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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:
key study
Study period:
09-Nov-2020 to 20-Nov-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin
EC Number:
941-364-9
Molecular formula:
Not applicable to UVCB substance
IUPAC Name:
Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin
Test material form:
other: Low visocosity, liquid hydrocarbon
Details on test material:
Batch number: 204377961
Physical description: Colourless or pale yellow coloured liquid
Purity: 100% UVCB
Expiry date: 20 August 2022 if kept under storge conditions
Source and site of characterisation: Repsol Refinery, Carretera de la Calzada s/n, Apartado de Correos 12, 13500 Puertollano, Ciudad Real, Spain
Storage conditions: Store under controlled humidity conditions and temperature (18-25 °C) in a sealed container, protecting the product from sunlight (opaque container or in a cabinet)

Method

Target gene:
S. typhimurium strains: his-operon
E. coli strains: tryptophan-operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene, Potassium dichromate
Details on test system and experimental conditions:
Preliminary assessment of solubility
A preliminary solubility assessment was conducted for the test item, Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9), at 100 mg per mL in dimethyl sulphoxide (DMSO). As the test item dissolved in DMSO at 100 mg per mL, solubility was not assessed with any other solvents and DMSO was used as the solvent for the test item throughout this study.

Mutagenicity tests
Test 1
A plate incorporation mutagenicity test was performed using Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, in both the presence and absence of S9 mix. In all cases there were three plates in the solvent control, test item and positive control groups.

Test 2
For Test 2, a liquid pre-incubation test was performed using Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101 in the presence and absence of S9 mix. In all cases there were three plates in the solvent control, test item and positive control groups.

Test item administration
The test item was administered in solvent (DMSO) at a volume of 100 μL per plate for the plate incorporation method and a volume of 50 μL per plate when the liquid pre-incubation method was employed, within a maximum of three hours of formulation.
Evaluation criteria:
Test acceptance criteria
A minimum of five analysable (scoreable) concentrations was required, with at least four showing no signs of cytotoxicity.

Evaluation
Cytotoxicity
A dose of the test item was judged to be toxic to a bacterial strain if the formation of microcolonies (background lawn) was reduced, or a relevant decrease in the number of revertant colonies was seen.

Mutagenicity
A test item was considered to be mutagenic if the following criteria were satisfied:
- For all five strains, the mean number of revertant colonies is equal to or greater than 2 times the concurrent solvent control mean value at one or more doses of the test item, with or without. In addition, for TA1535 and TA1537, the mean number of revertant colonies of one or more doses of the test item, with or without metabolic activation must be equal to or greater than 2 times the relevant historical mean value.
- There was a dose-related increase in the number of revertant colonies.
- A reproducible (at one or more doses) increase in numbers of revertant colonies per plate in at least one strain with or without metabolic activation.
Statistics:
The mean number of revertant colonies and standard deviations were calculated for all groups.
All valid data were plotted and analysed using a linear regression analysis programme.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Plate incorporation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: above 5000 μg/plate with metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Plate incorporation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: above 5000 μg/plate with metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Plate incorporation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: above 5000 μg/plate with metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Plate incorporation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: above 5000 μg/plate with metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Remarks:
Plate incorporation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Liquid pre-incubation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
above 500 μg/plate without metabolic activation; 1600 μg/plate with metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Liquid pre-incubation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
above 500 μg/plate without metabolic activation; 1600 μg/plate with metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Liquid pre-incubation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
above 500 μg/plate without metabolic activation; 1600 μg/plate with metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Liquid pre-incubation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
above 500 μg/plate without metabolic activation; 1600 μg/plate with metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Remarks:
Liquid pre-incubation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 μg/plate with metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Plate incorporation test
On the day of dosing, precipitate was seen at and above 500 μg per plate for all five strains in both the presence and absence of S9 mix. On the day of scoring, precipitate was seen at and above 1600 μg per plate for all five strains in both the presence and absence of S9 mix.
Evidence of cytotoxicity as indicated by reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies was seen at a dose of 5000 μg per plate for the four S. typhimurium strains in the absence of S9 mix.
No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.

Liquid pre-incubation test
On the day of dosing precipitate was seen at and above 500 μg per plate for all five strains in both the presence and absence of S9 mix. On the day of scoring, precipitate was seen at and above 1600 μg per plate for all five strains in both the presence and absence of S9 mix.
Evidence of cytotoxicity as indicated by reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies was seen at and above a dose of 500 μg per plate for the four S. typhimurium strains in the absence of S9 mix, at and above 1600 μg per plate for four S. typhimurium strains in the presence of S9 mix, and at 5000 μg per plate for the E. coli strain in the absence of S9 mix.
No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
no mutagenic potential

Any other information on results incl. tables

Table 1 – Test 1: Petroleum Gas Oil Fraction, Co-pressed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): plate incorporation method – with metabolic activation

Dose per plate

Number of revertant colonies per plate

S. typhimuriumLT2

E. coliWP2

 

TA1535

TA1537

TA98

TA100

uvrA/pKM101

μg

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

12

2.1

11

1.5

37

2.5

138

10.0

201

7.8

1.6

14

2.1

13

1.2

34

4.5

154

5.0

202

14.6

5

12

2.0

12

0.6

33

3.2

142

11.4

201

12.4

16

10

1.0

13

1.5

36

2.1

131

7.8

214

1.5

50

13

2.1

11

1.5

31

6.1

120

11.2

187

9.5

160

12

2.0

12

2.1

34

7.6

147

6.2

191

17.9

500

11

3.1

9

1.2

38

3.1

142

18.5

192

14.7

1600

11

4.7

10

3.2

39

2.6

150

11.4

194

14.0

5000

11

4.9

15

1.5

40

11.4

152

3.8

178

17.6

Positive control

176

7.5

152

4.5

1586

180.4

2455

20.9

1945

21.4

Positive controls

TA1535: 2-aminoanthracene 2 μg/plate
TA1537: 2-aminoanthracene 2 μg/plate
TA98: 2-aminoanthracene 2 μg/plate
TA100: 2-aminoanthracene 2 μg/plate
uvrA/pKM101: 2-aminoanthracene 20 μg/plate

 

Table 2 – Test 1: Petroleum Gas Oil Fraction, Co-pressed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): plate incorporation method – without metabolic activation

Dose per plate

Number of revertant colonies per plate

S. typhimuriumLT2

E. coliWP2

 

TA1535

TA1537

TA98

TA100

uvrA/pKM101

μg

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

15

1.5

11

0.6

27

0.6

128

2.0

170

4.2

1.6

14

2.1

11

2.0

28

2.0

122

2.6

156

10.0

5

13

2.0

9

3.2

25

5.6

113

10.6

165

2.0

16

14

1.5

10

0.6

24

7.1

104

2.5

173

3.0

50

11

1.7

9

3.8

23

6.1

110

3.2

155

4.2

160

12

1.2

11

1.5

23

7.0

110

9.7

150

13.9

500

14

2.6

12

1.7

26

4.5

113

12.8

141

8.3

1600

11

0.6

10

2.5

25

5.5

101

10.8

141

10.0

5000

10

2.0

9

1.5

24

2.1

100

8.2

145

5.7

Positive control

432

7.6

195

8.3

360

13.1

558

28.3

1000

57.3

Positive controls

TA1535: sodium azide 0.5 μg/plate
TA1537: 2-aminocridine.HCl 50 μg/plate
TA98: 2-nitrofluorene 1 μg/plate
TA100: sodium azide 0.5 μg/plate
uvrA/pKM101: potassium dichromate 25 μg/plate

 

Table 3 – Test 2: Petroleum Gas Oil Fraction, Co-pressed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): liquid pre-incubation method – with metabolic activation

Dose per plate

Number of revertant colonies per plate

S. typhimuriumLT2

E. coliWP2

 

TA1535

TA1537

TA98

TA100

uvrA/pKM101

μg

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

10

0.6

9

1.0

32

3.1

142

6.7

209

6.7

1.6

11

1.0

10

2.1

31

2.6

148

5.0

193

7.6

5

12

1.0

8

2.6

18

8.1

140

16.6

197

23.5

16

10

0.6

8

2.0

29

6.6

115

19.3

197

8.6

50

10

3.1

8

2.0

30

6.0

120

9.2

208

1.5

160

9

3.1

9

3.5

24

2.1

113

28.7

192

7.0

500

10

2.1

9

3.2

25

5.5

125

2.1

204

13.2

1600

7

2.0

6

2.1

25

5.6

116

11.8

195

12.7

5000

8

1.5

8

1.5

25

1.0

111

11.0

175

8.5

Positive control

126

1.5

102

6.1

1327

105.1

1769

35.4

1844

56.7

Positive controls

TA1535: 2-aminoanthracene 2 μg/plate
TA1537: 2-aminoanthracene 2 μg/plate
TA98: 2-aminoanthracene 2 μg/plate
TA100: 2-aminoanthracene 2 μg/plate
uvrA/pKM101: 2-aminoanthracene 20 μg/plate

 

Table 4 – Test 2: Petroleum Gas Oil Fraction, Co-pressed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): liquid pre-incorporation method – without metabolic activation

Dose per plate

Number of revertant colonies per plate

S. typhimuriumLT2

E. coliWP2

 

TA1535

TA1537

TA98

TA100

uvrA/pKM101

μg

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Solvent control

13

1.2

9

0.6

28

1.2

118

2.5

146

7.2

1.6

12

1.0

9

0.6

27

5.7

120

4.2

148

7.1

5

12

2.0

8

1.0

26

1.7

113

3.5

135

2.0

16

11

2.6

9

2.1

28

2.1

116

6.8

143

5.0

50

10

1.5

11

1.2

27

4.5

110

3.6

132

6.8

160

11

2.1

8

1.2

23

6.0

101

3.0

128

6.0

500

10

2.6

5

3.1

16

1.5

98

8.9

121

7.8

1600

5

1.2

4

0.6

17

3.1

76

14.5

122

17.4

5000

7

1.0

3

1.5

15

3.0

100

7.8

100

4.0

Positive control

435

22.0

175

15.0

343

27.4

444

28.0

939

29.7

Positive controls

TA1535: sodium azide 0.5 μg/plate
TA1537: 2-aminocridine.HCl 50 μg/plate
TA98: 2-nitrofluorene 1 μg/plate
TA100: sodium azide 0.5 μg/plate
uvrA/pKM101: potassium dichromate 25 μg/plate

Applicant's summary and conclusion

Conclusions:
It was concluded that Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was not mutagenic for Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay.
Executive summary:

Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was tested for mutagenic activity using genetically modified Salmonella typhimurium LT2 bacteria of strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101 as indicator organisms, according to the methods of Maron and Ames, 1983, Venitt et al, 1984, Mortelmans and Zeiger, 2000 and Mortelmans and Riccio, 2000.

A preliminary solubility test was conducted. Dimethyl sulphoxide was found to be suitable and was therefore used throughout this study as the solvent for the test item.

A mutagenicity test was conducted for Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) using the plate incorporation method (Test 1) for all five indicator strains in both the presence and absence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). The dose range used was 1.6 to 5000 μg per plate. As the result of Test 1 was clearly negative, a confirmatory test was carried out using the liquid pre-incubation method, Test 2, using a dose range of 1.6 to 5000 μg per plate in the presence and absence of S9 mix.

The test item showed evidence of cytotoxicity. The minimum dose level at which cytotoxicity was seen was 500 μg per plate. The maximum dose level scored for revertant colonies was 5000 μg per plate. The minimum dose level at which precipitate was seen on the test plates was 1600 μg per plate.

No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.

It was concluded that Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was not mutagenic for Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay.

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