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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2005-06-17 to 2005-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction by the because it was carried out in accordance with OECD Test Guideline 471 and is GLP compliant.
Justification for type of information:
The standard OECD 471 test is not suitable to test petroleum UVCBs, because it has a tendency to produce false negatives for these substances. Therefore, the petroleum industry has developed a Modified Ames assay, optimized to accurately identify positive results for this endpoint. This deviation from the prescribed testing procedure requires some further explanation which is given in the attached document. The document gives a brief history of the development of the Modified Ames test and outlines Concawe’s proposed work (as part of a wider testing strategy, see Annex 13) to further support the use of this test for PS.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
-Test Substance: CAS number: 8002-74-2 (100% purity)
Chemical name: aliphatic alkane
Other name: hydrocarbon wax
Appearance: solid, white, waxy
Batch/Lot Number: A053603
Molecular Formula: C(n)H(2n+2); Sasolwax 5203 contains alkanes ranging from C19 to C36. The man component is C26
Average Molecular Weight: 360
Melting point: 54°C
TNO Test Substance Number: 0500A9

Prior to administration, test substance was melted and extracted in DMSO at a concentration of 50 mg/ml Sasolwax 5203 for approximately 30 minutes at 60±2°C under agitation. After 30 minutes incubation, the wax was coagulated by incubation at 6°C for 8 minutes. The extract (DMSO aliquot) was collected. The test substance was melted again and extraction procedure was repeated. A light white extraction solution was obtained. The extracts were pooled and serial dilutions, separated by 3-fold intervals, in DMSO were prepared.

- Viscosity (3.5 mm2/s at 100 °C)
- Congealing point (54 °C)
- Color saybolt ASTM D156 (30)
- Oil content (0.2%)

Method

Target gene:
No data reported.
Species / strain
Species / strain / cell type:
other: S. typhimurium TA98, 100, 1535, 1537 and E. coli WP 2 uvrA
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Wistar rat S9 homogenate
Test concentrations with justification for top dose:
Just before use, the test substance was extracted with DMSO at 50 mg/ml. The test substance was melted at approximately 60°C and extracted with half of the volume of vehicle needed, for 30 minutes at 60 ± 5°C under agitation. After the 30 minutes incubation, the wax was coagulated by incubation at ± 6°C for ± 8 minutes. The extract (DMSO aliquot) was collected. The test substance was melted again and extraction procedure was repeated. A light white extraction solution was obtained. The extracts were pooled and serial dilutions, separated by 3-fold intervals, in DMSO were prepared. A highest dose 100 % extract was tested. In total test five dose levels, ranging from 1.23 to 100 % of the extract were tested, which is comparable to 62 to 5000 μg of the test substance per plate, assuming a complete extraction. Nominal concentrations were used.
Vehicle / solvent:
dimethyl sulphoxide
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: In the absence of S9, positive controls were sodium azide 1.0 µg/plate, 9-aminoacridine 80.0 µg/plate, 2-nitrofluorene 2.0 µg/plate, N-ethyl-N-nitrosourea 100 µg/plate. In the presence of the S9 mix, 2-aminoanthracene 2.0 µg/plate, benzo(a)pyrene 4.0 µg
Details on test system and experimental conditions:
Briefly, the mutagenicity assay was carried out as follows. To 2 millilitres molten top agar (containing 0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine HCl/0.05 mM biotin for the S. typhimurium strains, and supplemented with 0.05 mM tryptophane for the E. coli WP2 uvrA strain), maintained at approximately 46°C, were added subsequently: 0.1 millilitres of a fully grown culture of the appropriate strain, 0.1 millilitres of the test substance extract, or of the negative or the positive control substance solution, and 0.5 millilitres S9-mix for the experiments with metabolic activation or 0.5 millilitres sodium phosphate 100 millimolar (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed, and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). All determinations were made in triplicate. The plates were incubated at approximately 37°C for approximately 72 hours. Subsequently, the his+ and trp+ revertants were counted. Cytotoxicity is defined as a reduction (at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.
Evaluation criteria:
The mutagenicity study is considered valid if the mean colony counts of the control values of the strains were 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 substance is considered to be positive in the bacterial gene mutation test if there is a concentration-related increase in the mean number of revertants colonies on the test plates of if a reproducible two-fold or more increase is observed to that on the negative
control plates.

A test substance 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.
Statistics:
No statistic analyses were performed.

Results and discussion

Test results
Species / strain:
other: S. typhimurium strains TA 1535, TA 1537, TA 98, and TA 100; E. coli strain WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Extract of Sasolwax 5203 was slightly toxic, in the presence and absence of S9-mix, to TA 1537 at and above 33.3% of the extract, and to TA 98 at 100% of the extract, as was evidenced by a slightly less dense background lawn compared to the negative control. In both the absence and presence of S9-mix in all strains, the extract of Sasolwax 5203 did not cause a dose-related or more than two-fold 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 controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Dose

(% of extract)

TA 1535

TA 1537

TA 98

TA 100

E. coli

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0%

24

19

2

16

26

47

118

133

44

38

25

20

13

17

34

42

142

144

38

42

24

17

19

30

34

49

120

133

43

41

Mean

24

19

11

21

31

46

127

137

42

40

StDev

1

2

9

8

5

4

13

6

3

2

1.23%

34

16

17

22

34

38

103

130

35

38

26

24

17

28

28

46

143

136

34

32

25

25

***

24

28

50

118

144

29

40

Mean

28

22

17

25

30

45

121

137

33

37

StDev

5

5

0

3

3

6

20

7

3

4

3.70%

23

13

12

20

28

50

100

137

28

42

23

18

13

25

37

44

115

150

34

44

22

19

22

25

37

74

126

124

36

53

Mean

23

17

16

23

34

56

114

137

33

46

StDev

1

3

6

3

5

16

13

13

4

6

11.10%

24

16

10

20

31

37

129

154

35

50

25

11

16

34

37

53

121

154

40

43

32

18

12

14

43

40

107

150

38

36

Mean

27

15

13

23

37

43

119

153

38

43

StDev

4

4

3

10

6

9

11

2

3

7

33.30%

26

14

11

35

36

36

138

138

32

34

26

18

13

12

24

49

125

101

38

34

22

13

22

19

34

56

137

148

37

43

Mean

25

15

15

22

31

47

133

129

36

37

StDev

2

3

6

12

6

10

7

25

3

5

100%

30

16

7

20

28

41

151

121

35

37

25

14

8

24

32

50

129

109

31

40

29

13

13

13

30

55

151

136

44

34

Mean

28

14

9

19

30

49

144

122

37

37

StDev

3

2

3

6

2

7

13

14

7

3

Pos. Control

527

402

1631

165

889

730

728

1493

***

921

530

452

3461

159

***

691

722

1517

288

1129

539

380

3266

156

1086

500

694

1569

333

1058

Mean

532

411

2786

160

988

640

715

1526

311

1036

StDev

6

37

1005

5

139

123

18

39

32

106

Mean: Average number of revertants per plate (bolded)

StDev: Standard deviation

S9: Liver homogenate from rats treated with aroclor

Pos. Control Positive control

*** Not counted due to contamination

Underlined values have slightly less dense background lawn of bacterial

Applicant's summary and conclusion

Conclusions:
It is concluded that the results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that the extract of Sasolwax 5203 is not mutagenic under the conditions employed in this study.
Executive summary:

In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA 1535, TA 1537, TA 98, TA 100) andE. coli (WP 2 uvrA) were exposed to extracted Sasolwax 5203 in DMSO.  A highest dose of 100% extract was tested. In total, five dose levels ranging from 1.23 to 100% of the extract were tested, which is comparable to nominal concentrations of 62 to 5000 μg of the test substance per plate.  The positive controls induced the appropriate response in the corresponding strains.  There was no evidence of induced mutant colonies over background.

This study received a Klimisch score of 1 and is classified as reliable without restriction because it was carried out in accordance with OECD Test Guideline 471 and is GLP compliant.