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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:
other: experimental result from similar substance
Adequacy of study:
key study
Study period:
1991-07-01 to 1991-07-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with one minor deviation from current guideline. Only four strains were tested (instead of five).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No E. coli strain tested
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media:The test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983). 0.1 mL compound, 0.1 mL bacteria, 0.5 mL S9 mix or buffer, 2.0 mL soft agar, 45 degree C in water bath, max. 30 sec, transfer to petri dish with solid agar
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
First test: 5000, 1000, 200, 40 and 8 µg/plateRepeat tests: 800, 400, 200, 100, 50 and 25 µg/plate 70, 60, 50, 40, 30, 20 and 10 µg/plate TA 100 with 10 %, 30 %, 50 % S9
Vehicle / solvent:
The solvent employed for the test item was methanol and for the positive controls DMSO.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
10 µg per plate, only for strain TA 1535 w/o S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: nitrofurantoin
Remarks:
0.2 µg per plate, only for TA 100 w/o S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene diamine
Remarks:
10 µg per plate, only for TA 1537 w/o S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene diamine
Remarks:
0.5 µg per plate, only for strain TA 98 w/o S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
3 µg per plate, all strains with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation: 0.1 mL TS+0.1 mL bacteria+0.5 mL S9+2 mL soft agar: 30 sec at 45 °C- Incubation period: 48 hours at 37°CNUMBER OF REPLICATIONS: 4 plates/strain/concentrationDETERMINATION OF CYTOTOXICITY - Method: - background growth - marked and dose-dependent reduction in mutant count compared to negative controls - titer determination Acceptance criteria:a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratory's own historical datab) The positive controls had to show sufficient effects, as defined by the laboratory's experiencec) Titer determinations had to demonstrate sufficient bacterial density in the suspension.An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result.For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
Statistics:
N.A.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
TA 100: 1.6-fold increase at cytotoxic concentrations
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There was no indication of a bacteriotoxic effect of the test item at 8 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted. Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only be used to a limited extent up to 1000 µg per plate for assessment purposes.In the first trial, none of the four strains concerned showed a dose related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix.The negative findings for Salmonella typhimurium TA 1535, TA 1537 and TA 98 were confirmed by further repeat tests. In the TA 100 strain, a 1.6-fold increase in comparison of the negative controls was found in the second test. The findings of the second trial for Salmonella typhimurium TA 100 were confirmed by further repeat tests using 30 % and 50 % S9 mix. No relevant increase was found using 10 % S9 mix. The lowest dose at which this finding was reproducible was approximately 30 µg per plate for Salmonella typhimurium TA 100. Minimally increased mutation rates - below the threshold for positive effects ( 2 fold) - were obtained only with S9 mix containing 30 % and 50 % S9 fraction in S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of mean values without S9-mix from the pre-test

Pre-Test: µg/plate Strain
TA 1535 TA 100 TA 1537 TA 98
 
0 8 89 7 20
8 11 84 7 18
40 12 84 6 22
200 10 88 7 22
1000 1 20 4 19
5000 0 0 0 0
Na-azid 631
NF 382
4-NPDA     56 86

Table 2: Summary of mean values with S9-mix from the pre-test
Pre-Test: µg/plate Strain
TA 1535 TA 100 TA 1537 TA 98

30% S9-mix

0 17 117 10 34
8 16 137 8 28
40 20 145 10 39
200 8 148 12 40
1000 -- 11 3 30
5000 0 0 0 0
2-AA 99 817 134 427

Table 3: Summary of mean values without S9-mix from the repeat tests

Repeat-Test: µg/plate Strain
TA 1535 TA 100 TA 1537 TA 98
 
0 15 130 11 32
25 16 141 12 30
50 16 124 10 39
100 13 131 10 41
200 12 138 14 37
400 13 100 13 29
800 5 42 4 20
Na-azid 793
NF 416
4-NPDA     58 105

Table 4: Summary of mean values with S9-mix from the repeat tests
Repeat-Test: µg/plate Strain
TA 1535 TA 100 TA 1537 TA 98

30% S9-mix

0 34 190 17 53
25 27 241 21 71
50 28 303 20 74
100 13 197 20 64
200 6 106 20 50
400 -- 12 3 13
800 0 -- -- --
2-AA 242 1297 68 570

Table 5: Summary of mean values with different S9-mix concentrations tested in strain TA 100
 µg/plate Strain TA 100
30 % S9 10% S9 30% S9 50% S9
 
0 155 150 149 177
10 204 142 160 233
20 189 169 185 199
30 237 175 201 207
40 253 173 212 243
50 261 174 246 261
60 168 174 256 250
70 162 185 209 271
2-AA 534 1406 942 613

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negative without metabolic activationnegative with metabolic activationNo biologically relevant and dose dependent increase in the mutant count at non-bacteriotoxic concentrations in comparison to the negative controls was observed after treatment with the test item in the presence and absence of metabolic activation. Therefore, the test item is considered to be non-mutagenic with and without S9 mix in the Salmonella/microsome test.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 98 of S. typhimurium were exposed to the test item (98.5% purity) in methanol at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (first/pre-test) and at concentrations of 25, 50, 100, 200, 400, 800 µg/plate (repeat test) in the presence and absence of mammalian metabolic activation. 

The test item was tested up to the limit concentration (5000 µg/plate). Based on bacteriotoxic effects only the dose groups up to 1000 µg per plate were used to a limited extent for assessment purposes. The positive controls induced the appropriate responses in the corresponding strains. In the studies with metabolic activation, a slight increase (up to 1.6 fold) in the mutant count was observed. This slight increase was not dose-dependent and did not occur in all experiments. In the studies without metabolic activation no induction of mutant counts was observed. Therefore, the test item is considered to be non-mutagenic with and without S9 mix in the Salmonella/microsome test.

This study is classified as acceptable. The study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.