Octane

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 Octane. However, 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 read across genetic toxicity tests listed below had negative results for Octane.

 

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 (migrated information):
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 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 (migrated information):
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 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 Octane. However, data is available for structural analogues, Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics, Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics and Hydrocarbons, C10-C13, n-alkanes, <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.

 

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

 

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)

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:

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:

The justification for read across is provided 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 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:

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:

The justification for read across is provided 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, 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

Negative controls validity:

not examined

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 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

Study period:

1978

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

Route of administration:

inhalation: vapour

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

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

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

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

Duration of treatment / exposure:

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

Frequency of treatment:

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

Post exposure period:

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

Remarks:

Doses / Concentrations:
900 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
300 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

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

Control animals:

yes

Positive control(s):

triethylenemelamine

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

Tissues and cell types examined:

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

Statistics:

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

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

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

Executive summary:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There is no genetic toxicity data available for Octane. However, in vitro data is available for structural analogues Heptane and 2,2,4-trimethylpentane, and in vivo data is available for structural analogues Hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics, Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics and Hydrocarbons, C10-C13, n-alkanes, <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.

 

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.

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

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

  

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

The test material (Hydrocarbons, C10-C13, n-alkanes, < 2% aromatics) was tested in the mammalian bone marrow micronucleus assay using CD-1 mice (ExxonMobil, 1991). 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 cytogenicity/ erythrocyte micronucleus 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.

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 Octane as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).