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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Appearance: White to light yellow crystalline
- Storage Conditions: Stored in a cold and dark place.

Method

Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strainopen allclose all
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.
Controls
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.

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.