Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No studies are available for the registered substance Methylcyclopentane. Based on the read across approach, information om the structural analogue n-Hexane is used.

Three in vitro genetic toxicity studies were read across based on data for commercial hexane (52% n-hexane).

All in vitro genetic toxicity tests were negative.

Genetic Toxicity in vitro - Bacterial reverse mutation assay (OECD TG 471) - Negative

Genetic Toxicity in vitro - In vitro Mammalian Chromosome Aberration Test (OECD TG 473) - Negative

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

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-05-19 to 1989-07-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions because it is well documented and follows OECD Guideline 471.
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:
The method was modified to use the dessicator methodology in order to test the mutagenicity of the vapor phase of the test substance.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 from aroclor induced rat liver
Test concentrations with justification for top dose:
0, 600, 1000, 3000, 6000, 9000 ppm
Untreated negative controls:
yes
Negative solvent / vehicle controls:
other: no solvent used
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-nitrofluorene, sodium azide, 9-aminoacridine, 1,1-dichloroethene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Prepared plates were placed uncovered and inverted in 9l dessicators. An appropriate amount of test substance was placed in a glass petri dish was suspended at the bottom of the dessicator. A magnetic stirring bar was placed at the bottom of the dessicator, and served as a fan to ensure even distribution of the vapor.


DURATION
- Preincubation period: 48 hrs
- Exposure duration: 7-8 hrs at 37 degree C
- Expression time (cells in growth medium): There was an additional incubation time of 40 hrs after exposure.

NUMBER OF REPLICATIONS: 3


Evaluation criteria:
To be considered positive for mutation, at least a doubling of mean revertants per plate in at least one tester strain must be seen. This increase must be dose-related.
Statistics:
Mean number of revertant per plate and the standard deviation was calculated.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No positive responses were observed in any of the tester strains. The positive control substance, 2-aminoanthracene, did not produce any mutations in strain TA 1538 in one experiment in the presence of S9. As revertants were seen in the test of TA 1538 without S9, and revertants were seen in other strains exposed to positive controls with S9, the lack of revertants was most likely due to a technical error and the study is still considered valid.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Average Revertants per Plate (SD) - Experiment B1

Dose (ppm)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

Without S9

0.0

17 ± 1

105 ± 12

11 ± 1

7 ± 1

8

600

20 ± 5

113 ±  9

13 ± 4

6 ± 1

6

1000

16 ± 3

121 ± 8

14 ± 2

7 ± 1

6

3000

18 ± 4

112 ± 13

17 ± 3

5 ± 4

4

6000

24 ± 4

117 ± 14

12 ± 4

6 ± 4

8

9000

17 ± 3

112 ± 7

14 ± 5

9 ± 2

5

Positive control

227 ± 8

422 ± 44

349 ± 36

481 ± 135

452

With S9

0.0

24 ± 5

119 ± 10

17 ± 1

7 ± 0

12

600

30 ± 10

137 ± 12

23 ± 4

9 ± 4

15

1000

23 ± 5

126 ± 3

14 ± 2

11 ± 5

13

3000

22 ± 1

120 ± 19

16 ± 2

9 ± 2

9

6000

24 ± 2

103 ± 11

14 ± 4

7 ± 2

11

9000

30 ± 6

120 ± 3

17 ± 1

8 ± 2

14

Positive control

3142 ± 139

3902 ± 68

189±  22

351 ± 26

--

Positive vapor control

372 ± 52

Average Revertants per Plate (SD) - Experiment B2

Dose (ppm)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

Without S9

0.0

12 ± 2

107 ± 11

9 ± 5

5 ± 1

19

600

20 ± 1

103 ±  9

8 ± 4

7 ± 3

19

1000

14 ± 4

110 ± 6

12 ± 5

7 ± 2

19

3000

14 ± 1

124 ± 14

12 ± 2

7 ± 2

19

6000

22 ± 6

125 ± 14

15 ± 1

5 ± 2

16

9000

15 ± 6

114 ± 5

13 ± 3

5 ± 3

13

Positive control

444 ± 86

991 ± 67

758 ± 19

162 ± 51

754

With S9

0.0

21 ± 4

132 ± 16

17 ± 4

7 ± 3

26

600

25 ± 7

133 ± 8

18 ± 2

6 ± 1

25

1000

22 ± 2

155 ± 4

15 ± 7

8 ± 3

25

3000

20 ± 1

123 ± 23

16 ± 7

7 ± 3

24

6000

21 ± 4

151 ± 14

15 ± 1

6 ± 2

30

9000

25 ± 4

161 ± 8

11 ± 4

6 ± 1

27

Positive control

2187 ± 166

2229 ± 223

166 ±  32

230 ± 24

2195

Positive vapor control

394 ± 50

Conclusions:
Interpretation of results: negative

The test substance is not mutagenic.
Executive summary:

This study examined the mutagenicity of vapors of the test substance commercial hexane. Plates of S. typhimurium were exposed for 7 -8 hrs to test atmospheres of 0, 600, 1000, 3000, 6000, or 9000 ppm of test substance. 20,000 ppm of 1,1 -dichloroethene was used a vapor-phase positive control substance.

The test substance did not produce a positive response in any of the test strains. The test substance is not mutagenic.

Endpoint:
genetic toxicity in vitro, other
Remarks:
bacteria, mammalian cells
Type of information:
other: data from collection of data
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
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 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from livers of male rats or Syrian hamsters
Test concentrations with justification for top dose:
up to 1000 µg/plate
Details on test system and experimental conditions:
pre-incubation test
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined

In other experiments in vitro (with Escherichia coli, Bacillus subtilis, Salmonella typhimurium or Saccharomyces cerevisiae, as well as in the Chinese hamster cells) no genetic toxicity was found.

In the CHO-test sister chromatid exchange in the fraction with S9 -mix was reported, but no dose-response appeared.

Conclusions:
No in vitro toxicity found in assays using bacterial cells.
Executive summary:

No in vitro toxicity found in assays using bacterial cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-08-28 to 1990-04-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions because it is well documented and follows OECD Guideline 476.
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 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):

- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
other: K1-BH4
Metabolic activation:
with and without
Metabolic activation system:
S9 from aroclor induced rat liver
Test concentrations with justification for top dose:
0, 0.132, 0.098, 0.063, 0.0362, 0.0122 ul/ml (analytical)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate 0.2 ul/ml without metabolic activation, benzo(a)pyrene 4 ug/ml with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: 7.5 ml of test substance was added to 17.5 ml of DMSO and vortexed for 2 min. The solution was allowed to separate, and the bottom DMSO/test substance layer and diluted further with DMSO. Appropriate amounts were then added to the test medium.


DURATION
- Preincubation period: 18-24 hrs at 37 degree C
- Exposure duration: 5 hrs at 37 degree C
- Expression time (cells in growth medium): 18-24 at 37 degree C

NUMBER OF REPLICATIONS: 2


DETERMINATION OF CYTOTOXICITY
Replicates from each treatment concentration were pooled and cultured in triplicate (100 cells/60 mm dish). After 7 days of incubation, colonies were fixed with methanol , stained with Giemsa, and counted. Determination was based on relative cloning efficiency.

Evaluation criteria:
mutant frequency > 20 mutants per 10^6 clonable cells, at least twice the mutant frequency of negative and solvent controls, mutant frequency above negative and solvent controls of at least 11 mutants per 10^6 clonable cells
Statistics:
Students t-test (p <= 0.05)
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
None of the mutant frequencies in the test groups were significantly elevated over negative and solvent controls. The test substance was cytotoxic at concentrations of 0.063 ul/ml or greater.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

CHO/HGPRT Mutation Assay

Dose (µl/ml)

Total Colonies

Cloning Efficiency

Total Mutant Colonies

Mutants/106 Clonable Cells

Without S9

Negative control

310

1.03

19

18.4

Solvent Control

329

1.10

6

5.5

0.132

284

0.95

0

5.3

0.098

260

0.87

0

 5.8

0.063

321

1.07

0

0.9

0.0362

289

0.96

5

5.2

0.0122

294

0.98

12

12.2

Positive Control

282

0.94

227

241.5

With S9

Negative control

320

1.07

0

0.9

Solvent Control

330

1.10

1

0.9

0.132

299

1.00

0

1.0

0.098

299

0.99

0

1.0

0.063

256

0.85

4

4.7

0.0362

317

1.06

13

12.3

0.0122

293

0.98

13

13.3

Positive Control

239

0.80

91

114.2

Conclusions:
Interpretation of results: negative

The test substance is not mutagenic.
Executive summary:

This study determined the mutagenicity of commercial hexane to Chinese hamster ovary (CHO) cells. CHO cells were exposed to concentrations of 0, 0.132, 0.098, 0.063, 0.0362, or 0.0122 µL/mL both with and without metabolic activation for 5 hrs. The cells were then analyzed for mutation frequency. A concurrent cytotoxicity assay was performed.

 

Results of the mutagenicity assay show that the test substance is not mutagenic both in the presence and absence of metabolic activation. However, the test substance was cytotoxic at concentrations of 0.063 µl/mL or greater.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-08-24 to 1990-02-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it followed a protocol comparable to OECD Guideline 473.
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:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
other: CHO-K1 cells
Metabolic activation:
with and without
Metabolic activation system:
S9 from aroclor induced rat liver
Test concentrations with justification for top dose:
0.015, 0.034, 0.074, 0.123, 0.416 ul/ml without S9
0.014, 0.022, 0.056, 0.118, 0.251 ul/ml with S9
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: triethylenemelamine 0.5 ug/ml without S9, cyclophosphamide 50 ug/ml with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: 100 ul of dosing solutiong was added to the test medium


DURATION
- Preincubation period: 16-24 hrs at 37 degree C
- Exposure duration: 12 hrs without S9 at 37 degree in humidified air; 2 hrs with S9 at 37 degree in humidified air
- Expression time (cells in growth medium): For cells not treated with S9, two hours prior to cell harvest, treatment medium was removed and cells washed with PBS and refed with medium containing 0.1 ug/ml of Colcemid. For cells treated with S9, treatment medium was removed after exposure, cells were washed with PBS, refed, and returned to the incubator for 16 hrs. Colcemid was added at 0.1 ug/ml, and flasks incubated for two hrs.
- Fixation time (start of exposure up to fixation or harvest of cells): 14-20 hrs, collected by centrifugation



SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): 5% Giemsa


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 100 per concentration


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell cycle delay


OTHER EXAMINATIONS:
Chromatid and isochromatid breaks, exchange figures, chromosome breaks, fragments, pulverized chromosomes, chromatid and isochromatid gaps


OTHER:
Evaluation criteria:
In order for the test to be valid, there must be no more than 6% cells with chromosome aberrations in the negative and solvent control groups. Positive control must be statistically increased over untreated controls (p<=0.05, Fisher's exact test). A positive result is percentage of cells with aberrations was statistically increased over untreated controls (p<=0.05, Fisher's exact test).
Statistics:
Fisher's exact test was used to determine statistical significance. Cochran-Armitage test was used to test dose-responsiveness.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 0.074 ul/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity was observed at concentrations of 0.074 µL/mL or greater. No significant increase in chromosome aberrations was seen in treatment groups.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results of CHO Chromosome Aberration Assay

Dose (µl/ml)

Mitotic Index

Cells Scored

Aberrations per cell (mean ± SD)

% Cells with Aberrations

Without S9

Negative Control

5.7

100

0.010 ± 0.100

1

Solvent Control

4.9

100

0.010 ± 0.100

1

0.015

4.8

100

0.000 ± 0.000

0

0.034

4.5

100

0.010 ± 0.100

1

0.074

2.9

100

0.010 ± 0.100

1

0.123

0.2

8

0.000 ± 0.000

0

0.416

0.0

0

Positive Control

2.2

100

0.360 ± 1.124

21

With S9

Negative Control

5.3

100

0.010 ± 0.100

1

Solvent Control

5.6

100

0.010 ± 0.100

1

0.014

5.3

100

0.010 ± 0.100

1

0.022

5.9

100

0.010 ± 0.100

1

0.056

3.1

100

0.010 ± 0.100

1

0.118

0.2

2

0.000 ± 0.000

0

0.251

0.0

0

Positive Control

2.5

100

0.840 ± 1.819

40

Conclusions:
Interpretation of results: negative

The test substance is not clastogenic.
Executive summary:

This study examined the potential for commercial hexane to cause chromosome aberrations in Chinese Hamster Ovary (CHO) cells. CHO cells were exposed to concentrations of 0, 0.015, 0.034, 0.074, 0.123, and 0.416 µL/mL without metabolic activation and 0, 0.014, 0.022, 0.056, 0.118, and 0.251 µL/mL with metabolic activation. 0.5 µg/mL triethylenemelamine was used a positive control without metabolic activation and 50 µg/mL cyclophosphamide was used as a positive control with metabolic activation.

 

Negative and positive controls were valid. There was no significant increase in chromosome aberrations in any test group. The test substance was cytotoxic at concentrations of 0.074 µL/mL or greater. The test substance is not clastogenic.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There is no data available for Methylcyclopentane. Data is available from a structural analogue n-Hexane and used as read across.

In Vitro

The first in vitro study evaluated the mutagenicity of vapors of commercial hexane (52% n-hexane). According to the study report (API, 1989a; Klimisch score =1) plates of S. typhimurium were exposed for seven to eight hours to test atmospheres of 0, 600, 1000, 3000, 6000, or 9000 ppm of test substance. The test substance did not produce a positive response in any of the test strains.

In another bacterial reverse mutation assay (US DHHS, 1999), noin vitrotoxicity was found in assays using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 exposed to hexane at concentrations up to 1000 µg/plate in the presence and absence of metabolic activation.

Two in vitro studies determined the mutagenicity of commercial hexane (52% n-hexane) in Chinese hamster ovary (CHO) cells. In one

study CHO cells were exposed to concentrations of 0, 0.132, 0.098, 0.063, 0.0362, or 0.0122 µL/mL both with and without metabolic activation

for 5 hours (API, 1990d; Klimisch score =1). The cells were then analyzed for mutation frequency. The test substance was found not to be mutagenic both in the presence and absence of metabolic activation. However, the test substance was cytotoxic at concentrations of 0.063 µL/mL or greater.

In the second in vitro study CHO cells were exposed to concentrations of commercial hexane (52% n-hexane) of 0, 0.015, 0.034, 0.074,

0.123, and 0.416 µL/mL without metabolic activation and 0, 0.014, 0.022, 0.056, 0.118, and 0.251 µL/mL with metabolic activation (Daughtrey et al., 1984; Klimisch score = 1). 0.5 µg/mL triethylenemelamine was used a positive control without metabolic activation and 50 µg/mL cyclophosphamide was used as a positive control with metabolic activation. Negative and positive controls were found to be valid and no significant increase in chromosome aberrations were found. The test substance was however found to be cytotoxic at concentrations of 0.074 µL/mL or greater. The test substance is not clastogenic.

All studies were conducted in a manner similar or equivalent to currently established OECD guidelines.

Justification for classification or non-classification

There is no data available for Methylcyclopentane. Data is available from a structural analogue n-Hexane and used as read across.

Based on the negative results in the in vitro genotoxicity assays, Methylcyclopentane does not warrant classification for genetic toxicity under the new Regulation

(EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).