Cyclopentane, methoxy-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames study: negative

Chromosome aberration study: negative

In vitro gene mutation: negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 2001 - 02 July 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed in accordance with several official test guidelines including OCED, EC and US EPA, and in compliance with GLP; on this basis the study is considered reliable without restriction.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Analytical purity: 99.6%
- Lot/batch No.: 01529

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate derived from Aroclor 1254-induced rats (S-9 mix).

Test concentrations with justification for top dose:

Range-finding study: 5, 15, 50, 150, 500, 1500, and 5000 µg/plate (with and without metabolic activation)
Definitive study: 50, 150, 500, 1500, and 5000 µg/plate (with and without metabolic activation).

Vehicle / solvent:

Solvent: Dimethylsulfoxide (DMSO)
Solubility of the test material was assesed in both DMSO and water; the test substance was found to be insoluble in water, but soluble in DMSO. For this reason, DMSO was selected as the solvent.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control for TA1535 and TA100 in the absence of S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Positive control for TA1537 in the absence of S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Positive control for TA98 in the absence of S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

Positive control for WP2uvrA/pKM101 in the absence of S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control for TA1535 and WP2uvrA/pKM101 in the presence of S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Positive control for TA1537, TA98 and TA100 in the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:in agar (plate incorporation)

DURATION
- Preincubation period: 30 minutes (main test only)
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 Plates per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (appearance of background bacterial lawn)

Evaluation criteria:

Number of revertant colonies, relative to concurrent vehicle controls. A two-fold increase in the number of revertant colonies relative to the concurrent control group, with some evidence of a dose-relationship is considered significant.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
No signs of toxicity were observed towards the tester
strains in either mutation test.


The first (range-finding) test was a standard plate
incorporation assay; the second involved a pre-incubation
stage.

Conclusions:

Under the conditions of this bacterial reverse mutation assay, the substance showed no evidence of mutagenic activity in any of the strains tested, either in the presence or absence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2002 - 01 May 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with OECD, EC, US EPA, and Japanese test guidelines or methods, and in compliance with GLP, so the data is considered reliable without restriction.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

(Joint directives of JEPA, JMHW and JMITI (31 October 1997))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Analytical purity: 99.8%
- Lot/batch No.: 010912

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimal Essential medium

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate derived from Aroclor 1254 - induced rats (S9 mix).

Test concentrations with justification for top dose:

Range finding / preliminary test: 7.83, 15.65, 31.3, 62.6, 125.2, 250.4, 500.8, and 1001.6 µg/mL.
Second test: 62.6, 125.2, 250.4, 500.8, 1001.6 µg/mL

Vehicle / solvent:

Dimethylsulfoxide (DMSO)
DMSO was chosen on the basis that the sponsor advised that NZE was poorly soluble in water but freely soluble in DMSO and Acetone. In a solubility trial, NZE was found to be soluble in DMSO at 1M (100.16 mg/mL)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Positive control in the absence of S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Positive control in the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours in the preliminary test. In the second test, 3-hour exposure was used in the presence of S9 mix; a 15-hour continuous exposure was used in the absence of S9 mix.
- Expression time (cells in growth medium): 12 hours (except in the second test in the absence of S9 mix, which used continuous exposure without a dedicated expression period)
- Fixation time (start of exposure up to fixation or harvest of cells): 17 hours (15 hours exposure and / or expression time + 2 hours incubation)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: 2 cultures per concentration in each test.

NUMBER OF CELLS EVALUATED: 100 metaphase figures per culture; the incidence of polyploid cells out of 500 metaphase cells was also recorded.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

The assay was considered to be acceptable if the negative and positive control values lay within the recent historical control range for the laboratory.

The test substance was considered to cause a positive response if the following conditions were met:
Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) were observed at one or more test concentration.
The increases exceed the negative control range of the laboratory, taken at the 99% confidence limit.
The increases were reproducible between replicate cultures.
The increases were not associated with large changes in osmolality of the treatment medium or extreme toxicity.
Evidence of a dose-relationship was considered to support the conclusion.

A negative response was claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies were observed, at any dose level.

Statistics:

The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

(tested up to 10 mM)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
In the absence of S-9 mix in the first test, the test substance caused a statistically significant increase in the proportion of cells with chromosomal aberrations at dose level of 250.4 µg/ml, including gap-type aberrations only when compared with the solvent control. In the second test, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control. The statistical increase observed in the first test is not considered to be of biological importance as the significance of gap-type aberrations is questionable and the increase was not reproducible between tests.

Conclusions:

It was concluded that the substance showed no evidence of clastogenic activity in this in vitro cytogenetic test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2018-04-24 to 2018-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Batch No.: 5820146
Purity: 99.99%

Target gene:

Thymidine Kinase Gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

2000, 800, 320, 128, 51.2and 20.48 μg/mL

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 3E+05 cells/mL

DURATION
- Preincubation period:
- Exposure duration: 3 hours with S9, 3 hours and 24 hours without S9
- Expression time (cells in growth medium): 24 hours
- Selection time (if incubation with a selection agent): 11 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2000 cells / well

CELL COLONIES OBSERVATION:
For the PE plate, empty wells and total wells were counted.
For the TFT plate, wells with viable colony and total wells were counted. The number of empty wells resulted from the difference between total and colony-containing wells. In addition, for the TFT plates of the highest concentration, the solvent and positive controls, large and small colonies were counted, respectively.

Evaluation criteria:

When IMF(s) at one or several dose levels are more than the GEF of 1.26E-04, and the increase is concentration-related and/or replicated, the result is evaluated as positive.
The result is evaluated as negative, if none of the above criteria is met.
In case the criteria above are not met at the same time, the repeated test should be performed using modified experimental conditions, and the biological relevance of the result should be considered. If the result is still not clear, the result is concluded to be equivocal.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In this study, the results of the solvent and positive controls met all criteria, so the sensitivity of the assay and the efficacy of the S9 mix could be validated.
Under each treatment condition, no test item precipitation was observed in the cell cultures either at the beginning or the end of the treatment.
The test item produced dose-related suppression of cell growth, as demonstrated by the relative total growth (RTG), the key parameter representing overall cell proliferation and survival across all operation steps of the test: RTG includes the relative suspension growth during treatment (RSG: test culture vs. solvent control) and during the incubation time for mutant expression along with the relative plating/cloning efficiency (RPE: test culture vs. solvent control) at the time of mutant selection.
The results of mutant frequency showed that the IMF of all cultures exposed for 3h and 24h in the absence and present of S9 was less than 1.26E-04, the generally accepted average background mutant level for the ML assay making use of the version of microtiter plates (96 wells) for the determination of plating efficiency and mutant selection.

Conclusions:

The results of this study are negative, so it is concluded that the test item is non-mutagenic to the cultured L5178Y mouse lymphoma cells under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo mouse micronucleus test: negative

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:

experimental study

Adequacy of study:

key study

Study period:

21 November 2001 - 08 January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD, EC and US EPA test guidelines, and in compliance with GLP, so the data is considered reliable without restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Analytical purity: 99.96%
- Lot/batch No.: 011107

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 28 - 32 g (males) and 22 - 26 g (females)
- Assigned to test groups randomly: Yes
- Diet (e.g. ad libitum): Free access
- Water (e.g. ad libitum): Free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 55±15%
- Photoperiod (hrs dark / hrs light): 12 hours light per day

IN-LIFE DATES: From: 21 November 2001 To: 08 January 2002

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Corn oil
- Concentration of test material in vehicle: 100 or 50 mg/mL (preliminary study); 25, 50, or 100 mg/kg (Micronucleus test)
- Amount of vehicle (if gavage or dermal): 20 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions of the test substance in corn oil were prepared freshly on the day of use.

Duration of treatment / exposure:

A single oral dose was administered. Observation or sacrifice times were as follows:
48 hours (preliminary study)
24 hours (micronucleus test); in addition, additional animals in the negative control group and highest test group were sacrificed 48 hours after exposure

Frequency of treatment:

N/A (A single dose was administered)

Post exposure period:

Refer above for details.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Preliminary test - 2 males and 2 females per group (2 groups)
Micronucleus test - 7 males per dose in the 500 and 1000 mg/kg dose groups; 14 males in the control and 2000 mg/kg dose groups.

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Route of administration: Oral gavage (solution in purified water)
- Doses / concentrations: 12 mg/kg (solution concentration = 0.6 mg/mL)

Tissues and cell types examined:

Bone marrow from femurs

Details of tissue and slide preparation:

The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 ml of pre-filtered foetal calf serum. The cells were sedimented by
centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner (Schmid 1976). Three smears were made from each animal. The prepared smears were fixed in methanol (> 10 minutes). After air-drying the smears were stained for 10 minutes in 10% Giemsa (prepared by 1 : 9 dilution of Giemsa with purified water). Following rinsing in purified water and differentiation in buffered purified water, the smears were rinsed in purified water, air-dried and mounted with coverslips using DPX.

Evaluation criteria:

The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animal was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear.

Micronuclei were identified by the following criteria:
• Large enough to discern morphological characteristics
• Should possess a generally rounded shape with a clearly defined outline
• Should be deeply stained and similar in colour to the nuclei of other cells - not black
• Should lie in the same focal plane as the cell
• Lack internal structure, ie they are pyknotic
• There should be no micronucleus-like debris in the area surrounding the cell.

The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept as recommended by Schmid (1976).

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

A number of clinical signs were noted following administration at 1000 and 2000 mg/kg, but all animals in all groups survived to the scheduled sacrifice times.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Doses producing toxicity:
In the preliminary test, two groups of animals were dosed with the test substance at concentrations of 1000 and 2000 mg/kg. At 2000 mg/kg, clinical signs observed included underactivity, overactivity, flattened posture, abnormal gait, fast and irregular respiration, reduced righting reflexes. At 1000 mg/kg, clinical signs of underactivity, abnormal gait and fast respiration were observed. All animals survived to scheduled termination.

Results showed that a dose level of 2000 mg/kg was tolerated. This dose was therefore selected as an appropriate maximum for use in this test.

Observations: No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decreases in the proportion of immature erythrocytes were observed in mice treated with the test substance and killed 24 or 48 hours later, compared with the vehicle control values.

Conclusions:

It is concluded that the substance did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by gavage in this in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation:

A Bacterial reverse mutation test was conducted (Huntingdon Life Sciences, 2001, ZCE074) to examine the potential for the substance to cause gene mutation. The study was conducted according to OECD test guideline 471, EC method B13/14 and US EPA Guideline OPPTS 870.5100. The study was conducted in compliance with GLP.

Mutant strains of Salmonella Typhimurium (TA1535, TA1537, TA98, and TA100) and Escherichia Coli (WP2 uvrA pKM101) were exposed to the substance as a solution in Dimethylsulphoxide (DMSO) in both the presence and absence of metabolic activation.

The substance did not show significant signs of cytotoxicity at any of the levels tested, and showed no signs of mutagenic activity in any of the tester strains used, either in the presence or the absence of metabolic activation.

 

Chromosome aberration:

A study was conducted (Huntingdon Life Sciences, 2002, ZCE092) do determine the potential for the substance to cause chromosomal aberrations in Chinese Hamster Lung cells culturedin vitro. The study was conducted according to OECD Test Guideline 473, EU method B10, and US EPA Guideline OPPTS 870.5375, and in compliance with GLP.

A first test was conducted both in the presence and absence of metabolic activation, with 3 hours treatment with the substance, followed by 12 hours recovery. A second test was conducted using 15 hours continuous treatment in the absence of metabolic activity, and 3 hours treatment / 12 hours recovery in the presence of metabolic activation.

No significant increases in the incidence of chromosomal aberrations relative to the concurrent controls were observed at any dose level tested. It was concluded that the substance had not shown any evidence of clastogenic activity in this study.

 

In vitro gene mutation:

A study was performed to evaluate this substance for its ability to cause gene mutations in vitro in cultured mammalian cells after being exposed for 3 hours with and without metabolic activation, respectively, and exposed for 24 hours without metabolic activation. The method of this test was designed to be compatible with the OECD Guideline 490.

The mouse lymphoma L5178Y cells were exposed to six exposure concentrations including 2000, 800, 320, 128, 51.2 and 20.48 μg/ml at the three treatment conditions mentioned above.

The results of mutant frequency showed that the induced mutant frequencies (IMF) of all cultures exposed for 3h and 24h were consistently less than the average background mutant frequency of 1.26E-04. It is concluded that the test item, is non-mutagenic to the L5178Ymouse lymphoma cells under the test conditions of this study.

Mouse micronucleus test:

A study was conducted (Huntingdon Life Sciences, 2002, ZCE078) to assess the potential for the substsance to induce micronuclei in bone marrow cells in mice. The study was conducted according to OECD test guideline 474, EC method B12, and US EPA Guideline OPPTS 870.5395, and in compliance with GLP.

A preliminary toxicity study indicated that there was no difference in toxicity between sexes, and so the main micronucleus study was conducted in male mice. Groups of seven mice were administered a single oral dose of the substance in corn oil, then sacrificed 24 hours after dosing, and slides of bone marrow smears assessed. The dose groups assessed were 0 mg/kg (vehicle control), 500, 1000, and 2000 mg/kg. Seven additional mice were dosed in the negative control group and the highest dose group (2000 mg/kg) and sacrificed after 48 hours.

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decreases in the proportion of immature erythrocytes were observed in mice treated with the substance and killed 24 or 48 hours after dosing.

It was concluded that the substance did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by gavage in this test.

Justification for classification or non-classification

Of the four studies mentioned above, none indicated any evidence of mutagenic or clastogenic activity. On this basis it is concluded that there is no basis on which to classify the substance for mutagenic effects, this substance is not classified as mutagen according to CLP regulation 1272/2008 table 3.5.1.