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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All substance specific and read across genetic toxicity tests listed below had negative results for 2-methylbutane.

 

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

 

Genetic Toxicity in vitro – Mammalian chromosome aberration test (EU Method B.10)

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

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:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because no information regarding GLPs was provided; however, the study closely followed OECD 471 and EEC B.13/14.
Justification for type of information:
This study received a Klimisch score of 2 and is classified as reliable with restrictions because no information regarding GLPs was provided; however, the study closely followed OECD 471 and EEC B.13/14.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Ames test was modified for the gaseous chemical (did not affect results); strain TA1538 is not included in current Guidelines; only data from highest dose presented
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
not reported
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine auxotrophs
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: histidine auxotrophs
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-stimulated S9 mix
Test concentrations with justification for top dose:
10% v/v
8% v/v
5% v/v
2% v/v
1% v/v
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylene chloride
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not reported
- Exposure duration: 6 hours
- Selection time (if incubation with a selection agent): 40 to 45 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: not reported

OTHER: The number of his+ revertants on each plate was counted and recorded.
Evaluation criteria:
The number of his+ revertants on each plate was counted and compared to the controls to determine mutagenicity.
Statistics:
not reported
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: cytotoxic at concentrations of 10% and above
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: cytotoxic at concentrations of 10% and above
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: cytotoxic at concentrations of 10% and above
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: cytotoxic at concentrations of 10% and above
Vehicle controls validity:
not applicable
Untreated negative 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:
other: cytotoxic at concentrations of 10% and above
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: not reported

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Because 2-methylbutane was somewhat toxic at concentrations of 10% and above, additional tests were conducted at lower concentrations to determine mutagenicity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The results of the Ames test with the highest non-toxic dose are presented below. The positive control was mutagenic in strains TA98 and TA100, and was slightly mutagenic in strain TA1535. 2 -methylbutane was somewhat toxic at concentrations of 10% and above, so additional tests were conducted to determine whether the compound was mutagenic at lower concentrations (1, 2, 5, and 8%). Results show that 2 -methylbutane is not mutagenic at the lower concentrations.

Compound

Metabolic activation

Concentration of Gas in the Desiccator (V/V) %

Average histidine revertants per plate

 

 

 

TA1535

TA1537

TA1538

TA98

TA100

Negative control

-

 

15

12

10

29

138

 

+

 

16

18

30

38

155

Positive control (methylene chloride)

-

2

34

10

16

234

900

 

+

2

52

12

52

237

1066

2 -methylbutane

-

10

22

3

14

15

124

 

+

10

10

6

16

22

124

Conclusions:
Interpretation of results: negative

2-methylbutane was not found to be mutagenic at any concentration after evaluation via an Ames test.
Executive summary:

The genetic toxicity in bacteria of 2 -methylbutane was evaluated via an Ames test. 2 -methylbutane was not found to be mutagenic at any concentration in both the presence and absence of S9 mix. This study is classified as reliable with restrictions, Klimisch score 2, because information regarding GLPs was not provided. However, the study closely followed OECD 471 and EEC B.13/14. The standard plate test was modified due to the gaseous nature of the chemicals; these modifications did not affect the overall study results. One of the strains used (TA1538) is not included in the current Guidelines, and only data from the highest dose was presented in the study report. 

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: This study is classified as reliable with restrictions because no information regarding GLPs was provided; however, the study closely followed OECD 471 and EEC B.13/14.
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:
Ames test was modified for the gaseous chemical (did not affect results); strain TA1538 is not included in current Guidelines; only data from highest dose presented
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
not reported
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine auxotrophs
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: histidine auxotrophs
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-stimulated S9 mix
Test concentrations with justification for top dose:
50% , 25%, 10%, 8%, 5%, 2%, 1% v/v
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylene chloride
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no data
- Exposure duration: 6 hours
- Selection time (if incubation with a selection agent): 40 to 45 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: no data

OTHER: The number of his+ revertants on each plate was counted and recorded.
Evaluation criteria:
The number of his+ revertants on each plate was counted and compared to the controls to determine mutagenicity.
Statistics:
not reported
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: cytotoxic at concentrations of 25 and 50%
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: cytotoxic at concentrations of 25 and 50%
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: cytotoxic at concentrations of 25 and 50%
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: cytotoxic at concentrations of 25 and 50%
Vehicle controls validity:
not applicable
Untreated negative 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:
other: cytotoxic at concentrations of 25 and 50%
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
no data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Because n-pentane was toxic at concentrations of 25 and 50%, additional tests were conducted at lower concentrations to determine mutagenicity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The results of the Ames test with the highest non-toxic dose are presented below. The positive control was mutagenic in strains TA98 and TA100, and was slightly mutagenic in strain TA1535. n-Pentane was toxic at concentrations of 25 and 50%, so additional tests were conducted to determine whether the compound was mutagenic at lower concentrations (1, 2, 5, 8 and 10%). Results show that n-pentane is not mutagenic at the lower concentrations.

Compound

Metabolic activation

Concentration of Gas in the Desiccator (V/V) %

Average histidine revertants per plate

 

 

 

TA1535

TA1537

TA1538

TA98

TA100

Negative control

-

 

15

12

10

29

138

 

+

 

16

18

30

38

155

Positive control (methylene chloride)

-

2

34

10

16

234

900

 

+

2

52

12

52

237

1066

n-Pentane

-

10

25

1

8

26

138

 

+

10

14

6

22

18

116

Conclusions:
Interpretation of results: negative

n-Pentane was not found to be mutagenic at any concentration after evaluation via an Ames test.
Executive summary:

The genetic toxicity in bacteria of n-pentane was evaluated via an Ames test. The statistical methods used were appropriate. The test conditions complied with the guideline requirements for this study type. 

 

n-Pentane was not found to be mutagenic at any concentration in both the presence and absence of S9 mix

 

This study is classified as reliable with restrictions, Klimisch score 2, because no information regarding GLPs was provided. However, the study closely followed OECD 471 and EEC B.13/14. The standard plate test was modified due to the gaseous nature of the chemicals; these modifications did not affect the overall study results. One of the strains used (TA1538) is not included in the current Guidelines, and only data from the highest dose was presented in the study report. 

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:
1996-07-29 to 1997-03-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it is in compliance with OECD principles of GLP and E.U. Council Decision on GLP and adhered to the E.U. method B.10.
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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium containing 10% fetal bovine serum, 25 mM HEPES, and 2 mM L-glutamine (referred to as complete medium); McCoy's 5A medium containing 25 mM HEPES, 2 mM L-glutamine, and +S9 mix was used for metabolically activated assays (referred to as serum-free medium
- Properly maintained: yes (kept at 37±2 degrees C in 4-6% carbon dioxide in air)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Metabolic activation was prepared immediately prior to use from Aroclor-pretreated Sprague-Dawley rat ivers. S9 mix consisted of 0.015 mL rat S9 per mL serum-free medium and 0.060 mL cofactor mix (13.4 mg/mL NADP and 25 mg/mL DL-isocitric acid in water).
Test concentrations with justification for top dose:
Initial and confirmatory assay: 600, 1000, 1100, 1200, 1300, 1400, and 1500 μg/mL, +S9 and 300, 600, 900, 950, 1000, 1050, and 1100 μg/mL, -S9
Acetone (vehicle used for positive controls): 5 μL/mL
DMBA (positive control): 10 μg/mL
MNNG (positive control): 0.6 μg/mL

Vehicle / solvent:
Not applicable
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone was used as the vehicle for the positive controls
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-Methyl-N'-nitro-N-nitrosoguanidine (positive control -S9) and 7, 12-Dimethylbenz-a-anthracene (positive control, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Cultures were seeded at 6.0x10E5 cells (20 hour harvest) and 3.0x10E5 cells (44 hour harvest) the day prior to dosing. For both the initial and confirmatory assays, appropriate amounts of test material, acetone, MMNG (-S9), or DMBA (+S9) were injected into a flask containing complete or serum-free medium; cultures with metabolic activation were first washed once with warm phosphate buffered saline without Ca2+ or Mg2+. Cultures were incubated for 3±0.5 hours prior to study treatment; washed twice with warm phosphate buffered saline containing Ca2+ or Mg2+; refed with complete or serum-free medium (10 mL); and incubated for 20 hours (initial and confirmatory assay) or 44 hours (confirmatory assay). Cultures were then incubated with Colcemid (10 μg/mL) during the last 2-3 hours prior to harvest. Nontreated flasks contained 71 mL complete medium only. All other procedures were conducted in a similar manner to treatment flasks.

Prior to harvest, cells were evaluated for the percent confluency, morphology, and number of mitotic cells. Cells were harvested using 0.25% trysin-EDTA. Removed medium was returned to the flask to neutralize the trysin, and the resulting cell suspension was mixed, transferred into tubes and centrifuged. Cell suspensions were treated with 0.75 M KCl hypotonic solution, centrifuged, fixed twice with absolute methanol:glacial acetic acid (3:1 v/v) fixative, refrigerated, centrifuged, and resuspended in fixative prior to cell preparation. Based on the results from the initial assay, dose levels for the confirmatory assay were selected.

Slides were prepared by dropping harvested cells onto clean slides. Slides were then air dried, stained with 5% Giemsa, rinsed with distilled water, and dipped in xylene and mounted. Microscopic analysis of the slides included determining mitotic indices for 200 metaphase cells (100 per flask), each containing 19-23 chromosomes per treatment group (only 50 metaphase cells were scored for slides revealing 50% or more aberrant cells); counting the number of chromosomes; recording type of aberration; recording vernier readings for metaphase spreads with aberrations; and calculating the percent of aberrant cells and the frequency of aberrations per treatment group.

Evaluation criteria:
An assay was considered valid if (1) the percentage of cells with one or more aberrations was between 0-5% for the nontreated control; and (2) the positive control substances induced chromosomal aberration frequencies outside the normal range of the vehicle control that were at least twice the appropriate vehicle control. Positive results were considered to be either a statistically significant, dose-related increase in the mean percentage of aberrant cells, with the mean percentage of aberrant cells exceeding 5% in at least of the treatment groups or a reproducible, statistically significant response for at least one of the treatment groups with the mean percentage of aberrant cells exceeding 5%. If these criteria were not met, the results were considered negative.
Statistics:
The number of cells with at least one aberrant chromosome and the number of cells examined in each replicate were statistical analyzed. The number of aberrant individual chromosomes per cell was not statistically analyzed. Homogeneity of replicates in the nontreated control and treated groups were evaluated with the Fisher's Exact Test. Two times the sum of the log of the individual two-sided significance levels was compared to chi-square distribution with 2k degrees of freedom (k is the number of replicate pairs). If the test failed, further investigation was pursued, and the remaining analysis was not conducted. A 2x5 Fisher's Exact Test was performed to test the differences between the nontreated controls and treated groups. An individual 2x2 Fisher's Test was performed if differences at the 0.05 level or greater were detected. Positive control results were compared to the vehicle using the Fisher's Exact Test; dose-trends were evaluated using a permutation test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>50% reduction in confluency at 1250 μg/mL, ±S9, in the toxicity pretest
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The solubility test showed that the test substance was soluble in culture media three hours after dose at ≤313 μg/mL, +S9, and ≤156 μg/mL,-S9. The toxicity pretest showed a >50% reduction in confluency at 1250 μg/mL when compared to the nontreated control, both with and without metabolic activation. There were no decreases in mitotic indices at any dose level. Nominal concentrations first selected were 200, 400, 600, 700, 800, 900, and 1000 μg/mL; however, none of these dose levels produced a 50% reduction in cell confluency in the initial assay. Therefore, the initial assay was repeated with higher nominal dose levels because the European test guideline (EC, 1992) calls for greater than 50% reduction in cell confluency at the high dose. Including higher doses, however, resulted in evaluating concentrations in excess of 10 mM. This is the concentration limit recommended by the Working Group on in vitro Test for Chromosomal Aberrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

There were no statistically significant results in the initial 20 -hour assay,±S9, in the repeat 20 -hour assay, -S9, or in the 44 -hour assay,±S9. There was an increasing, dose-related trend (p<0.01) in aberrations in the 20-hour harvest with metabolic activation, with a significant increase in aberrant cells at 1200 (p<0.05) and 1500 (p<0.01) μg/mL; however, these increases were only observed at doses that may have exceeded the recommended 10 mM threshold above which artifacts are known to occur.  Increases also were not considered biologically significant because no increases in aberrant cells were observed in the initial 20-hour assay with metabolic activation conducted with the same dose concentrations.  Positive controls induced the appropriate response. 

 

Treatment (μg/mL)

Percent Mitotic Cellsa

Initial

Confirmatory

20 hour

20 hour

44 hour

+S9

0

8.4

12.8

11.0

600 +S9

9.5

13.8

8.9c

1000b

7.7

11.5

10.8

1200b

6.5

13.6

5.8

1500b

5.2

6.6

13.8

Acetone

10.6

13.8

---

DMBA

3.1

8.6

---

-S9

0 -S9

10.0

11.2

7.6

300

7.0c

7.0

7.0

600

9.4

9.8

7.3

900b

4.4

6.2

5.0

1100b

11.1

5.8

9.3

Acetone

13.5

13.3

---

MNNG

3.4

4.8

---

aPage 34 of study report.

bConcentration >10mM

cData based on one flask

 

Treatment (μg/mL)

Percent Aberrant Cellsa

Initial

Confirmatory

20 hour

20 hour

44 hour

+S9

0

3.5

1.8 (P)

2.5

600

6.5

3.0

3.0c

1000b

5.5

3.5

2.5

1200b

8.5

5.0*

1.5

1500b

5.7

9.5**

1.5

Acetone

2.0

1.5

---

DMBA

12.5**

6.3**

---

-S9

0

1.0

1.8

2.0

300

4.0c

1.5

0.5

600

1.0

1.0

2.0

900b

2.0

2.5

1.0

1100b

1.0

3.5

1.5

Acetone

0.5

1.5

---

MNNG

36.7**

49.6**

---

aPage 34 of study report.

bConcentration >10mM

cData based on one flask

* Statistically significantly higher than control (p<0.05)

** Statistically significantly higher than control (p<0.01)

(P) Statistically significant trend (p<0.01) by the permutation test

 

 

Treatment (μg/mL)

Percent Frequency of Aberrationsa

Initial

Confirmatory

20 hour

20 hour

44 hour

+S9

0

10.5

2.0

7.0

600

17.0

7.0

6.0c

1000b

11.0

9.0

2.5

1200b

26.5

12.0

2.0

1500b

8.9

14.5

1.5

Acetone

3.0

1.5

---

DMBA

13.5

6.8

---

-S9

0

1.0

2.3

2.0

300

5.0c

1.5

0.5

600

1.0

1.0

2.0

900b

2.0

3.0

1.0

1100b

2.0

3.5

1.5

Acetone

0.5

1.5

---

MNNG

54.0

92.8

---

aPage 34 of study report.

bConcentration >10mM

cData based on one flask

Conclusions:
Interpretation of results: negative

n-Pentane did not induce any biologically significant increase in chromosome aberrations in cultured CHO cells with or without metabolic activation under the conditions of this study. Positive, vehicle, and nontreated controls performed in an appropriate manner, indicating that the test system could detect both activation-dependent and direct-acting clastogens.
Executive summary:

In a mammalian cell chromosome aberration assay, Chinese hamster ovary cells were exposed to n-pentane (97.4% a.i., batch 2036924) at concentrations of 600, 1000, 1100, 1200, 1300, 1400, or 1500 μg/mL, +S9, and 300, 600, 900, 950, 1000, 1050, or 1100 μg/mL, -S9, for 20 hours (initial and confirmatory assay) and 44 hours (confirmatory assay).

 

The solubility test showed that the test substance was soluble in culture media three hours after dose at ≤313 μg/mL, +S9, and ≤156 μg/mL,-S9. The toxicity pretest showed a >50% reduction in confluency at 1250 μg/mL when compared to the nontreated control, ±S9. There were no decreases in mitotic indices at any dose level. Nominal concentrations first selected were 200, 400, 600, 700, 800, 900, and 1000 μg/mL; however, none of these dose levels produced a 50% reduction in cell confluency in the initial assay. Therefore, the initial assay was repeated with higher nominal dose levels because the European test guideline (EC, 1992) calls for greater than 50% reduction in cell confluency at the high dose. Including higher doses, however, resulted in evaluating concentrations in excess of 10 mM. This is the concentration limit recommended by the Working Group on in vitro Test for Chromosomal Aberrations.

 

There were no statistically significant results in the initial 20 -hour assay,±S9, in the repeat 20 -hour assay, -S9, or in the 44 -hour assay,±S9. There was an increasing, dose-related trend (p<0.01) in aberrations in the 20-hour harvest with metabolic activation, with a significant increase in aberrant cells at 1200 (p<0.05) and 1500 (p<0.01) μg/mL; however, these increases were only observed at doses that may have exceeded the recommended 10 mM threshold above which artifacts are known to occur.  Increases also were not considered biologically significant because no increases in aberrant cells were observed in the initial 20-hour assay with metabolic activation conducted with the same dose concentrations.  Positive controls induced the appropriate response. There was no biologically significant increase in chromosome aberrations over background.

This study received a Klimisch score of 1 because it is in compliance with OECD principles of GLP and E.U. Council Decision on GLP and adhered to E.U. Method B.10.

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:
The justification for read across is provided 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 had negative results for 2-methylbutane.

 

Genetic Toxicity in vivo – In Vivo Mammalian Erythrocyte Micronucleus Test (EU Method B.12)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1997-05-02 to 1997-07-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it is in compliance with OECD principles of GLP and E.U. Council Decision on GLP.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
MacGregor, J. Wehr, C., Henika, P., Shelby, M. 1990. The in vivo erythrocyte micronucleus test: measurement at steady state increases assay efficiency and permits integration with toxicity studies. Fundamental and Applied Toxicology 14:513–522.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
other: Crl:CDBR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Stone Ridge, New York
- Age at study initiation: 7 to 8 weeks (n-pentane treated and control groups); 15 to 16 weeks (positive control group)
- Weight at study initiation: 230 to 256 grams for n-pentane treated and control males; 433 to 470 grams for positive control males; 185 to 213 grams for n-pentane treated and control females; 270 to 295 grams for positive control females
- Assigned to test groups randomly: yes; using a computer-generated body weight sorting program that placed animals in groups that were within ±20% of the mean body weight of their sex
- Fasting period before study: no; however animals were fasted prior to clinical laboratory studies
- Housing: Individually housed in suspended stainless-steel and wire mesh cages
- Diet (e.g. ad libitum): ad libitum during non-exposure periods
- Water (e.g. ad libitum): ad libitum during non-exposure periods
- Acclimation period: 12 days for n-pentane treated and control animals; 13 days for positive control animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 18 to 22°C
- Humidity (%): 30 to 70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 1996-11-18 (n-pentane treated and control animals); 1997-05-05 (positive control animals) To: 1997-02-21 (n-pentane treated and control animals); 1997-05-08 (positive control animals)
Route of administration:
inhalation: vapour
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1.5 cubic meter stainless steel and glass whole body inhalation exposure chambers
- Source and rate of air: 300 liters per minute under a slight negative pressure to the room
- Temperature, humidity, pressure in air chamber: approximately 19 to 25°C with 40 to 70% humidity; chamber airflow, temperature, and relative humidity were continuously monitored throughout the exposure and recorded approximately every 30 minutes
- Other: Additional information regarding the generation of the test atmosphere is in the 90-day inhalation study (Exxon 157518).
Duration of treatment / exposure:
n-pentane: 6 hours plus chamber equilibrium (theoretical T99 = 23 minutes)
Cyclophosphamide (positive control): three days
Frequency of treatment:
n-pentane: 6 hours per day, 5 days per week for 13 weeks
Cyclophosphamide (positive control): once daily for three days
Post exposure period:
Not applicable
Remarks:
Doses / Concentrations:
n-pentane
Basis:
nominal conc.
5000; 10,000; and 20,000 mg/m3
Remarks:
Doses / Concentrations:
n-pentane
Basis:
analytical conc.
5097±97; 10,203±151; and 20,483±734 mg/m3
Remarks:
Doses / Concentrations:
positive control
Basis:
nominal conc.
20 mg/kg; dosing volume did not exceed 1.0 mL/100 grams per body weight
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent no treatment
Positive control(s):
cyclophosphamide (diluted with Reagent grade water)

- Justification for choice of positive control(s): cyclophosphamide is a known clastogen
- Route of administration: gavage
- Doses / concentrations: 20 mg/kg with a dosing volume of 10 mL/kg; individual doses were based on the animal’s day 0 body weight
Tissues and cell types examined:
It was noted that selected tissues were weighed, preserved, and examined microscopically in n-pentane treated and control animals. However, additional details, including what tissues were selected, are not provided. This information is presented in the 90-day inhalation study (Exxon 157518). Micronucleated polychromatic erythrocytes (MNEs), polychromatic erythrocytes (PCEs), and normochromatic erythrocytes (NCEs) were examined.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION: One femur per animal was removed per animal immediately after sacrifice. The bone marrow was aspirated, pooled, flushed in fetal bovine serum, and centrifuged. Resulting cell pellet was resuspended in any remaining supernatant after supernatant was decanted. Smears (2 slides per animal) were prepared from the resuspended cell pellet. Slides were stained using Acridine Orange, wet mounted, and labeled with the study and animal number.

METHOD OF ANALYSIS: Two thousand PCEs from each animal were examined for the presence of micronuclei. The ratio of polychromatic to total erythrocytes (by percent), number of polychromatic erythrocytes with micronuclei, and number of polychromatic erythrocytes scored were recorded. The percent of PCEs in the total population of erythrocytes was determined for each animal by counting the number of PCEs and NCEs per 1000 erythrocytes. Means and standard deviations of micronuclei data were calculated, and a statistical analysis to compare group means was conducted.
Evaluation criteria:
An assay was considered valid if (1) the mean incidence of MNEs per 1000 PCEs (calculated from the evaluation of 2000 PCEs) did not exceed 4 (or 0.4%) in the control group; and (2) the mean incidence of MNEs for the positive control group was significantly greater (p<0.05) than the control group.

A response was considered positive if there was (1) a dose-related statistical increase in the mean number of MNEs, including at least one dose point that is statistically (p<0.05) from the mean number of MNEs of the control group, that was outside the normal range (i.e., greater than 4); or (2) at least two dose points were statistically different (p<0.05) from the mean number of MNEs in the control group and greater than the normal range of the mean number of MNEs of the control group. A response was considered negative if the mean value for a statistically significant increase in MNEs was within the normal range of the control; a result was considered inconclusive if a single dose was statistically different from the control but there was no dose response.
Statistics:
A statistical analysis was conducted to compare group means using a one-way analysis of variance for each time period. Significant results were than analyzed using Duncan’s Multiple Range Test to compare the treated and control groups. A standard regression analysis was conducted to test for dose response.

Normality for the ANOVA residual was analyzed by either Wilk's Criterion or Kolmogorov Smirnov Statistic. For those not normally distributed in more than 25% of the analyses, nonparametric analyses were conducted. Nonparametric analyses included the Kruskal Wallis one-way analysis of variance, followed by Dunn's Summed Rank Test if significant differences were noted. Dose response was evaluated by Jonckheere’s test of Ordered response.

Sexes were analyzed separately.
Key result
Sex:
male/female
Genotoxicity:
negative
Remarks:
There were no increases in micronuclei formation
Toxicity:
no effects
Remarks:
There were no treatment-related effects observed regarding systemic toxicity.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY: A 5-day range finding study in rats was conducted, in which animals were exposed whole-body for 5 consecutive days. Additional detail regarding this range finding study is presented above under Details of tissue and slide preparation.

- Dose range: 5000; 10,000; and 20,000 mg/m3 (nominal); 5446; 10,680; and 21,418 mg/m3 (actual)
- Clinical signs of toxicity in test animals: There were no clinical signs of toxicity observed in any of the animals.
- Rationale for exposure: The high dose was selected because it half of the lower explosive limit and was the highest considered safe to test. Inhalation was chosen as the route of exposure because it is the mostly likely route for human exposure.

RESULTS OF DEFINITIVE STUDY (see table in Remarks on results including tables and figures)
- Induction of micronuclei, ratio of MNE/1000 PCE (for Micronucleus assay): N-pentane did not induce a statistically significant increase in micronuclei formation at any exposure level when compared to the control group. The positive control substance did induce a statistically significant (p<0.01) increase in the mean number of MNEs, indicating that this substance was clastogenic, the test system responded in the appropriate manner.
- Percent PCE (for Micronucleus assay): N-pentane did not induce a statistically significant decrease in the percent PCEs at any exposure level when compared to the control group. The positive control substance did induce a statistically significant (p<0.01) decrease in the percent PCEs, indicating that this substance was clastogenic, the test system responded in the appropriate manner.
- Appropriateness of dose levels and route: Dose levels and route of administration were appropriate.

There were no adverse effects with regard to clinical signs of toxicity, body weights, food consumption, pathology parameters, organ weights, postmortem observations, or microscopic changes. Only body weight information is presented in the study report. Additional information regarding clinical signs of toxicity, food consumption, pathology parameters, organ weights, postmortem observations, and microscopic changes are presented in the 90 -day inhalation study.

Summary of Micronucleus Data (mean ± s.d.)

 

Males - mg/m3(n)

0 (5)

5000 (5)

10,000 (5)

20,000 (5)

20 mg/kg Cyclophosphamide (5)

%PCE

42.76±2.37

43.92±2.75

47.68±3.17

39.94±6.67

39.94**±1.36

MNE/1000 PCE

2.6±2.6

2.0±2.3

1.0±0.6

2.7±1.8

35.9**±13.5

 

Females - mg/m3(n)

0 (5)

5000 (5)

10,000 (5)

20,000 (5)

20 mg/kg Cyclophosphamide (5)

%PCE

46.34±4.94

45.76±4.85

44.52±10.51

44.30±3.41

11.54**±4.74

MNE/1000 PCE

1.8±1.0

1.2±1.0

2.2±0.8

1.1±0.9

31.3**±2.3

a Data obtained from pages 25 to 26 in the study report.

** Statistically significantly higher than control (p<0.01)

Conclusions:
Interpretation of results: negative n-pentane did not induce an increase in micronuclei formation at any exposure level when compared to the control group.
There were no adverse effects observed in any of the n-pentane treated animals with regard to clinical signs of toxicity, body weights, food consumption, pathology parameters, organ weights, postmortem observations, or microscopic changes. n-Pentane did not induce an increase in micronuclei formation at any exposure level when compared to the control group. N-pentane was tested at an adequate dose because the high dose was half of the lower explosive limit and was the highest dose considered safe to test. The positive control induced the appropriate response. 
Executive summary:

In a Crl:CDBR rat bone marrow micronucleus assay, 5 animals/sex/dose were administered n-pentane via inhalation at nominal doses of 0; 5000; 10,000; or 20,000 mg/m3. Doses were selected based on a 5 -day range finding inhalation test in rats, and the test was part of the 90-day inhalation study. Rats were exposed to either n-pentane or air (control) 6 hours per day, 5 days per week for 13 weeks. A positive control group was administered 20 mg/kg of cyclophosphamide diluted in water at a constant volume of 1.0 mL/100 grams body weight for three treatments via gavage, 24 hours apart. Animals were examined for signs of toxicity during the study, and sacrificed on day after the last dose was administered. Bone marrow cells were harvested immediately after sacrifice. 

 

Actual doses received were 5097±97; 10,203±151; and 20,483±734 mg/m3. There were no signs of toxicity during the study. There were no adverse effects observed in any of the n-pentane treated animals with regard to clinical signs of toxicity, body weights, food consumption, pathology parameters, organ weights, postmortem observations, or microscopic changes. n-Pentane did not induce an increase in micronuclei formation at any exposure level when compared to the control group. n-Pentane was tested at an adequate dose because the high dose was half of the lower explosive limit and was the highest dose considered safe to test. The positive control induced the appropriate response. 

This study received a Klimisch score of 1 and is classified as reliable without restriction because it is in compliance with OECD principles of GLP and E.U. Council Decision on GLP.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Key in vitro genetic toxicity in bacteria data is available for 2-methylbutane. Additionally, data is also available for structural analogues, pentane (in vitro and in vivo) and 2,2,4-trimethylpentane (in vitro) and presented in the dossier. This data is read across to 2-methylbutane 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 Genetic Mutation in Bacteria

2-methylbutane

 

The genetic toxicity in bacteria of the test material (2-methylbutane) was evaluated via an Ames test (Kirwin et al., 1980). 2 -methylbutane was not found to be mutagenic at any concentration in both the presence and absence of S9 mix. This study is classified as reliable with restrictions, Klimisch score 2, because information regarding GLPs was not provided. However, the study closely followed OECD 471 and EEC B.13/14. The standard plate test was modified due to the gaseous nature of the chemicals; these modifications did not affect the overall study results. One of the strains used (TA1538) is not included in the current Guidelines, and only data from the highest dose was presented in the study report. 

 

Pentane

 

The genetic toxicity in bacteria of the test material (n-pentane) was evaluated via an Ames test (Kirwin et al., 1980). The statistical methods used were appropriate. The test conditions complied with the guideline requirements for this study type. n-Pentane was not found to be mutagenic at any concentration in both the presence and absence of S9 mix

 

This study is classified as reliable with restrictions, Klimisch score 2, because no information regarding GLPs was provided. However, the study closely followed OECD 471 and EEC B.13/14. The standard plate test was modified due to the gaseous nature of the chemicals; these modifications did not affect the overall study results. One of the strains used (TA1538) is not included in the current Guidelines, and only data from the highest dose was presented in the study report. 

In Vitro Chromosome Aberration in Mammalian Cells

Pentane

In a key in vitro mammalian cell chromosome aberration assay (ExxonMobil, 1997), Chinese hamster ovary cells were exposed to n-pentane (97.4% a.i., batch 2036924) at concentrations of 600, 1000, 1100, 1200, 1300, 1400, or 1500 μg/mL, +S9, and 300, 600, 900, 950, 1000, 1050, or 1100 μg/mL, -S9, for 20 hours (initial and confirmatory assay) and 44 hours (confirmatory assay).

 

The solubility test showed that the test substance was soluble in culture media three hours after dose at ≤313 μg/mL, +S9, and ≤156 μg/mL,-S9. The toxicity pretest showed a >50% reduction in confluency at 1250 μg/mL when compared to the nontreated control, ±S9. There were no decreases in mitotic indices at any dose level. Nominal concentrations first selected were 200, 400, 600, 700, 800, 900, and 1000 μg/mL; however, none of these dose levels produced a 50% reduction in cell confluency in the initial assay. Therefore, the initial assay was repeated with higher nominal dose levels because the European test guideline (EC, 1992) calls for greater than 50% reduction in cell confluency at the high dose. Including higher doses, however, resulted in evaluating concentrations in excess of 10 mM. This is the concentration limit recommended by the Working Group on in vitro Test for Chromosomal Aberrations.

 

There were no statistically significant results in the initial 20 -hour assay, ±S9, in the repeat 20 -hour assay, -S9, or in the 44 -hour assay,±S9. There was an increasing, dose-related trend (p<0.01) in aberrations in the 20-hour harvest with metabolic activation, with a significant increase in aberrant cells at 1200 (p<0.05) and 1500 (p<0.01) μg/mL; however, these increases were only observed at doses that may have exceeded the recommended 10 mM threshold above which artifacts are known to occur.  Increases also were not considered biologically significant because no increases in aberrant cells were observed in the initial 20-hour assay with metabolic activation conducted with the same dose concentrations.  Positive controls induced the appropriate response. There was no biologically significant increase in chromosome aberrations over background.

 

This study received a Klimisch score of 1 because it is in compliance with OECD principles of GLP and E.U. Council Decision on GLP and adhered to E.U. Method B.10.

In Vitro Gene Mutation study in Mammalian Cells

 

2,2,4-trimethylpentane

 

2,2,4-trimethylpentane (Iso-octane; 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).

In Vivo

Pentane

 

In a key in vivo Crl:CDBR rat bone marrow micronucleus assay (ExxonMobil, 1997), 5 animals/sex/dose were administered n-pentane via inhalation at nominal doses of 0; 5000; 10,000; or 20,000 mg/m3. Doses were selected based on a 5 -day range finding inhalation test in rats, and the test was part of the 90-day inhalation study. Rats were exposed to either n-pentane or air (control) 6 hours per day, 5 days per week for 13 weeks. A positive control group was administered 20 mg/kg of cyclophosphamide diluted in water at a constant volume of 1.0 mL/100 grams body weight for three treatments via gavage, 24 hours apart. Animals were examined for signs of toxicity during the study, and sacrificed on day after the last dose was administered. Bone marrow cells were harvested immediately after sacrifice. 

 

Actual doses received were 5097 ± 97; 10,203 ± 151; and 20,483 ± 734 mg/m3. There were no signs of toxicity during the study. There were no adverse effects observed in any of the n-pentane treated animals with regard to clinical signs of toxicity, body weights, food consumption, pathology parameters, organ weights, postmortem observations, or microscopic changes. n-Pentane did not induce an increase in micronuclei formation at any exposure level when compared to the control group. n-Pentane was tested at an adequate dose because the high dose was half of the lower explosive limit and was the highest dose considered safe to test. The positive control induced the appropriate response. 

 

This study received a Klimisch score of 1 and is classified as reliable without restriction because it is in compliance with OECD principles of GLP and E.U. Council Decision on GLP.

Justification for classification or non-classification

The negative results observed in substance specific and read across in vitro and in vivo genotoxicity assays do not warrant the classification of 2-methylbutane as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).