Nonane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES data is available for Nonane. Additional in vitro genetic toxicity data is available for structural analogues Heptane and 2,2,4-trimethylpentane. 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.

The genetic toxicity test listed below had a negative result for Nonane.

 

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

 

The read across genetic toxicity tests listed below had negative results for Nonane.

 

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

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

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

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

1982-1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study 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 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

No specific method or guideline was noted; similar to OECD guideline 471; limited documentation

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon (Salmonellla), Trp-operon (E. coli)

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

E. coli WP2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of livers from Aroclor1254-pretreated rats

Test concentrations with justification for top dose:

max. conc. tested: 250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tween80/ethanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: benzo[a]pyrene in DMSO, 4-nitroquinoline-N-oxide in DMSO, sodium azide, neutral red, potassium dichromate

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation


DURATION
- Preincubation period: no data
- Exposure duration: not applicable, preincubation method
- Expression time (cells in growth medium): 48-72 hours


DETERMINATION OF CYTOTOXICITY
- Method: other: toxicity screening test

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

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:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The addition of heptane at amounts up to 250 µg per mL to cultures of Escherichia coli WP2 and WP2 uvr A, Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, and TA 100 did not lead to an increase in the reverse gene mutation frequency in any of these strains, either in the presence or in the absence of rat liver S9 fraction.

Table: Relative Reverse Mutation Rate – E. coli

Concentration (µg/ml)	E. coliWP2 Assay 1	E. coliWP2 Assay 2	E. coliWP2 Assay 3	E. coliWP2 uvr A Assay 1	E. coliWP2 uvr A Assay 2
Without S9
3.91	-	1.6	0.9	-	0.9
7.81	-	2.3	1.0	-	0.9
15.6	1.2	1.3	0.8	0.5	0.8
31.3	1.1	1.4	0.7	0.8	0.6
62.5	1.0	1.0	0.8	0.9	0.5
125	0.7	0.6	1.2	0.3	0.5
250	0.5	0.8	1.0	0.2	0.5
4-nitroquinoline-N-oxide	31.7	7.7	4.8	1.9	6.3
With S9
3.91	-	1.5	0.9	-	1.0
7.81	-	2.4	0.9	-	1.4
15.6	0.7	3.1	1.0	1.8	0.9
31.3	0.7	2.8	0.8	0.8	0.9
62.5	1.0	2.4	0.6	1.0	0.9
125	0.9	2.2	1.1	2.0	0.9
250	0.7	1.3	0.9	-	0.8
4-nitroquinoline-N-oxide	1.0	3.9	0.7	9.0	19.7

Table: Relative Mutation Rate – S. typhimurium TA 1535, TA 1537, TA 1538

Concentration (µg/ml)	TA 1535 Assay 1	TA 1535 Assay 2	TA 1537 Assay 1	TA 1537 Assay 2	TA 1538 Assay 1	TA 1538 Assay 2	TA 1538 Assay 3
Without S9
3.91	-	-	-	-	-	-	-
7.81	-	1.2	-	0.7	-	0.8	1.0
15.6	1.6	1.2	1.3	0	0.5	1.0	1.0
31.3	0.1	0.6	0.4	0	0	0.4	0.7
62.5	0	0	0	0	0	0	0
125	0	0	0	0	0	0	0
250	0	0	0	0	0	0	0
Sodium azide 1.7 µg	48.0	73.1	-	-	-	-	-
Benzo(a)-pyrene 6.7 µg	-	-	-	-	1.4	1.1	0.9
Neutral red 6.7 µg	-	-	1.5	1.6	-	-	-
With S9
3.91	-	-	-	-	-	-	-
7.81	-	0.9	-	0.7	-	-	0.8
15.6	2.1	1.0	2.4	0.7	1.5	-	1.0
31.3	1.4	0.9	1.3	0.9	2.1	-	0.7
62.5	1.7	0.6	1.8	0.9	1.8	-	1.0
125	1.7	1.3	2.1	0.6	1.2	-	1.1
250	1.1	0.7	2.3	0.8	1.3	-	0.9
Sodium azide 1.7 µg	19.3	78.3	-	-	-	-	-
Benzo(a)-pyrene 6.7 µg	-	-	-	-	2.4	-	10.6
Neutral red 6.7 µg	-	-	3.0	6.0	-	-	-

Table: Relative Mutation Rate – S. typhimurium TA 98, TA 100

Concentration (µg/ml)	TA 98 Assay 1	TA 98 Assay 2	TA 100 Assay 1	TA 100 Assay 2
Without S9
3.91	-	0.5	-	1.1
7.81	-	0.1	-	1.0
15.6	0.6	0	1.1	0.8
31.3	0	0	1.1	0.1
62.5	0	0	1.0	0
125	0	0	1.2	0
250	0	0	0.6	0
Benzo(a)-pyrene 6.7 µg	0.8	1.2	1.6	1.0
With S9
3.91	-	0.8	-	1.0
7.81	-	1.1	-	0.9
15.6	0.9	1.1	0.9	1.0
31.3	0.9	1.0	0.8	1.1
62.5	0.8	0.9	0.7	0.9
125	0.7	0.8	0.9	1.2
250	0.4	0.5	0.9	0.9
Benzo(a)-pyrene 6.7 µg	12.6	4.7	2.8	5.4

Conclusions:

Interpretation of results: negative

The purpose of this study was to determine the mutagenicity of the test substance Normal-Heptane. A reverse mutation assay was done using S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli strains WP2 and WP2 uvr A. The strains were exposed to concentrations of 0, 3.91, 7.81, 15.6, 31.3, 62.5, 125, and 250 ug/mL for 48 -72 hrs both with and without metabolic activation. The number of revertant colonies was then counted.
No significant increases in the ratio of mutations over controls was seen. The test substance is not mutagenic in either the presence or absence of metabolic activation.

Executive summary:

The purpose of this study was to determine the mutagenicity of the test substance Normal-Heptane. A reverse mutation assay was done using S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli strains WP2 and WP2 uvr A. The strains were exposed to concentrations of 0, 3.91, 7.81, 15.6, 31.3, 62.5, 125, and 250 ug/mL for 48 -72 hrs both with and without metabolic activation. The number of revertant colonies was then counted.

No significant increases in the ratio of mutations over controls was seen. The test substance is not mutagenic in either the presence or absence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 97

Metabolic activation:

with and without

Metabolic activation system:

liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333, 10000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

sodium azide, 9-aminoacridine, 4-nitro-o-phenylenediamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

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:

valid

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:

valid

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:

valid

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Under the conditions of this assay, there was no evidence of mutagenic activity.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the conditions of this assay, there was no evidence of mutagenic activity.

Executive summary:

Under the conditions of this assay, there was no evidence of mutagenic activity.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

1982-1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study 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 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

No guideline stated; similar to OECD guideline 473; rat hepatocytes with intrinsic metabolic activity, therefore no metabolic activation system added, only 100 cells from each culture analyzed.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

hepatocytes: rat liver (RL4) cells

Details on mammalian cell type (if applicable):

The rat liver cell line, RL4, an epithelial-type cell line, was derived in this laboratory following the procedure described by Williams et al. (1971). RL4 was initiated in 1978 from a 10-day-old Wistar rat as described in Dean and Hodson-Walker (1979).

Metabolic activation:

not applicable

Metabolic activation system:

not necessary, liver cells

Test concentrations with justification for top dose:

2.5, 5, 10 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tween 80/ethanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 7,12-dimethylbenzanthracene in DMSO, ethyl methanesulphonate, cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 22 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours + 2 hours fixation


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


NUMBER OF REPLICATIONS: no data


NUMBER OF CELLS EVALUATED: 100 cells from each culture

Evaluation criteria:

Significant or dose-related increase in chromosome damage.

Key result

Species / strain:

hepatocytes: Rat liver (RL4) cells

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

There was no significant or dose-related increase of chromosome damage in any of the cultures exposed.

Table: Metaphase chromosome analysis of RL₄cells

	0 µg/ml	2.5 µg/ml	5.0 µg/ml	10.0 µg/ml	1.0 µg/ml 7,12-dimethylbenzanthracene
Polyploidy (% cells)	3.7	2.0	2.7	1.3	1.5
Chromatid gaps (% cells)	1.3	1.7	1.0	2.3	6.0
Multiple aberrations (% cells)	-	-	-	-	0.5
Severe damage (% cells)	-	-	-	-	0.5
Chromatid aberrations (% cells)	0.3	-	-	-	4.0
Chromosome aberrations (frequency per cell)	-	-	-	-	-
Chromatid gaps (frequency per cell)	0.013	0.017	0.010	0.027	0.085
Chromatid breaks (frequency per cell)	0.003	-	-	-	0.005
Chromatid exchange (frequency per cell)	-	-	-	-	0.05
Chromosome breaks (frequency per cell)	-	-	-	-	0.005
Chromosome exchange (frequency per cell)	-	-	-	-	-

Conclusions:

Interpretation of results: negative

This study examined the potential for the test substance Heptane to cause chromosomal aberrations in rat liver RL4 cells. Cells were exposed to concentrations of 0, 2.5, 5, and 10 ug/ml of test substance, and then examined for chromosomal aberrations including polyploidy, chromatid gaps, and chromatid exchanges. The positive control substance was 7,12 -dimethylbenzanthracene. A significant increase in chromatid gaps (7) was seen at the 10 ug/ml concentration. Though greater than the solvent control frequency, this was not accompanied by an increase in any other aberrations and is not considered to be treatment related. Under the conditions of this study, the test material was not clastogenic.

Executive summary:

This study examined the potential for the test substance Normal-Heptane to cause chromosomal aberrations in rat liver RL4 cells. Cells were exposed to concentrations of 0, 2.5, 5, and 10 ug/ml of test substance, and then examined for chromosomal aberrations including polyploidy, chromatid gaps, and chromatid exchanges. The positive control substance was 7,12 -dimethylbenzanthracene. A significant increase in chromatid gaps (7) was seen at the 10 ug/ml concentration. Though greater than the solvent control frequency, this was not accompanied by an increase in any other aberrations and is not considered to be treatment related. Under the conditions of this study, the test material was not clastogenic.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic 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:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

- using microtiter plates

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium supplemented with 15% heat-inactivated horse serum

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

with Aroclor induced-rat liver homogenate

Test concentrations with justification for top dose:

5 % v/v TMP in DMEM medium, administered undiluted or as 50 % (1:1 saturated to normal medium)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMEM medium

Untreated negative controls:

yes

Remarks:

with and without S9

Negative solvent / vehicle controls:

yes

Remarks:

DMEM medium

Positive controls:

yes

Remarks:

for S9 activated cultures

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 15 µM

Untreated negative controls:

yes

Remarks:

with and without S9

Negative solvent / vehicle controls:

yes

Remarks:

DMEM medium

Positive controls:

yes

Remarks:

for non-activated cultures

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: 0.2 mM

Details on test system and experimental conditions:

see "any other information on materials and methods"

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

TMP did not induce significant increases in the mutation frequency at the thymidine kinase locus.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cell survival in TMP-saturated medium with and without metabolic activation was greater than 50-60 %.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The elevated mutation frequencies of positive control compounds were as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cell survival in positive control treated cultures was 40 or 30% for benzo(a)pyrene and EMS, respectively

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

Based on the study design there is no incidence of increased genetic toxicity caused by the test substance.

Executive summary:

Based on the study design there is no incidence of increased genetic toxicity caused by the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There is no in vivo genetic toxicity data available for Nonane. However, data is available for structural analogues, Hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, Hydrocarbons, C7-C9, isoalkanes and 2,2,4-trimethylpentane. 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.

 

The read across genetic toxicity tests listed below had negative results for Nonane.

 

Genetic Toxicity in vivo – Mammalian Erythrocyte Micronucleus Test (equivalent/similar to OECD 474)

Genetic Toxicity in vivo – Rodent Dominant Lethal Test (equivalent/similar to OECD 478)

Genetic Toxicity in vivo – Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells (equivalent/similar to OECD 486)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: 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: Adopted according to OECD SIDS (public available peer reviewed source) and respective study summary. The original source is not available and has not been reviewed.

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

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil (dried)
- 10 mL/kg

Duration of treatment / exposure:

one single application

Frequency of treatment:

single dose

Post exposure period:

24 and 48 hours

Remarks:

Doses / Concentrations:
2000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Doses / concentrations: 65 mg/kg

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

see "Any other information on materials and methods"

Statistics:

Analysis of variance and Student's t-test on transformed data [square root transformation for micronucleated polychromatic erythrocytes and double arcsine transformation for % polychromatic erythrocytes].

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

No statistically or biologically significant increases in micronucleated PCE were observed at either sampling time.

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

NOEL > 2,000 mg/kg.
No effects were observed at either sampling time. There was no significant reduction in PCEs.
No statistically or biologically significant increases in micronucleated PCE were observed at either sampling time.
Cyclophosphamide induced the expected significant increases in micronucleated PCE.
No treatment-related effects were observed in mortality, in-life physical observations, or necropsy observations in any treated males.

Conclusions:

Interpretation of results: negative
Based on the study design the test substance showed no genetic toxicity.

Executive summary:

Based on the study design the test substance showed no genetic toxicity.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study 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 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

- limited documentation

GLP compliance:

not specified

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Kingston, N.Y.)
- Weight at study initiation: 200-300 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled
- Humidity (%): controlled
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: none

Duration of treatment / exposure:

one single dose

Frequency of treatment:

sinlge treatment

Post exposure period:

2, 12, 24 and 48 hours after dosing

Remarks:

Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

3 males/ time period

Control animals:

yes

Positive control(s):

dimethylnitrosamine (DMN), dissolved in water
- Route of administration: orally
- Doses / concentrations: 10 mg/kg

Tissues and cell types examined:

hepatocytes

Details of tissue and slide preparation:

see "any other information on materials and methods"

Statistics:

One-way analysis of variance was performed on UDS and S-phase data with multiple treatment groups. S-phase data were adjusted by square root transformation. Treatment means were compared to control means by Dunnett's multiple comparison test. Level of significance was <0.05.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

TMP did not induce unscheduled DNA synthesis in hepatocyte cultures from rats treated in vivo.

Toxicity:

no effects

Remarks:

Mean cell viabilities in preparations of hepatocytes were: 84 % at 2-12 hours after administration of 500 mg/kg TMP.

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see "Remarks on results"

Mean cell viabilities in preparations of hepatocytes were: 85 % at 24 hours after administration of water (negative control) and 84 % at 2-12 hours after administration of 500 mg/kg TMP.

No increase in UDS expressed as increase in NG or number of cells in repair was observed in hepatocytes isolated from male rats at 2, 12, 24, or 48 hours post-dose. A strong response was elicited by the DMN positive control, as expected.

Autoradiographic preparations of hepatocytes isolated from rats 24 hr after treatment with TMP revealed a significant increase in the number of cells in S phase (2.1% vs 0.35% in controls); this value dropped back to 0.55% at 48 hours. TMP also significantly increased RDS in mice at 24 hours; no 48 hour values were presented. However low serum alanine transaminase activity were observed at 24 and 48 hours after a single treatment with 500 mg/kg TMP indicating that TMP induced little if any hepatocellular necrosis which could trigger regenerative cell proliferation.

Following 11 consecutive daily treatments with 100 mg/kg/day TMP, a significant increase (p<0.05) in rat hepatic concentration of DNA (mg/g liver) was seen but the total hepatic DNA content per rat was comparable to controls. Liver weight relative to body weight was also comparable to controls. The demonstrated increase in RDS suggests that TMP may stimulate additive rather than regenerative cell proliferation but not of sufficient magnitude to significantly increase the total DNA content of the liver.

TMP was administered at a single 500 mg/kg concentration to evaluate UDS and RDS in conjunction with a multidose study for unleaded gasoline. When RDS was observed, a liver DNA content determination was performed in rats with treatment over 11 days to determine the type of proliferative response.

Conclusions:

Interpretation of results: negative
Based on this study design 2, 2, 4- trimethyl pentane does not induce unscheduled DNA synthesis in vivo by unchanged application via gavage.

Executive summary:

Based on this study design 2, 2, 4- trimethyl pentane does not induce unscheduled DNA synthesis in vivo by unchanged application via gavage.

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

AMES data is available for Nonane. Additionally, in vitro data is available for structural analogues Heptane and 2,2,4-trimethylpentane, and in vivo data is available for structural analogues, Hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, Hydrocarbons, C7-C9, isoalkanes and 2,2,4-trimethylpentane. 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.

 

In Vitro

 

In vitro gene mutation study in bacteria

 

Heptane

A bacterial reverse mutation assay (Ames test) (Shell Chemicals, 1983; Brooks et al., 1988) was conducted with normal-heptane following a protocol similar to OECD 471. The pre-incubation procedure was performed with Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and Escherichia coli strains WP2 and WP2 uvr A. The strains were exposed to concentrations of 0, 3.91, 7.81, 15.6, 31.3, 62.5, 125, and 250 µg/mL for 48 -72 h both with and without metabolic activation. No significant increases in the ratio of mutations over controls was seen. Therefore, the test substance was not mutagenic in either the presence or absence of metabolic activation.

 

Nonane

A bacterial reverse mutation assay (Ames test) (Zeiger et al., 1992) was conducted with nonane following a protocol similar to OECD 471. The pre-incubation procedure was performed with Salmonella typhimurium strains TA 1535, TA 1537, TA 97, TA 98 and TA 100. The strains were exposed to concentrations of 0, 100, 333, 1000, 3333, 10000 µg/plate both with and without metabolic activation for 20 min prior to addition of top agar and plating. The number of revertant colonies was counted 2 days later. Under the conditions of this assay, there was no evidence of mutagenic activity both in the presence and absence of metabolic activation.

 

In vitro Chromosome Aberration in Mammalian Cells

 

Heptane

The potential of normal-heptane to cause chromosomal aberrations in rat liver RL4 cells was tested with a method comparable to OECD 473 (Shell Chemicals, 1983; Brooks et al., 1988). Cells were exposed to concentrations of 0, 2.5, 5, and 10 µg/mL of test substance for 22 h, and then examined for chromosomal aberrations including polyploidy, chromatid gaps, and chromatid exchanges. No cytotoxicity was observed. A significant increase in chromatid gaps was seen at 10 µg/mL. However, this effect was neither dose-dependent nor accompanied by an increase in any other aberration type, and therefore not considered to be treatment-related. Under the conditions of this study, the test material was not clastogenic.

 

In vitro Gene Mutation study in Mammalian Cells

 

2,2,4-trimethylpentane

In a key study (Richardson et al., 1986), 2,2,4-trimethylpentane was tested in a mammalian cell gene mutation assay performed according to OECD 476. The test material was prepared by adding iso-octane at a final concentration of 5 % v/v in culture (DMEM) medium and stirred overnight at room temperature in a foil wrapped, capped parafilm-sealed bottle to saturate the medium. Human lymphoblastoid cells (TK6) were exposed to 100 or 50 % of this saturated DMEM medium with and without metabolic activation for 3 h and allowed for expression for 4 to 8 days. Both with and without metabolic activation, 2,2,4-trimethylpentane did not induce significant increases in the mutation frequency at the thymidine kinase locus and cell survival in 2,2,4-trimethylpentane-saturated medium was greater than 50-60 %. Based on the study design there was no incidence of increased genetic toxicity caused by the test substance. 

In Vivo

Hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics

A mouse micronucleus assay (comparable to OECD 474) was performed using the substance Hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics administered orally in a single dose of 2000 mg/kg with sampling of bone marrow at 24 and 48 h post-dose. No statistically significant increases in micronucleated polychromatic erythrocytes were observed. The test substance was not clastogenic to mouse bone marrow cells under the conditions of this assay (Fox, 2003).

 

Hydrocarbons, C7-C9, isoalkanes

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

 

2,2,4-trimethylpentane

As part of an unscheduled DNA synthesis (UDS) study performed in vitro, 2,2,4-trimethylpentane was administered to rats and mice orally at a single dose of 500 mg/kg. Primary hepatocytes were isolated from livers at sacrifice times 2, 12, 28 or 48 hours post-treatment and cultures were evaluated for unscheduled and replicative DNA synthesis. 2,2,4-trimthylpentane did not induce unscheduled DNA synthesis in hepatocyte cultures, and increases in replicative DNA synthesis were not of sufficient magnitude to increase the total DNA content of the liver (Loury et al., 1986).

Justification for classification or non-classification

The negative results in in vitro and in vivo genotoxicity assays from Nonane and structural analogues do not warrant the classification of Nonane as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).