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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:
2010-03-05 - 2010-04-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Oil shale, thermal processing residue
IUPAC Name:
Oil shale, thermal processing residue
Constituent 2
Reference substance name:
Oil shale, thermal processing waste
EC Number:
297-648-1
EC Name:
Oil shale, thermal processing waste
Cas Number:
93685-99-5
Molecular formula:
Not applicable (UVCB substance)
IUPAC Name:
tricalcium oxo[(oxoalumanyl)oxy]alumane oxo[(oxoferrio)oxy]iron silanedione carbonate oxidandiide sulfate
Details on test material:
- Name of test material (as cited in study report): Burnt Oil shale (BOS)
- Physical state: greish-reddish fine powder
- Analytical purity: no data
- Lot/batch No.: P.1082
- Expiration date of the lot/batch: not applicable
- Storage condition of test material: room temperature, avoid moisture
- Density: ca. 2.8 [g/m³]

Method

Target gene:
his-operon
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix (phenobarbital and β-naphthoflavone induced)
Test concentrations with justification for top dose:
0 (solvent control), 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
Aqua dest.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9: 4-nitroquinoline-N-oxide (10 µg/plate for TA 98, 40 µg/plate for Ta 1537), sodium azide (10 µg/plate), methylmethanesulfonate (1 µl/plate), +S9: 2-aminoanthracene (2.5 µg/plate; 10 µg/plate for TA 102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Two experiments were performed: in agar (plate incorporation) (experiment 1) and preincubation (Experiment 2)

DURATION
- Preincubation period: 60 min (experiment 1)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: background lawn or reduction in the number of revertants
Evaluation criteria:
Evaluation of cytotoxicity:
Cytotoxicity can be detected bay a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 to the solvent control.

Evaluation of mutagenicity:
The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control.
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if a tester strains TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 98, TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
Statistics:
No data

Results and discussion

Test results
Species / strain:
S. typhimurium, other:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Ames Test Results - Experiment 1

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

Base-pair substitution type

Frameshift type

TA 98

TA 100

TA 1535

TA 1537

TA 102

-

Neg. control (a. dest.)

22

115

17

11

301

-

31.6

21

98

14

14

290

-

100

20

95

11

9

288

-

316

23

111

16

12

367

-

1000

20

117

17

12

372

-

2500

27

138

14

15

396

-

5000

27

134

12

12

380

Positive

controls

- S9

Name

4-NOPD

NaN3

NaN3

4-NOPD

MMS

Concentrations

(μg/plate)

10

10

10

40

1 µl/plate

Number of colonies/plate

418

815

921

206

1666

 

TA 98

TA 100

TA 1535

TA 1537

TA 102

+

Neg. control (a. dest.)

28

131

10

9

204

+

31.6

35

124

10

18

212

+

100

29

117

10

13

211

+

316

30

125

10

11

176

+

1000

35

122

8

14

203

+

2500

28

141

11

13

250

+

5000

23

134

6

17

213

Positive

controls

+ S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5

2.5

2.5

2.5

10

Number of colonies/plate

2484

2251

201

439

1692

Table 2: Ames Test Results - Experiment 2

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

Base-pair substitution type

Frameshift type

TA 98

TA 100

TA 1535

TA 1537

TA 102

-

Neg. control (a. dest.)

19

117

10

6

325

-

31.6

26

128

11

7

331

-

100

31

138

12

12

348

-

316

32

137

12

5

354

-

1000

36

153

10

11

362

-

2500

27

152

7

12

407

-

5000

28

128

10

11

452

Positive

controls

- S9

Name

4-NOPD

NaN3

NaN3

4-NOPD

MMS

Concentrations

(μg/plate)

10

10

10

40

1 µl/plate

Number of colonies/plate

578

994

989

134

1678

 

TA 98

TA 100

TA 1535

TA 1537

TA 102

+

Neg. control (a. dest.)

37

115

8

7

290

+

31.6

31

117

6

10

280

+

100

28

120

13

11

310

+

316

35

116

8

11

268

+

1000

26

114

8

12

323

+

2500

33

113

7

9

326

+

5000

34

139

10

14

305

Positive

controls

+ S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5

2.5

2.5

2.5

10

Number of colonies/plate

2432

1411

95

207

1079

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions, Oil shale, thermal processing waste did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therfore, Oil shale, thermal processing waste is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In order to investigate the potential of Oil shale, thermal processing waste for its ability to induce gene mutations the plate incorporation test (experiment 1) and the pre-incubation test (experiment 2) were performed with Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. In 2 independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. Precipitation of the test item was observed in all tester strains used in experiment 1 and 2 (with and without metabolic activation).

No toxic effect of the test item were noted in any of the 5 tester strains used up to the highest dose group evaluated in both experiments with the exception of a toxic effect noted in strain TA 100 at a concentration of 5000 µg/plate in experiment 2.

No biologically relevant increases in revertant colony numbers of any of the 5 tester strains were observed following treatment with Oil shale, thermal processing waste at any concentration level, neither in the presence nor absence of metabolic activation.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiment.