Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation (Bacterial reverse mutation assay / Ames test), Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 & E. coli WP2 uvr A: negative with and without metabolic activation [EU Method B.13/14, GLP]

- Chromosome aberration, Chinese hamster lung fibroblasts (V79): negative with and without metabolic activation [EU Method B.10, GLP]

- Gene mutation (mammalian cell gene mutation assay), Chinese hamster (Cricetulus griseus) ovary cell line CHO-K1: negative [OECD 476, GLP]

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-04-22 - 2015-11-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented GLP OECD 476 guideline study without deviations on the registered substance itself.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
OECD Guideline No. 476 for testing of chemicals, “In Vitro Mammalian Cell Gene Mutation Test”, adopted: July 21, 1997 (OECD, 1997)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Government of India, Department of Science and Technology, National Good Laboratory Practice (GLP) Compliance Monitoring Authority; No GLP/C-065/2014
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine phosphoribosyl transferase (HGPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
Ham’s F-12 medium supplemented with sodium bicarbonate, antibiotics, and L-glutamine was the basic medium.
Basic medium supplemented with 10% fetal bovine serum (FBS) was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells.
Basic medium without serum was the treatment medium and was used for target cell exposure to the test item and controls.
Cloning medium was basic medium supplemented with 20 % FBS and was used for the determination of cell viability or plating/cloning efficiency.
Selective medium was basic medium supplemented with 20 % FBS and the selective agent 6-Thioguanine (6-TG) at 35 µM and was used for the selection of mutants.

- Periodically checked for Mycoplasma contamination: This cell line was screened for the absence of mycoplasma contamination at the Mycoplasma Laboratory, Statens Serum Institut, Copenhagen, Denmark and was certified free of mycoplasma contamination on 08 August 2014.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 with the co-factors
Test concentrations with justification for top dose:
0, 2.5, 5, 10, 20, 40, 80, 160, 320 and 500 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is one of the organic vehicles compatible with this test system. Hence, based on the results of solubility test, DMSO was selected as the vehicle of choice.
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Remarks:
8 µg/ml 3-MCA +S9, 600 µg/ml EMS -S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): 9 days

SELECTION AGENT (mutation assays): 6-Thioguanine (6TG)

NUMBER OF REPLICATIONS: Triplicates (CE); 5 replicates (mutant phenotype determination)

NUMBER OF CELLS EVALUATED: n/a

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
There are several criteria for determining a positive result, such as a concentration related, or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response.
A test item, for which the results do not meet the above criteria is considered non mutagenic in this system.
Statistics:
A power transformation procedure (Snee, R.D., and Irr, J.D. (1981). Design of a statistical method for the analysis of mutagenesis at the hypoxanthene-guanine phosphoribosyl transferase locus of cultured chinese hamster ovary cells. Mutation Research 85: 77-93.) with which the observed mutant frequency was transformed using the formula:

Y = (X + A) B
where,
Y = transformed mutant frequency
X = observed mutant frequency
and A, B = constants.

Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p ≤ 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p ≤ 0.05).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH ways not altered by the test item
- Effects of osmolality: osmolality was not altered by the test item
- Water solubility: test item did not precipitate in the test medium at any of the tested concentrations
- Precipitation: test item did not precipitate in the test medium at any of the tested concentrations

RANGE-FINDING/SCREENING STUDIES:
At the end of 3-hour exposure within the cytotoxicity test, the test item did not precipitate in the test medium at any of the tested concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA:
The frequency of mutants in the DMSO control was within the range of the in-house historical control data.

Results

S9 Characterization

a.   Sterility Check

      

      The S9 homogenate was found to be sterile.

 

b.   Metabolic Activation

 

      The S9 homogenate was found to be active as evidenced by its ability to metabolize the promutagens, 2-Aminoanthacene and Benzo(a)pyrene to mutagens usingS. typhimuriumstrainTA 100.

 

c.    Protein Content

 

The protein content of the S9 homogenate was 30 and 27.75 mg/mL for batches 37 and 38, respectively.

 

SolubilityTest and Justification for the Selection of Vehicle

The test item was insoluble in water and Ethanol. The test item formeda workable suspension in DMSO at 50 mg/mL.

 

DMSO is one of the organic vehicles compatible with this test system. Hence, based on the results of solubility test, DMSO was selected as the vehicle of choice to prepare the stock and dilutions of the test item as well as the positive controls.

 

Cytotoxicity Testand Justification for the Selection of Test Doses

At the end of 3-hour exposure, the test item did not precipitate in the test medium at any of the tested concentrations.

 

At the end of 3-hour exposure, the pH of the test medium in the presence of metabolic activation ranged from 7.34 to 7.39 with 7.28 in the DMSO control while in the absence of metabolic activation it was between 7.42 and 7.49 with 7.40 in the DMSO control.

 

 

At the end of 3-hour exposure, in the presence of metabolic activation, osmolality of the test medium at the highest test item treatment level
(500
mg/mL) was 0.403 OSMOL/kg, whereas the corresponding osmolality in the DMSO control was 0.402 OSMOL/kg.

 

Similarly, at the end of 3-hour exposure, in the absence of metabolic activation, osmolality of the test medium at the highest test item treatment level (500mg/mL) was 0.407 OSMOL/kg, whereas the corresponding osmolality in the DMSO control was 0.396 OSMOL/kg.

 

The Relative Cloning Efficiency (RCE) at the highest tested concentration of 500 µg/mL was 48 and 42% of the DMSO control, in the presence and absence of metabolic activation, respectively.

 

The test item did not show evidence of significant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to vehicle control) at any of the tested concentrations either in the presence or absence of metabolic activation.

 

Based on the results of cytotoxicity and the limit of solubility, a maximum of 500 µg/mL was tested in the gene mutation assay.

 

Stability of Test Item in the Vehicle and Analytical Verification of Dosing Formulations

Dose formulations of the test item at 5000 and 50000 µg/mL were found to be stable in DMSO for 24 hours at room temperature.

 

The dose formulation concentration analysis results for the initial gene mutation assay indicate that the mean of % agreement with claimed concentrations were 112.59 and 114.22 % (with an RSD of 1.60 and 0.88 %, respectively) of their respective claimed concentrations of 6300 and 50000 µg/mL,confirming that the concentration of the test item was within the acceptable limits (± 15% of claimed concentrations and < 10 % RSD). No test item was detected in the vehicle control.

 

The concentration analysis results of the dose formulation samples of the confirmatory gene mutation assayindicate that the mean of % agreement with claimed concentrations were 102.99 and 98.77 % (with an RSD of 2.93 and 1.56 %, respectively) of their respective claimed concentrations of 5000 and 50000 µg/mL,confirming that the concentration of the test item was within the acceptable limits. No test item was detected in the vehicle control.

 

These results from the initial and confirmatory mutation assays indicate that the respective top concentrations were achieved and support the validity of the study conclusion.

 

Gene Mutation Assay

Initial Gene Mutation Assay: Experiment 1 & 2 - Presence and Absence of Metabolic Activation

 

There was no evidence of excessive cytotoxicity (i.e., <10 % RCE) at any of the tested concentrations either in the presence or absence of metabolic activation. The RCE values in the presence of metabolic activation, ranged from 47.8 to 82.1 % while in the absence of metabolic activation, ranged from 41.2 to 78.8 % compared to the vehicle control.

 

The frequency of mutants in the DMSO control was within the range of the in-house historical control data.

 

The test item did not cause a significant increase in the frequencies of mutants compared to the vehicle control in the presence or absence of metabolic activation at any of the tested concentrations.

 

Under similar conditions the positive controls 3-methylcholanthrene (3-MCA) and ethyl methanesulphonate(EMS) both inducedstatisticallysignificant increases in the mutant frequency as compared with the vehicle control.

 

 

Confirmatory Gene Mutation Assay: Experiment 3 & 4 – Absence and Presence of Metabolic Activation

 

There was no evidence of excessive cytotoxicity (<10 % RCE) at any of the tested concentrations either in the presence or absence of metabolic activation. The RCE values in the presence of metabolic activation, ranged from 46.2 to 83.7 % while in the absence of metabolic activation, ranged from 42.8 to 81.5 % compared to the vehicle control.

 

The frequency of mutants in the DMSO control was within the range of the in-house historical control data.

 

The test item did not cause a significant increase in the frequencies of mutants compared to the vehicle control in the presence or absence of metabolic activation at any of the tested concentrations.

Under similar conditions the positive control 3-methylcholanthrene (3-MCA) and ethylmethanesulphonate(EMS) both induced astatisticallysignificant increase in the mutant frequency as compared with the vehicle control.

Conclusions:
The study was performed according to the OECD Guideline 476 without deviations and according to the principles of the good laboratory practice and therefore considered to be of the highest quality (reliability Klimisch 1). The vehicle and the positive control substances fulfilled validity criteria of the test system. The test material did not induce gene mutations at the HPRT locus in CHO-K1 cells. The test material was considered to be non-mutagenic under the conditions of the test.
Executive summary:

The genotoxic potential of the test item the test item to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO-K1) cells in an OECD 476 guideline study.

 

The study consisted of a preliminary toxicity test, an initial gene mutation assay, and a confirmatory gene mutation assay. Each of these mutation assays comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

The test item formed a workable suspension in Dimethyl sulphoxide (DMSO) at 50 mg/mL. Dose formulations of the test item at 5040 and 50152 µg/mL were found to be stable in DMSO for 24 hours at room temperature.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the test item did not show evidence of significant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to vehicle control) at any of the tested concentrations either in the presence or in the absence of metabolic activation. The test item did not precipitate in the test medium at any of the tested concentrations and did not show any appreciable change in the pH and osmolality of the test medium. Based on these observations, a maximum of 500 µg/mL was tested in the gene mutation assay. 

 

In the initial gene mutation assay, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 63, 125, 250 and 500 µg/mL of the medium for 3 hours in the presence and absence of metabolic activation. Similarly, in the confirmatory gene mutation assay, CHO-K1 cells were exposed to the test item in duplicate at 50, 108, 232 and 500 µg/mL test concentrations in the presence and absence of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and appropriate positive controls i.e., 3-methylcholanthrene in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation were also tested in duplicate.

 

The results of the concentration analysis of the dose formulation samples of the gene mutation assay confirmed that the respective top concentrations were achieved and the results support the validity of the study conclusion.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. Under identical conditions, the respective positive controls produced a statistically significant increase in the frequencies of mutants, demonstrating the sensitivity of the assay procedure used.

The results of the forward gene mutation assay at the hprt locus with the test item indicated that the test item was non-mutagenic under the conditions of this study.

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

Additional information

In Vitro: There are all three stipulated (according to REACH Annex VIII) genotoxicity studies available, covering the endpoints gene mutation in bacteria, chromosome aberrations in mammalian cells, and gene mutations in mammalian cells. All studies gave consistently negative results. Hence, the database is of high quality, no data gaps were identified, no additional testing is required and the substance does not need to be classified according to Regulation 1272/2008.

Justification for classification or non-classification

The test material does not meet the criteria for classification and will not require labelling as a mutagen in accordance with European Regulation (EC) No. 1272/2008.