Reaction mass of 1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one and 1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative
In vitro chromosome aberration test (OECD TG 473): negative
Genemutation in mammalian cells (OECDTG 490): negative

Link to relevant study records

Reference

Reference 1

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:

18 June, 2015 - 31 August, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(2014)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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 from healthy adult male volunteers by venapuncture 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.
- Average Generation Time (AGT): 12.8 - 13.0 h
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) 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 (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight)

Test concentrations with justification for top dose:

Dose range finding test:
With and 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
First cytogenetic test:
With and without S9-mix, 3 h exposure time, 24 h fixation time: 5, 50, 75, 100, 125, 150, 175 and 200 µg/mL
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix, 3 h exposure, 24 h fixation time: 5, 100 and 125 µg/ mL
With S9-mix, 3 h exposure, 24 h fixation time: 5, 100 and 150 µg/ mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 50, 75, 100, 125 and 150 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 5, 10, 20, 30, 40, 50, 75 and 100 µg mL
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 75 and 100 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 10, 30 and 75 µg mL

Vehicle / solvent:

- Vehicle used: dimethyl sulfoxide
- Justification for choice of vehicle: A solubility test was performed to select the appropriate vehicle. The substance did not form a homogeneous suspension in culture medium. Consequently, GALBASCONE was dissolved in dimethyl sulfoxide of spectroscopic quality and dimethyl sulfoxide is accepted and approved by authorities and international guidelines. GALBASCONE concentrations were used within 2.5 hours after preparation.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Mitomycin C (in HBSS) was used at a final concentration of 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 a 48 h exposure period.

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide (in HBSS) was used at a final concentration of 10 μg/mL for a 3 h exposure period (24 h fixation time).

Remarks:

with S9

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

ENVIRONMENTAL CONDITIONS:
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 65 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 -37.4°C).

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

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:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.

A test substance is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) All results are inside the 95% control limits of the negative historical control data range.

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 Fisher’s exact test.
In case the Fisher’s exact test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

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
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 200 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 164 µg/mL and above in the absence and presence of S9, 3 hr treatment/24 hr fixation; at dose levels of 52 µg/mL and above in the absence of S9 for the continuous treatment of 24 hr and at dose levels of 17 µg/mL and above for the continuous treatment of 48 hr.

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.

Conclusions:

A chromosome aberration study with the substance was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that the substance is not clastogenic in human lymphocytes.

Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles.

In the first cytogenetic assay, the substance was tested up to and including cytotoxic concentrations of 125 and 150 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9 -mix, respectively. In the second cytogenetic assay, the substance was tested up to and including the cytotoxic concentration of 100 μg/mL for a 24 h exposure time with a 24 h fixation time and up to and including the cytotoxic concentration of 75 μg/mL for a 48 h exposure time with a 48 h fixation time in the absence of S9 -mix.

Reliable positive and negative controls were included. The substance Galbascone 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. Although in the second cytogenetic assay at the highest dose of 100 μg/ml at the 24 h exposure time the number of chromosomal aberrations was statistically different compared to the control, the number of aberrations was within the historical data range (7 cells with aberrations per 300 metaphases compared with control limits (+ gaps) -1.79 – 3.51 per 100 metaphases and (- gaps) -1.51 – 2.84 per 100 metaphases) and therefore this increase was considered not biologically relevant. No effects of the substance 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 the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

Finally, it is concluded that the substance is not clastogenic in human lymphocytes.

 

Reference 2

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:

13 July 2005 - 26 August 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day)

Test concentrations with justification for top dose:

- Dose range finding test:
TA 100 and WP2uvrA (without and with S9): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

- Experiment 1 and experiment 2:
TA 98, TA 100, TA 1535 and TA 1537 (without and with S9): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
WP2uvrA (without and with S9): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be miscible in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a postive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was not observed up to and including the concentration of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES: The test material was toxic at and above 500 µg/plate to strain TA100 but was not toxic to WP2 uvrA.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella strains initially at 500 µg/plate. The test material was not toxic to E.coli strain WP2 uvrA.

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent direct plate experiments up to and including 5000 µg/plate, in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose range finding test (TA100 >= 500 µg/plate, WP2uvrA was not toxic). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiment. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.

Reference 3

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:

12 October, 2015 - 07 December, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium: medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- 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, 3 hours treatment: 5.4, 17, 52, 164 and 512 µg/mL
Without S9-mix, 24 hours treatment: 5.4, 17, 52, 164 and 512 µg/mL

Experiment 1:
Without S9-mix, 3 hours treatment: 0.1, 1, 10, 20, 30, 35, 40, 45, 50 and 60 µg/mL
With S9-mix, 3 hours treatment: 0.1, 0.5, 1, 5, 10, 25, 50, 55, 60, 65, 70 and 80 µg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus :
Without S9-mix, 3 hours treatment: 1, 10, 20, 30, 35, 40, 45 and 50 µg/mL
With S9-mix, 3 hours treatment: 0.1, 0.5, 1, 5, 10, 50, 55 and 60 µg/mL

Experiment 2
Without S9-mix, 24 hours treatment: 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 75 and 100 µg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus:
Without S9-mix, 24 hours treatment: 0.1, 1, 5, 10, 30, 40, 50 and 60 µg/mL

Vehicle / solvent:

- Solvent used: DMSO.
- Justification for choice of solvent: The test item was dissolved in dimethyl sulfoxide. DMSO has been accepted and approved by authorities and international guidelines. No correction was made for the purity/composition of the test item.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate in DMSO at concentrations of 15 and 5 μg/mL for the 3 and 24 hours treatment periods, respectively.

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide in HBSS at a final concentration of 7.5 μg/mL.

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: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and 32-180 (24 hours treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). At least 40% of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10^-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if:none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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 data.
- Effects of osmolality: No data.
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 164 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES: GALBASCONE (multi-constituent) precipitated in the exposure medium at concentrations of 164 μg/mL and above, the concentration used as the highest test item concentration for the dose range finding test was 512 μg/mL.
In the absence of S9-mix after 3 hours treatment, the relative suspension growth was 11% at the test item concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 164 μg/mL and above.
In the presence of S9-mix after 3 hours treatment, the relative suspension growth was 73% at the test item concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 164 μg/mL and above.
In the presence of S9-mix after 24 hours treatment, the relative suspension growth was 24% at the test item concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 164 μg/mL and 512 μg/mL after 24 hours treatment and no cells survived the 24 hours subculture period.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First experiment:
In the absence of S9-mix, the dose levels of 0.1 and 1 μg/mL showed no cytotoxicity. Therefore, the dose level of 0.1 μg/mL was not regarded relevant for mutation frequency measurement. The dose level of 60 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing. The relative total growth of the highest test item concentration was 11% compared to the total growth of the solvent controls.
In the presence of S9-mix, the dose levels of 25 and 65 to 80 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing or showed an inconsistent RSG (25 μg/mL). The relative total growth of the highest test item concentration was 14% compared to the total growth of the solvent controls.
Second experiment:
The dose levels of 20 and 30 μg/mL showed similar cell growth delay, therefore, the dose level of 20 μg/mL was not regarded relevant for mutation frequency. The dose levels of 75 and 100 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The relative total growth of the highest test item was 20% compared to the total growth of the solvent controls.

Conclusions:

A mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.
It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of the study.

Executive summary:

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

In the first experiment, the substance was tested up to and including concentrations of 50 and 60 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. The relative total growth (RTG) was 11 and 14% in the absence and presence of S9-mix, respectively.

In the second experiment, the substance was tested up to a concentration of 60 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 20%.

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the substance did not induce a significant increase in the mutation frequency.

It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent direct plate experiments up to and including 5000 µg/plate, in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose range finding test (TA100 >= 500 µg/plate, WP2uv rA was not toxic). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiment. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.

Chromosome aberration:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles.

In the first cytogenetic assay, the substance was tested up to and including cytotoxic concentrations of 125 and 150μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9 -mix, respectively. In the second cytogenetic assay, the substance was tested up to and including the cytotoxic concentration of 100μg/mL for a 24 h exposure time with a 24 h fixation time and up to and including the cytotoxic concentration of 75μg/mL for a 48 h exposure time with a 48 h fixation time in the absence of S9 -mix.

Reliable positive and negative controls were included. The substance 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. Although in the second cytogenetic assay at the highest dose of 100μg/ml at the 24 h exposure time the number of chromosomal aberrations was statistically different compared to the control, the number of aberrations was within the historical data range (7 cells with aberrations per 300 metaphases compared with control limits (+ gaps) -1.79 – 3.51 per 100 metaphases and (- gaps) -1.51 – 2.84 per 100 metaphases) and therefore this increase was considered not biologically relevant. No effects of the substance 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 the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

Finally, it is concluded that the substance is not clastogenic in human lymphocytes.

Mouse lymphoma:

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

In the first experiment, the substance was tested up to and including concentrations of 50 and 60 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. The relative total growth (RTG) was 11 and 14% in the absence and presence of S9-mix, respectively.

In the second experiment, the substance was tested up to a concentration of 60 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 20%.

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the substance did not induce a significant increase in the mutation frequency.

It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system.

 

Justification for classification or non-classification

Based on the results of the Ames, chromosome aberration and mouse lymphoma studies, the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and GHS.