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Genetic toxicity in vitro

Description of key information

A range of in-vitro and in-vivo genotoxicity assays were identified for hydrodesulphurised kerosine. All the in-vitro assays were negative for genotoxicity, including modified Ames, mouse lymphoma and SCE tests.

In-vivo assays were negative for genotoxicity with the exception of one SCE test in male mice. This result was considered equivocal in view of the results in both the in-vivo and in-vitro assays and the evident toxicity at higher dose levels.

Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions because it was carried out in a method equivalent/similar to OECD TG 479.
Justification for type of information:
For read across, see read across justification in Section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
GLP compliance:
yes
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham’s F-12 or McCoy’s 5A medium supplemented with 10% foetal bovine serum (FBS)
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Without metabolic activation: 0.007, 0.013, 0.025, and 0.05 µl/ml
With metabolic activation: 0.05, 0.1, 0.2, and 0.4 µl/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: None reported
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: without activation: triethylenemelamine; with activation: cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 16 to 24 hours
- Exposure duration: Without activation: 25 hours; with activation: 2 hours


NUMBER OF REPLICATIONS: Two


NUMBER OF CELLS EVALUATED:Twenty-five cells from each replicate flask


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth


Evaluation criteria:
The test material was considered positive if it induced a doubling in SCE frequency over the solvent control at a minimum of three consecutive dose levels or if a dose responsive and statistically significant increase was observed over a minimum of three dose levels.
Statistics:
Student's t-test
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Preliminary studies tested for the cytotoxicity of the test compound in acetone. In the absence of metabolic activation, concentrations greater than 0.1 µl/ml inhibited the relative growth and mitotic indices. In the presence of metabolic activation, a concentration of 1 µl/ml was needed to inhibit the relative growth by 50% or more.


Remarks on result:
other: other: Chinese Hamster Ovary cells
Remarks:
Migrated from field 'Test system'.

The test material 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. Only the results of the second study with and without S9 are summarized in the following table.


Replicate flask

SCEs/ chromosome

Flask mean SCEs/cell

Group mean SCEs/cell

Assay in absence of exogenous activation

   

 

(±SD)

(±SD)

Untreated cells

 

 

 

A

0.42

 8.4±3.16

 

B

 0.42

 8.28±2.57

8.34±2.85

Acetone

 

 

 

A

 0.48

 9.44±3

  

B

 0.45

 8.96±2.35

 9.20±2.68

API 81-07 0.007 µl/ml

 

 

 

A

 0.44

 8.80±2.87

 

B

 0.43

 8.68±2.54

 8.74±2.69

API 81-07 0.013 µl/ml

 

 

 

A

 0.47

 9.4±2.96

 

B

 0.42

 8.32±2.58

 8.86±2.80

API 81-07 0.025 µl/ml

 

 

 

A

 0.47

 9.36±2.96

 

B

 0.48

 9.64±3.12

 9.50±3.01

API 81-07 0.05 µl/ml

 

 

 

A

 NE (a)

 

 

B

 NE

 

 

TEM

 

 

 

A

 1.53

 30.6±6.81

 

B

 1.75

 34.92±7.60

32.76±7.47**

Assay in presence of exogenous activation

Untreated cells

 

 

 

A

 0.5

 10.16±2.98

 

B

 0.55

 11.04±2.76

 10.6±2.88

Acetone

 

 

 

A

 0.47

 9.36±3.94

 

B

 0.45

 8.96±2.99

 9.16±3.47

API 81-07 0.05 µl/ml

 

 

 

A

 0.63

 12.52±4.09

 

B

 0.58

 11.64±3.38   

12.08±3.74**

API 81-07 0.1 µl/ml

 

 

 

A

 0.48

 9.56±3.8

 

B

 0.51

 10.36±4.27

 9.96±4.02

API 81-07 0.2 µl/ml

 

 

 

A

 0.5

 10.04±3.23

 

B

 0.44

 8.84±3.5

 9.44±3.39

API 81-07 0.4 µl/ml

 

 

 

A

 0.5

 9.96±3.25

 

B

 0.58

 11.56±3.57

 10.76±3.47*

CP

 

 

 

A

 1.91

 38.2±7.06

 

B

 2.01

 40.2±12.18

 39.2±9.91**

(a)      Not evaluated due to absence of second-division metaphase cells

*      P</=0.05

**      P</=0.01

 

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. API 81-07 did cause a significant increase in SCEs at two non adjacent doses 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 API 81-07 was negative in the SCE assay.

Conclusions:
Interpretation of results (migrated information):
negative

The test compound was not genotoxic.
Executive summary:

Hydrodesulfurised kerosine was tested in the sister chromatid exchange assay using Chinese hamster ovary cells. The assay was conducted with Aroclor-induced rat liver S-9 activation system at concentrations of 0.05, 0.1, 0.2, or 0.4 µl/mL or without at concentrations of 0.007, 0.013, 0.025, and 0.05 µl/mL. The high doses were selected based on the cytotoxicity of the test compound, which was tested in a preliminary study. A small but statistically significant increase in the frequency of sister chromatid exchanges was observed at the high and low concentrations with metabolic activation. These increases appeared to be random and of no biological significance. There were no significant increases observed at any concentration in the absence of metabolic activation. Under the conditions of the study, hydrodesulfurised kerosine is considered to be negative in the sister chromatid exchange assay with Chinese hamster ovary cells.

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

Additional information

Additional information from genetic toxicity in vitro:

Data on related substances have been used to 'read-across' and predict the hazard properties. A 'read-across' justification document can be found in section 13.

In vitro gene mutation studies in bacteria

Samples of hydrodesulphurised kerosine proved negative in a range of in-vitro genotoxicity assays, including Modified Ames, mouse lymphoma and sister chromatid exchange tests.

 

In vitro gene mutation in mammalian cells

In an in vitro gene mutation studies in mammalian cells (API, 1984), cultures of mouse lymphoma cells were exposed to hydrodesulfurised kerosine with or without metabolic activation by Aroclor 1254-induced rat liver S9 fraction. Under non-activation conditions the test material induced a good range of toxicities for evaluation (relative growths ranged from 2.8% to 65.3%). None of the assays induced a mutant frequency that exceeded the minimum criterion (40.8 x 10-6). The test material was not mutagenic under non-activation conditions. In the presence of metabolic activation a wide range of toxicities was induced (6.1 to 107.9% relative growths). The minimum criterion mutant frequency of 69.0 x 10-6 was not exceeded. The test material was therefore considered non mutagenic under activation conditions. 

  

In vitro cytogenicity in mammalian cells

Hydrodesulfurised kerosine was tested in the sister chromatid exchange assay using Chinese hamster ovary cells (API, 1988a).  The assay was conducted with Aroclor-induced rat liver S-9 activation system. A small but statistically significant increase in the frequency of sister chromatid exchanges was observed at the high and low concentrations with metabolic activation.  These increases appeared to be random and of no biological significance. There were no significant increases observed at any concentration in the absence of metabolic activation. Under the conditions of the study, hydrodesulfurised kerosine is considered to be negative in the sister chromatid exchange assay with Chinese hamster ovary cells.

 

In vivo cytogenicity

In a mouse sister chromatid exchange assay hydrodesulfurised kerosine (API, 1988b) was administered to mice and considered equivocal for genotoxicity. Although the test is considered valid, the following remarks can be made. Regarding the SCE assay with hydrodesulfurised kerosine no statistically significant effect was observed in the female mice, whereas the female mice responded positively to administration of both cyclophosphamide (10 mg/kg) and a carcinogenic heavy fuel oil (4000 mg/kg) which were used as positive control. Furthermore, the doses of the hydrodesulfurised kerosine were rather high (400, 2000, and 40000 mg/kg) and the males in the highest dose groups showed signs of toxicity (lethargy and weight loss) on the day of the administration of the kerosine and the day after (when they were sacrificed).

 

In vivo gene mutation

In a sperm cell dominant lethal mutation assay (API 1973), deodorised kerosine was administered subcutaneously to 10 male Swiss-Webster mice in corn oil vehicle or intraperitoneally to 10 Long-Evans rats undiluted at a dose of 1.0 mL/kg. Males were mated with females, and no pattern of decreased pregnancy rate or increased embryo loss was observed in the females.

 

In conclusion, the body of data available indicate that hydrodesulphurised kerosine is not mutagenic or genotoxic.

 

Justification for classification or non-classification

The body of in-vitro and in-vivo data do not indicate that hydrodesulphurised kerosine in genotoxic/mutagenic. Therefore the material is not classified as mutagenic under the EU CLP Regulation (EC No. 1272/2008).

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