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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
From 9 september 1992 To 15 october 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: white powder
- Storage condition of test material: room temperature under silica gel



Method

Target gene:
Histidine gene (S. Typhimurium)
Tryptophane gene (E. Coli (WP2uvrA-))
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium: TA1535, TA1537, TA98, and TA100. Escherichia coli: WP2uvrA-
Details on mammalian cell type (if applicable):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 from induced Aroclor rat liver
Test concentrations with justification for top dose:
0, 8, 40, 200, 1000, and 5000 µg/plate (experiment 1)
0, 312.5, 625, 1250, 2500 and 5000 µg/plate (experiment 2)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: not specified
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: N-Ethyl-N'-nitro-N-nitrosoguanidine 3 µg/plate for TA100 and 5 µg/plate for TA1535, 9-aminoacridine 80 µg/plate for TA1537,4-Nito-o-phenylenediamine 5 µg/plate for TA 1538 and 4-Nitroquinoline-1-oxide 0.2 µg/plate for TA98. + S9: 2-Aminoanthracene a
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 48 h at 37 °C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method:
The cytotoxicity of the test material was determined using TA 100 or WP2 uvrA-. Five doses(0, 8, 40, 200, 1000, and 5000 µg/plate ) of ITC 288 were tested in duplicate. After 48 hours incubation at 37°C the plates were scored for revertant colonies and examined for a thinning of the background lawn.







Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate.
Statistics:
No data

Results and discussion

Test results
Species / strain:
other: TA1535, TA1537, TA1538, TA98 and TA100
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:
not applicable
Positive controls validity:
valid
Additional information on results:
none
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1:Number of revertants per plate (mean of 3 plates), (TA 100, TA 1535 and WP2uvrA- +/- S9 in experiment 1)

 

[Strain TA 100]

[Strain TA 1535]

WP2uvrA-

Conc.
[µg/plate]

— MA

+ MA

Cytotoxicity

— MA

+ MA

Cytotoxicity

— MA

+ MA

Cytotoxicity

0*

 170

 186.3

 no

 17

 17.7

 no

 17.7

 18.3

 no

8.0

 156.7

 162.0

 no

 16.3

 20.0

 no

 16.3

18.7

 no

40

 167.7

 153.7

 no

 18.7

 15.0

 no

 10.3

14.0

 no

200

 151.7

 149.0

 no

 17.7

 17.3

 no

 17.7

14.0

 no

1000

 165.7

 144.0

 no

 21.3

 15.7

 no

 11.3

13.3

 no

5000

 170.7

 168.7

 no

 14.7

 13.0

 no

 15

 14.7

 no

Positive control

 695

 421.7

 no

 348.7

 306.7

 no

 447.7

 187.0

 no

*solvent control with water.

Table 7.6.1/2:Number of revertants per plate (mean of 3 plates), (TA98 and TA 1537 +/- S9 in experiment 1)

 

[Strain TA 98]

[Strain TA 1537]

Conc.
[µg/plate]

— MA

+ MA

Cytotoxicity

— MA

+ MA

Cytotoxicity

0*

20

28

no

14.3

11.7

no

8.0

18.3

23.7

no

 9.0

14.7

no

40

21.7

23.3

no

13.7

10.3

no

200

20.3

26.7

no

12.3

11.3

no

1000

16.7

31.0

no

9.7

11.7

no

5000

16.3

21.3

no

13.0

12.0

no

Positive control

183.3

263.3

no

454

144.0

no

*solvent control with water.

Table 7.6.1/3:Number of revertants per plate (mean of 3 plates), (TA 100, TA 1535 and WP2uvrA- +/- S9 in experiment 2)

 

[Strain TA 100]

[Strain TA 1535]

WP2uvrA-

Conc.
[µg/plate]

— MA

+ MA

Cytotoxicity

— MA

+ MA

Cytotoxicity

— MA

+ MA

Cytotoxicity

0*

 160.0

 174.7

 no

 11.0

 12.0

 no

 16

 11.7

 no

312.5

 154.7

 141.7

 no

 14.0

 9.5

 no

 13.7

11.3

 no

625

 127.3

 159.0

 no

 14.3

9.0

 no

 11.0

15.3

 no

1250

 163.5

 148.0

 no

 13.0

 11.7

 no

 13.0

15.7

 no

2500

 124.3

 169.3

 no

 14.0

 13.3

 no

 15.7

11.7

 no

5000

 158.3

 136.7

 no

 11.0

 12.3

 no

 13.0

 15.0

 no

Positive control

 508.3

 436.3

 no

 161.3

 142

 no

 599.0

 111.3

 no

*solvent control with water.

Table 7.6.1/4:Number of revertants per plate (mean of 3 plates), (TA98 and TA 1537 +/- S9 in experiment 2)

 

[Strain TA 98]

[Strain TA 1537]

Conc.
[µg/plate]

— MA

+ MA

Cytotoxicity

— MA

+ MA

Cytotoxicity

0*

17.3

22.3

no

6.0

9.3

no

312.5

22.3

20.0

no

 8.7

6.7

no

625

25.0

19.7

no

10.7

13.0

no

1250

22.0

17.7

no

7.7

4.0

no

2500

22.0

22.7

no

8.0

8.3

no

5000

20

17.7

no

8.7

7.7

no

Positive control

203.7

158.0

no

301

161.0

no

*solvent control with water.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the conditions of this test, ITC 288 was found to be non-mutagenic in Bacteria.
Executive summary:

In a reverse gene mutation assay in bacteria (Thompson, 1992), Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with ITC 288 by the Ames plate incorporation method at five dose levels, in triplicate, both with and without S9. The dose range determined in a preliminary toxicity assay was 8 to 5000 µg/plate using TA100 or WP2 uvrA-. In the mutation study, The dose range used were 8 to 5000 µg/plate in the first experiment and 312.5 to 5000 µg/plate in the second experiment. The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. ITC 288 caused no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of salmonella and escherichia used with and without S9. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of ITC 288, either with or without metabolic activation (experiment 1 + experiment 2). Under the conditions of this test, ITC 288 was found to be non-mutagenic in Bacteria.

This study is classified as acceptable and satisfies the requirement for test Guideline OECD 471 for in vitro mutagenesis (bacterial reverse gene mutation) data.