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Genetic toxicity in vitro

Description of key information

There is an in vitro bacterial reverse mutation assay data available for Hydrocarbons, C14-C17, n-alkanes, <2% aromatics. Data is also available for other in vitro genetic toxicity tests for structural analogues Hydrocarbons, C12 -C16, n-alkanes, isoalkanes, cyclics, <2% aromatics, isohexadecane, and Hydrodesulfurized Kerosene. This data is read across to Hydrocarbons, C14 -C17, n-alkanes, <2% aromatics based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

All read across genetic toxicity tests listed below had negative results for Hydrocarbons, C14-C17, n-alkanes, <2% aromatics.

 

Genetic Toxicity in vitro - Bacterial reverse mutation assay (OECD 471)

Genetic Toxicity in vitro - Mammalian Chromosome Aberration Test (OECD TG 473)

Genetic Toxicity in vitro - Mammalian Cell Gene Mutation Test (OECD TG 476)

Genetic Toxicity in vitro - Sister Chromatid Exchange Assay in Mammalian Cells (OECD TG 479)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 June 1985 to 15 july 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Ames test (5 Salmonella strains), GLP. Substance analytical certificate available provided by the manufacturer. Substance identification: commercial name 98.8% purity
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
according to Ames test
Principles of method if other than guideline:
Guideline principles
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Reverse gene mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: S. typhimurium TA 1538 (see below Table 7.6.1/1)
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix was prepared in laboratory from liver of CD rat (Sprague-Dawley derived from Charles River Ltd, UK) induced by Aroclor 1254 and stored at -80 °C as aliquots.
Test concentrations with justification for top dose:
5, 50, 500, 5000 µg/plate in dose range-finding test (see below Table 7.6.1/2)
50, 50, 500, 1500, 5000 µg/plate in the main mutation tests
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
yes
Remarks:
Sterile test: plates without the addition of bacteria are prepared in order to assess the sterility of test substance, the S9 mix and the vehicle
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
yes
Remarks:
Sterile 0.1 M sodium phosphate buffer (pH 7.4)
Positive controls:
yes
Positive control substance:
other: See below Table 7.6.1/3
Remarks:
See freetext "Any other information on materials and methods"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) Two independent experiments (range-finding test as first and main test as second test) in agar by direct plate incorporation for both experiments with and without S9 mix.
Pre-incubation test substance activation was not performed in presence of S9-mix.

DURATION
- Preincubation period: no
- Exposure duration: 72 h

SELECTION AGENT (mutation assays): histidine deficient agar

NUMBER OF REPLICATIONS: 3 measurements/plate) with a Biotran Automatic colony counter.


DETERMINATION OF CYTOTOXICITY
- Method: revertant colony counts during the dose-range finding assay


OTHER: scoring (3 measurements/plate) with a Domino automated counter
Evaluation criteria:
The mean number and standard deviation of revertants are calculated for all groups. The means for all treatment groups are compared with those obtained for the negative (solvent) and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.
Statistics:
A compound is deemed to provide evidence of mutagenic potential if:
- a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments, and
- the increase in the number of revertant colonies is at least twice the concurrent solvent control value.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with Petrepar 147 at any dose level, either in the presence or absence of metabolic activation (S9).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/4: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

12

0.6

12

3.8

9

1.5

18

3.0

70

4.2

0**

9

0

10

1.2

7

1.0

21

7.0

66

3.5

50

9

2.0

12

3.6

9

1.2

17

1.0

72

6.1

150

8

3.1

7

1.5

9

1.2

15

4.2

69

4.9

500

8

2.0

11

3.2

5

0

15

3.5

72

3.8

1500

10

3.8

10

3.5

9

2.1

17

3.8

63

3.1

5000

9

2.1

11

3.5

12

1.0

18

3.5

63

5.0

Positive control***

65

5.5

1750

89.1

54

3.8

76

6.7

236

21.2

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

Table 7.6.1/5: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (First test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

11

1.5

20

5.1

11

4.0

21

1.2

78

13

0**

9

2.3

21

2.1

9

1.5

14

1.5

69

4.0

50

7

0.6

11

1.0

9

4.2

16

3.6

72

5.1

150

8

2.0

10

2.1

9

2.3

13

1.5

69

6.0

500

6

2.0

12

3.0

9

3.1

14

3.5

71

4.7

1500

11

3.5

11

3.2

12

3.1

13

3.1

69

4.6

5000

6

1.0

9

3.8

9

1.5

13

2.3

61

2.9

Positive control***

130

9.0

103

17.4

408

47.5

372

52.4

542

15.9

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

Table 7.6.1/6: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (Second test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

14

2.0

14

3.8

5

1.2

14

3.2

63

2.9

0**

7

2.5

17

0.6

7

3.5

17

1.7

67

7..0

50

7

2.3

17

4.0

6

1.5

17

1.0

70

6.1

150

9

1.2

18

1.2

11

4.0

17

1.5

67

3.8

500

6

1.0

15

1.5

7

0

14

3.2

71

3.5

1500

6

2.1

19

2.0

8

2.9

16

2.6

72

4.0

5000

7

1.5

17

4.0

11

5.0

13

2.6

68

9.3

Positive control***

10

9.3

-

-

56

8.2

49

18.2

228

34.6

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

Table 7.6.1/7: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (Second test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 1538

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

10

2.6

14

3.8

9

2.6

19

2.6

88

17.9

0**

9

2.1

17

0.6

12

2.6

18

3.6

101

11.0

50

9

1.2

17

4.0

12

3.6

18

2.1

81

13.0

150

9

1.0

18

1.2

11

1.5

16

3.2

76

11.3

500

9

1.7

15

1.5

10

4.5

19

2.0

79

6.1

1500

9

3.1

19

2.0

11

4.0

16

2.6

79

9.1

5000

10

2.5

17

4.0

11

2.6

15

2.0

80

16.9

Positive control***

84

5.9

41

9.0

46

12.3

142

46.0

232

7.5

* Solvent control = acetone

** Sterile 0.1 M sodium phosphate buffer (pH 7.4) instead of test substance dilution

*** Mutagens positive controls: see Table 7.6.1/3

Conclusions:
Interpretation of results:
negative

Under the test conditions, Petrepar-147 did not demonstrate any in vitro mutagenic activity in the Salmonella test system up to 5000 µg/plate.
Executive summary:

The mutagenic potential of Petrepar 147 was assessed in the Salmonella typhimurium microsomal assay according to the Ames test in compliance with Good Laboratory Practice.

The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100 mutants were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels according to the direct incorporating plate method. After 72 hours of incubation at 37°C, the revertant colonies were scored.


A preliminary toxicity assay was performed according to the direct incorporating method to define the 5 dose levels to be used in the main test. The test substance was then tested in another experiment performed in the same way as the range-finding test.


The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies.
The test substance was dissolved in acetone. Dose levels used in the main assay were 0, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Two independent main tests were performed. Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments and compared.

No toxicity was observed in any of the strains in the absence and in the presence of S-9 mix up to the highest dose tested in the main test. No increase in the mean number of revertant colonies for any S. typhimurium strains with and without S9-mix in both tests (pre-test and main test).
Positive controls gave the expected increases in the number of revertants, with and without S-9 mix.

Under the conditions of this study, Petrepar 147 did not demonstrate any in vitro mutagenic activity in this bacterial test system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August to 21 September 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Few details on test material (no certificate of analysis)
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No certificate of analysis
Principles of method if other than guideline:
Guideline principles
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A culture medium supplemented with 10% fetal calf serum, 1% L-glutamine, and 1% penicillin and streptomycin, at about 37°C, in an atmosphere of about 5% C02 in air.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 from male Sprague-Dawley rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Range finding assay: half-log series of concentrations of 0.0835 to 2500 µg/mL
Main experiment:
- without metabolic activation: 3.13, 6.26, 9.35 and 12.5 µg/mL with 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL with 20-h harvest
- with metabolic activation: 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test material was insoluble in water and dimethylsulfoxide. A clear and homogeneous stock solution of 201 mg/mL with ethanol could be maintained.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: without metabolic activation: 7.25 and 17 h for 10 and 20 h assay, respectively; with metabolic activation: 2 h
- Expression time (cells in growth medium): with metabolic activation: 7.75 and 17.75 h for 20 and 10 h assay, respectively;
- Time in 0.1 µg/mL Colcemid: without metabolic activation: 1 and 0.5 h for 20 and 10 h assay, respectively; with metabolic activation: 2.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): 10 h and 20 h without and without metabolic activation

STAIN (for cytogenetic assays): 5% Giemsa solution and BrdUrd (5-bromodeoxyuridine) at 10 µM

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 cells for test substance; at least 25 cells for positive controls

CYTOTOXICITY: visual observations based on confluence of monolayer and floating dead cells
Evaluation criteria:
Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 ± 2 were analyzed.
The following factors were taken into account in the evaluation of the chromosomal aberrations data: the overall chromosomal aberration frequencies, the percentage of cells with any aberrations, the percentage of cells with more than one aberration, any evidence for increasing amounts of damage with increasing dose.
Chromatid and isochromatid gaps were not considered as they may be due to toxicity.
Statistics:
Fisher's exact test with an adjustment of multiple comparisons
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Range-finding without metabolic activation:
A very unhealthy cell monolayer, -70% reduction in the cell monolayer confluence, floating dead cells, and severe reduction in the number of visible mitotic cells were observed in the culture dosed with 25.0 µg/mL. Slight reductions in the number of visible mitotic cells and -15% reduction in the cell monolayer confluence were observed in the cultures dosed with 2.50 and 8.35 µg/mL.
Range-finding with metabolic activation:
An unhealthy cell monolayer, -85% reduction in the cell monolayer confluence, floating dead cells and debris, and severe reduction in the number of visible mitotic cells were observed in the culture dosed with 835 µg/mL. Reductions of -15% in the cell monolayer confluence were observed in the cultures dosed with 25.0 and 83.5 µg/mL.

Chromosomal aberrations assay without metabolic activation (Table 1):
In the 10 h assay, no toxicity was observed in any of the test cultures. These cultures were not analyzed for chromosomal aberrations as four dose levels were available for analysis from the 20 h assay. In the 20 h assay, an unhealthy cell monolayer, -70% and -45 % reduction in the cell monolayer confluence, floating dead cells and debris, and a severe reduction in visible mitotic cells were observed at 75.0 and 50.0 µg/mL, respectively. Toxicity was evident on the slides prepared from these cultures by the very sparse numbers of metaphases available for analysis.

Chromosomal aberration assay with metabolic activation (Tables 2 and 3):
In the 10 h assay, slight reductions in the numbers of visible mitotic cells were observed in the cultures dosed at 563 and 751 µg/mL.
In the 20 h assay, severe toxicity was exhibited on the slides prepared from the cultures dosed with 562 and 750 µg/mL by the presence of many dead cells and the sparse numbers of metaphases available for analysis. Reductions of -15% in the cell monolayer confluence were observed in the cultures dosed with 99.7, 187, 281, 375, 562, and 750 µg/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Chromosome aberrations in CHO cells fixed 20 h after exposure to MRD-90-843 without metabolic activation (results from pooled duplicate cultures)

 

Number and type of aberration

 

 

 

Not computed

Simple

Complex

% cells with aberrations

 

Concentration (µg/mL) 

Chromatid gap

Chromosome gap

 

 

 

Negative (vehicle)

-

7

1

 

 

0.0

Positive (Mitomycin C)

0.04

7

 

4

7

28.0*

Test article

25.0

15

2

 

 

0.0

37.5

7

3

1

1

0.5

50.0

8

1

 

1

0.5

75.0

19

2

4

 

0.5

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Table 2: Chromosome aberrations in CHO cells fixed 10 h after exposure to MRD-90-843 with metabolic activation (results from pooled duplicate cultures)

 

Concentration (µg/mL)

Number and type of aberration

 

 

 

Not computed

Simple

Complex

% cells with aberrations

 

 

Chromatid gap

Chromosome gap

 

 

 

Negative (vehicle)

-

2

 

 

0.0

Positive (Cyclophosphamide)

25.0

1

 

8

13

44.0*

Test article

282

7

1

 

 

0.0

375

3

1

0.5

563

4

 

1

0.5

751

3

3

 

1.0

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Table 3: Chromosome aberrations in CHO cells fixed 20 h after exposure to MRD-90-843 with metabolic activation (results from pooled duplicate cultures)

 

Concentration (µg/mL)

Number and type of aberration

 

 

 

Not computed

Simple

Complex

% cells with aberrations

 

 

Chromatid gap

Chromosome gap

 

 

 

Negative (vehicle)

-

7

 1

0.0

Positive (Cyclophosphamide)

12.5

1

 

17

31

80.0*

Test article

281

15

2

 

1.0

375

16

6

1

1

1.0

562

3

1

1

1.0

750

10

1

1.0

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Conclusions:
Interpretation of results:
negative

MRD-90-843 was found not to increase chromosome aberrations in CHO cells with and without metabolic activation.
Executive summary:

In an in vitro chromosome aberration test, Chinese Hamster Ovary cells were exposed to MRD-90-843 at concentrations of 3.13, 6.26, 9.35 and 12.5 µg/mL for 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL for 20-h harvest, for 7 and 17 h, without metabolic activation and 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest, for 2 h, with metabolic activation.

Positive controls (mitomycin C without metabolic activation and cyclophosphamide with metabolic activation) induced the appropriate response. As there was no evidence of chromosome aberration induced over background, MRD-90-843 is not classified according to the criteria of Annex VI to Directive 67/548/EEC and the CLP Regulation (1272/2008).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Equivalent or similar to OECD Guideline 476.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
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:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
na
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate
Test concentrations with justification for top dose:
Without activation: 6.25 nl/ml to 37.5 nl/ml
With activation: 3.91 nl/ml to 62.5 nl/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [ethanol]
- Justification for choice of solvent/vehicle:The test material was miscible with ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 hours

SELECTION AGENT (mutation assays): BrdU

NUMBER OF REPLICATIONS: Variable with or without activation

NUMBER OF CELLS EVALUATED: 3x10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 40.8 x 10^-6
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material induced a good range of toxicities for evaluation of the test material (percent relative growths, 65.3% to 2.8%). The toxicities did show some variability between replicate samples. In the presence of metabolic activation, no indication of mutagenic activity was observed. The average cloning efficencies for the solvent and untreated negative controls varied from 119.1% without activation to 82.7% with activation which demonstrated very good cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive compounds yielded normal mutant frequencies that were greatly in excess of the background.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

It is concluded in this study that the test material is not a mutagenic agent with or without activation. Classification is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

The test material was examined for mutagenic activity in the mouse lymphoma forward mutation assay in the absence and presence of a liver S9 fraction for metabolic activation. The test material did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Treatments up to 37.5 nl/ml without activation and 62.5 nl/ml with activation were assayed and high toxicities were induced without inducing significant increases in the mutant frequency. It is concluded in this study that the test material is not a mutagenic agent with or without activation. Classification is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

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

Genetic toxicity in vivo

Description of key information

Further testing is not required based on the negative results obtained in in vitro chromosome aberration test, bacterial reverse mutation assay, mammalian cell gene mutation test and sister chromatid exchange assay tests conducted. Hydrocarbons, C14-C17, n-alkanes, <2% aromatics is therefore not classified as a mutagen. Furthermore, there is no evidence from read across repeat dose toxicity studies that Hydrocarbons, C14-C17, n-alkanes, <2% aromatics is able to induce hyperplasia or pre-neoplastic lesions.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There is an in vitro bacterial reverse mutation assay available for Hydrocarbons, C14-C17, n-alkanes, <2% aromatics. Additionally, data is available for structural analogues Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics, Isohexadecane, and Hydrodesulfurized Kerosene. This data is read across to Hydrocarbons, C14-C17, n-alkanes, <2% aromatics based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

In Vitro

 

In vitro gene mutation study in bacteria

Hydrocarbons, C14-C17, n-alkanes, <2% aromatics

In a key study (Petroquimica, 1985), the test material (Hydrocarbons, C14-C17, n-alkanes, <2% aromatics) was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 1538, 98 and 100 and the tryptophan requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a liver S9 fraction for metabolic activation. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies. The test substance was dissolved in acetone. Dose levels used in the main assay were 0, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Two independent main tests were performed. Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments and compared. A preliminary toxicity assay was performed according to the direct incorporating method to define the 5 dose levels to be used in the main test. The test substance was then tested in another experiment performed in the same way as the range-finding test. No toxicity was observed in any of the strains in the absence and in the presence of S-9 mix up to the highest dose tested in the main test. No increase in the mean number of revertant colonies for any S. typhimurium strains with and without S9-mix in both tests (pre-test and main test). Positive controls gave the expected increases in the number of revertants, with and without S-9 mix. Under the conditions of this study, Petrepar 147 did not demonstrate any in vitro mutagenic activity in this bacterial test system.

Isohexadecane

In a reverse gene mutation assay in bacteria (Poth, 1990) and in compliance with Good Laboratory Practice, strains TA98, TA100, TA1535 and TA1537 of S. typhimurium were exposed to Isohexadecane at concentrations of 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate in the presence and absence of mammalian metabolic activation. No cytotoxicity was observed with all the dose tested. Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used (+/- S9). The positive controls induced the appropriate responses in the corresponding strains. Under the test conditions, Isohexadecan did not induce in vitro mutagenic activity in the bacterial test system in the presence and the absence of S9 activation system.


In Vitro Chromosome Aberration in Mammalian Cells

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a key OECD Guideline 473 study (ExxonMobil, 1991), the potential of the test material (Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics) to cause chromosome aberration was investigated in cultured human lymphocytes with and without the metabolic activation S9 system. Negative and positive control substance. Chinese Hamster Ovary cells were exposed to MRD-90-843 at concentrations of 3.13, 6.26, 9.35 and 12.5 µg/mL for 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL for 20-h harvest, for 7 and 17 h, without metabolic activation and 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest, for 2 h, with metabolic activation. Positive controls (mitomycin C without metabolic activation and cyclophosphamide with metabolic activation) induced the appropriate response. As there was no evidence of chromosome aberration induced over background, MRD-90-843 is not classified according to the CLP Regulation (1272/2008).

 

In vitro Gene Mutation study in Mammalian Cells

Hydrodesulfurized Kerosene

In a key study (API, 1984), exposure to eight graded doses of the test material (Hydrodesulfurized Kerosene) in the presence of and in the absence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Therefore Hydrodesulfurized Kerosene is not considered to be mutagenic in this test system.

 

In vitro Sister Chromatid Exchange Assay in Mammalian Cells

Hydrodesulfurized Kerosene

In a supporting study (API, 1987), the test material (Hydrodesulfurized Kerosene) was soluble at all concentrations tested. The study in both the presence and absence of S9 was repeated since there was a poor metaphase cell yield. The responses to the positive and negative control materials fulfilled the requirements for the assays. The test material did not cause an increase in SCEs in the absence of exogenous activation. The test material did cause a increase in SCEs at two non adjacent doses (0.05 and 0.4 uL/mL) in the activation assay. However, the increased activity was only seen in one of two treatment flasks. These increases appeared to be random and of no biological significance. It was concluded that the test material was negative in the SCE assay.

In Vivo

Further testing is not required based on the negative results obtained in in vitro chromosome aberration test, bacterial reverse mutation assay, mammalian cell gene mutation test and sister chromatid exchange assay tests conducted. Hydrocarbons, C14-C17, n-alkanes, <2% aromatics is therefore not classified as a mutagen. Furthermore, there is no evidence from read across repeat dose toxicity studies that Hydrocarbons, C14-C17, n-alkanes, <2% aromatics is able to induce hyperplasia or pre-neoplastic lesions.

Justification for classification or non-classification

The negative results using in vitro substance specific and read across genotoxicity assays from structural analogues do not warrant the classification of Hydrocarbons, C14-C17, n-alkanes,<2% aromatics as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).