for TA 1535 and TA 100
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 10 µg/plate in DMSO for TA98 and 50 µg/plate in DMSO for TA1537
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2.5 µg/plate in DMSO for TA1535, TA1537, TA98 and TA100 and 10 µg/plate in DMSO for TA102
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 in agar (plate incorporation) and experiment 2 preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
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 the historical data
-the positive control substances should produce a significant increase in mutant colony frequencies

EVALUATION OF RESULTS
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:
Not performed.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
tested up to the highest recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at the dose level of 5000 µg/plate in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- In experiment I with metabolic activation, the data in the untreated control of strain TA 102 were slightly above the historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate.
Conclusions:
Interpretation of results (migrated information):
negative

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay, performed according to OECD 471 guideline and GLP principles.
Executive summary:

The genetic toxicity of C-SAT 090103 was assessed using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains with and without metabolic activation, in accordance with OECD 471 guideline and GLP principles. Precipitation was observed at the top dose of 5000 µg/plate in the presence of S9 -mix. No toxicity was observed in all strains.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with C-SAT 090103 up to the highest concentration of 5000 µg/plate tested, neither in the presence nor absence of metabolic activation (S9 mix) in two independently repeated experiments.

There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Based on the results it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

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:
14 January, 2015 - 24 April, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(2014)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Stability at higher temperatures: Yes
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture from healthy non-smoking volunteers (18 - 35 years) using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
The Average Generation Time (AGT) of the cells was determined (December 2014) 12.8 - 13.0 hours.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 3hr exposure; 24 hr fixation: 5.4, 17, 52, 164 and 512 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 5.4, 17, 52, 164 and 512 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 5.4, 17, 52, 164 and 512 µg/mL

First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 52, 164 and 512 µg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 5, 50, 100, 125, 150, 175, 200, 225, 250 and 275 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 50, 100, 200, 225, 250, 275, 300 and 350 µg mL
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix, 24 hr exposure; 24 hr fixation: 5, 125 and 175 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 50, 225 and 275 µg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: Mitomycin C in Hanks’ Balanced Salt Solution: 0.5 and 0.75 μg/ml for a 3 h exposure period, 0.2 and 0.3 μg/ml for a 24 h exposure period and 0.1 and 0.15 μg/ml for and 0.15 μg/ml for a 48 h exposure period.
Remarks:
without S9
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

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

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 150 metaphase chromosome spreads per culture.
Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Acceptability of the assay:
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

Data evaluation and statistical procedures:
A test substance is considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
24 h and 48 h continuous exposure time without S9-mix.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation in the exposure medium was observed at the highest dose level of 512 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 164 and 512 µg/mL in the absence of S9 for the continuous treatment of 24 and 48 hr.
No toxicity was observed up to and including the highest precipitating tested dose of 512 µg/mL in the absence and presence of S9, 3 hr treatment/24 hr fixation.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring in the absence of S9 for the continuous treatment of 24 and 48 hr.
Conclusions:
Interpretation of results (migrated information):
negative

A chromosome aberration study with GABEPRO® GPM-800 was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that GABEPRO® GPM-800 is not clastogenic in human lymphocytes.
Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of GABEPRO® GPM-800 (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles. In the first cytogenetic assay, GABEPRO® GPM-800 was tested up to and including the precipitating concentration of 512 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. In the second cytogenetic assay, GABEPRO® GPM-800 was tested up to and including cytotoxic concentrations of 50 and 275 μg/mL for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time respectively in the absence of S9-mix. GABEPRO® GPM-800 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of GABEPRO® GPM-800 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that GABEPRO® GPM-800 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

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:
March 18, 2010 - July 30, 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)
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-Guanine Phosphoribosyl Transferase (HPRT) in V79 cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts, Neomycin (5 µg/mL) and Amphotericin B (1 %).
- Properly maintained: yes
- 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:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 4 hours treatment: 32.8, 65.6, 131.3, 262.5, 525, 1050, 2100 and 4200 µg/mL
Without S9-mix, 24 hours treatment: 32.8, 65.6, 131.3, 262.5, 525, 1050, 2100 and 4200 µg/mL
Experiment 1:
Without S9-mix, 4 hours treatment: 7.8, 15.6, 31.3, 62.5, 125, 187.5 and 250 µg/mL
With S9-mix, 4 hours treatment: 15.6, 31.3, 62.5, 125, 500, 750 and 1000 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 3.1, 6.3, 12.5, 25, 50, 75, 100 and 125 µg/mL
With S9-mix, 4 hours treatment: 31.3, 62.5, 125, 250, 500 and 700 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test substance was dissolved in acetone. The solvent was chosen according to its solubility properties and its relative non-toxicity to the cells.

Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate (1.2 mM)
Remarks:
without S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 7,12-dimethylbenz(a)anthracene (4.3 µM)
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 4 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): 11 µg/mL thioguanine (6-TG)

NUMBER OF REPLICATIONS:
- Treatment groups and controls: Duplicate cultures

NUMBER OF CELLS EVALUATED: 1.0 x 10E6 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the cloning efficiency
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10E6 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 %.

EVALUATION OF RESULTS
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 SYSTAT11 (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.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
pH solvent control: 7.39
pH 4200 µg/mL: 7.41
- Effects of osmolality: No
Osm. solvent control: 373 mOsm
Osm. 4200 µg/mL: 332 mOsm
- Precipitation: In experiment I precipitation of the test item occurred at 62.5 µg/mL and above in the absence of metabolic activation and at 500.0 µg/mL and above in the presence of metabolic activation. In experiment II in the presence of metabolic activation, phase separation was observed at 250.0 µg/mL and above and precipitation was observed at 700.0 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was only observed at the highest dose level after the prolonged treatment period.

COMPARISON WITH HISTORICAL CONTROL DATA:
The highest solvent control exceeded the historical range of solvent control slightly. However, this effect was judged as irrelevant since it is very minor and the corresponding solvent control remained well within the range of historical controls. The positive controls showed a distinct increase in induced mutant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I, toxicity was observed at the highest precipitating dose levels with and without S9-mix.
In experiment II, toxicity was observed at the highest precipitating dose levels with S9-mix and highest non-precipitating dose levels without S9-mix.
Remarks on result:
other: strain/cell type: supplied by Laboratory for Mutagenicity Testing; Technical University, Germany
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that C-SAT 090103 is not mutagenic in the gene mutation test with V79 Chinese hamster cells, performed according to OECD 476 guideline and GLP principles.
Executive summary:

A Gene Mutation Assay in Chinese Hamster V79 Cells (V79/HPRT) with C-SAT 090103 with and without metabolic activation was performed according to OECD 476 guideline and GLP principles.

The dose ranges of the two main experiments were limited by precipitation and toxicity of the test substance. 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.

In conclusion it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, C-SAT 090103 is considered to be non-mutagenic in the HPRT assay.

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

Additional information

Ames test:

The genetic toxicity of C-SAT 090103 was assessed using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains with and without metabolic activation, in accordance with OECD 471 guideline and GLP principles. Precipitation was observed at the top dose of 5000 µg/plate in the presence of S9 -mix. No toxicity was observed in all strains.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with C-SAT 090103 up to and including the highest concentration of 5000 µg/plate tested, neither in the presence nor absence of metabolic activation (S9 mix) in two independently repeated experiments.

There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Based on the results it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Chromosome aberration test:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of GABEPRO® GPM-800, in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles. In the first cytogenetic assay, GABEPRO® GPM-800 was tested up to and including the precipitating concentration of 512 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. In the second cytogenetic assay, GABEPRO® GPM-800 was tested up to and including cytotoxic concentrations of 50 and 275 μg/mL for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time respectively in the absence of S9-mix. GABEPRO® GPM-800 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of GABEPRO® GPM-800 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that GABEPRO® GPM-800 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

Gene mutation assay:

A Gene Mutation Assay in Chinese Hamster V79 Cells (V79/HPRT) with C-SAT 090103 with and without metabolic activation was performed according to OECD 476 guideline and GLP principles.

The dose ranges of the two main experiments were limited by precipitation and toxicity of the test substance. 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.

In conclusion it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, C-SAT 090103 is considered to be non-mutagenic in the HPRT assay.


Justification for selection of genetic toxicity endpoint
No study was selected, since all three in vitro studies (according to OECD guidelines and in accordance with GLP principles) were negative.

Justification for classification or non-classification

Based on the available data on genetic toxicity, the substance needs not to be classified for genotoxicity according to Regulation (EC) No. 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.