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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In response to the Final Decision Letter (dated 03 July 2020) CCH- D-2114515259 -48 -01/F two new in vitro genetic toxicity studies (Ames test (OECD 471) and mouse micronucleus assay (OECD 487)) were conducted on the target substance. As the results from both studies were negative, an OECD 490 study has been planned to be conducted, fulfilling all information requirements.

In addition, two read-across in vitro studies were identified for a structurally related material; there was no evidence of genotoxic activity in-vitro either the modified Ames test or mouse lymphoma assay.

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
S. typhimurium strains: his-operon
E. coli strains: tryptophan-operon
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)
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.
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

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

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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-Jan-2021 to 25-Mar-2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9
Test concentrations with justification for top dose:
The final concentration ranges tested from which micronuclei were analysed for were:
3h +S9 treatment schedule: 296.3 to 1500 µg/mL
3h -S9 treatment schedule : 666.7 to 1500 µg/mL
continuous treatment schedule (24h -S9): 131.7 to 666.7 µg/mL

The following levels of cytotoxicity were observed, recommended maximum level of cytotoxicity (55 ± 5%) was observed in the continuous and 3h -S9 treatment schedules:
3h +S9 treatment schedule: 42.67% at 1500 µg/mL
3h -S9 treatment schedule: 52.84% at 1500 µg/mL
continuous treatment schedule (24h -S9): 55.22% at 666.7 µg/mL

For a test item exhibiting apparent cytotoxicity, the aim was that the highest concentration selected was that which yielded cytotoxicity of ~55 ± 5%. Further doses were selected from those yielding decreasing levels of cytotoxicity, as far as a no-effect dose (little or no cytotoxicity by CBPI).

For test items that did not yield apparent cytotoxicity, the three highest test concentrations were selected for further microscopic analysis.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Test system
The study was designed to meet the requirements of OECD Guideline 487 (2016) and was agreed with the sponsor prior to commencement of the study. Since this study utilised isolated human lymphocytes and hence used cytokinesis block protocols, the specific guidance (in OECD 487) relating to lymphocytes and studies performed in the presence of cytoB, was followed.

Experimental conditions
Human lymphocytes were isolated from pooled anti-coagulated fresh blood obtained from two healthy non-smoking males and allowed to proliferate for approximately 44 to 48 hours in the presence of the mitogen, phytohaemagglutinin (PHA). Cultures of proliferating lymphocytes were exposed to the test item in the presence of S9 mix for 3 hours before cells were washed and then further incubated (humidified atmosphere of 5% CO2 at a temperature of 37oC) in fresh medium containing cytoB for approximately 21 hours. Treatments in the absence of S9 mix were performed for a 3-hour period (followed by cell washing and incubation in fresh medium containing cytoB for approximately 21 hours) and for a continuous exposure period for 24 hours. In the continuous treatment condition, cytoB was added concurrently with the test item. Duplicate cultures were treated for each test item concentration and positive control. For the solvent-treated controls, four cultures were dosed with DMSO.
Evaluation criteria:
Criteria for a clearly positive response:
- at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical solvent control range (Poisson-based 95% control limits)

Criteria for a clearly negative response
- none of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- there was no concentration-related increase when evaluated using an appropriate trend test
- all results were inside the distribution of the historical negative control data (Poisson-based 95% control limits)

Criteria for an equivocal response
Although most experiments are expected to yield clearly positive or negative results, in some cases the data preclude making a definitive judgement about the activity of the test item. Such equivocal responses may occur regardless of the number of times the experiment was repeated.
Statistics:
The number of micronuclei analysed from 2000 binucleated cells for each selected test item dose was compared with that from the concurrent solvent control. Pair-wise statistical analysis employing a one-sided Fisher’s Exact test were used to evaluate statistical significance (p < 0.05). A linear trend test was employed (Cochran-Armitage) in order to confirm there was no dose related increase (p < 0.05).
Key result
Species / strain:
lymphocytes: micronuclei
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
no mutagenic potential

Micronucleus data

Table 1 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): 3 hour exposure testing in the presence of S9 mix

Dose (μg/mL)

3h +S9 treatment

CBPI

% Cytostasis

% MN per dose

p-value

Solvent (DMSO)

1.599

N/A

1.0

N/A

296.3

1.550

8.15

1.2

ns

1000

1.481

19.67

1.1

ns

1500P

1.343

42.67

1.0

ns

CPA 4

1.418

30.15

4.3

2.942×10-11

CBPI = cytokinesis block proliferation index
MN per dose = micronucleated binucleates/2000 binucleates
CPA = cyclophosphamide
P = precipitate observed at the end of the treatment period
ns = not statistically significant (by Fisher’s exact test), % - individual percentages of micronucleated binucleated cells,Cochran-Armitage treated test p-value = 8.445
×10-1, therefore not statistically significant

Table 2 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): 3 hour exposure testing in the absence of S9 mix

Dose (μg/mL)

3h +S9 treatment

CBPI

% Cytostasis

% MN per dose

p-value

Solvent (DMSO)

1.490

N/A

1.1

N/A

666.7

1.426

13.05

1.3

ns

1000

1.315

35.63

1.1

ns

1500P

1.231

52.84

1.0

ns

MMC 50 ng/mL

1.443

9.56

4.6

1.006×10-11

CBPI = cytokinesis block proliferation index
MN per dose = micronucleated binucleates/2000 binucleates
MMC = mitomycin
P = precipitate observed at the end of the treatment period
ns = not statistically significant (by Fisher’s exact test), % - individual percentages of micronucleated binucleated cells,Cochran-Armitage treated test p-value = 5.309
×10-1, therefore not statistically significant

Table 3 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): continuous (24 hour) exposure testing in the absence of S9 mix

Dose (μg/mL)

3h +S9 treatment

CBPI

% Cytostasis

% MN per dose

p-value

Solvent (DMSO)

1.460

N/A

1.1

N/A

131.7

1.501

-8.91

1.1

ns

296.3

1.335

27.10

1.0

ns

444.4

1.298

35.22

1.2

ns

666.7

1.206

55.22

1.2

ns

MMC 30 ng/mL

1.376

18.26

4.5

1.043×10-11

COL 7.5 ng/mL

1.458

0.43

3.9

2.275×10-11

CBPI = cytokinesis block proliferation index
MN per dose = micronucleated binucleates/2000 binucleates
MMC = mitomycin
COL = colchicine
P = precipitate observed at the end of the treatment period
ns = not statistically significant (by Fisher’s exact test), % - individual percentages of micronucleated binucleated cells,Cochran-Armitage treated test p-value = 6.801
×10-1, therefore not statistically significant

Cytotoxicity assessment

Table 4 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): cytotoxicity assessment in cytokinesis blocked lymphocytes from 3 hour exposure testing in the presence of S9 mix

Concentration
µg/mL

Number of nucleated cells

CBPI

% Cytostasis

Mono-

Bi-

Multi-

Total scored

Solvent (DMSO)

208

286

7

501

1.599

0.00

5.138

sns

sns

sns

sns

ND

ND

7.707

sns

sns

sns

sns

ND

ND

11.56

sns

sns

sns

sns

ND

ND

17.34

sns

sns

sns

sns

ND

ND

26.01

sns

sns

sns

sns

ND

ND

39.02

sns

sns

sns

sns

ND

ND

58.53

sns

sns

sns

sns

ND

ND

87.79

sns

sns

sns

sns

ND

ND

131.7

sns

sns

sns

sns

ND

ND

197.5

sns

sns

sns

sns

ND

ND

296.3

231

263

6

500

1.550

8.15

444.4

250

250

1

501

1.503

16.00

666.7

251

244

5

500

1.508

15.16

1000

265

231

5

501

1.481

19.67

1500P

334

162

5

501

1.343

42.67

CPA 4 µg/mL

316

200

10

526

1.418

30.15

Mono = mononucleated cells
Bi = binucleated cells
Multi = multinucleated cells
total scored = sum of mono-, bi- and multi-
CBPI = cytokinesis block proliferation index
CPA = cyclophosphamide
sns = slide not scored/analysed
ND = not determined
P = precipitate observed at the end of the treatment period

Table 5 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): cytotoxicity assessment in cytokinesis blocked lymphocytes from 3 hour exposure testing in the absence of S9 mix

Concentration

µg/mL

Number of nucleated cells

CBPI

% Cytostasis

Mono-

Bi-

Multi-

Total scored

Solvent (DMSO)

283

260

4

547

1.490

0.00

5.138

sns

sns

sns

sns

ND

ND

7.707

sns

sns

sns

sns

ND

ND

11.56

sns

sns

sns

sns

ND

ND

17.34

sns

sns

sns

sns

ND

ND

26.01

sns

sns

sns

sns

ND

ND

39.02

sns

sns

sns

sns

ND

ND

58.53

sns

sns

sns

sns

ND

ND

87.79

sns

sns

sns

sns

ND

ND

131.7

sns

sns

sns

sns

ND

ND

197.5

257

241

3

501

1.493

-0.63

296.3

274

226

0

500

1.452

7.74

444.4

301

205

0

506

1.405

17.31

666.7

288

211

1

500

1.426

13.05

1000

344

156

1

501

1.315

35.63

1500P

386

116

0

502

1.231

52.84

MMC 50 ng/mL

281

218

2

501

1.443

9.56

Mono = mononucleated cells
Bi = binucleated cells
Multi = multinucleated cells
total scored = sum of mono-, bi- and multi-
CBPI = cytokinesis block proliferation index
MMC = mitomycin C
sns = slide not scored/analysed
ND = not determined
P = precipitate observed at the end of the treatment period


Table 6 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): cytotoxicity assessment in cytokinesis blocked lymphocytes from continuous (24 hour) exposure testing in the absence of S9 mix

Concentration
µg/mL

Number of nucleated cells

CBPI

% Cytostasis

Mono-

Bi-

Multi-

Total scored

Solvent (DMSO)

270

230

0

500

1.460

0.00

5.138

sns

sns

sns

sns

ND

ND

7.707

sns

sns

sns

sns

ND

ND

11.56

sns

sns

sns

sns

ND

ND

17.34

sns

sns

sns

sns

ND

ND

26.01

sns

sns

sns

sns

ND

ND

39.02

sns

sns

sns

sns

ND

ND

58.53

sns

sns

sns

sns

ND

ND

87.79

240

259

2

501

1.525

-14.12

131.7

253

245

3

501

1.501

-8.91

197.5

325

174

2

501

1.355

22.76

296.3

335

164

2

501

1.335

27.10

444.4

355

141

4

500

1.298

35.22

666.7

397

103

0

500

1.206

55.22

1000

399

101

0

500

1.202

56.09

1500P

450

50

1

501

1.104

77.44

MMC 30 ng/mL

314

184

2

500

1.376

18.26

Col 7.5 ng/mL

286

199

15

500

1.458

0.43

Mono = mononucleated cells
Bi = binucleated cells
Multi = multinucleated cells
total scored = sum of mono-, bi- and multi-
CBPI = cytokinesis block proliferation index
MMC = mitocyin C
sns = slide not scored/analysed
ND = not determined
P = precipitate observed at the end of the treatment period


Conclusions:
It was concluded that Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) did not induce the formation of micronuclei (MN) in human lymphocytes in the absence and presence of S9, under the test conditions used. The criteria for a negative response were met.
Executive summary:

Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was tested for its potential to induce micronucleus formation in thein vitro micronucleus test with manual scoring on microscope slides. In all tests none of the treatment schedules resulted in significant increases in micronucleus formation in test item treated cultures. It was concluded that Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) did not induce the formation of micronuclei (MN) in human lymphocytes in the absence and presence of S9, under the test conditions used. The criteria for a negative response were met.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recognised method in GLP compliant laboratory
Justification for type of information:
Read across justification included in Section 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: Modified Ames Test according to ASTM E 1687
Principles of method if other than guideline:
14 Gas oils were examined for mutagenic activity in one histidine dependent auxotroph of Salmonella typhimurium, strain TA98, using a modification of the pour-plate assay designed to detect mutagenicity mediated by polynuclear aromatic compounds derived from petroleum, based upon the principles of the ASTM Standard Test Method E 1687.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
His- (reverse mutation to histidine-independence)
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
S. typhimurium TA98 hisD3052 rfa uvrB pKM101
Metabolic activation:
with
Metabolic activation system:
Aroclor 1254-induced S9
Test concentrations with justification for top dose:
Test substance
0, 12, 24, 36, 48, 60 microlitres per plate

Positive control
0, 3, 6, 9, 12, 15 microlitres per plate
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
concurrent DMSO extract of the Reference oil No. 1, a straight-run naphthenic vacuum distillate of known mutagenic activity
Details on test system and experimental conditions:
Preparation of test substance extract
Extracts of the test substance in dimethyl sulphoxide (DMSO) were prepared as follows:
Three separate aliquots (1 mL) of the test substance, warmed to approximately 45°C, were mixed thoroughly with 5 mL of DMSO. The mixtures were vortexed intermittently (every five minutes) for a total of thirty minutes. The mixtures were then centrifuged at 200 g for ten minutes, and the extract (lower phase) was removed. Only one extract of Reference oil No. 1 was prepared. All extracts were prepared on the day of use, and stored in the dark until required.
Dosing solutions were prepared for each test substance extract and for the reference oil extract by dilution with DMSO (the reference oil extract was diluted 1:3 with DMSO before preparation of the dosing solutions).

Mutation test procedure
The following sequence of steps was used in the performance of this test:
The S9 mix was prepared and placed on ice (for not longer than 2 hours before use).
“Top agar", consisting of 0.4% bacteriological grade agar and 0.5% NaCl in purified water (prepared by reverse osmosis) was melted and placed in a 45°C water-bath.
Dosing solutions for the test substance and Reference oil No. 1 were prepared as described above. Two (for each of the test extracts) or three (for the reference extract) sterile glass test tubes were dosed with 60 micro-L of each of these solutions and were allowed to stand for at least 30 minutes.
To each of the dosing tubes were added in order 0.5 mL of S9 mix and 0.1 mL of bacterial suspension, taking care to introduce both the S9 mix and the bacteria into the bottom of the tube. The tube was gently swirled after each addition. When each set of tubes (one test article including the solvent control) was completed, it was transferred to the gyratory incubator at 150 rpm and incubated at 37°C for 30 minutes.
The tubes were removed from the gyratory incubator. To each tube, beginning with the solvent controls and proceeding to higher doses, 2.0 mL of the top-agar, cooled to ca 40°C and supplemented with 10 mL of 0.5 mM histidine/0.5 mM biotin solution per 100 mL, was added. Immediately after addition, the mixture was vortex-mixed and poured onto previously prepared Petri dishes containing 25 mL minimal agar. Care was taken to obtain an evenly distributed and level top-agar layer. When the top-agar had hardened, the plates were inverted and incubated at 37°C for 48 hours. The plates were then removed from the incubator and the number of colonies determined by either automated or manual counting. Each Petri dish was individually labelled with a unique code recorded in the study file, identifying the contents of the dish.
Evaluation criteria:
For a test to be considered valid, the mean revertant colony counts obtained for Reference Oil No. 1 (diluted 1:3) must reach, in a dose-responsive manner, at least a two-fold increase over the mean solvent control count, and no more than three doses should produce mean revertant counts more than 15% below the following representative values:

Dose (µL/plate) 0 3 6 9 12 15

Mean revertants 46 54 69 73 81 93


The mean revertant count for the solvent controls should be in the range 30 - 60. Excursions from this range were considered acceptable only if there was no significant change in the slopes of the curves. The curve should be linear over at least four doses.

The Mutagenicity Index (MI), defined as the slope of the curve (revertants per µL DMSO extract), was calculated for all test samples.

Thresholds for interpretation of MI values defined in ASTM E 1687 are as follows:
Test substances with MI values <1 are considered to have a high probability of being non-carcinogenic in a mouse skin-painting bio-assay.
Test substances with MI values >1 but <2 may or may not be non-carcinogenic in a mouse skin-painting bio-assay.
Test substances with MI values >2 are considered to have a high probability of being carcinogenic in a mouse skin-painting bio-assay.

A lower threshold value of 0.4 has been selected as the cut-off for Residual Aromatic Extracts (s), based on the results of skin-painting studies (Blackburn et al, 1996): s with a MI >0.4 demonstrated carcinogenic potential upon dermal application to mouse skin with chronic exposure, whereas s with a MI <0.4 did not demonstrate a carcinogenic potential.
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.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
MI = 0.33
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix and test substance extracts.
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 obtained for Reference Oil No. 1 (diluted 1:3) reached, in a dose-responsive manner, at least a two-fold increase over the mean solvent control count, and mean revertant counts were within the acceptable range.

It was, therefore, confirmed that the tests were valid.
Remarks on result:
other:
Remarks:
negative with metabolic activation

 

Reference Oil No. 1

 

 

 

 

 

 

 

 

 

 

 

 

 

Dose (µL)

Counts

Mean

SD

 

 

 

 

 

 

 

 

 

 

 

 

0

41

44

45

43.3

2.1

 

 

 

3

56

55

51

54.0

2.6

 

 

 

6

66

70

68

68.0

2.0

 

 

 

9

77

74

76

75.7

1.5

 

 

 

12

79

83

79

80.3

2.3

 

 

 

15

87

91

94

90.7

3.5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Con#16(i)

 

 

 

 

 

 

 

 

 

 

 

 

 

Dose (µL)

Counts A

Counts B

Counts C

Mean

SD

 

 

 

 

 

 

 

 

 

0

42

44

41

46

44

43

43.3

1.8

12

54

55

52

55

54

50

53.3

2.0

24

55

61

61

59

55

57

58.0

2.8

36

62

62

59

59

61

58

60.2

1.7

48

63

59

64

61

61

64

62.0

2.0

60

67

63

63

66

61

62

63.7

2.3

 

 

 

 

 

 

 

 

 

 

Con#16(i) MI value: 0.33

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation

The test substance had a Mutagenicity Index (MI) of 0.33

Test substances with MI values <1 are considered to have a high probability of being non-carcinogenic in a mouse skin-painting bio-assay.
Executive summary:

The test substance had a Mutagenicity Index (MI) of 0.33

Test substances with MI values <1 are considered to have a high probability of being non-carcinogenic in a mouse skin-painting bio-assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: no information on GLP. Study report lacks some details but nevertheless provides useful supporting information
Justification for type of information:
Read across justification included in Section 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
L5178Y mouse lymphoma cells
Test concentrations with justification for top dose:
0.125, 0.188, 0.25, 0.375, 0.5 uL/ml - non activation study
0.064, 0.125, 0.188, 0.25, 0.375, 0.5 uL/ml - activation study
Vehicle / solvent:
acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ACETONE
Positive controls:
yes
Positive control substance:
other: ethylmethane sulphonate and dimethyl nitrosamine
Details on test system and experimental conditions:
L5178Y MOUSE LYMPHOMA CELLS
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
both with and without activation
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Diesel fuel was not mutagenic in the mouse lymphoma assay
Executive summary:

The genotoxic activity of diesel fuel was evaluated in a mouse lymphoma assay using L5178Y cells. Assays both in the presence and absence of a mouse liver mirosome fraction were conducted. Dose levels ranged between 0.064 and 0.5 uL/ML.

Results showed no evidence of of genotoxic activity, either in the presence or absence of metabolic activation. The positive control materials both showed expected results.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Three read-across in-vivo were identified for a structurally related material. In a dominant lethal assay in male mice there was no evidence of heritable genotoxic effects. In a well conducted bone marrow chromosomal aberration assay by the oral route there was no evidence of genotoxic activity. In a further bone marrow chromosomal aberration study by the intraperitoneal route weak activity was observed. Effects were only seen at high dose levels (2.0 and 6.0 ml/kg) and the magnitude of effect was small and restricted to chromosomal fragments.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because, though it does not follow a prescribed guideline, it is well conducted and reported.
Justification for type of information:
Read across justification included in Section 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Principles of method if other than guideline:
No specific GLP guideline was followed. The study is well conducted and reported.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Doses / Concentrations:
1.0, 2,5, 5,0 g/kg
Basis:
no data
No. of animals per sex per dose:
15 male 15 female / dose level
Tissues and cell types examined:
bone marrow erythrocytes
Details of tissue and slide preparation:
bone marrow smears harvested at 24, 48, 72 hrs post exposure
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
The study authors concluded that home heating oil did not exhibit a positive response when tested for micronucleus induction.
Executive summary:

In a micronucleus assay, fifteen male and female CD-1 mice were treated with 1.0, 2.5, or 5.0 g/kg of home heating oil dissolved in corn oil via oral gavage. A concurrent control group received only corn oil, while another group served as positive control and was treated with 0.04 g/kg cyclophosphamide.

Five male and female mice from each dose group were sacrificed 24, 48 or 72 hours after test material administration. Bone marrows were removed and examined for the presence of micronuclei. Mice treated with cyclophosphamide were sacrificed 24 hours after administration. The micronucleus assay was conducted using the Schmid method. Erythrocytes numbering 1,000, were counted for each animal bone marrow slide and the number of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. Frequency of micronucleus induction was determined by examining the number of micronuclei per 1,000 PCEs. 

There was no increase in the frequency of micronuclei for the test material. In addition, there was no evidence of bone marrow depression. Cyclophosphamide, the positive control, exhibited appropriate results and the vehicle control result was within the normal range. Based on these results the study authors concluded home heating oil did not exhibit a positive response. 

This study received a Klimisch rating of “reliable with restrictions” because, though it does not follow a prescribed guideline, it is well conducted and reported.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: other: dominant lethal assay
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No information on GLP Compliance, No guideline stated. Study report lacks some details but nevertheless provides useful supporting information
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
no guideline available
Principles of method if other than guideline:
Male mice were exposed by inhalation to diesel fuel, 6 hrs/day, 5 days / week for 8 weeks prior to mating with untreated female mice. Dominant lethal outcome was assessed by monitoring successful uterine implants and also number of dead implants.
GLP compliance:
not specified
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
CD-1
Sex:
male
Route of administration:
inhalation
Vehicle:
filtered air
Duration of treatment / exposure:
8 weeks
Frequency of treatment:
6 hours / day, 5 days / week
Remarks:
Doses / Concentrations:
100 and 400 ppm
Basis:

No. of animals per sex per dose:
12 males / dose
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine (TEM)
Sex:
male
Genotoxicity:
negative
Remarks:
NO EFFECT ON MALE FERTILITY
Positive controls validity:
valid


The results were as follows:

                                Dose Group
                 100        400        Negative     Positive
  Parameter        ppm        ppm        control      control

 Fertility index  
    Week 1        0.727       0.750        0.708      0.458
    Week 2        0.955       0.792        0.833      0.792


Implants/female
    Week 1        11.06       10.72        11.18      8.36
    Week 2        10.81       11.68        10.95      7.42


Resorptions/female
    Week 1        0.375       0.5         0.77        4.73
    Week 2        0.71        0.32        0.55        4.9


Proportion of females with 1 or more dead implants
    Week 1        0.32        0.39        0.41        1.0
    Week 2        0.52        0.26        0.55        1.0

Proportion of females with 2 or more dead implants
    Week 1        0.06        0.11        0.24        1.0
    Week 2        0.14        0.05        0           0.95


Dead implants/Total implants
    Week 1        0.03        0.04        0.07        0.57
    Week 2        0.07        0.03        0.05        0.66


Living implants/pregnant female
    Week 1        10.7        10.2        10.4        3.6
    Week 2        10.1        11.4        10.4        2.5

Conclusions:
Interpretation of results (migrated information): negative for effects on male fertility
Inhalation exposure of male mice to diesel fuel showed no evidence of an effect on male fertility.
Executive summary:

The effects of inhalation exposure to diesel fuel on male fertilty were investigated in mice exposed 6 hrs/day, 5 days /week for 8 weeks. Following exposure, mice were mated with untreated female mice. Levels of exposure were 100 and 400 ppm. Effect on male fertility was evalueted on the basis of number of succesful uterine implants and viability of the implanted embryos.

There was no effect on the number of succesful impants, pre or post implant loss or death of embryos. The positive control material (TEM ) gave the expected response.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
Read across justification included in Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 10-12 weeks
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ] yes
- Fasting period before study:
- Housing: 5/cage
- Diet (e.g. ad libitum): Purina lab chow
- Water (e.g. ad libitum):
- Acclimation period: 10 days

Route of administration:
intraperitoneal
Details on exposure:
animals dosed and 2 hrs before sacrifice administered 4mg/kg colchicine to arrest metaphase
Duration of treatment / exposure:
Up to 48 hours and 5 days
Frequency of treatment:
either single dose or 5 exposures 24 hrs apart
Post exposure period:
Single dose - 6 24 or 48 hrs post dose
Repeat dose - 6hrs after last treatment
Remarks:
Doses / Concentrations:
0.6, 2 and 6 ml/kg
Basis:

No. of animals per sex per dose:
Acute - 15 animals / dose
Repeat dose - 5 animals / dose
Control animals:
other: saline
Positive control(s):
triethylene melamine
Tissues and cell types examined:
bone marrow smears
Details of tissue and slide preparation:
femeur and tibia bone marrow cells were collected and tranferred to Hank's balanced salt solution. Cells were centrifuged and the cell pellet resuspended in potassium chloride solution prior to fixation in Carnoy's fixative.
Slides were prepared and satined with 5% Giesma solution. Slides were examined microscopically for metaphases.
Sex:
male
Genotoxicity:
positive
Remarks:
2.0 & 6.0 ml/kg gave weak positive responses (acute study)
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

Similar aberrations were observed throughout the treatment and negative  

control groups, the only difference being the frequency of aberration.
The results of the evaluation are summarized in the following table.
Group   Time      No        Cells with      No. animals        MI**
        (hrs)     of        1 or more       without                
                cells       aberrations    aberrations*                

Saline   6       53         0 (0%)          2 (2)               0.7
        24       225        6 (2.6%)        2 (5)               3.2
        48       200        1 (0.5%)        3 (4)               3.6
        5 doses  250        2 (0.8%)        4 (5)               10.6        

TEM     24       144        68 (47.2%)      0 (5)               1.4

Diesel   6       189        5 (2.6%)        0 (5)               5.9
  0.6   24       48         1 (2.1%)        4 (5)               1.7
ml/kg   48       186        1 (0.5%)        4 (5)               2.3
     5 doses     250        4 (1.6%)        2 (5)               3.6


Diesel   6        228       8 (3.5%)        1 (5)               6.3
2.0     24        86        5 (5.8%)        2 (5)               1.7
ml/kg   48        216       2 (0.9%)        3 (5)               4.5
   5 doses        250        1 (0.4%)        4 (5)              7.4

Diesel   6        100       1 (1.0%)       1 (2)                4.0
6        24       227       5 (2.2%)       2 (5)                4.7
ml/kg    48       93        5 (5.4%)       2 (4)                3.8
        5 doses   200       10 (5.0%)      0 (4)                6.0

*         ( ) Number in parenthesis is No. of animals examined.
**        Mitotic Index

For each dose group, the total number of cells with aberrations were  

combined and the mean % aberrations was estimated. These data formed the  

basis for the report's conclusion, that at a dose level of 2 and 6 ml/kg  

diesel fuel was clastogenic.

Group             Total No. cells       Mean %
                with aberrations        aberrations
Control                 9                   1.0
TEM                     68                  47.2
Diesel 0.6 ml/kg        11                   1.7
Diesel 2.0 ml/kg        16                   2.7
Diesel 6.0 ml/kg        21                   3.4

Conclusions:
Interpretation of results (migrated information): positive weak positive response at 2.0 and 6.0 ml/kg
Diesel fuel was clastogenic in the rat following IP injection at levels of 2.0 and 6.0 mL / Kg bw. The % increase in abberration rate following a single exposure was small, ranging from 0.9 to 5.8%, and the predominant aberration seen was chromosomal fragements. Following repeated exposure small % increases were seen and the findings were not dose related. This suggests that the clastogenic activity of diesel fuel is at most weak.
Executive summary:

The genotoxic activity of diesel fuel was investigated in the rat following IP injection at 0.6, 2,0 or 6.0 ml/kg bw. Chromosomal aberrations in bone marrow cells were assessed following acute (single) or repeated exposure.

A small increase in the % of cells with aberrations (0.9 -5.8%), predominantly showing chromosomal fragements, was seen following acute exposure to diesel fuel at 2.0 and 6.0 mL/kg. The % increase at 0.6 mL/kg was within the range seen in control animals. Chromosomal fragements are not heritable changes and few 'rearrangement' aberrations were seen. Smaller % increases in cells with aberrations (0.4 -5.0%) were seen in the repeat dose study and the findings were not dose related.

These results suggest that diesel fuel may be at most weakly clastogenic in vivo but that changes observed are unlikley to be heritable.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Based on substance specific data and read-across to data on structurally related materials, the substance is not considered to be genotoxic. There was no evidence of activity in four in-vitro assays and two out of three in-vivo studies. In one chromosomal aberration study, weak activity was seen following intraperitoneal treatment at high doses. The magnitude of effects was small and was restricted to small increases in chromosomal fragments.


Justification for selection of genetic toxicity endpoint
key in-vivo study, selected from 4 in-vitro and 3 in-vivo assays.

Justification for classification or non-classification

Some oil products containing relatively high concentrations of polycyclic aromatic compounds (PAC) are considered genotoxic carcinogens, and, consequently, are classified and labelled as carcinogenic, Cat. 1A or 1B (H350) or Cat. 2 (H351) according to the EU CLP Regulation (EC) 1272/2008. This classification as carcinogenic does not automatically imply that these substances need also to be classified as mutagenic as defined by the CLP Regulation. The EU legislation aims primarily to classify substances as mutagenic if there is evidence of producing heritable genetic damage, i.e. evidence of producing mutations that are transmitted to the progeny or evidence of producing somatic mutations in combination with evidence of the substance or relevant metabolite reaching the germ line cells in the reproductive organs. The PAC in oil products are poorly bioavailable due to their physico-chemical properties (low water solubility and high molecular weight), making it unlikely that the genotoxic constituents can reach and cause damage to germ cells (Roy, 2007; Potter, 1999). Considering their poor bioavailability, oil products which have been classified as carcinogenic do not need to be classified as mutagenic unless there is clear evidence that germ cells are affected by exposure, consistent with the CLP Regulation. For example, based on in vivo micronucleus tests on home heating oil as well as for read-across substances that were all negative for genotoxicity, vacuum distillate fuels are not classified as mutagens according to the EU CLP Regulation (EC) 1272/2008.