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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All read across genetic toxicity tests listed below had negative results for Isoheptane.

 

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

 

Genetic Toxicity in vitro – Mammalian chromosome aberration test (OECD 473)

 

Genetic Toxicity in vitro – Mammalian cell gene mutation assay (OECD 476)

 

Genetic Toxicity in vitro – Sister chromatid exchange assay in mammalian cells (OECD 479)

 

Genetic Toxicity in vitro – Unscheduled DNA synthesis in mammalian cells (OECD 482)

Link to relevant study records

Referenceopen allclose all

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 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:
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:
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.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study meets generally accepted scientific principles, acceptable for assessment.
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)
Deviations:
yes
Remarks:
- no A-T base pair tester strains included (e.g. TA102 or E.coli); limited documentation
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
- no A-T base pair tester strains included (e.g. TA102 or E.coli); limited documentation
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his-operon
Species / strain / cell type:
S. typhimurium, other: TA 98, TA100, TA1535, TA1537, TA1538
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomes from male Sprague Dawley rats treated with a single intraperitoneal injection of Aroclor 1254 5-days before sacrifice.
Test concentrations with justification for top dose:
20, 60, 560, 1670, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: N-methl-N'-nitro-N-nitrosoguanidine
Remarks:
positive control for TA100 and TA1535 (5 µg/plate)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
positive control for TA98 and TA1538 (50 µg/plate) Migrated to IUCLID6: in absence of S9-mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
positive control for TA1537 (75 µg/plate)
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle 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:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle 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:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle 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:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: TA 98, TA100, TA1535, TA1537, TA1538
Remarks:
Migrated from field 'Test system'.

TA98: Revertants Test

Test substance Test 1 (without S9)  Test 2 (without S9)  Test 1 (with S9) Test 2 (with S9) 
solvent control  22 -26 22 -26  22 -26  18 -20 
2 -Nitrofluorene  1508 -1637 2131 -2282 
2 -Aminoanthracene   -  - 2213 -2247  1913 -2020 
7 µg/plate isooctane - 15 -27  24 -46 
20 µg/plate isooctane 140 -191  19 -23 145 -206   29 -31
60 µg/plate isooctane 112 -124 19 -26  64 -117   24 -33
190 µg/plate isooctane 37 -55 19 -24  17 -28   12 -25

Retest (test 2) of TA98 without and with S9 at dose levels for 7, 20 and 60 µg/plate evidenced no difference from solvent controls.

Observed 5 -10 fold increase of TA98 revertants without and with S9 at 20 and 60 µg/plate in initial test was not confirmed in retest of TA98 without and with S9 at dose levels 7, 20 and 60 µg/plate.

In all experiments, no increase in revertants for other frame shift strains (TA1537 and TA 1538) was observed. Therefore the in test 1 observed genotoxic effects of isooctane in strain TA 98 were rather false positive.

Conclusions:
Interpretation of results: negative

Under the test conditions, the experimental compound, Isooctane, did not exhibit a positive response and is, therefore, considered not be mutagenic in this system.
Executive summary:

Under the test conditions, the experimental compound, Isooctane, did not exhibit a positive response and is, therefore, considered not be mutagenic in this system.

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.

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

Read across genetic toxicity tests listed below has negative results for Isoheptane.

 

Genetic Toxicity in vivo – rodent dominant lethal assay (OECD 478)

 

Genetic Toxicity in vivo – unscheduled DNA synthesis (OECD 486)

Link to relevant study records

Referenceopen allclose all

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.

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 in vitro or in vivo genetic toxicity data available for Isoheptane. However, data is available for structural analogues, Heptane; Hydrocarbons, C7-C9, isoalkanes; and 2,2,4-trimethylpentane and presented in the dossier. This data is read across to Isoheptane 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 in Bacteria

 

Heptane

 

The in vitro genotoxicity of n-heptane (CAS No. 142-82-5) has been assessed in different test systems. A bacterial reverse mutation assay (Ames test) was conducted with n-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 (Shell Chemicals, 1983; Brooks et al., 1988).

 

2,2,4-trimethylpentane

 

A bacterial reverse mutation assay (Ames test) was conducted with iso-octane following a protocol similar to OECD 471. Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were exposed to the test substance at 20, 60, 560, 1670, and 5000 µg/plate in the absence and in the presence of a metabolic activator. Cytotoxic effects were observed at 5000 µg/plate. Iso-octane did not induce mutations in the bacterial mutation test in either the absence or presence of metabolic activator in any strain tested. Under the test conditions, the experimental compound, iso-octane, did not exhibit a positive response and is, therefore, considered not be mutagenic in this system (Chevron Phillips, 1982).

 

In Vitro Chromosome Aberration in Mammalian Cells

 

Heptane

 

The potential of n-heptane to cause chromosomal aberrations in rat liver RL4 cells was tested with a method comparable to OECD 473. 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 (Shell Chemicals, 1983; Brooks et al., 1988).

 

In Vitro Gene Mutation in Mammalian Cells

 

2,2,4-trimethylpentane

 

Isooctane (CAS No. 540-84-1) 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, iso-octane did not induce significant increases in the mutation frequency at the thymidine kinase locus and cell survival in iso-octane-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 (Richardson et al., 1986).

 

Isooctane was also tested in a Mouse Lymphoma Forward Mutation Assay performed according to a protocol similar to OECD TG 476. At concentrations ranging from 20 to 500 µg/mL (limit of solubility), iso-octane did not induce gene mutations in the mouse lymphoma L5178Y heterozygous TK+/-cells in the presence of metabolic activation, resulting in cytotoxicity at 165 µg/mL and above. Without metabolic activation, cytotoxicity at 500 µg/mL and an equivocal to weakly mutagenic response were observed (Chevron Phillips, 1982).

 

In Vitro Sister Chromatid Exchange in Mammalian Cells

 

2,2,4-trimethylpentane

 

Human lymphoblastoid cells exposed to iso-octane for 3 h as described above were also tested for the induction of sister chromatid exchange (SCE) according to OECD TG 479. Following exposure, the cells were incubated in the dark in the presence of 5'Bromo-deoxyuridine (10 µM) for 36 h, and metaphases were analysed microscopically thereafter. Both with and without metabolic activation, iso-octane did not induce an increased incidence of SCEs in TK6 cells (Richardson et al., 1986; Wilmer et al., 1981).

 

No statistically significant increase in number of SCEs (with and without metabolic activation) was also observed in another study compliant with OECD TG 479. Chinese hamster Ovary (CHO) cells were exposed to 25, 84, 250 µg/mL iso-octane in DMSO and 1400 and 3500 µg/mL iso-octane without vehicle. No cell growth occurred at the 250 µg/mL, without activation. Based on the study design there was no increased mutagenic response caused by isooctane in CHO cells with and without metabolic activation (Chevron Phillips, 1982).

 

In Vitro Unscheduled DNA Synthesis in Mammalian Cells

 

2,2,4-trimethylpentane

 

The potential of isooctane to induce unscheduled DNA synthesis (UDS) as an indicator of genotoxicity was tested in rat hepatocytes in a study comparable to OECD TG 482. Primary cultures were prepared from hepatocytes isolated from a rat. After attachment to coverslips and acclimatisation, cells were treated with [3H] thymidine (10 µCi/mL) and undiluted iso-octane at 0.33, 1.00, 3.33 % (v/v). The cells were then incubated for 18 hours. The highest concentration of 3.33% v/v was lethal to cell cultures. At the lower concentrations, isooctane did not induce unscheduled DNA synthesis in vitro by direct application to primary hepatocyte cultures. Based on the study design there was no incidence of increased genetic toxicity caused by the test substance (Loury et al. 1986).

 

In Vivo

 

Hydrocarbons, C7-C9, isoalkanes

 

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

 

2,2,4-trimethylpentane

 

As part of an unscheduled DNA synthesis (UDS) study performedin vitro, iso-octane 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. Iso-octane 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 read across in vitro and in vivo genotoxicity assays do not warrant the classification of Isoheptane as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).