CGL 210

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test, a HPRT assay and a chromosome aberration test was performed according OECD guideline 471, 476 and 473 to evaluate the genotoxic potential of the test substance. The test item did not induce mutations or chromosome aberrations in vitro. Therefore, the substance is considered as non-genotoxic under the conditions of these tests.

Link to relevant study records

Reference

Reference 1

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

- purity of test item not mentioned

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his, trp

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvr-def., rfa-def. R-factor

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from aroclor induced rat liver

Test concentrations with justification for top dose:

33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

TA1535, TA100

Positive control substance:

other: sodium azide, NaN3

Remarks:

without metabolic activation

Positive controls:

yes

Remarks:

TA1537, TA98

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

without metabolic activation

Positive controls:

yes

Remarks:

WP2 uvrA

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Positive controls:

yes

Remarks:

all strains

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in overlay agar (plate incorporation)

DURATION
Precultures
From the thawed ampoules of the strains 0.5 ml suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 pi ampicillin (25 pg/ml) was added to the strains TA 98 and TA 100. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

SELECTION AGENT (mutation assays): ampicillin

NUMBER OF CELLS EVALUATED: colonies were counted using the AUTOCOUNT

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

minor toxic effects in TA 1535/1537/98 at 1000 or 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.
In the pre-experiment the concentration range of the test item was 3 - 5000 pg/plate. The pre-experiment is reported as part of experiment I since no relevant toxic effects were observed and 5000 pg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. The following concentrations were tested: 33; 100; 333; 1000; 2500; and 5000 pg/plate.

Conclusions:

The test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

The plates incubated with the test item showed nonnal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Minor toxic effects (below the factor of 0.5), evident as a reduction in the number of revertants, were observed in strain TA 1535 at 1000 and 5000 µg/plate with metabolic activation and in strain TA 1537 at 2500 and 5000 pg/plate with and without metabolic activation in experiment I. In experiment II, a minor toxic effect was also observed in strain TA 98 at 5000 µg/plate with metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment 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 ofthe strains used.

Reference 2

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79

For cell lines:
- Absence of Mycoplasma contamination: periodically checked for Mycoplasma contamination
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes, cells have a stable karyotype

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: MEM with 10 % FCS (complete medium)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/p-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

20, 40, 80, 160 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: with/out S9-mix: 4h exp. I, 18 and 28h without S9-mix exp. II and 4h with S9-mix exp. II
- Fixation time (start of exposure up to fixation or harvest of cells): 15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 pg/ml culture medium) to the cultures.

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa (E. Merck, D-64293 Darmstadt)

NUMBER OF CELLS EVALUATED: 100 well spread metaphase plates per culture were scored for cytogenetic damage, In addition, the number of polyploid cells in 500 metaphase cells per culture was determined

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: no

Evaluation criteria:

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 our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps) 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 our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps) and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (10) (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not cleariy 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 criteria is valid: A test item can be classified as aneugenic if the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
In the pre-experiment, precipitation of the test item in culture medium was observed after 4 hrs treatment with 78 pg/ml and above in the absence and the presence of S9 mix and after 24 hrs continuous treatment with 156 pg/ml and above in the absence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 347 mOsm, pH 7.3 versus 330 mOsm and pH 7.4 at 5000 pg/ml).

COMPARISON WITH HISTORICAL CONTROL DATA
The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.0 - 1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

Conclusions:

The test item is considered to be non-clastogenic in this chromosome aberration test neither with nor without S9 mix.

Executive summary:

The test item, dissolved 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. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 µg/ml) was chosen with respect to the current OECD Guideline 473. Since no relevant toxicity was observed in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473.

No clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. 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 structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79

For cell lines:
- Absence of Mycoplasma contamination: periodically checked for Mycoplasma contamination
- Methods for maintenance in cell culture: properly maintained
- Periodically checked for karyotype stability: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: MEM

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

112.5, 225.0, 450.0, 900.0, 1800.0, 3600.0 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: EMS without metabolic activation, DMBA with metabolic activation

Remarks:

with/out metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 or 24h
- Expression time (cells in growth medium): 7d
- Selection time (if incubation with a selection agent): 8d

SELECTION AGENT (mutation assays): 6-TG
NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation 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.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. 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.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: from 450 ug/ml onward
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: pre-test on toxicity and dose selection test

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Conclusions:

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

Executive summary:

The study was performed to investigate the potential of the test item 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 4 hours with and 24 hours without metabolic activation. The highest concentration of the pre-experiment and the main experiments (3600 µg/mL) was limited by the solubility properties of the test item. The test item was dissolved in acetone. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system 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

Performance and observations

There are three reliable, GLP-conform in vitro studies available to assess the potential of the end product for gene mutations in bacteria and cytogenicity in mammalian cells.

 

An Ames test was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the S. typhimurium strains TA1535, TA 1537, TA 98, and TA 100, and the E. coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate. Minor toxic effects (below the factor of 0.5), evident as a reduction in the number of revertants, were observed in strain TA 1535 at 1000 and 5000 µg/plate with metabolic activation and in strain TA 1537 at 2500 and 5000 pg/plate with and without metabolic activation in experiment I. In experiment II, a minor toxic effect was also observed in strain TA 98 at 5000 µg/plate with metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment 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.

 

In a chromosome aberrations assay, test item, dissolved 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. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 µg/ml) was chosen with respect to the current OECD Guideline 473. Since no relevant toxicity was observed in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473. No clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

 

At last, a HPRT assay in V79 cells was conducted to to investigate the potential of the test item 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 4 hours with and 24 hours without metabolic activation. The highest concentration of the pre-experiment and the main experiments (3600 µg/mL) was limited by the solubility properties of the test item. The test item was dissolved in acetone. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.

 

Discussion

In conclusion, it can be stated that under the conditions of the ames test, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Furthermore, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells in vitro. At last, it can be stated that the test item did not induce gene mutations at the HPRT locus in V79 cells. Thus, the test substance is not mutagenic and not clastogenic.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 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.