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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames, Ogura (1997)

Under the conditions of the study, the test material displayed no reverse mutagenic potential.

Chromosome Aberration, Ajimi (1998)

Under the conditions of the study, it was considered that the test material had no ability of inducing chromosomal aberration.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 February 1997 to 28 March 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: Standards for Toxicity Investigations
Version / remarks:
Japan's Ministry of Labor, No.77, September 1, 1988
Deviations:
no
Qualifier:
according to
Guideline:
other: Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances
Version / remarks:
Notification No. 700 of the Planning and Coordination Bureau, EA, No. 1039 of the Pharmaceutical Affairs Bureau, MHW & No. 1014 ( 1986) of the Basic Industries Bureau, MITI, December 5, 1986
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. B.N. Ames, University of California, U.S.A., on June 20, 1990
- Storage of cells: After 0.045 mL of spectrophotometric grade of dimethyl sulfoxide (DMSO) was added to 0.5 mL of the bacterial culture, it was stored at -80 °C until use.
-The amino acid requirements were confirmed using histidine. The presence or absence of R-factor were confirmed by ampicillin resistance, and mutations in membrane and DNA repair were examined by sensitivity to crystal violet and UV sensitivity, respectively.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Japan Bioassay Research Centre on January 7, 1997.
- Storage of cells: After 0.045 mL of spectrophotometric grade of dimethyl sulfoxide (DMSO) was added to 0.5 mL of the bacterial culture, it was stored at -80 °C until use.
-The amino acid requirements were confirmed using tryptophan. The presence or absence of R-factor were confirmed by ampicillin resistance, and mutations in membrane and DNA repair were examined by sensitivity to crystal violet and UV sensitivity, respectively.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
- 5000, 2500, 1250, 625 and 313 μg/plate.
- The results of the dose-range-finding test showed that both growth inhibition and increases in the revertant colonies were not observed at 1000, 500, 100, 50, 10 and 5 μg/plate and so 5000 µg/plate was used as the highest dose in the main test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dehydrolysed DMSO
- The test material was dissolved in dehydrolysed DMSO (Lot No. DN072), to make 5 w/v % concentration and diluted with the same solvent to make lower concentrations. DMSO was dehydrated with Molecular Sieves 3A 1/8.
- The test material was prepared just before use and used within 0.5 hour. The test material solutions were kept at room temperature until use.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylarnide (AF-2), 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine.2HCl (ICR-191) and 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre-incubation
- After 0.1 mL of the test material solution, 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) or S9 mix, and 0.1 mL of the bacterial culture were added to a tube, the mixtures were incubated for 20 min at 37 ± 0.5 °C. Two mL of the soft agar was then added to each tube and poured onto a minimal glucose agar plate. After incubation for 48 hours at 37 ± 0.5 °C, the number of revertant colonies was counted.
- As the sterility test, 0.1 mL of each bacterial strain, test material solution, and S9 mix or 0.1 M sodium phosphate buffer (pH 7.4) were smeared on a minimal glucose agar plate, which was incubated at 37 ± 0.5 °C for 48 hours to check the bacterial contamination. Dehydrolysed DMSO was used as a negative control, and appropriate positive controls were used for each bacterial strain.

NUMBER OF REPLICATIONS: Three plates were used for the negative control and two plates for the test material and positive controls.

OBSERVATION AND COLONY COUNTING
- Microscopic Observation: The state of revertant colonies (size and number of colonies), deposition of the test material and the growth inhibition were examined with a stereo microscope.
- Colony Counting: The number of colonies was counted with a manual counter or a colony analyser. Correction for counting errors was made for measurements with the colony analyser. Each plate was measured three times, and the average of these three measurements was adopted as the number of revertant colonies on the plate. The average for each dose was calculated from the values of the plates used. Decimals of the average figures were rounded off.
Evaluation criteria:
JUDGEMENT CRITERIA OF TEST RESULTS
- The test material was judged to be positive when the number of revertant colonies increased twice or more that of the negative control in a dose-dependent manner, and the reproducibility of the test results was also obtained.
- It was judged to be negative in other cases.
Statistics:
Any statistical procedures were not applied.
Key result
Species / strain:
other: TA98, TA100, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- The test results demonstrated that the number of the revertant colonies in all the test strains was less than twice that of the negative control with and without S9 mix.
- The deposition of the test material was observed at more than 1000 μg/plate with and without S9 mix, however, the deposition did not interfere with counting the revertant colonies.
- The positive controls induced significant increases in the revertant colonies, and the number of revertant colonies in the positive controls and the negative control was found to be within a range of the background data in the testing laboratory.
- There were no fluctuations that affected the test results, since it was confirmed that there was no contamination in the test system by the sterility test.

Table 1: Summary of Results of the Main Test

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

313

625

1250*

2500*

5000*

124

127

124

113

112

120

10

12

10

12

14

11

29

28

32

32

32

39

23

20

26

22

28

23

10

8

8

10

9

6

+

Solvent

313

625

1250*

2500*

5000*

126

108

112

124

108

117

9

11

10

11

9

11

29

29

30

33

36

30

32

32

35

31

29

31

20

17

16

13

14

15

Positive Controls

-

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Concentration (µg/plate)

0.01

0.5

0.01

0.1

1

Mean no. colonies/plate

544

338

152

469

2223

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

1

2

10

5

2

Mean no. colonies/plate

883

170

651

361

162

* deposition of test material

AF-2 = 2-(2-Furyl )-3-(5 -nitro-2-furyl)acrylamide

2AA = 2-aminoanthracene

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine · 2HCI

NaN3 = Sodium azide

Conclusions:
The test material was not mutagenic under the conditions of the study.
Executive summary:

The mutagenicity of the test material was examined in Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 as well as Escherichia coli strain WP2 uvrA using the pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). The testing was performed under GLP conditions.

The test results demonstrated that the number of the revertant colonies in all the test strains was less than twice that of the negative control with and without S9 mix. The deposition of the test material was observed at more than 1000 μg/plate with and without S9 mix, however, the deposition did not interfere with counting the revertant colonies.

The positive controls induced significant increases in the revertant colonies, and the number of revertant colonies in the positive controls and the negative control was found to be within a range of the background data in the testing laboratory.

There were no fluctuations affected the test results, since it was confirmed that there was no contamination in the test system by the sterility test.

Under the conditions of this study, the test material displayed no reverse mutagenic potential.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 1998 to 23 October 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances
Version / remarks:
Notification No. 700 of the Planning and Coordination Bureau, EA, No. 1039 of the Pharmaceutical Affairs Bureau, MHW & No. 1014 (1986) of the Basic Industries Bureau, MITI, December 5, 1986 and Notification No. 287 of the Planning and Coordination Bureau, EA, No. 127 of the Environmental Health Bureau, MHW & No. 2 of the Basic Industries Bureau, MITI, October 31, 1997
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):
CELLS USED
- Source of cells: National Institute of Hygienic Sciences. Cells of passage number 18 were supplied.
- Suitability of cells: Chinese hamster lung fibroblasts (CHL cells, clone No. 11) were used in this test because of their high sensitivity to induce chromosomal aberrations and available data have already been accumulated.
- The modal number of chromosome is 25 and the doubling time is about 15 hours.
- The passage number of the cells was 41 in the chromosomal aberration test.
- Cells were cultured in a humidified atmosphere of 5% CO2 in air at 37°C. A 100 mm diameter petri dish (Nunc) and a 60 mm diameter petri dish (Iwaki Glass Co., Ltd.) were used for passage and experiments, respectively.

MEDIA USED
- Eagle's minimum essential medium (Lot No. 403803, Nissui Seiyaku Co., Ltd.) was used, with which supplemented newborn calf serum (Lot No. NBQ07, Mitsubishi Chemical Corporation) to the extent of 10 v/v%. This medium is hereinafter referred to as 10% NCS/MEM.
- Distilled water used for preparation of the medium was obtained with a glass still (WG220, Yamato Scientific Co., Ltd.).
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Based on the results of the dose range finding test, the following concentrations were set for each treatment method:
Short-term treatment method:
- without metabolic activation system: 1250, 2500 and 5000 μg/mL
- with metabolic activation system: 750, 1500 and 3000 μg/mL
Continuous treatment method:
- 1250, 2500 and 5000 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 0.5% methylcellulose
- Justification for choice of solvent/vehicle: The test material was insoluble in water, dissolved in DMSO at about 100 mg/mL, acetone at about 250 mg/mL, and suspended in 0.5% methylcellulose (MC, Lot No. AY01 , Tokyo Kasei Kogyo Co., Ltd.) solution with good condition. Thus, 0.5% MC was selected and used in the study.
- The test material was broken into fragments with an agate mortar and a quantity measured, and suspended in 0.5% MC solution to make stock solution. The solution was diluted with the same solvent to make the required test concentrations.
- No denaturation of test solution, including colour change, heat generation and others, was confirmed until 2 hours after preparation. The test material was prepared just before use, and used within 2 hours. They were stored at room temperature until practice.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
DOSE RANGE FINDING TEST
- Pre-culture: Five mL of 10% NCS/MEM containing 0.5 x 10^4 cells/mL were added to each dish, and cells were pre-cultured for three days.
- Treatment: In the short-term treatment method, cells were treated with either 3 mL of 10% NCS/MEM including the test material or 3 mL of S9 mix/MEM including the test material for 6 hours following removal of the medium and the maximum concentration of test material was 5,000 μg/mL. The medium was removed after treatment, and the cells were washed three times with 2 mL of the phosphate buffer saline without Ca2+, Mg2+(PBS(-)) and cultured for 18 hours with the 5 mL of fresh 10% NCS/MEM. In the continuous treatment method, cells were treated with 5 mL of 10% NCS/MEM including the test material for 24 or 48 hours. Two dishes were used for each dose. A solvent-treated group was used as a negative control. The presence and absence of precipitations of the test material was observed by the naked eye at the end of the treatment period.
- Cells count and specimens preparation: Colcemid was added to the medium to give a final concentration of 0.1 μg/mL on 2 hours before the end of incubation in any treatment methods. The cells were detached from the dish with 2 mL of 0.25% trypsin following removal of the medium at the end of incubation, and the number of cells was counted using a Microcell Counter (F-500, Toa Medical Electronics Co., Ltd.) to determine cell growth rate. One specimen was prepared per each dose using remaining cells, and the presence of mitotic cells and chromosome aberration were observed.
- Microscopic observation of specimens: Specimens were observed for the presence of mitotic cells was examined. More than 50 cells were observed per dose level.

CHROMOSOMAL ABERRATION TEST
- Methods: Cell culture and treatments in the short-term treatment method and the continuous treatment method were performed using the same procedure as the dose range finding test to prepare chromosome specimens. Cell growth rates were calculated in the same way as the dose range-finding test except the positive control group.
- Negative control: For the negative control, solvent was used.
- Positive control: Based on the historical data of the testing laboratory, 0.1 μg/mL of MMC was applied for the short-term treatment without S9 mix, 10 μg/mL of CPA for the short-term treatment with S9 mix and 0.05 μg/mL of MMC for the continuous treatment method were used.
- Replicates: Two dishes were used for each dose.
- Preparation of Microscope Slides: Cells were removed from each dish with 2 mL of 0.25% trypsin and collected by centrifugation (1000 r.p.m. for 5 min). Hypotonic treatment was carried out by treating the cells with 0.075M KCl at 37°C for 15 min. The cells were fixed with Carney's solution (methanol: acetic acid = 3: 1) to make a slightly cloudy cell suspension, dropped onto slides and stained with 2% Giemsa solution. Two slides per dish (four slides per dose) were prepared.
- Observation and Scoring of Chromosomal Aberrations: One hundred metaphase cells with 25 ± 2 chromosomes per slide were observed. The number of cells which has chromatid or chromosomal structural aberrations (such as breaks, exchanges, etc.), and the types of structural aberrations were classified. The cell with aberration was counted as one aberrant cell, and the incidences of aberrant cells were obtained by observing 200 cells per dose. A gap was scored when a clear discontinuity (larger than a chromatid width) was excluded structural aberrations, and recorded apart from other aberrations. The incidence of numerical aberrations (polyploid, more than triploid) was recorded by observing 200 cells. Slides were all coded and blind-tested by microscopy. Specimens of the short-term treatment group were observed at first. So as the results were evaluated negative, specimens of the continuous treatment group were observed.
Evaluation criteria:
The incidence of cells with aberrations was judged:
less than 5% = negative
5% or more and less than 10% = equivocal
10% or more = positive
The test material was judged to be positive when the incidence of aberrant cells was 10% and its increase was in a dose-dependent manner, or the incidence of it was 5% or more and it was reproducible.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING TEST
- Fifty percent of growth inhibition concentrations (IC50) by the test material were about 2,400 μg/mL in the short-term treatment method with S9 mix, and about 3,600 μg/mL in the continuous treatment method for 48 hours. Furthermore, cytotoxicity was so weak that it could not be found at IC50 less than 5,000 μg/mL in the short-term treatment method without S9 mix and continuous treatment method for 24 hours. The maximum concentration sufficient to assess chromosomal aberration was 5,000 μg/mL in the short-term treatment method without S9 mix and continuous treatment methods, and 3,000 μg/mL in the short-term treatment method with S9 mix.
- According to the preliminary observation of the specimens, the clear increase of structural and numerical aberrations were not seen in any treatment methods.

ACCEPTABILITY OF THE TEST
- The values for two dishes were not markedly different. The incidence of cells with any aberration did not exceed 5% in negative control, structural aberrations excluding gap was 20% or more in the positive control, and there were no fluctuations in the test conditions. Therefore, the test was judged to be conducted appropriately.

CHROMOSOMAL ABERRATION TEST
Short-Term Treatment Method
- Without metabolic activation: The cell growth rate at 1250, 2500 and 5000 μg/mL were 94.3, 83.3 and 76.1%, respectively. The incidences of structural aberrations were 1.0% at 1250, 2500 and 5000 μg/mL, respectively. The incidence of the negative control was 0.5%, which was within the normal range, and the incidence of the positive control treated with MMC was 39.5%, which showed a clear induction of chromosomal aberrations. The incidence of polyploid cells at any doses was less than 5% in any treatment groups.
- With metabolic activation: The cell growth rate at 750, 1,500 and 3,000 μg/mL were 92.0, 91.0 and 37.2%, respectively. The incidences of structural aberrations were 2.0, 0 and 0.5% at 750, 1500 and 3000 μg/mL, respectively. The incidence in the negative control was 0.5%, which was within the normal range, and the incidence of the positive control treated with CPA was 33.5%, which showed a clear induction of chromosomal aberrations. The incidence of polyploid cells at any doses was less than 5% in any treatment groups.

Continuous Treatment Methods
- 24 hours treatment: The cell growth rate at 1250, 2500 and 5000 μg/mL were 101.7, 91.7 and 126.7%, respectively. The incidences of structural aberrations were 1.0% at 1250, 2500 and 5000 μg/mL, respectively. The incidence in the negative control was 1.0%, which was within the normal range, and the incidence in the positive control treated with MMC was 57.5%, which showed a clear induction of chromosomal aberrations. The incidence of polyploid cells at any doses was less than 5% in any treatment groups.
- 48 hours treatment: The cell growth rate at 1250, 2500 and 5000 μg/mL were 93.5, 77.6 and 60.5%, respectively. The incidences of structural aberrations were 1.0% at 1250, 2500 and 5000 μg/mL, respectively. The incidence in the negative control was 1.0%, which was within the normal range, and the incidence in the positive control treated with MMC was 48.0%, which showed a clear induction of chromosomal aberrations. The incidence of polyploid cells at any doses was less than 5% in any treatment groups.
Conclusions:
Under the conditions of this study, it was considered that the test material had no ability of inducing chromosomal aberration.
Executive summary:

The genetic toxicity of the test material was investigated in a chromosome aberration test. The test was performed under GLP conditions.

The effect of the test material on chromosomal aberrations was investigated in Chinese hamster lung fibroblasts cells (CHL/IU cells). Cells were treated with the test material for 6 hours both with and without metabolic activation system (S9 mix) in a short-term treatment method, and for 24 and 48 hours in a continuous treatment method.

According to the results of a dose range finding test, chromosomal aberration tests were carried out at the concentration of 1250, 2500 and 5000 μg/mL in the short-term treatment method without S9 mix and the continuous treatment method, 750, 1500 and 3000 μg/mL in the short-term treatment method with S9 mix.

The test material induced no chromosomal aberration in both short-term and continuous treatment methods at the concentrations tested.

Cyclophosphamide monohydrate and Mitomycin C used as positive control significantly induced chromosomal aberrations.

The cell growth rate increased at the highest concentration in the 24 hour continuous treatment method. It was also observed in the dose finding test and appearance of disruptive cells was repressed adequately. Thus, it was considered to be depend on the influence of precipitation of the test material.

Under the conditions of this study, it was considered that the test material had no ability of inducing chromosomal aberration.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames, Ogura (1997)

The mutagenicity of the test material was examined in Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 as well as Escherichia coli strain WP2 uvrA using the pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). The testing was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test results demonstrated that the number of the revertant colonies in all the test strains was less than twice that of the negative control with and without S9 mix. The deposition of the test material was observed at more than 1000 μg/plate with and without S9 mix, however, the deposition did not interfere with counting the revertant colonies.

The positive controls induced significant increases in the revertant colonies, and the number of revertant colonies in the positive controls and the negative control was found to be within a range of the background data in the testing laboratory.

There were no fluctuations affected the test results, since it was confirmed that there was no contamination in the test system by the sterility test.

Under the conditions of the study, the test material displayed no reverse mutagenic potential.

Chromosome Aberration, Ajimi (1998)

The genetic toxicity of the test material was investigated in a chromosome aberration test. The test was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The effect of the test material on chromosomal aberrations was investigated in Chinese hamster lung fibroblasts cells (CHL/IU cells). Cells were treated with the test material for 6 hours both with and without metabolic activation system (S9 mix) in a short-term treatment method, and for 24 and 48 hours in a continuous treatment method.

According to the results of a dose range finding test, chromosomal aberration tests were carried out at the concentration of 1250, 2500 and 5000 μg/mL in the short-term treatment method without S9 mix and the continuous treatment method, 750, 1500 and 3000 μg/mL in the short-term treatment method with S9 mix.

The test material induced no chromosomal aberration in both short-term and continuous treatment methods at the concentrations tested.

Cyclophosphamide monohydrate and Mitomycin C used as positive control significantly induced chromosomal aberrations.

The cell growth rate increased at the highest concentration in the 24 hour continuous treatment method. It was also observed in the dose finding test and appearance of disruptive cells was repressed adequately. Thus, it was considered to be depend on the influence of precipitation of the test material.

Under the conditions of the study, it was considered that the test material had no ability of inducing chromosomal aberration.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.