Hydrocarbons, C9-C11, n-alkanes, isoalkanes,

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No genotoxicity studies are available for the registered substance, Hydrocarbons, C9-C11 aliphatics, <2% aromatics (GS180). However, some reliable in vitro data are available for related Fischer-Tropsch process-derived products in the relevant carbon number range: GS160 (C8-C11) and GS170 (C9-C12). These are read across to Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics.

Bacterial reverse mutation assay: read-across from Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics; negative with and without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA (OECD Test Guideline 471) (WIL Research, 2014k).

Bacterial reverse mutation assay: read-across from Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics; negative with and without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA (OECD Test Guideline 471) (WIL Research, 2014l).

Mammalian Chromosome Aberration Test: read-across from Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics; negative with and without metabolic activation in human lymphocytes (OECD Test Guideline 473) (Harlan Cytotest Cell Research GmbH, 2014a).

Mammalian Chromosome Aberration Test: read-across from Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics; negative with and without metabolic activation in human lymphocytes (OECD Test Guideline 473) (Harlan Cytotest Cell Research GmbH, 2014b).

Mammalian Cell Gene Mutation Test (OECD TG 476): read-across from Hydrocarbons C10-C13, isoalkanes and isododecane; Negative (INEOS, 1996 and Chevron, 1982).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04-Aug-2014 to 28-Aug-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. Hypothesis for the analogue approach:
The hypothesis for the analogue approach is that both the registration substance, Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics (target substance), and the test substance, Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics (source substance), are produced from the same Fischer-Tropsch substance, GTL Gasoil, by fractional distillation. The substances are C9-C14 Aliphatics [≤2% Aromatics] Hydrocarbon Solvents.

The source substances contain the all of the constituents of the target substance. The substances have constituents that are part of the same homologous series and have many constituents in common. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies).

2. Source and target chemical(s)
The source substance Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics is composed of linear, branched and cyclic hydrocarbons of chain length C8-C11.

The target substance, Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics, is composed of linear, branched and cyclic hydrocarbons of chain length C9-C11.

3. Analogue approach justification
The constituents of the source and target substances are all hydrocarbons. Identical constituents have identical toxicological properties. The source substances cover the full carbon chain length of the target substance.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9: 650 µg/plate in DMSO for TA100

Positive control substance:

2-nitrofluorene

Remarks:

without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9: 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

sodium azide

Remarks:

without S9: 5 µg/plate in saline for TA1535

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitated (observed as droplets) on the plates was observed at the top dose of 5000 μg/plate


RANGE-FINDING/SCREENING STUDIES:
The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed, except in tester strain TA100 in the presence of S9-mix at the top dose of 5000 µg/plate where a moderate reduction in the number of revertants was observed.

SECOND EXPERIMENT:
The bacterial background lawn was not reduced at any of the concentrations tested. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the presence of S9-mix at 1600 and 5000 µg/plate.
COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Conclusions:

Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics has been tested for mutagenicity to bacteria in a reliable study conducted according to OECD Test Guideline 471 and in compliance with GLP. The test substance was negative in bacterial strains Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA when tested with and without metabolic activation (Aroclor 1254-induced rat liver S9) up to cytotoxic or the highest required concentrations in both the initial experiment using the plate incorporation method and the independent repeat pre-incubation assay. Appropriate positive controls were included and showed the expected increase in the number of revertants. Therefore it is concluded that Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics is not mutagenic to bacteria.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04-Aug-2014 to 28-Aug-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. Hypothesis for the analogue approach:
The hypothesis for the analogue approach is that both the registration substance, Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics (target substance), and the test substance, Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics (source substance), are produced from the same Fischer-Tropsch substance, GTL Gasoil, by fractional distillation. The substances are C9-C14 Aliphatics [≤2% Aromatics] Hydrocarbon Solvents.

The source substances contain the all of the constituents of the target substance. The substances have constituents that are part of the same homologous series and have many constituents in common. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies).

2. Source and target chemical(s)
The source substance Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics, is composed of linear, branched and cyclic hydrocarbons of chain length C9-C12

The target substance, Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics, is composed of linear, branched and cyclic hydrocarbons of chain length C9-C11.

3. Analogue approach justification
The constituents of the source and target substances are all hydrocarbons. Identical constituents have identical toxicological properties. The source substances cover the full carbon chain length of the target substance.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512 and 1600 µg/plate
Experiment 2:
Without and with S9-mix: 17, 52, 164, 512 and 1600 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9: 650 µg/plate in DMSO for TA100

Positive control substance:

2-nitrofluorene

Remarks:

without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9: 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

sodium azide

Remarks:

without S9: 5 µg/plate in saline for TA1535

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation (droplets of the test substance) was observed at dose levels of 1600 and 5000 µg/plate in the dose range finding and the first mutation experiment



RANGE-FINDING/FIRST MUTATION EXPERIMENT:
- In tester strain TA100, toxicity was observed at dose levels of 164μg/plate and above in the absence and at dose levels of 1600μg/plate and above in the presence of S9-mix. In all other strains, no toxicity was observed up in any of the concentrations tested.

SECOND MUTATION EXPERIMENT:
- Cytotoxicity was observed in tester strain TA100 in the absence and presence of S9-mix, where slight to moderate reductions of the revertant colonies was observed at test substance concentrations of 164 µg/plate and above in the absence of S9-mix and at 1600 µg/plate in the presence of S9-mix. And in tester strain TA1537, where an extreme reduction of the revertant colonies was observed at the test substance concentrations of 1600 µg/plate in the absence of S9-mix. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the other tester strains in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.
 
In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 In the second mutation experiment, no dose level with precipitate or toxicity was tested in tester strains TA1535, TA98, WP₂uvrA and TA1537 with S9-mix.

Evaluation:Although precipitation of the test substance on the plates was observed at concentrations of 1600 and 5000 µg/plate in the dose range finding and 1600 µg/plate in the first mutation experiment, no precipitation was observed at 1600 µg/plate in the second experiment.Since clearnegative responses were observed in all tester strain tested and in both the dose range finding test and the first mutation experiment the test substance was tested up to or beyond precipitation, the lack of a dose level with precipitate or toxicityhad no influence on the study.

Conclusions:

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics has been tested for mutagenicity to bacteria in a reliable study conducted according to OECD Test Guideline 471 and in compliance with GLP. The test substance was negative in bacterial strains Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA when tested with and without metabolic activation (Aroclor 1254-induced rat liver S9) up to cytotoxic or the highest required concentrations in both the initial experiment using the plate incorporation method and the independent repeat pre-incubation assay. Appropriate positive controls were included and showed the expected increase in the number of revertants. Therefore it is concluded that Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics is not mutagenic to bacteria.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-07-09 to 2014-09-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

According to OECD guideline 473 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

1. Hypothesis for the analogue approach:
The hypothesis for the analogue approach is that both the registration substance, Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics (target substance), and the test substance, Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics (source substance), are produced from the same Fischer-Tropsch substance, GTL Gasoil, by fractional distillation. The substances C9-C14 Aliphatics [≤2% Aromatics] Hydrocarbon Solvents.

The source substances contain the all of the constituents of the target substance. The substances have constituents that are part of the same homologous series and have many constituents in common. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies).

2. Source and target chemical(s)
The source substance Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics is composed of linear, branched and cyclic hydrocarbons of chain length C8-C11

The target substance, Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics, is composed of linear, branched and cyclic hydrocarbons of chain length C9-C11.

3. Analogue approach justification
The constituents of the source and target substances are all hydrocarbons. Identical constituents have identical toxicological properties. The source substances cover the full carbon chain length of the target substance.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 8.8, 15.3, 26.9, 47.0, 82.3, 143.9, 251.9, 440.8, 771.4, 1350.0 µg/mL
Experiment II: 26.9, 47.0, 82.3, 143.9, 251.9, 440.8, 771.4, 1350.0 µg/mL

Without metabolic activation:
Experiment I: 8.8, 15.3, 26.9, 47.0, 82.3, 143.9, 251.9, 440.8, 771.4, 1350.0 µg/mL
Experiment II: 8.8, 15.3, 26.9, 47.0, 82.3, 143.9, 251.9, 440.8, 771.4, 1350.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: about 1.5

NUMBER OF CELLS EVALUATED: 100 per culture, except for the positive control in Experiment I in the presence of S9 mix and Experiment II in the absence of S9 mix, where only 50 metaphases were evaluated

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment I in the presence of S9 mix and Experiment II in the absence of S9 mix, where only 50 metaphases were evaluated.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Key result

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: tested up to cytotoxic or the highest required concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics, dissolved in ethanol, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment I in the presence of S9 mix and Experiment II in the absence of S9 mix, where only 50 metaphases were evaluated. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 1350.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
No visible precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment I at 82.3 µg/mL and above in the absence of S9 mix and at 143.9 µg/mL and above in the presence of S9 mix at the end of treatment. In Experiment II phase separation was observed at 251.9 µg/mL and above in the absence of S9 mix and at 143.9 µg/mL and above in the presence of S9 mix at the end of treatment.
No relevant influence on pH value was observed. The osmolarity was decreased in Experiment I at the highest applied concentration.
In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II after continuous treatment in the absence of S9 mix clear cytotoxic effects were observed at the highest evaluated concentration (41.6 % of control) (Table 3 and Table 4).
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed (see Table 6, Table 7, Table 9 and Table 10). The aberration rates of the cells after treatment with the test item (0.5 – 2.5 % aberrant cells, excluding gaps) did not exceed the range of the solvent control values (1.5 – 3.0 % aberrant cells, excluding gaps) and were within the range of the laboratory historical solvent control data (see Appendix 2).
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (660.0 µg/mL) or CPA (7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table2     Summary of results of the chromosomal aberration study with
Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics

Exp.	Preparationinterval	Test itemconcentration in µg/mL	Mitotic indices in % of control		Aberrant cells in %
					incl. gaps*		excl. gaps*		carrying exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control^1		100.0		3.0		2.5		0.0
		Positive control^2		52.8		13.5		13.0^S		2.5
		47.0		90.4		1.5		1.0		0.0
		440.8^PS		94.3		1.5		1.0		0.0
		771.4^PS		94.0		2.0		2.0		0.0
		1350.0^PS		87.6		2.0		2.0		0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1		100.0		2.5		2.5		0.0
		Positive control^2#		44.7		37.0		37.0^S		6.0
		8.8		101.5		1.5		1.5		0.0
		15.3		75.2		3.0		2.5		0.5
		26.9		41.6		2.5		2.5		0.0
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control^1		100.0		3.0		3.0		0.0
		Positive control^3#		82.0		35.0		35.0^S		8.0
		82.3		108.4		2.0		2.0		0.0
		440.8^PS		113.9		3.0		2.5		0.5
		771.4^PS		112.8		2.0		2.0		0.0
		1350.0^PS		104.6		1.5		1.5		0.0
II	22 hrs	Solvent control^1		100.0		2.5		1.5		0.0
		Positive control^3		55.0		9.0		9.0^S		1.5
		82.3		93.6		2.0		2.0		0.0
		440.8^PS		82.2		1.5		1.5		0.0
		771.4^PS		97.0		1.0		0.5		0.0
		1350.0^PS		81.2		1.5		1.5		0.0

*   Including cells carrying exchanges

^#    Evaluation of 50 metaphases per culture

^PS  Phase separation occurred at the end of treatment

^S    Aberration frequency statistically significant higher than corresponding control values

¹    Ethanol         0.5% (v/v)

²     EMS          660.0 µg/mL

³    CPA               7.5 µg/mL

Conclusions:

Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics has been tested for clastogenicity in a reliable study conducted according to OECD Test Guideline 473 and in compliance with GLP. In Experiment I in the absence and presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration (1350.0 μg/mL). In Experiment II after continuous treatment in the absence of metabolic activation clear cytotoxic effects were observed at the highest evaluated concentration. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural aberrations. In conclusion, it is concluded that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest required concentration.

Executive summary:

The test item Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics, dissolved in ethanol, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment I in the presence of S9 mix and Experiment II in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (1350.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473. The rationale for the dose selection is reported in section3.5.1. The chosen treatment concentrations are reported inTable 1and the results are summarised inTable 2.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II after continuous treatment in the absence of S9 mix clear cytotoxic effects were observed at the highest evaluated concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-07-09 to 2014-09-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

According to OECD guideline 473 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

1. Hypothesis for the analogue approach:
The hypothesis for the analogue approach is that both the registration substance, Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics (target substance), and the test substance, Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics (source substance), are produced from the same Fischer-Tropsch substance, GTL Gasoil, by fractional distillation. The substances are C9-C14 Aliphatics [≤2% Aromatics] Hydrocarbon Solvents.

The source substances contain the all of the constituents of the target substance. The substances have constituents that are part of the same homologous series and have many constituents in common. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies).

2. Source and target chemical(s)
The source substance Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics, is composed of linear, branched and cyclic hydrocarbons of chain length C9-C12

The target substance, Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics, is composed of linear, branched and cyclic hydrocarbons of chain length C9-C11.

3. Analogue approach justification
The constituents of the source and target substances are all hydrocarbons. Identical constituents have identical toxicological properties. The source substances cover the full carbon chain length of the target substance.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 9.7, 16.9, 29.6, 51.9, 90.8, 158.9, 278.0, 486.5, 851.4, 1490.0 µg/mL
Experiment II: 29.6, 51.9, 90.8, 158.9, 278.0, 486.5, 851.4, 1490.0 µg/mL

Without metabolic activation:
Experiment I: 9.7, 16.9, 29.6, 51.9, 90.8, 158.9, 278.0, 486.5, 851.4, 1490.0 µg/mL
Experiment II: 9.7, 16.9, 29.6, 51.9, 90.8, 158.9, 278.0, 486.5, 851.4, 1490.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: about 1.5

NUMBER OF CELLS EVALUATED: At least 100 per culture, except for the positive control in Experiment I, in the presence of S9 mix and in Experiment II in the absence of S9 mix, where only 50 metaphases were evaluated

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Key result

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: tested up to cytotoxic or precipitating or the highest required concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics, dissolved in ethanol, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment I, in the presence of S9 mix and in Experiment II in the absence of S9 mix, where only 50 metaphases were evaluated. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 1490.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
Visible precipitation of the test item in the culture medium was observed in Experiment I in the presence of S9 mix at 158.9 µg/mL and above at the end of treatment. Phase separation was observed in Experiment I at 486.5 µg/mL and above in the absence of S9 mix. In Experiment II phase separation was observed at 51.9 µg/mL and above in the absence of S9 mix and at 90.8 µg/mL and above in the presence of S9 mix at the end of treatment.
No relevant influence on pH value and osmolarity was observed.
In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration (42.5 % of control) (Table 3 and Table 4).
Either with or without metabolic activation no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item (Table 6, Table 7, Table 9 and Table 10). The aberration rates of the cells after treatment with the test item (0.5 – 3.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.0 – 3.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data (see Appendix 2). However, in Experiment I in the presence of S9 mix one single statistically significant increase (2.5 % aberrant cells, excluding gaps), clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) was observed after treatment with 51.9 µg/mL.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (660.0 µg/mL) or CPA (7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table2     Summary of results of the chromosomal aberration study with
Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics

Exp.	Preparationinterval	Test itemconcentration in µg/mL	Mitotic indices in % of control		Aberrant cells in %
					incl. gaps*		excl. gaps*		carrying exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control^1		100.0		1.0		0.5		0.0
		Positive control^2		65.6		13.5		13.0^S		2.5
		278.0		101.9		0.5		0.5		0.0
		486.5^PS		110.0		2.5		1.5		0.0
		851.4^PS		92.9		0.5		0.5		0.0
		1490.0^PS		111.9		1.0		1.0		0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1		100.0		3.5		3.0		0.0
		Positive control^2#		35.8		37.0		37.0^S		6.0
		9.7^##		95.4		3.3		3.0		0.0
		16.9^##		105.5		3.8		3.5		0.0
		29.6		42.5		1.5		1.0		0.0
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control^1		100.0		1.5		0.0		0.0
		Positive control^3#		129.2		35.0		35.0^S		8.0
		51.9		95.7		2.5		2.5^S		0.0
		90.8		114.2		1.0		1.0		0.0
		158.9^P		108.6		1.0		1.0		0.0
II	22 hrs	Solvent control^1		100.0		1.0		0.5		0.0
		Positive control^3		41.3		9.0		9.0^S		1.5
		51.9		100.7		1.5		1.5		0.0
		486.5^PS		95.2		1.5		0.5		0.0
		851.4^PS		100.4		0.5		0.5		0.0
		1490.0^PS		89.6		0.5		0.5		0.0

*   Including cells carrying exchanges

^#    Evaluation of 50 metaphases per culture

^##  Evaluation of 200 metaphases per culture

^P   Precipitation occurred at the end of treatment

^PS  Phase separation occurred at the end of treatment

^S    Aberration frequency statistically significant higher than corresponding control values

¹    Ethanol       0.5 % (v/v)

²     EMS       660.0 µg/mL

³    CPA            7.5 µg/mL

Conclusions:

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics has been tested for clastogenicity in a reliable study conducted according to OECD Test Guideline 473 and in compliance with GLP. In Experiment I in the absence and presence of metabolic activation and in Experiment II in the presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration (1490.0 μg/mL). In Experiment II in the absence of metabolic activation, clear cytotoxicity was observed at the highest evaluated concentration. Either with or without metabolic activation no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of metabolic activation one single statistically significant increase (2.5 % aberrant cells, excluding gaps), clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) was observed after treatment with 51.9 μg/mL. No evidence of an increase in polyploidy metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, it is concluded that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or precipitating or the highest required concentrations.

Executive summary:

The test item Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics, dissolved in ethanol, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitroin two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment I, in the presence of S9 mix and in Experiment II in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (1490.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473. The rationale for the dose selection is reported in section3.5.1. The chosen treatment concentrations are reported inTable 1and the results are summarised inTable 2.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration.

Either with or without metabolic activation no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix one single statistically significant increase (2.5 % aberrant cells, excluding gaps), clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) was observed after treatment with 51.9 µg/mL.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read across genetic toxicity test listed below had negative results for Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics.

 

Genetic Toxicity in vivo – micronucleus assay (OECD 474)

 

Genetic Toxicity in vivo – Rodent Dominant Lethal Test (OECD TG 478)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1978 - January 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline with acceptable restrictions.

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 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Principles of method if other than guideline:

Similar to OECD 478, only two dose levels were used.

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Massachusetts
- Age at study initiation: males: 10 weeks at initiation of pre-treatment mating period; females: 9 weeks at initiation of week 1 of pre-treatment mating period; females: 8 weeks at initiation of week 1 of post-treatment mating period
- Housing: males: individually housing during the treatment period; females: individually housing during the pre- and post-mating period
- Diet: Purina Laboratory Chow; ad libitum; except chamber-exposed groups during the treatment period
- Water: ad libitum; except chamber-exposed groups during the treatment period

Route of administration:

inhalation

Vehicle:

none

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The stainless steel and glass chambers had a total volume of one cubic metre with an effective volume of 760 L.
- Air flow rate: 132 L/min
- Air change rate: every 7.6 min
- The test substance was placed in fritted-bottom, gas-washing bottles. Dry air, at various flow rates, was passed through the test material and the vapour-air mixtures entered the chamber air inlets and were further diluted with room air to provide the desired concentrations.


TEST ATMOSPHERE
Atmospheric sampling was performed using a Wilks Scientific Corp., Miran IA Ambient Air Analyzer (long pathlength infrared). On each exposure day three samples were drawn from each exposure chamber (at 1, 3 and 5 hours) and the exposure concentrations calculated by comparing the absorption of this sample to the standard curve.

Duration of treatment / exposure:

8 consecutive weeks

Frequency of treatment:

6 hours/day, 5 days/week

Post exposure period:

4 weeks

Remarks:

Doses / Concentrations:
400 and 1200 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

only males were exposed: 10/group

Control animals:

yes, sham-exposed

Positive control(s):

triethylenemelamine
- Route of administration: intraperitoneally on a single day, at least 2 hours prior to mating
- Doses level/ volume: 0.5 mg/kg body weight; 1 mL/kg body weight
- Vehicle: 0.9 % injectable normal saline solution

Tissues and cell types examined:

Males: testes, seminal vesicles, epididymides, prostate tissues
Females: uteri

Details of tissue and slide preparation:

METHOD OF ANALYSIS:
Males: Seminal vesicles, epididymides, prostate and any abnormal lesion or tissue masses were preserved in 10 % neutral buffered formalin. Testes were stored for 48 hours in Bouin´s solution and subsequently placed in formalin. Tissues from 5 randomly chosen males from each group were examined.
Females: The uteri from all females (pregnant and non-pregnant) were appropriately identified and preserved intact in a 10 % neutral buffered formalin solution.

Statistics:

Comparisons were made during the treatment and post-treatment periods between negative control, positive control and test substance-treated groups by the Chi-square test. Absolute data were compared by using the F-test and Student´s t-test. When variances differed significantly, Student´s t-test was appropriately modified using Cochran´s approximation (t´). Corpora lutea and implantation data were evaluated by the one-tailed test.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

At dose level of 400 and 1200 ppm the test substance was not mutagenic in rats by the dominant-lethal test.

Toxicity:

no effects

Vehicle controls validity:

other: no vehicle was used

Negative controls validity:

valid

Positive controls validity:

valid

No treatment-related effects were observed for mortality, in-life physical or necropsy examinations and for body weights in males.

No treatment effect was indicated from microscopic evaluation of testes, seminal vesicles, epididymides and prostate tissues from 5 randomly chosen males from each exposure group.

In one animal of the 400 ppm exposure group a caseous mass was observed in the left epididymis. This caseous mass was seen microscopically to be a spermatic granuloma. Since this was observed in a single animal, this is not considered to be treatment related. Several animals (one in control, one in 100 ppm group, one in 1200 ppm group) presented a purulent prostatitis which was of probable bacterial etiology.

Pregnancy rates, implantation data and implantation efficiency values and fetal death data for females mated to treated males were considered comparable to data for females mated to negative control males for each week of the post-treatment mating period. At dose level of 400 and 1200 ppm the test substance was not mutagenic in rats by the dominant-lethal test.

Positive control:

Females mated to the positive control males had fewer implants and lower implantation efficiency values (indicative of pre-implantation loss) than females mated to the negative control males. Fetal death parameters (mean and expressed as a percentage of total implants) for these same females were significantly increased during the post-treatment mating interval.

Conclusions:

Interpretation of results: negative

Under the conditions of the test no negative effects were induced by the test substance, hydrocarbons, C7-C9, isoalkanes.

Executive summary:

Under the conditions of the test no negative effects were induced by the test substance, hydrocarbons, C7 -C9, isoalkanes.

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report equivalent or similar to OECD guideline 478.

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 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: Males (7-8 weeks); females pre-treatment mating period (8-9 weeks); females post treatment mating period (7-8 weeks)

- Housing: males were house individiually during the treatment period and housed with two females per week during the 2 week pretreatment mating period and the 6 week post-treatment mating period. Females were housed individually during the pre-mating and post-mating periods and housed with males in a 2:1 ratio during mating.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum

Route of administration:

inhalation: vapour

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
MRD-77-44 was transferred from a reservoir using a metering pump into a heated flask and flash evaporated. A stream of clean air was also passed through the flask and the vapor laden air transferred to a port in the chamber air inlet where it was diluted with normal chamber intake air to give the desired concentration.
- Exposure apparatus: inhalation chamber
- Rate of air: 125 liters/minute

- Air flow rate: 125 liters/minute
- Air change rate: 8 minutes
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: Wilks Scientific Corp, Miran IA Ambient Air Analyzer (long path infrared)
- Samples taken from breathing zone: no

Duration of treatment / exposure:

MRD-77-44 was administered by vapor inhalation for six hours/day for five consecutive days.
Negative control (chamber air) was administered 6 hours/day for five consecutive days
Triethylenemelamine was administered by intraperitoneal injection (normal saline) as a single dose.

Frequency of treatment:

MRD-77-44 was administered by vapor inhalation for six hours/day for five consecutive days.
Negative control (chamber air) was administered 6 hours/day for five consecutive days
Triethylenemelamine was administered by intraperitoneal injection (normal saline) as a single dose.

Post exposure period:

Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks. The females were sacrificed 12 days after the last day of cohabitation

Remarks:

Doses / Concentrations:
900 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
300 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

Negative control: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
Positive control: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
300ppm MRD-77-44: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
900ppm MRD-77-44: 10 males; 120 females during six week post-treatment mating period (two females/male/week)

Control animals:

yes

Positive control(s):

triethylenemelamine

- Route of administration: Intraperitoneal injection
- Doses / concentrations: 0.5mg/kg/bw

Tissues and cell types examined:

Males: seminal vesicle, epididymides, prostate, and any abnormal lesion or tissue masses, testes.
Females: reproductive tissues examined (uterine horns preserved, implantation sites, resorption sites)

Statistics:

Comparisons were made during the treatment and post-treatment periods between negative control, positive control and test substance-treated groups by the chi-square method where applicable. Absolute data were compared using the F-test and Students t-test. When variances differed significantly, Students T-test was appropriately modified using Cochran’s approximation.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative
When administered by vapor inhalation, MRD-77-44 is not mutagenic by the dominant lethal test. This finding does not warrant classification of the test material as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations.

Executive summary:

In a dominant lethal assay, MRD-77-44 was administered by vapor inhalation for six hours/day for five consecutive days to male rats at dose levels of 300 and 900 ppm to test for mutagenic potential.  Included in the study was a negative (chamber exposed) control group and a positive control group.  The latter received 0.5mg/kg of triethylenemelamine administered intraperitoneally on a single day, two hours prior to mating.  Each group contained 10 proven fertile rats.  Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks.  The females were sacrificed 12 days after the last day of cohabitation.  Exposure of males to MRD-77-44 produced no adverse effects on mortality or body weight gain during the post-treatment mating period. Overall, the number of pregnant females, number of implantations per litter, number of live fetuses, number of dead implantations, and the number of resorptions were unaffected by MRD-77-44 exposure.  Exposures to male rats had no effect on their ability to mate and impregnate females, and to produce live fetuses.  Based on these data, MRD-77-44 when administered by vapor inhalation to male rats is not considered mutagenic by the dominant lethal test.  This finding does not warrant the classification of MRD-77-44 as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations.

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report equivalent or similar to OECD guideline 478.

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 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: Males (7-8 weeks); females pre-treatment mating period (8-9 weeks); females post treatment mating period (7-8 weeks)
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: males were house individiually during the treatment period and hosed with two females per week during the 2 week pretreatment mating period and the 6 week post-treatment mating period. Females were housed individually during the pre-mating and post-mating periods and housed with males in a 2:1 ratio during mating.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum

Route of administration:

inhalation: vapour

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
MRD-77-44 was transferred from a reservoir using a metering pump into a heated flask and flash evaporated. A stream of clean air was also passed through the flask and the vapor laden air transferred to a port in the chamber air inlet where it was diluted with normal chamber intake air to give the desired concentration.
- Exposure apparatus: inhalation chamber
- Rate of air: 125 liters/minute



- Air flow rate: 125 liters/minute
- Air change rate: 8 minutes
- Method of particle size determination:
- Treatment of exhaust air:


TEST ATMOSPHERE
- Brief description of analytical method used: Wilks Scientific Copr, Miran IA Ambient Air Analyzer (long path infrared)
- Samples taken from breathing zone: no

Duration of treatment / exposure:

Six hours /day

Frequency of treatment:

five days
Triethylenemelamine was administered by intraperitoneal injection (normal saline) as a single dose.

Post exposure period:

Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks. The females were sacrificed 12 days after the last day of cohabitation

Remarks:

Doses / Concentrations:
900 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
300 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

Negative control: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
Positive control: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
300ppm MRD-77-43: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
900ppm MRD-77-43: 10 males; 120 females during six week post-treatment mating period (two females/male/week)

Control animals:

yes

Positive control(s):

triethylenemelamine

- Route of administration: Intraperitoneal injection
- Doses / concentrations: 0.5mg/kg/bw

Tissues and cell types examined:

Males: seminal vesicle, epididymides, prostate, and any abnormal lesion or tissue masses, testes.
Females: reproductive tissues examined (uterine horns preserved, implantation sites, resorption sites)

Statistics:

Comparisons were made during the treatment and post-treatment periods between negative control, positive control and test substance-treated groups by the chi-square method where applicable. Absolute data were compared using the F-test and Students t-test. When variances differed significantly, Students T-test was appropriately modified using Cochran’s approximation.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative
When administered by vapor inhalation, MRD-77-43 is not mutagenic by the dominant lethal test. This finding does not warrant classification of (the test material as a genotoxin 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:

In a dominant lethal assay,  MRD-77-43 was administered by vapor inhalation for six hours/day for five consecutive days to male rats at dose levels of 300 and 900 ppm to test for mutagenic potential.  Included in the study was a negative (chamber exposed) control group and a positive control group.  The latter received 0.5mg/kg of triethylenemelamine administered intraperitoneally on a single day, two hours prior to mating.  Each group contained 10 proven fertile rats.  Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks.  The females were sacrificed 12 days after the last day of cohabitation.  Exposure of males to MRD-77-43 produced no adverse effects on mortality or body weight gain during the post-treatment mating period. Overall, no treatment related effects were observed on the number of pregnant females, number of implantations per litter, number of live fetuses, number of dead implantations, and the number of resoprtions.  Exposures to male rats had no effect on their ability to mate and impregnate females, and to produce live fetuses.  Based on these data, MRD-77-43 when administered by vapor inhalation to male rats is not considered mutagenic by the dominant lethal test.  This finding does not warrant the classification of MRD-77-43 as a genotoxin 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)

Additional information

No genotoxicity studies are available for the registered substance, Hydrocarbons, C9-C11 aliphatics, <2% aromatics (GS180). However, some reliable in vitro data are available for related Fischer-Tropsch process-derived products in the relevant carbon number range: GS160 (C8-C11) and GS170 (C9-C12). These are read across to Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics.

In Vitro

In vitro gene mutation study in bacteria

Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics has been tested for mutagenicity to bacteria in a reliable study conducted according to OECD Test Guideline 471 and in compliance with GLP. The test substance was negative in bacterial strains Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA when tested with and without metabolic activation (Aroclor 1254-induced rat liver S9) up to cytotoxic or the highest required concentrations in both the initial experiment using the plate incorporation method and the independent repeat pre-incubation assay. Appropriate positive controls were included and showed the expected increase in the number of revertants. Therefore it is concluded that Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics is not mutagenic to bacteria (WIL Research, 2014k).

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics has been tested for mutagenicity to bacteria in a reliable study conducted according to OECD Test Guideline 471 and in compliance with GLP. The test substance was negative in bacterial strains Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA when tested with and without metabolic activation (Aroclor 1254-induced rat liver S9) up to cytotoxic or the highest required concentrations in both the initial experiment using the plate incorporation method and the independent repeat pre-incubation assay. Appropriate positive controls were included and showed the expected increase in the number of revertants. Therefore it is concluded that Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics is not mutagenic to bacteria (WIL Research, 2014l).

In Vitro Chromosome Aberration in Mammalian Cells

Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics has been tested for clastogenicity in a reliable study conducted according to OECD Test Guideline 473 and in compliance with GLP. In Experiment I in the absence and presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration (1350.0 μg/mL). In Experiment II after continuous treatment in the absence of metabolic activation clear cytotoxic effects were observed at the highest evaluated concentration. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural aberrations. In conclusion, it is concluded that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest required concentration (Harlan Cytotest Cell Research GmbH, 2014a).

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics has been tested for clastogenicity in a reliable study conducted according to OECD Test Guideline 473 and in compliance with GLP. In Experiment I in the absence and presence of metabolic activation and in Experiment II in the presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration (1490.0 μg/mL). In Experiment II in the absence of metabolic activation, clear cytotoxicity was observed at the highest evaluated concentration. Either with or without metabolic activation no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of metabolic activation one single statistically significant increase (2.5 % aberrant cells, excluding gaps), clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) was observed after treatment with 51.9 μg/mL. No evidence of an increase in polyploidy metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, it is concluded that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or precipitating or the highest required concentrations.

In vitro Gene Mutation study in Mammalian Cells

C10-C13 isoalkanes

In a key mammalian cell gene mutation assay (Chevron Phillips, 1982), exposure to eight graded doses of the test material (C10-C13 isoalkanes) 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 C10-C13 isoalkanes are not considered to be mutagenic in this test system.

Isododecane

In a key mammalian cell gene mutation assay (INEOS, 1996), exposure to the test material in the presence of and in the absence of metabolic activation did not increase the induction of mutations. Therefore isododecane was not considered to be mutagenic in this test system.

In Vivo

Hydrocarbons, C7-C9, isoalkanes, <2% aromatics

Hydrocarbons, C7-C9, isoalkanes tested in a dominant lethal study (similar to OECD 478) showed no evidence of genotoxicity in the germ cells of treated male rats exposed to 400 or 1200 ppm by inhalation (ExxonMobil Chemical,1980).

Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, < 2% aromatics

In a key dominant lethal assay, the test material (Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, < 2% aromatics) was administered by vapor inhalation for six hours/day for five consecutive days to male rats at dose levels of 300 and 900 ppm to test for mutagenic potential (ExxonMobil, 1978).  Included in the study was a negative (chamber exposed) control group and a positive control group.  The latter received 0.5mg/kg of triethylenemelamine administered intraperitoneally on a single day, two hours prior to mating.  Each group contained 10 proven fertile rats.  Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks.  The females were sacrificed 12 days after the last day of cohabitation.  Exposure of males to the test material produced no adverse effects on mortality or body weight gain during the post-treatment mating period. Overall, the number of pregnant females, number of implantations per litter, number of live fetuses, number of dead implantations, and the number of resorptions were unaffected by exposure to the test material.  Exposures to male rats had no effect on their ability to mate and impregnate females, and to produce live fetuses.  Based on these data, the test material when administered by vapor inhalation to male rats is not considered mutagenic by the dominant lethal test.  This finding does not warrant the classification of Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, < 2% aromatics as a genotoxin under EU GHS guidelines.

Hydrocarbons, C10-C12, isoalkanes, <2% aromatics

In a key in vivo dominant lethal assay (ExxonMobil Corp., 1978), the test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) was administered by vapor inhalation for six hours/day for five consecutive days to male rats at dose levels of 300 and 900 ppm to test for mutagenic potential.  Included in the study was a negative (chamber exposed) control group and a positive control group.  The latter received 0.5mg/kg of triethylenemelamine administered intraperitoneally on a single day, two hours prior to mating.  Each group contained 10 proven fertile rats.  Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks.  The females were sacrificed 12 days after the last day of cohabitation.  Exposure of males to the test material produced no adverse effects on mortality or body weight gain during the post-treatment mating period. Overall, no treatment related effects were observed on the number of pregnant females, number of implantations per litter, number of live fetuses, number of dead implantations, and the number of resoprtions. Exposures to male rats had no effect on their ability to mate and impregnate females, and to produce live fetuses.  Based on these data, the test material when administered by vapor inhalation to male rats is not considered mutagenic by the dominant lethal test. 

Justification for classification or non-classification

All in vitro and in vivo tests on test substances relevant to the registration substance were negative; Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics is considered not to be mutagenic and does not require classification for mutagenicity according to Regulation (EC) No 1272/2008.