Hydrocarbons, C6-C7, n-alkanes, isoalkanes, cyclics, <5% n-hexane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is no in vitro genetic toxicity data available for Hydrocarbons, C6 -C7, n-alkanes, isoalkanes, cyclics, <5% n-hexane. However, data is available for structural analogues Hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics and 2,2,4 -trimethylpentane. This data is read across to Hydrocarbons, C6 -C7, n-alkanes, isoalkanes, cyclics, <5% n-hexane 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 Hydrocarbons, C6 -C7, n-alkanes, isoalkanes, cyclics, <5% n-hexane.

 

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:

September - October 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure according to national standards

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:

reference to same study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

- limited documentation of cytotoxicity data

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium strains: his-operon
E. coli strains: tryptophan-operon

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli, other: WP2, WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate S9 activation, 10 % S9 liver homogenate from Aroclor treated rats

Test concentrations with justification for top dose:

TA 1538 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 1538 (-S) mix): 0, 15.62, 31.25, 62.5, 125, 250, 500, 1000; (+S9 mix): 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 1537 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 1537 (-S9 mix): 0, 3.9, 7.81, 15.62, 31.25, 62.5, 125; (+S9 mix): 0, 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL
TA 1535 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 100 (-S9 mix): 0, 0.31, 0.62, 1.25, 2.5, 5, 10; (+S9 mix): 0, 7.81, 15.62, 31.25, 62.5, 125, 250 µg/mL
TA 100 (-S9 mix): 0, 31.25, 62.5, 125, 250, 500; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 100 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 98 (-S9 mix): 0, 0.31, 0.62, 1.25, 2.5, 5, 10; (+S9 mix): 0, 7.81, 15.62, 31.25, 62.5, 125, 250 µg/mL
TA 98 (-S9 mix): 0, 3.9, 7.81, 15.62, 31.25, 62.5, 125; (+S9 mix): 0, 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL
TA 98 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
EC WP2 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
EC WP2uvrA (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: TA 1538, TA 98, TA 100: benzo(a)pyrene; TA 1537: neutral red; TA 1535: Sodium azide; and E. coli strains: 4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 min
- Incubation period: 48-72 hours

NUMBER OF REPLICATIONS: All test were carried out in triplicate.

Two replicate assays were carried out on different days to confirm reproducibility of results.

Statistics:

none

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.

Cytotoxicity / choice of top concentrations:

other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix

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

Remarks:

Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.

Cytotoxicity / choice of top concentrations:

other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.

Cytotoxicity / choice of top concentrations:

other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix

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

Remarks:

Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.

Cytotoxicity / choice of top concentrations:

other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix

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

Remarks:

Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.

Cytotoxicity / choice of top concentrations:

other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix

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

Remarks:

Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.

Cytotoxicity / choice of top concentrations:

other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli, other: WP2, WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of E. coli strains either in the presence or absence of a rat liver microsomal activation system

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no evidence of test compound precipitation in the assay system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table: Relative Mutation Rate – Bacterial Experiment I without Metabolic Activation

Concentration	E. coli WP2	E. coli WP2 uvr A	S. typhimurium TA 1535	S. typhimurium TA 1537	S. typhimurium TA 1538	S. typhimurium TA 98	S. typhimurium TA 100
Without S9
3.91	-	-	-	-	-	-	-
7.81	-	-	-	-	-	-	-
15.6	-	-	-	-	-	-	-
31.3	-	-	-	-	-	-	-
62.5	0.8	0.7	1.2	0.1	0.7	0.2	1.4
125	0.9	0.6	0.7	0	0.4	0	0.7
250	0.7	0.8	0.6	0	0	0	0.3
500	0.7	0.7	0.9	0	0	0	0.1
1000	0.8	0.5	0.9	0	0	0	0
2000	0.7	0.6	0.7	0	0	0	0
4000	0.8	0.6	0.6	0	0	0	0
8000	0.6	0.5	0.7	0	0	0	0
Sodium azide 2 µg	-	-	89.8	-	-	-	-
Benzo(a)-pyrene 7 µg	-	-	-	-	1.0	0.7	0.9
4-nitroquinoline-N-oxide 3 µg	39.8	3.2	-	-	-	-	-
Neutral red 7 µg	-	-	-	1.8	-	-	-

Table: Relative Mutation Rate – Bacterial Experiment I with Metabolic Activation

Concentration	E. coli WP2	E. coli WP2 uvr A	S. typhimurium TA 1535	S. typhimurium TA 1537	S. typhimurium TA 1538	S. typhimurium TA 98	S. typhimurium TA 100
Without S9
31.3	-	-	-	-	-	-	-
62.5	0.7	1.3	1.4	0.3	1.5	0.7	1.0
125	0.8	1.0	1.1	0.2	1.3	1.3	0.8
250	0.9	1.0	0.8	0.6	1.5	1.2	1.0
500	0.7	0.8	1.0	0.2	1.0	0.7	1.3
1000	0.7	0.7	1.0	0.4	0.7	0.2	1.4
2000	1.1	0.9	1.1	0	0.4	0	1.2
4000	1.0	1.0	1.0	0	0.7	0	1.1
8000	0.8	1.0	0.5	0	0.7	0	1.1
Sodium azide 2 µg	-	-	15.2	-	-	-	-
Benzo(a)-pyrene 7 µg	-	-	-	-	3.7	5.3	3.1
4-nitroquinoline-N-oxide 3 µg	1.2	3.6	-	-	-	-	-
Neutral red 7 µg	-	-	-	4.4	-	-	-

Table: Relative Mutation Rate – Bacterial Experiment II without Metabolic Activation

Concentration	E. coli WP2	E. coli WP2 uvr A	S. typhimurium TA 1535	S. typhimurium TA 1537	S. typhimurium TA 1538	S. typhimurium TA 98	S. typhimurium TA 100
Without S9
3.91	-	-	-	1.1	-	0.8	-
7.81	-	-	-	0.6	-	0.9	-
15.6	-	-	-	0.7	0.9	0.8	-
31.3	-	-	-	0.4	1.0	0.8	1.1
62.5	1.0	1.0	1.2	0.4	0.7	0.4	1.0
125	1.0	1.0	0.9	0	0.5	0	0.9
250	0.7	0.8	1.4	-	0.2	-	1.0
500	0.9	0.7	1.1	-	0	-	0.7
1000	1.0	0.8	0.9	-	0	-	-
2000	0.7	0.9	0.7	-	-	-	-
4000	0.9	0.8	0.9	-	-	-	-
8000	0.8	0.7	0.9	-	-	-	-
Sodium azide 2 µg	-	-	86.6	-	-	-	-
Benzo(a)-pyrene 7 µg	-	-	-	-	1.1	1.1	1.3
4-nitroquinoline-N-oxide 3 µg	36.7	5.3	-	-	-	-	-
Neutral red 7 µg	-	-	-	1.2	-	-	-

Table: Relative Mutation Rate – Bacterial Experiment II with Metabolic Activation

Concentration	E. coli WP2	E. coli WP2 uvr A	S. typhimurium TA 1535	S. typhimurium TA 1537	S. typhimurium TA 1538	S. typhimurium TA 98	S. typhimurium TA 100
Without S9
31.3	-	-	-	1.1	0.9	0.8	-
62.5	0.9	0.9	0.7	1.2	1.1	1.0	1.1
125	1.1	1.0	1.2	1.1	1.1	0.9	1.0
250	1.4	0.8	0.8	1.4	1.1	0.8	1.1
500	1.2	1.0	0.9	1.0	1.1	0.8	1.2
1000	1.0	1.1	0.8	0.5	0.9	0.4	0.9
2000	0.8	1.0	1.1	0	0.9	0.1	0.9
4000	0.8	0.9	0.8	-	0.5	-	0.9
8000	1.0	0.7	0.7	-	0.4	-	0.9
Sodium azide 2 µg	-	-	18.2	-	-	-	-
Benzo(a)-pyrene 7 µg	-	-	-	-	9.7	6.1	6.5
4-nitroquinoline-N-oxide 3 µg	1.1	2.8	-	-	-	-	-
Neutral red 7 µg	-	-	-	5.2	-	-	-

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The purpose of this study was to determine the mutagenicity of the test substance hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics. An Ames reverse mutation assay was done on S. typhimurium strains, TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli strains WP2 uvr A and WP2, both with and without metabolic activation. Species were tested at concentrations ranging from 0.31 to 8000 ug/plate. The test plates were incubated for 48 -72 hrs, and then the number of colonies were counted. For all species, there was no significant increase in the number of revertants as compared to negative controls. Positive controls showed a significant increase in mutations. The test substance is not mutagenic.

Executive summary:

The purpose of this study was to determine the mutagenicity of the test substance hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics. An Ames reverse mutation assay was done on S. typhimurium strains, TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli strains WP2 uvr A and WP2, both with and without metabolic activation. Species were tested at concentrations ranging from 0.31 to 8000 ug/plate. The test plates were incubated for 48 -72 hrs, and then the number of colonies were counted. For all species, there was no significant increase in the number of revertants as compared to negative controls. Positive controls showed a significant increase in mutations. The test substance is not mutagenic.

Reference 2

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:

September - October 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure according to national standards.

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:

reference to same study

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)

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Rat liver epithelial-type line (RL4) cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock culture media and assay media was Minimal Essential Medium supplemented with 10% fetal calf serum and 1% non-essential amino acids.

Additional strain / cell type characteristics:

other: metabolic competent

Metabolic activation:

without

Metabolic activation system:

RL4 cells are metabolically competent.

Test concentrations with justification for top dose:

0, 2.5, 5.0, and 10 mg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

see "Any other information on materials and methods"

Key result

Species / strain:

mammalian cell line, other: RL4 cells, metabolic competent

Metabolic activation:

not applicable

Genotoxicity:

negative

Remarks:

There was no significant increase in chromosome damage

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exposure of RL4 cells to 20 µg/mL test substance resulted in inhibition of cell proliferation by 50 %. Therefore final test concentrations of 2.5, 5, and 10 µg/mL were chosen

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A maximum dose level of 10 µg/mL was determined in the chromosome assay. There was no significant increase beyond control limits in the frequency of chromatid breaks or total chromatid aberrations in rat liver cells exposed to the test material up to 10 µg/mL indicating that the compound did not induce chromosome damage in rat liver cells. A small increase in the percentage of cells showing gaps in exposed cultures was well within historical control ranges. Cultures exposed to the positive control material, DMBA, showed an increase in the frequency of chromosome damage.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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)	1.3	1.3	1.3	1.3	0.5
Chromatid gaps (% cells)	0.3	0.7	0.9	1.0	5.5
Multiple aberrations (% cells)	-	-	-	-	-
Severe damage (% cells)	-	-	-	-	-
Chromatid aberrations (% cells)	0.7	-	0.9	-	4.0
Chromosome aberrations (% cells)	-	-	-	-	1.5
Chromatid gaps (frequency per cell)	0.003	0.007	0.009	0.010	0.060
Chromatid breaks (frequency per cell)	0.007	-	0.004	-	-
Chromatid exchange (frequency per cell)	-	-	0.004	-	-
Chromosome breaks (frequency per cell)	-	-	-	-	0.015
Chromosome exchange (frequency per cell)	-	-	-	-	-

Conclusions:

Interpretation of results (migrated information):
negative

This study examined the potential for the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, to cause chromosomal aberrations in rat liver RL4 cells. Cells were exposed to concentrations of 0, 2.5, 5.0, 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. No significant increase in the frequency of chromosomal damage was observed. Based on the study design the test substance showed no genetic toxicity.

Executive summary:

This study examined the potential for the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, to cause chromosomal aberrations in rat liver RL4 cells. Cells were exposed to concentrations of 0, 2.5, 5.0, 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. No significant increase in the frequency of chromosomal damage was observed. Based on the study design the test substance showed no genetic toxicity.

Reference 3

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 (migrated information):
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 Hydrocarbons, C6 -C7, n-alkanes, isoalkanes, cyclics, <5% n-hexane. However, data is available for structural analogue Hydrocarbons, C7 -C9, isoalkanes. This data is read across to Hydrocarbons, C6 -C7, n-alkanes, isoalkanes, cyclics, <5% n-hexane 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 Hydrocarbons, C6 -C7, n-alkanes, isoalkanes, cyclics, <5% n-hexane.

 

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

Link to relevant study records

Reference

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

There is no genetic toxicity data available for Hydrocarbons, C6 -C7, n-alkanes, isoalkanes, cyclics, <5% n-hexane. However, data is available for structural analogues Hydrocarbons, C7-C9, isoalkanes, 2,2,4 -trimethylpentane and Hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics. This data is read across to Hydrocarbons, C6 -C7, n-alkanes, isoalkanes, cyclics, <5% n-hexane based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

 

In Vitro

 

In vitro gene mutation study in bacteria

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

In a key study (Shell, 1993; Brooks, 1988) the mutagenicity of the test substance hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics was determined. An Ames reverse mutation assay was done on S. typhimurium strains, TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli strains WP2 uvr A and WP2, both with and without metabolic activation. Species were tested at concentrations ranging from 0.31 to 8000 ug/plate. The test plates were incubated for 48 -72 hrs, and then the number of colonies were counted. For all species, there was no significant increase in the number of revertants as compared to negative controls. Positive controls showed a significant increase in mutations. The test substance is not mutagenic.

In vitro Chromosome Aberration in Mammalian Cells

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

A key study (Shell, 1983; Brooks, 1988) examined the potential for the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, to cause chromosomal aberrations in rat liver RL4 cells. Cells were exposed to concentrations of 0, 2.5, 5.0, 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. No significant increase in the frequency of chromosomal damage was observed. Based on the study design the test substance showed no genetic toxicity.

 

In vitro Gene Mutation study in Mammalian Cells

 

2,2,4 -trimethylpentane

In a key study (Richardson et al, 1986), the mutagenicity of test substance 2,2,4 -trimethylpentane to human lymphoblastoid cells (TK6). Two 75 ml aliquots of TK6 cells (4x10E5 cells/mL) were exposed to 100 or 50 % TMP saturated DMEM medium. Metabolic activation was supplied by adding S9 mixture (5% v/v S9 homogenate). Exposure was 3 hrs at 37 °C in a 500 mL tightly sealed glass bottle. Following the 3 hr exposure period, cells were washed, resuspended in 75 mL RPMI 1640 supplemented with 15 % heat-inactivated horse serum and 10 U/mL penicillin. Cells were maintained in 75 mL cultures at 37 °C and 6 % CO2 in air with daily dilutions to 4x10E5 cells/mL for 4 to 8 days. Cells were plated twice on 2 consecutive days to determine mutant fraction and cloned in 96 well microtiter dishes. Mutation dishes contained 4x10E4 cells/well and 1 µg/mL trifluorothymidine (TFT). Dishes to measure colony-forming ability (2/culture) contained 2 cells/well. Dishes were incubated at 37 °C and 6 %CO2 in air for 10 to 14 days. Colony formation efficiency was calculated by dividing the negative log of the fraction of negative wells by the average number of cells/well. Mutant frequency was calculated by dividing the colony forming efficiency of TFT containing plates by the colony formation efficiency of plates without selection. The positive control compounds were administered to 25 mL of cells at 4x10E5 cells/mL. All experiments were repeated at least twice on different days. Results of the mutagenicity assy showed that the test substance was not mutagenic both in the presence and the absence of metabolic activation.

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

 

In Vivo

In vivo Rodent Dominant Lethal Test

Hydrocarbons, C7 -C9, isoalkanes

In a rat dominant lethal assay (Exxon, 1980), 10 rat/group were treated via inhalation at doses of 400 or 1200 pm Hydrocarbons, C7 -C9, isoalkanes for 6 hours per day, 5 days per week, for 8 weeks. Each male was then caged with 2 virgin females for one week, these females were removed and 2 new virgin females were caged with each male for an additional week (pre-treatment mating). The same procedure was done for 2 consecutive weeks following exposure of the male animals (post-treatment mating).

There were no treatment-related effects observed for mortality, in-life physical or necropsy examinations and for bodyweights in males. There were no treatment-related effect 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.

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

Justification for classification or non-classification

The negative results of in vitro and in vivo genotoxicity assays from structural analogues do not warrant the classification of Hydrocarbons, C6-C7, n-alkanes, isoalkanes, cyclics, <5% n-hexane as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).