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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:
05 December 2001 to 10 December 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997 (Only a single experiment was conducted, which was clearly negative. In accordance with TG471 the lack of a repeat experiment is fully justified in the study report.)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance: straw/brown liquid

Method

Target gene:
Mutation in Genes for Histidine or Tryptophan amino acids:
TA 98: His D3052
TA 100: His G46
TA 1535: His G46
TA 1537: His C3076
WP 2 uvrA: Trp (E. Coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate (induced with Aroclor)
Test concentrations with justification for top dose:
0, 62, 185, 556, 1667, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water

- Additional information on solvent/vehicle: In vitro genotoxicity assays are aqueous based test systems. In this assay test substance is added to an aqueous mix of bacterial culture, buffer/S9 mix and molten soft agar, before pouring onto a solid (aqueous-based) agar plate. The study report indicates preparation of the MBTC formulations (in water) immediately prior to addition to the test system. Precipitation was recorded at the highest concentration but the lower concentrations remained fully soluble. It is known that an equilibrium exists between strong complexes of water with MBTC and chemical species where one or several chloride ions are abstracted, therefore it is considered highly unlikely that there would be any degradation of MBTC in the aqueous media during exposure of test systems. It can therefore be concluded that most of the examined concentrations appeared to be fully soluble in the assay system, and the test system was exposed to soluble MBTC.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: N-ethyl-N-nitrosourea (WP2 uvrA without S9) and 2-aminoanthracene (TA 98, TA 100, TA 1535 and WP2uvrA with S9)
Details on test system and experimental conditions:
SYSTEM OF TESTING - 
- Deficiences/Proficiences: TA 98, TA 100, TA 1535 and TA 1537 are his-deficient strains. WP2 uvrA is a trp-deficient strain.  
- Metabolic activation system: S9 supernatant of liver homogenate from male Wistar rats treated with Aroclor 1254. One dose of 500 mg/kg bw in soya bean oil (20% w/v) was injected intraperitoneally, and the rats were sacrificed five days later. The livers were homogenised in 0.15 M KCl for 10 minutes at 9,000 g and the supernatant (S9) collected and frozen before storing it at -60 °C. 

ADMINISTRATION: 
- Dosing: 62, 185, 556, 1667, 5000 µg/plate.
- Number of replicates: 3.
- Positive and negative control groups and treatment: The negative control groups were exposed to water. The positive control substances were sodium azide (TA 100, TA 1535), 9-aminoacridine (TA 1537),  2-nitrofluorene (TA 98) and N-ethyl-N-nitrosourea (WP2 uvrA) for assays without S9.  The positive control substances were benzo(a)pyrene (TA 1537) and 2-aminoanthracene (TA 98, TA 100, TA 1535, and WP2 uvrA) for assays with S9. All plates were incubated at 37 °C for 72 hrs.
Evaluation criteria:
The study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the results of the positive  controls meet the criteria for a positive response, and if no more than 5 % of the plates are lost through contamination or other unforeseen events.

A test material is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates  is increased in a concentration-related manner, or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test material is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

In case of an inconclusive first assay, a second independent assay was conducted. The first mutagenicity assay was regarded as inconclusive if a positive or equivocal response at only one concentration is observed or if a positive or equivocal responses at several concentrations without a concentration-related increase is observed.

Omission of the second assay under these conditions is acceptable as a  single assay does not or hardly results in false negative conclusions. Positive results from the bacterial reverse mutation test indicate that a test material induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test material is not mutagenic in the tested strains.

Both numerical significance and biological relevance were considered in the evaluation.
Statistics:
No statistical analysis was performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS:
- With metabolic activation: Negative.
- Without metabolic activation: Negative.

PRECIPITATION CONCENTRATION: 5000 µg/plate.

CYTOTOXIC CONCENTRATION:
- With metabolic activation: 1667 and 5000 µg/plate for TA 100 and WP2 uvrA. 556, 1667 and 5000 µg/plate for TA 1537.
- Without metabolic activation: 185, 556, 1667 and 5000 µg/plate for TA 100. 1667 and 5000 µg/plate for WP2 uvrA.

- The test material was toxic to the E. coli strain and to TA 100 at the two higher doses (1667 and 5000 µg/plate) in both the absence and presence of S9 mix, and to TA 100 also at 185 and 556 µg/plate in the absence of S9 mix, as was evidenced by a decrease in the mean number of revertant colonies. In addition, the test material was toxic to TA 1537 at 556, 1667 and 5000 µg/plate in the presence of S9 mix.
- In both the presence and the absence of S9 mix and in all strains the test material did not cause a more than two-fold or a dose-related increase in the mean number of revertant colonies appearing in the test plates, compared to the background spontaneous reversion rate observed with the negative control.
- The mean number of his+ and trp+ revertant colonies of the negative control were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.
- It is concluded that the test material was not mutagenic under the conditions employed in this study.

STATISTICAL RESULTS: No statistical analysis was performed.

Any other information on results incl. tables

Table 1: Summary of Experiment

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

62

185

556

1667

5000

158

138

100

117

69

83

22

21

18

13

19

20

34

26

23

23

21

19

25

19

19

19

21

18

9

13

8

3

6

5

+

Solvent

62

185

556

1667

5000

159

162

131

149

117

95

14

10

16

12

11

12

32

25

26

21

15

16

38

35

31

40

36

30

17

16

15

9

9

8

Positive Controls

-

Name

SA

SA

NENN

2NF

9AA

Mean no. colonies/plate

598

365

263

760

408

+

Name

2AA

2AA

2AA

2AA

BP

Mean no. colonies/plate

978

278

758

601

235

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

SA = Sodium azide

NENN = N-ethyl-N-nitrosourea

2NF = 2-Nitrofluorene

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material was not mutagenic in bacterial reverse mutation tests conducted in four strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli WP2 uvrA with or without metabolic activation.
Executive summary:

The test material was examined for mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, the tryptophan-requiring Escherichia coli strain WP2 uvrA, and a liver fraction of Aroclor 1254 -induced rats for metabolic activation (S9 mix). The test was performed in accordance with the standardised guidelines OECD 471 and EPA OPPTS 870.5100, under GLP conditions.

Five different concentrations of the test material were used ranging from 62 - 5000 µg/plate. Vehicle and positive controls were run simultaneously.

The test material was toxic to E. coli WP2 uvrA strain and to TA 100 at the two highest doses, 1667 and 5000 ug/plate, in both the absence and presence of S9 mix, and also, to TA 100 at 185 and 556 ug/plate in the absence of S9 mix, evidenced by a decrease in the mean number of revertant colonies. It was also toxic to TA 1537 at 556, 1667, and 5000 µg/plate in the presence of the S9 mix. The test material did not cause a more than 2 -fold or a dose-related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate in the negative control. The mean number of his+ and trp+ revertant colonies in the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.

Under the conditions of this study, the test material was not mutagenic in bacterial reverse mutation tests conducted in four strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli WP2 uvrA with or without metabolic activation.