Cyclopentene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential of Cyclopentene (CAS 142-29-0/EC 205-532-9) and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9) was determined in an relevant OECD-GLP study. Based on the results of this study it is concluded that Cyclopentene is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 08 Jun 2018 to 11 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Based on laboratory historical data the deviations are considered not to affect the study integrity.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

Based on laboratory historical data the deviations are considered not to affect the study integrity.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TEST MATERIAL INFORMATION
- Identification: Cyclopentene
- Test Facility test item number: 209393/A
- Purity/Composition correction factor: No correction factor required
- Chemical name (IUPAC, synonym or trade name): Cyclopentene
- CAS / EC number: 142-29-0 / 205-532-9
- Appearance:Clear colourless liquid
- Batch: 0000015386
- Molecular formula: C5H8
- Molecular weight: 68.117 g/mol
- Purity/Composition: 98.9% (Certificate of Analysis)
- Test item storage: In refrigerator (2-8°C)
- Stable under storage conditions until: 9 March 2021 (expiry date)
- Test item handling: The test item is volatile and evaporates rapidly at room temperature. Do not leave the test item at room temperature longer than 30 minutes.
- Stability at higher temperatures: Stable at temperatures << 44°C Not stable at higher temperatures, potential to polymerize and form peroxides
- Solubility in vehicle: Dimethyl sulfoxide: Not indicated
- Stability in vehicle: Dimethyl sulfoxide: Not indicated

Target gene:

STRAIN HISTIDINE MUTATION MUTATION TYPE
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535, TA1537, TA98, TA100; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA)]
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC).

MEDIA USED
- Type and identity of media:
- Enriched nutrient broth (Oxoid LTD, Hampshire, England)
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

The test material Cyclopentene was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix.

Based on the Range Finder results, the first mutation experiment concentrations with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix were 52, 164, 512, 1600 and 5000 μg/plate.

Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 µg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Vehicle / solvent:

VEHICLE:
- Chemical name: Dimethyl sulfoxide
- CAS number: 67-68-5
- Purity ≥ 99.9 %
- Batches K49824131817 and K49431131805 (Merck, Darmstadt, Germany).

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

PREPARATION OF CELL CULTURE
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/mL). Freshly grown cultures of each strain were used for testing.

AGAR PLATES
Agar plates (ø 9 cm) containing 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained
15 µg/plate tryptophan (Sigma).

TOP AGAR
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

ENVIRONMENTAL CONDITIONS
The temperature was continuously monitored throughout the experiment in order to be at 37.0 ± 1.0°C (actual range 33.0 - 38.5°C).

METABOLIC ACTIVATION SYSTEM
Rat liver microsomal enzymes (S9 homogenate) (Trinova Biochem GmbH, Giessen, Germany) were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight). Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

PREPARATION OF S9
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Evaluation criteria:

The experiment is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data

b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

E. coli WP2 uvr A

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:

RANGE FINDER EXPERIMENT
Precipitation
Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain.

Citotoxicity
Cytotoxicity (reduction of the bacterial background lawn) was observed in all tester strains in the absence and presence of S9-mix at the top dose level, except for tester strain WP2uvrA in the absence of S9-mix where no toxicity was observed at any of the dose levels tested.

Mutagenicity
No increase in the number of revertants was observed under all conditions tested.

MAIN EXPERIMENT
Precipitation
Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain.

Citotoxicity
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. Except for tester strain TA98 in the absence of S9-mix where a slight reduction in the bacterial background lawn was observed at the highest dose level tested.

Mutagenicity
No increase in the number of revertants was observed under all conditions tested.

Remarks on result:

other: No increase in the number of revertants was observed upon treatment with Cyclopentene under all conditions tested.

Remarks:

Not mutagenic

Table 1. Dose-Range Finding Test: Mutagenic Response of Cyclopentene in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay
Dose (µg/plate)	Mean number of revertant colonies / 3 replicate plates (± S.D.) with oneSalmonella typhimuriumand oneEscherichia colistrain
	TA 100	WP2uvrA
Without S9-mix
Positive Control	960 ± 40	1208 ± 40
Solvent Control	117 ± 6	37 ± 10
1.7	128 ± 19	30 ± 1
5.4	127 ± 14	31 ± 12
17	136 ± 19	32 ± 4
52	136 ± 12	29 ± 9
164	128 ± 5	30 ± 5
512	132 ± 1	27 ± 8
1600	126 ± 16^n	21 ± 3^n
5000	99 ± 6^sNP	20 ± 8^sNP
With S9-mix^1
Positive Control	1440 ± 33	324 ± 24
Solvent Control	124 ± 21	28 ± 5
1.7	141 ± 14	28 ± 6
5.4	143 ± 4	35 ± 8
17	141 ± 15	33 ± 17
52	134 ± 17	41 ± 10
164	134 ± 15	34 ± 5
512	130 ± 12	34 ± 9
1600	118 ± 18^n	35 ± 10
5000	89 ± 11^sNP	24 ± 4^nNP

¹Plate incorporation assay (5% S9)

NP No precipitate

n Normal bacterial background lawn

s Bacterial background lawn slightly reduced

Experiment 1: Mutagenic Response of Cyclopentene in the Salmonella typhimurium Reverse Mutation Assay
Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA 1535	TA 1537	TA 98
Without S9-mix
Positive Control	935 ± 36	812 ± 38	1087 ± 16
Solvent Control	10 ± 6	6 ± 2	17 ± 2
52	13 ± 11	5 ± 2	20 ± 5
164	12 ± 2	3 ± 2	13 ± 2
512	11 ± 1	5 ± 3	14 ± 4
1600	13 ± 5^n	3 ± 2^n	18 ± 3^n
5000	4 ± 1^sNP	7 ± 4^sNP	5 ± 3^sNP
With S9-mix^1
Positive Control	334 ± 16	712 ± 86	1184 ± 104
Solvent Control	8 ± 3	6 ± 3	20 ± 8
52	10 ± 2	5 ± 2	21 ± 10
164	9 ± 5	3 ± 2	20 ± 2
512	9 ± 3	7 ± 4	19 ± 5
1600	9 ± 8^n	4 ± 3^n	14 ± 8^n
5000	9 ± 2^sNP	4 ± 4^sNP	17 ± 3^sNP

¹Plate incorporation assay (5% S9)

NP No precipitate

n Normal bacterial background lawn

s Bacterial background lawn slightly reduced

Experiment 2: Mutagenic Response of Cyclopentene in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay
Dose (µg/plate)	Mean number of revertant colonies / 3 replicate plates (± S.D.) with different strains ofSalmonella typhimurium and one Escherichia coli strain
	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
Without S9-mix
Positive Control	809 ± 68	1104 ± 130	1145 ± 72	856 ± 19	1412 ± 70
Solvent Control	9 ± 3	3 ± 2	12 ± 2	118 ± 9	22 ± 5
275	7 ± 6	6 ± 1	12 ± 3	131 ± 9	15 ± 4
492	6 ± 2	3 ± 2	17 ± 4	132 ± 21	20 ± 10
878	8 ± 4	3 ± 2	11 ± 2	107 ± 15	23 ± 8
1568	9 ± 3	5 ± 2	12 ± 2	128 ± 8	19 ± 1
2800	7 ± 4	5 ± 2	9 ± 1	126 ± 13	16 ± 2
5000	9 ± 2^n NP	4 ± 2^n NP	7 ± 2^n NP	109 ± 10^n NP	23 ± 1^n NP
With S9-mix^1
Positive Control	146 ± 19	172 ± 31	847 ± 60	372 ± 25	279 ± 25
Solvent Control	13 ± 6	3 ± 2	16 ± 5	71 ± 17	22 ± 9
275	6 ± 3	4 ± 1	18 ± 5	69 ± 14	26 ± 4
492	8 ± 7	4 ± 2	14 ± 7	71 ± 7	19 ± 4
878	9 ± 2	4 ± 2	15 ± 8	69 ± 5	20 ± 7
1568	9 ± 5	4 ± 1	15 ± 3	84 ± 12	27 ± 5
2800	9 ± 3	6 ± 5	15 ± 3	67 ± 11	23 ± 4
5000	10 ± 2^n NP	4 ± 3^n NP	12 ± 1^n NP	60 ± 13^n NP	27 ± 6^n NP

¹Plate incorporation assay (10% S9)

NP No precipitate

n Normal bacterial background lawn

Conclusions:

Based on the results of this study, the test material Cyclopentene is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The aim of this study was to assess the potential of the test material Cyclopentene (CAS 142-29-0/EC 205-532-9) and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The study was performed according to the OECD 471and EC No. 440/2008 Guidelines, and under GLP conditions.

The vehicle of the test item was dimethyl sulfoxide (DMSO). The positive control were sodium azide (SA) (TA1535), ICR-191 (TA1537), 2 -nitrofluorene (NF) (TA98), methylmethanesulfonate (MMS)(TA100), 4 -nitroquinoline N-oxide (4 -NQO) (WP2 uvrA).

In the dose-range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. No precipitate was observed on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in both tester strains in the absence and presence of S9-mix at the top dose level, except for tester strain WP2uvrA in the absence of S9-mix.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 52 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. No precipitate on the plates at any of the dose levels tested was observed. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix at the highest dose level tested.

In a follow-up experiment, the test item was tested at a concentration range of 275 to 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. No precipitation on the plates at any of the dose levels tested was observed. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Except for tester strain TA98 in the absence of S9-mix where a slight reduction in the bacterial background lawn was observed at the highest dose level tested. The acceptance criteria were met and the controls (positive and negative) were considered valid.

The test item Cyclopentene did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. 

Based on the results of this study, the test material Cyclopentene (CAS 142-29-0/EC 205-532-9) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The available data indicated that classification for genotoxicity is not warranted.

Cyclopentene is not classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).