Hydrocarbons, C14-C16, n-alkanes, isoalkanes,

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in bacterial mutagenicity assay (OECD TG 471).

Negative in chromosome aberration assay (OECD TG 473)

Negative in Mammalian Cell Gene Mutation Test (OECD TG 476)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-Aug-2014 to 25-Aug-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 1254

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

Of
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 1, 3, 10, 33, 66 and 100 µg/plate
With S9-mix: 1, 3, 10, 33, 100 and 200 µg/plate.
Experiment 2:

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

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

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 (small droplets of test substance) was observed.
Dose range finding test: at the start of the incubation period at concentrations of 512 µg/plate and upwards and at the end of the incubation period at 1600 and 5000 µg/plate.
First mutation experiment: at the start and end of the incubation period at 5000 µg/plate.
Second mutation experiment: at the start of the incubation period at a concentration of 1600 and 5000 µg/plate and at 5000 µg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Conclusions:

An Ames test was performed with Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics according to OECD 471 and GLP. Strains tested were Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli strain WP2 uvrA, both with and without metabolic activation (Aroclor 1254-induced rat liver S9). The test was negative both with and without metabolic activation.

Executive summary:

Hydrocarbons, C14-C16, 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-21 to 2014-10-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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.

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 IA:13.8, 24.1, 42.2, 73.8, 129.2, 226.0, 395.6, 692.2, 1211.4, 2120.0 µg/mL
Experiment II: 73.8, 129.2, 226.0, 395.6, 692.2, 1211.4, 2120.0 µg/mL

Without metabolic activation:
Experiment IA: 13.8, 24.1, 42.2, 73.8, 129.2, 226.0, 395.6, 692.2, 1211.4, 2120.0 µg/mL
Experiment IB: 24.1, 42.2, 73.8, 129.2, 226.0, 395.6, 543.9, 692.2, 951.8, 1211.4, 2120.0 µg/mL
Experiment II: 13.8, 24.1, 42.2, 73.8, 129.2, 226.0, 395.6, 692.2, 1211.4, 2120.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- 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:

Three independent experiments were performed. In Experiment IA the exposure period was 4 hours with and without S9 mix. In Experiment IB the exposure period was 4 hours 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. 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


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.

Statistics:

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

Key result

Species / strain:

lymphocytes: human

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:

The test item Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, 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.
Three independent experiments were performed. In Experiment IA the exposure period was 4 hours with and without S9 mix. In Experiment IB the exposure period was 4 hours 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. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 2120.0 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 at the end of treatment at 73.8 µg/mL and above in Experiment IA and at 42.2 µg/mL and above in Experiment II in the absence of S9 mix. In Experiment IB in the absence of S9 mix and Experiment II in the presence of S9 mix phase separation was observed at 129.2 µg/mL and above. In Experiment IA in the presence of S9 mix phase separation was observed at 226.0 µg/mL and above.
No relevant influence on osmolarity or pH value was observed.
In this study no relevant cytotoxicity, indicated by reduced mitotic indices could be observed up to the highest applied concentration (Table 3 – Table 5).
In Experiment IA in the absence of S9 mix, statistically significant increases in chromosomal aberrations were observed after treatment with 42.2 and 692.2 µg/mL (3.0 and 5.5 % aberrant cells, excluding gaps). The first value is in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. The second value exceeded the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) (Table 7). However, no dose-dependency was observed. In the confirmatory Experiment IB in the absence of S9 mix, in Experiment II in the absence of S9mix after continuous treatment and in Experiment IA and II in the presence of S9 mix no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 – 2.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.0 – 1.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data (see Appendix 2). The chosen treatment concentrations are reported in Table 1 and the results are summarised in Table 2.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Either EMS (660.0 or 770.0 µg/mL) or CPA (2.5 or 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, C14-C16, 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
IA	22 hrs	Solvent control^1	100.0	1.0	0.5	0.0
		Positive control^2	90.2	10.0	10.0^S	2.0
		42.2	114.0	3.5	3.0^S	0.0
		395.6^PS	92.4	2.5	2.5	0.5
		692.2^#PS	96.0	5.5	5.5^S	0.0
		1211.4^PS	96.3	2.5	2.5	0.0
		2120.0^PS	107.9	2.0	2.0	0.0
IB	22 hrs	Solvent control^1	100.0	1.0	1.0	0.0
		Positive control^3	71.6	12.0	12.0^S	2.0
		73.8	69.4	1.5	1.0	0.0
		543.9^PS	80.4	1.5	1.5	0.0
		692.2^PS	79.0	1.5	1.5	0.0
		951.8^PS	64.6	0.5	0.5	0.0
		1211.4^PS	76.8	1.5	1.5	0.0
		2120.0^PS	70.8	1.5	1.5	0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1	100.0	2.0	1.5	0.0
		Positive control^3	32.8	24.0	24.0^S	6.5
		24.1	91.1	2.0	2.0	0.0
		692.2^PS	85.0	2.0	2.0	0.0
		1211.4^PS	86.6	1.0	1.0	0.0
		2120.0^PS	79.8	0.5	0.5	0.0

*   Including cells carrying exchanges

^#    Evaluation of 200 metaphases per culture

^PS  Phase separation occurred at the end of treatment

^S    Aberration frequency statistically significant higher than corresponding control values

¹    Acetone        0.5 % (v/v)

²     EMS       770.0 µg/mL

³     EMS       660.0 µg/mL

Table 2, cont.  Summary of results of the chromosomal aberration study with
Hydrocarbons, C14-C16, 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 with S9 mix
I	22 hrs	Solvent control^1	100.0	1.5	1.5	0.0
		Positive control^2	76.6	9.5	9.5^S	1.5
		129.2	106.6	1.0	1.0	0.0
		692.2^PS	100.0	1.0	1.0	0.0
		1211.4^PS	102.9	1.5	1.5	0.0
		2120.0^PS	102.9	1.5	1.0	0.0
II	22 hrs	Solvent control^1	100.0	1.0	1.0	0.0
		Positive control^3	48.7	17.0	17.0^S	3.0
		73.8	91.2	1.5	1.5	0.0
		692.2^PS	100.4	0.5	0.5	0.0
		1211.4^PS	101.3	2.0	2.0	0.0
		2120.0^PS	106.6	1.5	1.5	0.0

*   Including cells carrying exchanges

^PS  Phase separation occurred at the end of treatment

^S    Aberration frequency statistically significant higher than corresponding control values

¹    Acetone        0.5 % (v/v)

²    CPA            2.5 µg/mL

³    CPA            7.5 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.

Executive summary:

The test item Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. IA & IB	Exp. II	Exp. IA & 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.

The highest applied concentration in this study (2120.0 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 the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In Experiment IA in the absence of S9 mix, statistically significant increases in chromosomal aberrations were observed after treatment with 42.2 and 692.2 µg/mL (3.0 and 5.5 % aberrant cells, excluding gaps). The first value is in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. The second value exceeded the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps). However, no dose-dependency was observed. In Experiment IB these findings could not be confirmed. In Experiment II in the absence of S9mix after continuous treatment and in Experiment IA and II in the presence of S9 mix no relevant increases in chromosomal aberrations were observed.

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 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted study according to sound scientific principles.

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)

Deviations:

no

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

Chinese Hamster V79 cells in vitro (V79/HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Chinese Hamster V79 cells in vitro (V79/HPRT)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor

Test concentrations with justification for top dose:

without S9: 0.1, 0.3, 0.6, 1, 2, 3 ug/mL
with S9: 100, 500, 1000, 5000 ug/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without metabolic activation 0.6 ug/mL; with metabolic activation 5000 ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative with and without metabolic activation

Exposure to test material in the presence of and in the absence of metabolic activation did not increase the induction of mutations. Therefore isododecane is not considered to be mutagenic in this test system.

Executive summary:

Exposure to test material in the presence of and in the absence of metabolic activation did not increase the induction of mutations. Therefore isododecane is not considered to be mutagenic in this test system.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted study according to sound scientific principles.

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)

Deviations:

no

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK+/ phenotype

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

TK+/ phenotype of L5178Y mouse lymphoma cells from subline 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Aroclor

Test concentrations with justification for top dose:

up to was 1000 ug/mL in dimethylsulfoxide (maximum dose)

Vehicle / solvent:

dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

This assay was performed with the TK+/ phenotype of L5178Y mouse lymphoma cells from subline 3.7.2C using a minimum of eight test compound doses with and without metabolic activation by an Aroclor induced rat liver microsomal fraction. Appropriate negative, solvent, and positive controls were included with each assay. The test compound dose levels were determined by a preliminary multidose ranging study with the highest dose targeted to give approximately fifty to ninety percent inhibition of suspension cell growth depending on the solubility of the compound. C10-C13 isoalkanes achieved a homogeneous mixture at approximately 100 mg/ml in dimethylsulfoxide. The maximum dose selected for the mutagenicity test was 1000 ug/ml because it represents the limits of solubility of the test material.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Exposure to eight graded doses of the test material 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative with and without metabolic activation

Exposure to eight graded doses of the test material 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.

Executive summary:

Exposure to eight graded doses of the test material 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.

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, C14-C16, 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 somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1990/10/24 - 1990/11/30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to or similar to OECD Guideline 474. GLP

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 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Charles River Breeding Laboratories, Inc.
Sex: Male (65), Female (65)
Age at study initiation: Approximately 9-10 weeks
Weight at study initiation: 23-39g
Housing: Individually
Diet (e.g. ad libitum): Purina Certified Rodent 5002 chow (pellets), ad libitum
Water (e.g. ad libitum): Automatic watering system, ad libitum
Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
Temperature (°F): 68-76
Humidity (%): 40-70%
Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Corn oil was used. Dose volume did not exceed 1.0 ml/100 g bw.

Details on exposure:

The test material and the carrier were administered by oral gavage as a single dose. The carrier was dosed at a volume equal to the test material dose volume. The individual animal dose volumes did not exceed 1.0 ml/100 g body weight; animals were administered 1.0, 2.5, 5.0 g test material/ kg body weight. The positive control, cyclophosphamide was administered as a single dose of 40 mg/kg using water as a carrier.

Duration of treatment / exposure:

Animals were sacrificed 24, 48, and 72 hours after dose administration.

Frequency of treatment:

One dose was given at either 1.0, 2.5, 5.0 g test material/ kg body weight. Cyclophosphamide was dosed at 40 mg/kg.

Post exposure period:

Animals were sacrificed 24, 48, and 72 hours after dose administration.

No. of animals per sex per dose:

Male (65), Female (65) ; 5 Males and 5 Females per treatment group

Positive control(s):

The positive control, cyclophosphamide was administered as a single intraperitoneal injection (40 mg/kg) using water as a carrier.

Tissues and cell types examined:

Erythrocytes derived from femur bone marrow.

Details of tissue and slide preparation:

Immediately following the sacrifice of the animals, both femurs were removed and the bone marrow was removed and suspended in fetal bovine serum. After the suspension was centrifuged the pellet was resuspended and smears were prepared (two slides per animal).

Evaluation criteria:

Slides were stained using acridine orange; polychromatic erythrocytes (PCE) stained red/orange, nonchromatic erythrocytes (NCE) are unstained (dull green), and micronuclei stain bright yellow. Additional criteria for scoring micronuclei are a circular appearance and a diameter between 1/20 and 1/5 of the cell’s diameter. 1000 PCE from each animal were examined for the presence of micronuclei and the ratio of PCE to NCE was determined for each animal by counting 1000 erythrocytes (PCE and NCE).

Statistics:

Calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period (ANOVA). When ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan’s Multiple Range Test.

A standard regression analysis was performed to test for a dose response.
Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion. The residuals were normally distributed (values were greater than 0.01 level of significance). Therefore nonparametric analysis was not performed.

Sexes were analyzed separately.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The positive control (cyclophosphamide) induced a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes, indicating that the positive control was clastogenic and was responding in an appropriate manner. Carrier control values for the mean percent of polychromatic erythrocytes and for the mean number of micronucleated polychromatic erythrocytes are within the normal range for the corn oil control. MRD-90-874 did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes which is a measure of bone marrow toxicity. MRD-90-874 did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes. MRD-90-874 did not induce a significant increase in the mean number of micronucleated polychromatic erythrocytes. MRD-90-874 was not cytotoxic at doses up to 5.0 g/kg to mouse bone marrow under the conditions of this test.

Conclusions:

Interpretation of results: negative
These data indicate that MRD-90-874 is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/kg of body weight.

Executive summary:

The test material, MRD-90-874 was tested in the mammalian bone marrow micronucleus assay using CD-1 mice.  MRD-90-874 was tested at 24, 48, and 72 hour intervals following exposure and did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes or an increase in the mean number of micronucleated polychromatic erythrocytes.  Both the positive (cyclophosphamide) and the negative (carrier) controls behaved in an appropriate manner.  These data indicate that MRD-90-874 is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/kg.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report equivalent or similar to OECD guideline 474: GLP

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 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

According to US EPA Guideline 84-2

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: ca. 8-9 weeks
- Weight at study initiation: 21-40 grams
- Assigned to test groups randomly: [no/yes, under following basis: computer generated, body weight sorting program
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 28 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): not to exceed 1ml/100 grams bw
- Purity: assumed to be 100% pure

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was weighted out and on the day of dosing, mixed with the carrier to provide stock solutions such that individual animal dose volumes did not exceed 1ml/100grams body weight. The mice were administered 1.25, 2.5, or 5.0 grams of test material/kg of body weight. Corn oil served as the carrier for the test material and was dosed at the same volume as the test material.

Duration of treatment / exposure:

Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing.
Positive control animals were sacrificed 24 hours after injection

Frequency of treatment:

Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing

Post exposure period:

Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing

Remarks:

Doses / Concentrations:
5.0 g/kg/bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2.5g/kg/bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1.25 g/kg/bw
Basis:
nominal conc.

No. of animals per sex per dose:

30 animals (5 male; 5 female)/dose; 10/timepoint

Positive control(s):

cyclophosphamide;

- Route of administration: intraperitoneal injection
- Doses / concentrations:40 mg/kg using water as the carrier

Tissues and cell types examined:

Bone marrows were collected and extracted, smear preparations made and stained. Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were scored for each animal.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Range finding study was performed using 5.0, 2.5, and 1.0 gram of test material per kg of body weight. Two males and two females were used for each dose group. All animals survived and were sacrificed 24 hours after dosing. bome marrow was removed and slides were prepared. Slides were evaluated for percent of polychromatic erythrocytes in 1000 erythrocytes and for number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes.




DETAILS OF SLIDE PREPARATION: After sacrifice, both femurs were removed. The bone marrow was then removed and suspended in fetal bovine serum. After the suspension was centrifuged, the pellet was resuspended and smears were prepared (two slides per animal). Slides were labeled with blind coding. Slides were stained using acridine orange. 1000 polychromatic erythrocytes from each animal were examined for the presence of micronuclei, and the ratio of PCE’s to NCE’s determined


METHOD OF ANALYSIS: staining color, and circular appearance and a diameter between 1/20 and 1/5 of the cell's diameter

Statistics:

Statistical analysis included calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period. When the ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan’s Multiple Range Test. A standard regression analysis was performed to test for a dose response. Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion. The residuals were normally distributed (values were greater than 0.01 level of significance) in more than 25% of the analyses. Therefore nonparametric analysis were not performed. Sexes were analyzed separately

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No deaths or clinical signs of toxicity were observed in animals dosed with the test material up to the maximum recommended dose of 5g/kg

Conclusions:

Interpretation of results: negative
The in vivo micronucleus assay of MRD-89-582 in mice was negative. This finding does not warrant the 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:

MRD-89-582 was examined for its potential to induce chromosomal damage in bone marrow erythrocytes in mice dosed by oral gavage at concentrations of 5.0,2.5, and 1.25 g/kg. Vehicle and positive control animals received corn oil and cyclophosphamide, respectively.  Bone marrow samples were collected and evaluated for micronucleus formation 24, 48 and 72 hours after dosing.  MRD-89-582 did not induce a statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. The positive control material (cyclophosphamide) produced a marked increase in the frequency of micronucleated PCE when compared to the concurrent vehicle control group The test material was considered to be non-genotoxic and non-clastogenic under the conditions of the test. This finding does not warrant the 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 guidelines.

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

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: 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

In Vitro

In vitro gene mutation study in bacteria

Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics has been tested for mutagenicity to bacteria in a study conducted according to OECD TG 471 and in compliance with GLP (WIL Research, 2014d). No evidence of substance induced increase in the frequency of revertants was noted in the presence or absence of metabolic activation in Salmonella typhimurium strains TA1535, TA1537, TA98 and Escherichia coli WP2 uvrA when tested up to limit concentrations. Similar results were obtained in two independent experiments using plate incorporation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

In Vitro Chromosome Aberration in Mammalian Cells

Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics has been tested for potential for cytogenicity in a chromosome aberration study conducted according to OECD TG 473 and in compliance with GLP in two independent experiments (Harlan, 2014). The study was carried out using human lymphocytes in the presence of metabolic activation (four hours exposure) and in the absence of metabolic activation (four and twenty two hours (continuous) exposure). After four hours exposure in the absence of metabolic activation in the first test, significant increases in chromosomal aberrations were observed at the lowest and median concentrations (out of five concentrations tested). The increase at the lowest concentration was within the range of historical control data, and therefore considered to be biologically irrelevant. The increase at the median concentration exceeded the range of historical control data. In the absence of dose dependency and as the findings were not observed when the experiment was repeated using the same exposure times, nor with continuous exposure in the absence of metabolic activation, it is concluded that this increase did not represent a potential for cytogenicity. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for clastogenicity under the conditions of the test.

In vitro Gene Mutation study in Mammalian Cells

There is no in vitro mammalian genetic toxicity data available for Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics. However, data is available for structural analogues, Isododecane and, Hydrocarbons, C10-C13, isoalkanes, <2% aromatics. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

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, 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. 

 

Hydrocarbons, C10-C13, n-alkanes, <2% aromatics

In a key in vivo study (ExxonMobil, 1991), the test material (Hydrocarbons, C10-C13, n-alkanes, < 2% aromatics) was tested in the mammalian bone marrow micronucleus assay using CD-1 mice. The test material was tested at 24, 48, and 72 hour intervals following exposure and did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes or an increase in the mean number of micronucleated polychromatic erythrocytes. Both the positive (cyclophosphamide) and the negative (carrier) controls behaved in an appropriate manner. These data indicate that Hydrocarbons, C10-C13, n-alkanes, < 2% aromatics is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/Kg.

 

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a key in vivo study (ExxonMobil, 1991), the test material (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics) was examined for its potential to induce chromosomal damage in bone marrow erythrocytes in mice dosed by oral gavage at concentrations of 5.0,2.5, and 1.25 g/Kg. Vehicle and positive control animals received corn oil and cyclophosphamide, respectively. Bone marrow samples were collected and evaluated for micronucleus formation 24, 48 and 72 hours after dosing. The test material did not induce a statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. The positive control material (cyclophosphamide) produced a marked increase in the frequency of micronucleated PCE when compared to the concurrent vehicle control group. The test material was considered to be non-genotoxic and non-clastogenic under the conditions of the test. This finding does not warrant the classification of Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics as a genotoxin under EU GHS guidelines.

Justification for classification or non-classification

The negative results observed in in vitro and in vivo genotoxicity assays do not warrant the classification of Hydrocarbons, C14-C16 n-alkanes, isoalkanes, <2% aromatics as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).