Yellow PE 3260

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three Klimisch-1-rated studies have been conducted, one in-vitro mammalian cells mutagenicity test, one in-vitro mammalian chromosome aberration test, and one bacterial Ames Test. With the exception of the chromosome aberration test, all study results were negative. No conclusion for this endpoint is possible. One in-vivo study mammalian micronucleous mutagenicity test was conducted.

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:

1996

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:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Revised Chemical Substance Law (1987) according to the notification of December 9, 1986 by EA, Environmental Agency (no. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014), Japan.

Deviations:

no

GLP compliance:

yes

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):

- Type and identity of media: MEM (Minimal Essential Medium) supplemented with 10 % fetal calf serum (FCS).
- Properly maintained: yes (Cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 4.5
% carbon dioxide (95.5 % air))
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 mix from Aroclor 1254-induced rat liver
- source of S9: obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm which received a single i.p. injection of 500 mg Aroclor 1254 per kg body weight in olive oil 5 days previously.
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0. 75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µl/ml S9 mix contained in serum-free medium together with different conc. of the test article
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

Concentration range in the main test (experiment I, with metabolic activation): 10, 30, 100, 1000 µg/ml;
Concentration range in the main test (experiment II, with metabolic activation): 10, 30, 100, 3000 µg/ml;
Concentration range in the main test (experiment I, without metabolic activation): 3, 10, 30 µg/ml;
Concentration range in the main test (experiment II, without metabolic activation): 10, 30, 50 µg/ml.

Based on a dose range finding pre-test.

Vehicle / solvent:

- Deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1x10^4 -6x10^4 cells per chamber
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 18 and 28 hours
Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hours
All cultures were incubated at 37° C in a humidified atmosphere with 4.5 % CO2 (95.5 % air).

PREPARATION OF THE CULTURES:
15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.5 h later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).
Additionally, two cultures per treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the substance is given as reduction of % cells as compared to the negative control.


METHODS FOR MEASUREMENT OF CYTOTOXICITY/GENOTOXICITY
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" (9)) using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosome aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosome aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test. However, both biological and statistical significance should be considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations: - Precipitation occured 4 hours after treatment at concentrations >= 100 µg/ml with S9-mix and at >= 30 µg/ml without S9-mix.


RANGE-FINDING/SCREENING STUDIES (if applicable):
The highest concentration used in the pre-test was 5000 µg/ml (highest guideline­recommended concentration. Severe toxic effects (no surviving cells) were observed in a concentration range from 300 - 5000 µg/ml in the absence of S9 mix.
With S9 mix 1000 and 5000 µg/ml reduced the cell number to 29.4 % and 12.6 %, respectively, of the solvent control.
Precipitation of the test article in the culture medium was observed starting at concen­trations of 30 µg/ml (without S9 mix) and 100 µg/ml (with S9 mix).


HISTORICAL CONTROL DATA
The aberration rates of the cells after treatment with the test article (0.0 % - 2.0 %) were in the range of the solvent control values (0.5 % -3.0 %) and in the range of the laboratory historical control data: 0.0 %-4.0 %.

Conclusions:

As determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro, it can be stated that in the study described and under the experimental conditions reported, the substance induced reproducibly structural chromosome aberrations in the presence of S9 mix within a concentration range where precipitation of the test article was observed.
Therefore, the substance is considered to be mutagenic in this chromosome aberration test.

Executive summary:

The substance, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments according to First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 473, adopted May 26, 1983, „ In vitro Mammalian Cytogenetic Test" and EEC Directive 92/69, L 383 A, Annex V, B 10, dated December 29,1992.

The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosome aberrations.

Substantial reductions of the mitotic indices were observed in the absence of S9 mix, in experiment I after treatment with 10 and 30 µg/ml at preparation interval 18 hours. In the presence of S9 mix the mitotic indices were reduced after treatment with the highest evaluated concentrations (1000 µg/ml in experiment I and 3000 μg/ml in experiment II). In the presence of S9 mix, there were biologically relevant and statistically significant increases in cells carrying structural chromosome aberrations after treatment with 1000 μg/ml of the test article at fixation interval 28 h in experiment I as well as after treatment with 3000 μg/ml (18 h and 28 h fixation interval) in experiment II.

In the absence of S9 mix, no biologically relevant increase in cells carrying structural chromosome aberrations was observed.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells carrying structural chromosome aberrations.

In conclusion, as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro, it can be stated that in the study described and under the experimental conditions reported, the registration substance induced reproducibly structural chromosome aberrations in the presence of S9 mix within a concentration range where precipitation of the test article was observed.

Therefore, the substance is considered to be mutagenic in this chromosome aberration test.

 

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:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Revised Chemical Substance Law (1987) according to the notification of December 9, 1986 by EA, Environmental Agency (no. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014), Japan.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella thyphimurium:
TA1537: his C 3076; rfa-; uvrB-: frame shift mutations
TA 98: his D 3052; rfa.-; uvrB-;R-factor: frame shift mutations
TA1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA 100: his G 46; rfa-; uvrB-;R-factor: base-pair substitutions

Escherichia coli
WP2: trp; uvrA: base substitutions and others

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:

Phenobarbital / beta-Naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 33.3, 100, 333.3, 1000, 2500, 5000 µg/plate;
Concentration range in the main test (without metabolic activation): 33.3, 100, 333.3, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

Solvent: - aqua bidest.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

Remarks:

Without metabolic activation: sodium azide (TA 1535, TA 100), 4-nitro-o-phenylene-diamine 4-NOPD (TA 1537, TA 98), methyl methane sulfonate MMS (WP2 uvrA). With metabolic activcation: 2-aminoanthracene 2-AA (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (experiment I) and preincubation (experiment II) protocol

DURATION
- Preincubation period: 60 minutes

NUMBER OF REPLICATIONS: 3 plates per concentration / control

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (number of spontaneous revertants or clearing of the bacterial background lawn)

Evaluation criteria:

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a reproducibly positive response at any one of the test points is considered non-mutagenic in this system.

A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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:

CYTOTOXIC EFFECTS
Slight toxic effects in TA 1535 (2500 and 5000 µg/plate -S9), TA1537 (5000 µg/plate -S9, 333.3-5000 µg/plate +S9), and TA100 (2500 and 5000 µg/plate -S9) were noted using the plate incorporation test whereas in the pre-incubation test slight toxic effects were observed in strain TA 1537 (1000-5000 µg/plate +S9).

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test article occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100. 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 experiment I (plate incorporation test). Toxicity of the test article results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Dose Selection: According to the results of this pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered two logarithmic decades. Two independent experiments were performed. According to the dose selection criteria the test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000; 2500; and 5000 µg/plate. As the results of the pre-experiment were in accordance with the evaluation criteria , these data are reported as a part of the main experiment.

COMPARISON WITH HISTORICAL CONTROL DATA:
The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Conclusions:

Under the experimental conditions reported, the substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The substance was assessed for its potential to induce gene mutations using the plate incorporation test (experiment I) and the pre-incubation test (experiment II) with Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA according to the OECD Guidelines 471/472 and EC Method B.13/14.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test article was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate

In experiment I, slight toxic effects, evidenced by a reduction in the number of revertants were observed in strains TA 1535 (at 2500 and 5000 µg/plate without S9 mix) TA 1537 (5000 µg/plate without S9 mix and 333.3 up to 5000 µg/plate with S9 mix), and TA100 (2500 and 5000 µg/plate without S9 mix). The comparatively low factors of strain TA 98 at all concentrations with S9 mix are a result of the relatively large number of revertants of the corresponding solvent control and do not indicate a true toxic effect. In experiment II, slight toxic effects were observed in strain TA 1537 from 1000 up to 5000 µg/plate (with S9 mix).

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 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. They 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 registration substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

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:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- 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:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 19.5, 39.0, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0 µg/mL
with metabolic activation: 7.8, 15.6, 31.3, 62.5, 125.0, 3750.0, 5000.0 µg/mL
Experiment II:
without metabolic activation: 19.5, 39.0, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0 µg/mL
with metabolic activation: 7.8, 15.6, 31.3, 62.5, 125.0, 3750.0, 5000.0 µg/mL

Vehicle / solvent:

water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

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 to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation frequency 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.
In a case by case evaluation this decision depends on the level of the corresponding solvent control data.

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. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: see field "any other information on results incl. tables"

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Evaporation from medium: not examined
- Water solubility: soluble
- Precipitation:
The culture medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at concentrations from 78.1 µg/mL to 625.0 µg/mL in the absence of metabolic activation (4 hours treatment). Following continuous treatment (24 hours) without metabolic activation, precipitation was observed from 156.3 µg/mL to 1250.0 µg/mL. In the presence of metabolic activation precipitation could be observed at 78.1 µg/mL and above.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:

In the range finding pre-experiment test item concentrations between 39.0 and 5000.0 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Cytotoxic effects could be observed in the presence of metabolic activation at test item concentrations of 2500 µg/mL and above. Cytotoxicity occurred after 24 hours continuous treatment without metabolic activation at the highest test item concentration (5000.0 µg/mL) in Experiment II.

COMPARISON WITH HISTORICAL CONTROL DATA: complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Following pulse treatment in the presence of metabolic activation relevant cytotoxicity was observed in the highest evaluated dose group of 3750,0 µg/mL. Additionally, the cloning efficiency I and the cell density was relevantly reduced following 24 hours treatment in the absence of metabolic activation in culture II at a concentration of 2500.0 µg/mL.

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, the registration substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

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

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

The maximum concentration of the test item in the main experiments was chosen with respect to the current OECD guideline.

Precipitation occurred in the presence of S9 mix at a test item concentration of 62.5 µg/mL and above in both independent experiments. Following treatment in the absence of metabolic activation precipitation occurred in the middle concentration range from 39.0 to 625.0 µg/mL in experiment I and from 78.1 to 1250.0 µg/mL in experiment II.

Following pulse treatment in the presence of metabolic activation relevant cytotoxicity was observed in the highest evaluated dose group of 3750,0 µg/mL. Additionally, the cloning efficiency I and the cell density was relevantly reduced following 24 hours treatment in the absence of metabolic activation in culture II at a concentration of 2500.0 µg/mL.

 

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration required by the guideline. However, one single mutant frequency exceeded the historical range of solvent controls in experiment II culture 2 without S9 mix. This observation is judged to be biologically not relevant as the mutant frequency remained well within the laboratory’s historical data range in the respective culture 1. The induction factor did not exceed the threshold of three times the corresponding solvent control.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 11.2 up to 29.0 mutants per 10⁶cells; the range of the groups treated with the test item was from 9.2 up to 33.0 mutants per 10⁶cells.

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

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

Genetic toxicity in vivo

Description of key information

Four Klimisch-1-rated studies have been conducted, one in-vivo mammalian micronucleus mutagenicity test, one in-vitro mammalian cells mutagenicity test, one in-vitro mammalian chromosome aberration test, and one bacterial Ames Test. With the exception of the chromosome aberration test, all study results were negative. As outcome of the weight of evidence approach, the registration substance is considered not genotoxic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 Füllingsdorf
- Age at study initiation: 9-13 weeks
- Weight at study initiation: males mean value 31.3 ± 3.4 g, females mean value 24.3 ± 1.7 g
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon Type I cages with wire mesh top and granulated soft wood bedding
- Fasting period before study: approximately 18 hours
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 7 °C
- Humidity (%): 15-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: unspecified

Vehicle:

- Vehicle used: Deionised water
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test article was dissolved in deionised water.

Duration of treatment / exposure:

Single administration.

Frequency of treatment:

Single oral administration of test item and controls, respectively.

Post exposure period:

Sampling of the bone marrow was done 24 (all dose groups) and 48 hours (only high dose group) after treatment, respectively.

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

670 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males and 6 females per dose/control group

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control substance: cyclophosphamide
- Route of administration: orally
- Doses / concentrations: 40 mg/kg bw at 10 mL/kg bw in deionised water

Tissues and cell types examined:

femural bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w., the substance dissolved in deionised water. As no severe toxic reactions were observed within 48 after exposure, 2000 mg/kg bw was estimated to be suitable as high dose concentration.

DETAILS OF SLIDE PREPARATION:
The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald /Giemsa. Cover slips were mounted with Eukitt. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

Significant increase can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: limit test with 2000 mg/kg bw
- Solubility: well soluble at the tested concentration
- Clinical signs of toxicity in test animals: reduction of spontaneous activity in 4 of 4 animals (fully reversible within 48h), eyelid closure in 2 of 4 animals (fully reversible within 24h), apathy in 2 of 4 animals (fully reversible within 24h),

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 as compared to the mean value of NCEs of the vehicle control. However, the enhancement of the mean value was caused mainly by the results of two males (62 = 2160 NCEs per 1000 PCEs; 63 = 1377 NCEs per 1000 PCEs). Therefore, it cannot be concluded that the test article induced cytotoxic effects. Mean data are given in Table 1.
- Statistical evaluation: In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with the substance were in the range of the vehicle control group. Cyclophosphamide administered was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Conclusions:

The substance is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed according to the First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted May 26, 1983, „Micronucleus Test" and the method laid down in EEC Directive 92/69, L 383, Annexe V, B 12, dated December 29, 1992 to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was dissolved in deionised water. Deionised water was used as vehicle control. The volume administered orally was 20 ml/kg bw 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670 and 2000 mg/kg bw

48 h preparation interval: 2000 mg/kg bw

The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions.

After treatment with the test article at preparation interval 48 h the mean number of normochromatic erythrocytes was increased as compared to the mean value of NCEs of the vehicle control. However, the enhancement of the mean value was caused mainly by the results of two males (62 = 2160 NCEs per 1000 PCEs; 63 = 1377 NCEs per 1000 PCEs). Therefore, it cannot be concluded that the test article induced cytotoxic effects.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, the substance is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In-vivo micronucleus study:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

 

In-vitro mammalian cells mutagenicity test:

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

 

In-vitro mammalian chromosome aberration test:

In conclusion, as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro, it can be stated that in the study described and under the experimental conditions reported, the registration substance induced reproducibly structural chromosome aberrations in the presence of S9 mix within a concentration range where precipitation of the test article was observed. Therefore, the registration substance is considered to be genotoxic in this chromosome aberration test.

 

Bacterial reverse mutation study (Ames Test):

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the registration substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

Weight of evidence approach:

Four Klimisch-1-rated studies have been conducted on genotoxicity. The mammalian chromosome aberration test gave a positive result in the presence of metabolic activation, while all of the three other studies revealed a negative outcome. As the majority of study results are not indicating genotoxic properties of the test item, and in particular because the in-vivo micronucleus study gave a negative result, the registration substance is considered as not genotoxic.

Justification for classification or non-classification

The registration substance shall be considered as not genotoxic, thus no classification applies.