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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:
weight of evidence
Study period:
8 December 2000 to 21 December 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Japan Ministry of Agriculture, Forestry and Fisheries. (1985) Notification of Director General, Agricultural Production Bureau. NohSan No. 4200.
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Joint Directives of J EPA, J MHW and J MITI. (31 October 1997) Kanpoan No. 287, Eisei No. 127 and Kikyoku No. 2 (31 October 1997).
Deviations:
not specified
Principles of method if other than guideline:
In addition, the study was also designed to comply with the following guidelines:
-JMHW Genotoxicity Testing Guideline, PAB Notification No. 1604 (1 November 1999)
-Official Notice of J MOL. (8 February 1999)
-ICH (1995 & 1997)
-followed the recommendations of the United Kingdom Environmental Mutagen Society (Gatehouse et al 1990).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Physical state: Grey powder
- Storage conditions: Room temperature

Method

Target gene:
S. typhimurium: histidine locus
E. coli: tryptophan locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Batches of the strains were obtained from master stocks held in liquid nitrogen. The test batches were aliquots of nutrient broth cultures and were stored at -80 °C. Dimethyl sulphoxide (DMSO) was added to the cultures at 8 % v/v as a cryopreservative. Each batch of frozen strain was tested, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light and the pKM101 plasmid, which confers resistance to ampicillin. The responses of the strains to a series of diagnostic mutagens were also assessed.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: the test material was suspended in purified water (obtained by reverse-osmosis) containing 0.15 % agar.
- Justification for choice of solvent/vehicle: the test material was found to be insufficiently soluble in all compatible solvents.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water contining 0.15 % agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
furylfuramide
other: 2-aminoanthracene
Details on test system and experimental conditions:
PREPARATION OF S9 METABOLIC ACTIVATION SYSTEM: S9 fraction was prepared from a group of ca. 10 male Sprague-Dawley rats. Mixed function oxidase systems in the rat livers were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in corn oil at a dosage of 500 mg/kg bw. On the fifth day after injection, following overnight fasting, the rats were killed by cervical dislocation and their livers aseptically removed. Under aseptic conditions, at 0 - 4 ºC, the livers were placed in 0.15 M KCl (3 mL KCl:1 g liver) before being transferred to a homogeniser. Following preparation, the homogenate was centrifuged at 9000 x gravity for 10 minutes. The supernatant fraction (S9 fraction) was stored at -80 ºC until required. Each batch of S9 mix was tested for sterility and efficacy.
PREPARATION OF S9 MIX: S9 fraction (10 % v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6 -phosphate (5 mM), NADPH (4 mM), NADH (4 mM). All the cofactors were filter-sterilised before use.

METHOD OF APPLICATION: in medium; the range finding test was a standard plate incorporation assay; the definitive test included a preincubation step.

DURATION
- Preincubation period: 30 minutes at 37 °C
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: test concentrations were performed in triplicate

EVALUATION PROCEDURE: following the total incubation period the plates were examined for the lack of microbial contamination and evidence that the test was valid, i.e. there was a background lawn on the solvent control plates and on the plates for (at least) the lower concentrations of test material, and that the positive controls had responded as expected. All plates were counted using a Domino automated colony counter.
Evaluation criteria:
For a test to be considered valid, the mean of the solvent/vehicle control revertant colony numbers for each strain should lie within the 99 % confidence limits of the historical control range of the laboratory (previous 2 - 5 years). Also, positive control compounds must cause at least a doubling of mean revertant colony numbers over the negative control.

The mean number of revertant colonies for each treatment group was compared with those obtained for the solvent/vehicle control groups. The mutagenic activity of the test material was assessed by applying the following criteria:

- If treatment with a test material produces an increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-response relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9-mix, the material will be considered to show evidence of mutagenic activity in the test system.

- If treatment with a test material does not produce reproducible increases of at least 1.5 times the concurrent solvent/vehicle controls in either mutation test, the test material will be considered to show no evidence of mutagenic activity in the test system.

- If the results obtained fail to satisfy a clear positive or negative response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.
Statistics:
If required, the procedure used to determine the statistical significance of any increases in revertant colony numbers was analysis of variance followed by Dunnett's test.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
-The absence of colonies on sterility check plates confirmed that no microbial contamination took place.
-The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.
-The mean revertant colony counts for the solvent controls were within the 99 % confidence limits of the current historical control range of the laboratory (except strain TA 98, definitive test, where counts were slightly higher; this was not considered to affect the integrity of the study). Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

RANGE FINDING TEST
-No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at any concentration in either the presence or absence of S9 mix.
-No visible thinning of the background lawn of the non-revertant cells was obtained following exposure to the test material. A maximum exposure concentration of 5000 µg/plate was therefore selected for use in the definitive study.

DEFINITIVE TEST
-No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test material at any concentration in either the presence of absence of S9 mix.
-No visible thinning of the background lawn of non-revertant cells was obtained following exposure to test material.
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 Second Test

+/-

S9 Mix

Concentration

(µg/plate)

Mean number of revertant colony counts

TA100

TA1535

WP2uvrA

TA98

TA1537

-

-

-

-

-

-

Solvent Control

50

150

500

1500

5000

124

121

122

135

134

128

17

17

14

20

25

21

130

138

124

93

109

109

42

43

39

36

43

45

14

14

14

14

13

11

+

+

+

+

+

+

Solvent Control

50

150

500

1500

5000

152

134

138

143

147

126

19

12

16

11

12

12

159

150

139

147

161

155

50

51

54

48

47

50

21

16

19

17

18

13

                                                       Positive Controls

 

-

Name

SA

SA

AF2

2NF

9AA

Concentration (µg/plate)

0.5

0.5

0.05

1

30

Mean no. colonies

534

362

483

227

423

 

+

Name

BP

2AA

2AA

BP

BP

Concentration (µg/plate)

5

2

10

5

5

Mean no. colonies

743

196

395

496

115

SA = sodium azide 2-NF = 2-nitrofluorene BP = benzo[a]pyrene 2AA = 2-aminoanthracene AF-2 = furylfuramide 9AA = 9-aminoacridine

Applicant's summary and conclusion

Conclusions:
Under the conditions of the assay according to OECD 471, the test material gave a negative response (i.e. non-mutagenic), in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2uvrA/pKM101. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The potential of the test material tantalum metal (purity: 99.9 %) to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100. Four strains of Salmonella typhimurium (TA98, TA100, TA1535, 1537) and E.coli strain WP2uvrA/pKM101 were treated in the presence and absence of a rat liver derived metabolic activation system (S9 mix).

Under the conditions of the test, the test material did not induce any significant, reproducible increases in the observed number of revertant colony numbers in any of the tester strains used. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It was therefore concluded that, under the test conditions employed, the test material showed no evidence of mutagenic activity in this bacterial system.