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Genetic toxicity in vitro

Description of key information

Cyclacet tested for gene mutation in bacteria (OECD 471, GLP): Not mutagenic .

Cyclacet tested for gene mutations in mammalian cells (OECD 476, GLP): Not mutagenic

Cyclacet for cytogenicity is based on read across from Cyclobutanate, which was tested in OECD 473: Non-clastogenic.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2-23-2007 - 3-26-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
His-gene: Amino acid histidine, and Trp-gene: amino acid tryptophan
Species / strain / cell type:
other: S. typhimurium TA97a, TA98, TA100, TA 102, TA1535 and E. coli WP2 uvrA (328)
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: S. typhimurium TA97a, TA98, TA100: rfa, ∆uvrB, pKM101 S. typhimurium TA1535: rfa, ∆uvrB S. typhimurium TA102: rfa, pAQ1 Plasmid, pKM101 E. coli WP2 uvrA (328): ∆uvrB
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 induced by Aroclor 1254
Test concentrations with justification for top dose:
Plate incorporation assay: 5, 10, 50, 100, 500, 1000, 2500, and 5000 μg/plate
Preincubation assay: 5, 10, 50, 100, and 250 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Selection of the appropriate solvent and diluent for the test substance was based on solubility information provided by the Sponsor and on a solubility assessment at the testing facility.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
2-nitrofluorene
sodium azide
other: Without S9: ICRI91 Acridine mutagen: TA97a; N-Methyl-N'-nitro-N-nitroguanidine: E. coli WP2 uvrA. With S9: 2-Aminoanthracene TA98, TA100, TA1535, E.Coli
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3 plates per concentration

DETERMINATION OF CYTOTOXICITY
- Method: determination of a concentration related reduction in the mean number of revertants per plate and/or the reduction of the microcolony background lawns

OTHER EXAMINATIONS:
- Other: The dosage regimen was determined from a solubility study conducted prior to the experimental start.
Evaluation criteria:
A test substance was classified as positive if either of the first two conditions and the third condition were met:
1) The mean number of revertants in Salmonella strains TA97a, TA98, TA100, TA102 or Escherichia coli WP2 uvrA (328) at any test substance concentration was at least two times greater than the mean in the concurrent vehicle control.
2) The mean number of revertants in Salmonella strains TA1535 or TA1537 at any test substance concentration was at least three times greater than the mean in the concurrent vehicle control.
3) For a positive classification in any strain, there must have been a concentration-related increase in the mean revertants per plate in that same strain.

A test substance was classified as negative (e.g., nonmutagenic in bacteria) if the following conditions were met:
1) There were no test substance concentrations in Salmonella strains TA97a, TA98, TA100, TA102 or Escherichia coli WP2 uvrA (328) with a mean number of revertants that were at least two times greater than the mean in the concurrent vehicle control.
2) There were no test substance concentrations in Salmonella strains TAl535 or TAl537 with a mean number of revertants that were at least three times greater than the mean in the concurrent vehicle control.
3) For a negative classification in any strain, there could not be a concentration related increase in the mean revertants per plate in that same strain. In consultation with the sponsor, negative results may be confirmed as needed.

Results not meeting criteria for positive or negative classification were evaluated using scientific judgment and may have been reported as equivocal. In a two trial study, if one trial is positive and the other is negative, additional trials may be conducted.
Statistics:
Trials were evaluated independently. For each selected test strain, the average number of revertants and the standard deviation at each concentration level with and without the exogenous metabolic activation system was calculated.
Key result
Species / strain:
other: S. typhimurium TA97a, TA98, TA100, TA 102, TA1535 and E. coli WP2 uvrA (328)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: There was no evidence of substance-related precipitate at any concntration level


RANGE-FINDING/SCREENING STUDIES: Due to test substance associated toxicity in the plate incorporation assay, test substance concentrations were lowered for the confirmatory assay.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean number of revertants observed in the negative controls (DMSO) for each of the test strains was within acceptable historical negative control ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Plate incorporation assay: S. typhimurium TA97a, TA98, TA100 and TA1535 (± S9), and E. coli WP2 uvrA (–S9): ≥ 500 ug/plate; E. coli WP2 uvrA (+S9): ≥ 1000 µg/plate.
- Preincubation assay (–S9): S. typhimurium TA98: ≥ 100 ug/plate; S. typhimurium TA97a, TA100, TA1535, and E. coli WP2 uvrA (328): 250 µg/plate.
Conclusions:
Under the conditions of this study, no evidence of mutagenic activity was detected for the test substance, Cyclacet, in Salmonella typhimurium test strains TA97a, TA98, TA100, and TA1535 along with Escherichia coli strain WP2 uvrA (328). The findings of this study show the test substance to be negative for the induction of mutagenicity in the bacterial reverse mutation test.
Executive summary:

The test substance was evaluated in a bacterial reverse mutation assay performed according to OECD 471 and following GLP employing Salmonella typhimurium strains TA97a, TA98, TA100, and TA1535 along with Escherichia coli strain WP2 uvrA (328) both in the presence and absence of an exogenous metabolic activation system. The test substance was evaluated using an initial plate incorporation assay and a confirmatory preincubation procedure. The solvent, diluent and negative control used in this assay was dimethyl sulfoxide (DMSO). Test substance concentrations of 5, 10, 50, 100, 500, 1000, 2500, and 5000 ug per plate were assessed with respect to negative (solvent) controls in the plate incorporation assay. Due to test article associated toxicity, test substance concentrations were lowered for the confirmatory assay. Concentrations of 5, 10, 50, 100, and 250 ug per plate were assessed with respect to negative (solvent) controls in the confirmatory preincubation assay. In the plate incorporation assay, test substance associated toxicity, as evidenced by a concentration related reduction in the mean number of revertants per plate and/or the reduction of the microcolony background lawns, was observed at 500 µg per plate and above in S. typhimurium tester strains TA97a, TA98, TA100, and TA1535 with and without exogenous metabolic activation along with E. coli strain WP2 uvrA (328) without exogenous metabolic activation. Toxicity was observed at 1000 µg per plate and above in E. coli strain WP2 uvrA (328) with exogenous metabolic activation. Test substance associated precipitate was not observed at any concentration level. In the preincubation assay, toxicity was observed at 100 µg per plate and above in S. typhimurium test strain TA98 without exogenous metabolic activation and at 250 µg per plate in S. typhimurium strains TA97a, TA100, and TA1535 along with E. coli strain WP2 uvrA (328) without exogenous metabolic activation. No toxicity was observed at the tested concentrations for any strain with exogenous metabolic activation. The mean number of revertants observed in the negative controls (DMSO) for each of the test strains was within acceptable historical negative control ranges. All test strains demonstrated appropriate phenotypic characteristics. Under the conditions of this study, no evidence of mutagenic activity was detected for the test substance, Cyclacet, in S. typhimurium test strains TA97a, TA98, TA100, and TA1535 along with E. coli strain WP2 uvrA (328). The findings of this study show the test substance to be negative for the induction of mutagenicity in the bacterial reverse mutation test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 1, 2008 - February 10,2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
July 1997
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMlo Medium
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 (from Aroclor 1254 treated male Sprague Dawley rats)
Test concentrations with justification for top dose:
Range finding test: 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, 1000, and 5000 µg/mL (With and without S9)
Definitive Mutation Assay: 19, 28, 37, 46, 56, 65, 74, 83, 92, 102, 120, 130, 140, 150, 160, and 170 µg/mL (without S9); 109, 131, 175, 200, 225, 250, 275, and 300 µg/mL (with S9).
Repeat Definitive Mutation Assay: 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, and 500 µg/m (with S9)
Confirmatory Mutation Assay: 1.0, 5.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, and 140 µg/mL (without S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A miscibility test was performed using water and DMSO. The test article was not miscible in water and was readily miscible in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours except for the confirmatory mutation assay, which was 24 hours
- Expression time (cells in growth medium): Approx. 20 hours (confirmatory mutation assay) and 44 hours (other assays)
- Selection time: 11-12 days

SELECTION AGENT: Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Relative Suspension Growth (RSG, i.e., growth in culture after treatment compared to the growth in culture of the corresponding solvent control cultures)

OTHER EXAMINATIONS:
- Other: Colony sizes were determined by varying the size-setting potentiometer on an ARTEK 880 Colony Counter.
Evaluation criteria:
Validity criteria:
- The results for the solvent control were considered acceptable if the average CE of the solvent control cultures was 65% or higher, the average MF of the solvent controls was between 35 to 140 per 1E6 viable cells, and the suspension growth for the solvent controls was between 8 and 32 for the definitive assay and 23 to 180 for the Confirmatory assay
- The results for the positive control cultures were considered acceptable if at least one of the positive control-treated cultures had a MF that was three times or greater than the average MF of its solvent cultures and their solvent controls had an average CE of 65% or greater

A response was considered positive if at least one culture had a Mutation Frequency (MF = mutants per 1E6 viable cells cloned) that was two times or greater than the average MF of the corresponding solvent control cultures and the response was dose dependent.
A response was considered negative if:
- All of the cultures exhibiting a Total Growth (TG) of approximately 10% and greater had MFs that were less than twice that of the mean MF of the corresponding solvent control cultures, and
- There was no evidence of a dose-dependent response.
A response was considered equivocal if it did not fulfill the criteria of either a negative or a positive response, and/or the Study Director did not consider the response to be either positive or negative.
Statistics:
No statistical methods were used in analyzing the data.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: The solvent controls' Mutation frequency (MF) values were within an acceptable range of SITEK's historical solvent control values. The size distribution for the cultures treated with the positive controls, MMS and DMBA, exhibited an acceptable positive response and colony size distribution.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The Relative Suspension Growth (RSG) in the Range Finding Test for cultures treated with Cyclacet without activation indicated that Cyclacet was toxic at 50 ug/mL and above. Cultures treated with 50 µg/mL had 16% RSG. The cultures treated with higher concentrations had 0% RSG. The RSG for cultures treated with Cyclacet with S-9 activation indicated that Cyclacet was completely toxic, i.e., 0% RSG, at 500 µg/mL and above. The culture treated with 100 ug/mL had 74% RSG.
- In the Definitive Mutation Assay the Relative Total Growth (RTG) for the cloned cultures ranged from 17% to 99% for cultures treated without activation and from 42% to 90% for cultures treated in conjunction with exogenous activation. Since it is ideal to have some cloned cultures that have between 10% and 30% RTG for evaluating a test articles mutagenic potential, a repeat assay with activation was conducted. The RTG for these cultures ranged from 0% to 104%.
- In the Confirmatory Mutation Assay, the RTG for the cloned cultures ranged from 3% to 59%.
Conclusions:
The test article was tested in the L5178Y TK+ Mouse Lymphoma Mutagenesis Assay both in the presence and absence of exogenous metabolic activation. All of the cloned cultures treated with the test substance in both the Definitive and Confirmatory Mutation Assays had mutant frequencies that were similar to that of their corresponding solvent control cultures. Therefore, under the test conditions, the test article is negative in the L5178Y TK+ Mouse Lymphoma Mutagenesis Assay.
Executive summary:

The test article was tested for its potential to induce mutations at the thymidine kinase locus of L5178Y TK+ mouse lymphoma cells in an OECD 476 study performed according to GLP. The concentrations of test article tested with and without S-9 activation in the Range Finding Test were 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, 1000, and 5000 µg/mL. Relative Suspension Growth (RSG) was used to measure toxicity. The RSG for cultures without activation indicated that the test substance was toxic at 50 µg/mL and above. Cultures treated with 50 µg/mL had 16% RSG. The cultures treated with higher concentrations had 0% RSG. The RSG for cultures treated with S-9 activation indicated that the test substance was completely toxic, i.e., 0% RSG, at 500 µg/mL and above. The culture treated with 100 µg/mL had 74% RSG.

The Definitive Mutation Assay was performed using a 4-hour treatment period at test article concentrations ranging from 19 to 170 µg/mL without activation and from 109 to 300 µg/mL with S-9 activation. Cultures were selected for cloning for mutant selection based on their RSG. All of the cloned cultures, both with and without activation, had Mutant Frequencies (MF) that were similar to the average MF of their concurrent solvent control cultures. The Relative Total Growth (RTG) for the cloned cultures ranged from 17% to 99% for cultures treated without activation and from 42% to 90% for cultures treated in conjunction with exogenous activation. Since it is ideal to have some cloned cultures that have between 10% and 30% RTG for evaluating a test articles mutagenic potential, a repeat assay with a 4-hour exposure period with activation was conducted. Cultures were treated with concentrations ranging from 200 to 500 µg/mL with 20 µg/mL increments between doses. The results for the repeat of the with S-9 activation portion of the Definitive Mutation Assay also showed that the treated cultures all had MFs that were similar to the average MF of the solvent control cultures. The RTG for these cultures ranged from 0% to 104%. Under the test conditions, the results of the Definitive Mutation Assay are considered negative. The Confirmatory Mutation Assay was conducted without activation with a 24-hour exposure period. Cultures were treated with concentrations between 1.0 and 140 µg/mL. The cultures treated with 30 to 140 µg/mL were cloned for mutant selection. All of the cultures had MFs that were similar to the average MF of the solvent controls. The RTG for the cloned cultures ranged from 3% to 59%. Under the test conditions, the results of the Definitive and Confirmatory Mutation Assays are considered negative (i.e. the test substance is not mutagenic).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 January 2002 - 27 May 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not relevant
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagles Minimal Essential Media
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Male Sprague-Dawley rat liver S9 induced by Phenobarbitone/β-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1: 0*, 4.35, 8.7*, 17.3*, 34.5*, 51.7, 68.9 µg/mL (-S9); 0*, 68.9, 137.7*, 275.4*, 550.8*, 826.15, 1101.5 µg/mL (+S9)
Experiment 2: 0*, 2.175, 4.35, 8.7*, 17.3*, 34.5*, 51.7 µg/mL (-S9, 24 hours); 0*, 2.175, 4.35*, 8.7*, 17.3*, 34.5, 51.7 µg/mL (-S9, 48 hours); 0*, 68.9, 137.7*, 275.4*, 550.8*, 661.0, 826.15 µg/mL (+S9)
* Dose levels selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material soluble at 10 mM concentration
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1 (Short Term Treatment Test): 6-hour exposure, both with and without S9 mix.
Experiment 2 (Continuous and Short Term Treatment Test): 24-hour continuous exposure without S9 mix; 48-hour continuous exposure without S9 mix, and a repeat of the 6-hours exposure with S9 mix.
- Fixation time (start of exposure up to fixation or harvest of cells):
Experiment 1 (Short Term Treatment Test): 18 hours
Experiment 2 (Continuous and Short Term Treatment Test): 24 and 48 hours (continuous treatment), and 18 hours (repeat of short term treatment test with S9 mix).

SPINDLE INHIBITOR: Colcemid, 0.1 μg/mL, last 2 hours of incubation.
STAIN: Giemsa solution

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads from each culture.

DETERMINATION OF CYTOTOXICITY
- Method: Cell Growth Inhibition Test: a preliminary toxicity test was performed on cell cultures using 24 and 48-hour continuous exposure times without S-9 mix, and a 6-hour exposure period both with and without S-9 mix, followed by an 18-hour recovery period. The dose range used was 8.7 to 2203 µg/mL. Precipitate observations were noted at the beginning and end of the treatment period. Growth inhibition was estimated by counting the number of cells at the end of the culture period on an electronic cell counter, and expressing the cell count as a percentage of the concurrent vehicle control value. Slides were also prepared to check for the presence, number and quality of cells in metaphase. Selected dose levels were scored for mitotic index.

OTHER EXAMINATIONS:
- Determination of polyploidy: Cells with 38 or more chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported.
- Determination of endoreplication: Endoreduplicated cells are recorded and are included in the polyploid cell total number. If there was a dose-related increase in endoreduplicated cells then they are reported separately.
Evaluation criteria:
Where increases in the frequency of cells with aberrations were seen, statistical comparisons were made with the vehicle. A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test or Chi-squared test if the control frequency of cells with aberrations was 5 or greater. If the study gives a positive response then a D20 value will be calculated, which is the presumed dose level of the test substance that is required to induce aberrations in 20% of metaphases.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Short Term Treatment Test - Experiment 1: A precipitate of the test material was not observed at the end of the treatment period in either treatment group. This may have been because the fine precipitate of test material was not easily observable with the naken eye. Therefore, the maximum dose selected for metaphase analysis was based on toxicity and was 34.5 µg/mL for the treatment without S9 and 550.8 µg/mL for with S9 exposure group.
- Continuous and Short Term Treatment Test - Experiment 2: A precipitate of the test material was observed at the end of the treatment period in the 6(18)-hour group at and above 550.8 µg/mL, whereas no precipitate was observed at the dose levels used in the 24- and 48-hour groups.

RANGE-FINDING/SCREENING STUDIES: Cell Growth Inhibition Test: In all cases the test substance showed evidence of cell toxicity. A precipitate of the test material was seen at and above 275.4 µg/mL. Metaphases were present at dose levels up to 34.5 µg/mL in the 6(18)-hour without-S9 exposure group, and at up to 550.8 µg/mL in the 6(18)-hour with-S9 exposure group. The maximum dose with metaphases present in the 24-hour and 48-hour continuous exposures was 34.5 µg/mL. Due to the toxic nature of the test substance, cultures were discarded at and above 137.7 µg/mL in the groups without metabolic activation and at and above 1101.5 µg/mL in the groups with metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: The aberration levels in the solvent controls were normal for the laboratory. The frequencies of breaks were within the normal historical control level for this laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In both Experiment I and II Cyclobutanate demonstrated similar toxicity to that observed in the Cell Growth Inhibition Test and the data demonstrates a steep toxicity curve.
- Short Term Treatment Test - Experiment 1: The cell count data show that more than 50% growth inhibition was achieved at 34.5 µg/mL in the absence of S9, and an approximate 50% growth inhibition was achieved at 550.8 µg/mL in the presence of S9. Therefore, it was considered that adequate toxicity had been achieved.
- Continuous and Short Term Treatment Test - Experiment 2: The test substance did not achieve an approximate 50% growth inhibition in the 24-hour exposure group but the maximum scorable dose level was 34.5 µg/mL and there were no scorable metaphases at 51.7 µg/mL. In the 48-hour exposure group an approximate 60% reduction in growth index was observed in the cell count data and again at 34.5 µg/mL, but there were no scorable metaphases. In the 6(18)-hour treatment, dose selection was as in Experiment 1. The inclusion of additional intermediate dose levels was used to attempt to achieve an approximate 50% inhibition ideal. A steep toxicity curve was observed, with 44% growth inhibition of the cell count data. It was considered that adequate toxicity had been achieved for all exposure groups in Experiment 2.
Conclusions:
The test substance did not induce any toxicologically significant, dose-related increases in the frequency of cells with chromosome aberrations, either in the presence or absence of a liver enzyme metabolising system, or after various exposure times. The test substance was therefore considered to be non-clastogenic to CHL cells in vitro.
Executive summary:

In this in vitro chromosome aberration test performed according to OECD 473 and following GLP, the potential chromosomal mutagenicity of the test substance on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line was determined. Duplicate cultures of CHL cells were treated with the test substance at several dose levels, together with vehicle (DMSO) and positive controls. Five exposure groups were used: Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolising system; Experiment 2 included a 24-hour continuous exposure without metabolic activation, a 48-hour continuous exposure without metabolic activation and a repeat of the 6(18)-hours exposure with metabolic activation. The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 8.70 to 34.5 µg/mL for the 6(18)-hour exposure, without S9, 137.7 to 550.8 µg/mL for the with-S9 exposure, in both Experiment 1 and 2, and 4.35 to 34.5 µg/mL for the 24 and 48 -hour treatments. The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any toxicologically significant increases in the frequency of cells with aberrations in any of the exposure groups. The test substance was shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all exposure groups. The test substance was shown to be non-clastogenic to CHL cells in vitro.

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

Additional information

In this section the experimental information on genotoxicity of Cyclacet and Cyclobutanate is presented first. Thereafter the read across justification is presented.

Cyclacet in Ames
The test substance was evaluated in a bacterial reverse mutation assay performed according to OECD 471 and following GLP employing Salmonella typhimurium strains TA97a, TA98, TA100, and TA1535 along with Escherichia coli strain WP2 uvrA (328) both in the presence and absence of an exogenous metabolic activation system. The test substance was evaluated using an initial plate incorporation assay and a confirmatory preincubation procedure. The solvent, diluent and negative control used in this assay was dimethyl sulfoxide (DMSO). Test substance concentrations of 5, 10, 50, 100, 500, 1000, 2500, and 5000 ug per plate were assessed in the plate incorporation assay and due to test article associated toxicity concentrations of 5, 10, 50, 100, and 250 ug per plate were assessed in the confirmatory preincubation assay. In both assays, test substance associated toxicity, as evidenced by a concentration related reduction in the mean number of revertants per plate and/or the reduction of the microcolony background lawns, was observed. The mean number of revertants observed in the negative controls for each of the test strains was within acceptable historical negative control ranges. All test strains demonstrated appropriate phenotypic characteristics. Under the conditions of this study, no evidence of mutagenic activity was detected for the test substance, Cyclacet, in S. typhimurium test strains TA97a, TA98, TA100, and TA1535 along with E. coli strain WP2 uvrA (328). The findings of this study show the test substance to be negative for the induction of mutagenicity in the bacterial reverse mutation test.

Cyclacet in vitro gene mutation assay with mammalian cells

The test article was tested for its potential to induce mutations at the thymidine kinase locus of L5178Y TK+ mouse lymphoma cells in an OECD 476 study performed according to GLP. The concentrations of test article tested with and without S-9 activation in the Range Finding Test were 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, 1000, and 5000 µg/mL. Relative Suspension Growth (RSG) was used to measure toxicity. The RSG for cultures without activation indicated that the test substance was toxic at 50 µg/mL and above. Cultures treated with 50 µg/mL had 16% RSG. The cultures treated with higher concentrations had 0% RSG. The RSG for cultures treated with S-9 activation indicated that the test substance was completely toxic, i.e., 0% RSG, at 500 µg/mL and above. The culture treated with 100 µg/mL had 74% RSG.

The Definitive Mutation Assay was performed using a 4-hour treatment period at test article concentrations ranging from 19 to 170 µg/mL without activation and from 109 to 300 µg/mL with S-9 activation. Cultures were selected for cloning for mutant selection based on their RSG. All of the cloned cultures, both with and without activation, had Mutant Frequencies (MF) that were similar to the average MF of their concurrent solvent control cultures. The Relative Total Growth (RTG) for the cloned cultures ranged from 17% to 99% for cultures treated without activation and from 42% to 90% for cultures treated in conjunction with exogenous activation. Since it is ideal to have some cloned cultures that have between 10% and 30% RTG for evaluating a test articles mutagenic potential, a repeat assay with a 4-hour exposure period with activation was conducted. Cultures were treated with concentrations ranging from 200 to 500 µg/mL with 20 µg/mL increments between doses. The results for the repeat of the with S-9 activation portion of the Definitive Mutation Assay also showed that the treated cultures all had MFs that were similar to the average MF of the solvent control cultures. The RTG for these cultures ranged from 0% to 104%. Under the test conditions, the results of the Definitive Mutation Assay are considered negative. The Confirmatory Mutation Assay was conducted without activation with a 24-hour exposure period. Cultures were treated with concentrations between 1.0 and 140 µg/mL. The cultures treated with 30 to 140 µg/mL were cloned for mutant selection. All of the cultures had MFs that were similar to the average MF of the solvent controls. The RTG for the cloned cultures ranged from 3% to 59%. Under the test conditions, the results of the Definitive and Confirmatory Mutation Assays are considered negative (i.e. the test substance is not mutagenic).

Cyclobutanate In vitro cytogenicity assay with mammalian cells

No cytogenicity study was available for Cyclacet. However, an available study on the structural similar substance Cyclobutanate could be used for read across.

In this in vitro chromosome aberration test performed according to OECD 473 and following GLP, the potential chromosomal mutagenicity of the test substance on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line was determined. Duplicate cultures of CHL cells were treated with the test substance at several dose levels, together with vehicle (DMSO) and positive controls. Five exposure groups were used: Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolising system; Experiment 2 included a 24-hour continuous exposure without metabolic activation, a 48-hour continuous exposure without metabolic activation and a repeat of the 6(18)-hours exposure with metabolic activation. The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 8.70 to 34.5 µg/mL for the 6(18)-hour exposure, without S9, 137.7 to 550.8 µg/mL for the with-S9 exposure, in both Experiment 1 and 2, and 4.35 to 34.5 µg/mL for the 24 and 48 -hour treatments. The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any toxicologically significant increases in the frequency of cells with aberrations in any of the exposure groups. The test substance was shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all exposure groups. The test substance was shown to be non-clastogenic to CHL cells in vitro.

Cyclacet (EC no.911-369-0)and its absence ofcytogenicity in mammalian cells using read across fromCyclobutanate(EC no.441-420-8)

Introduction and hypothesis for the read across

Cyclacet has a tricyclodecenyl fused ring backbone structure to which an ethyl (acetic) ester is attached.For this substance gene mutation data in bacteria and gene mutation data in mammalian cells, but no cytogenicity data in mammalian cells is available. Therefore, additional information is used in accordance with Article 13 of REACH where it is said thatlacking information can be used by methods different from experimental testing such as (Q)SARs, grouping and read-across. In the present case read across is applied from Cyclobutanate.

Hypothesis:Cyclacet has the same in vitro cytogenicity results as Cyclobutanate.

Available information: For Cyclacet the gene mutation information is available (OECD TG 471 and 476). For Cyclobutanate a well conducted chromosome aberration test (OECD TG 473, Klimisch 1) is available.

Target and Source chemical(s):

The information on substance target and the analogue information from substance source are presented in the data matrix. Also, relevant physico-chemical properties are listed there.

Purity / Impurities:

Cyclacet is a reaction masses containing a mixture of two very similar isomers (5-yl and 6-yl). The impurities are all below 1%.

Analogue justification

According REACH Annex XI 1.5, an analogue approach can be used to replace testing when information from different sources provides sufficient evidence to conclude that this substance has or does not have a particular dangerous property. The result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation, which is presented below.

Analogue selection: For Cyclacet the analogue Cyclobutanate is selected based on similarity in chemical structure and for Cyclobutanate an in vitro chromosome aberration test is available.

Structural similarities and differences: The target and the source chemical both have a tricyclodecenyl fused ring structure with an unsaturated bond in the outside ring, which can be on the 5yl or 6 yl position. Both have an ester bond as a functional group with a short alkyl chain. The alkyl chain of Cyclacet is an ethyl chain while the source Cyclobutanate has a butyl chain, this difference is further assessed in the reactivity section.

Kinetic profile: Both Cyclacet and Cyclobutanate have a similar kinetic profile based on chemical structure, functional groups and physico-chemical properties.

Reactivity: The Cyclacet ethyl alkyl chain compared to the Cyclobutanate butyl alkyl chain will be similarly insensitive to genotoxicity, because the protrusive effect of two additional CH groups of the source chemical towards the ester bond is expected to be minimal. The QSAR Application Toolbox further supports the non-DNA binding.

Remaining uncertainties:There are no remaining uncertainties other than those presented above.

Conclusions on in vitro cytogenicity

For Cyclacet a gene mutation test in bacteria (OECD 471) and in mammalian cells (OECD 476) is available, which are both negative. A (in vitro) cytogenicity, however, is missing. For the structural analogue Cyclobutanate such information is available. This information can be used for read across. When using read across the information should be accompanied with adequate and reliable documentation and this is presented in the current document. Cyclobutanate is negative in an in vitro cytogenicity study OECD 473) and this can be applied to Cyclacet.

Final conclusion: Cyclacet is negative for in vitro cytogenicity.

 

Data matrix Cyclacet in vitro cytogenicity read across from Cyclobutanate

 

Name

Cyclacet

Cyclobutanate

 

Target

Source

Molecular structure

 

 

Cas no.

2500-83-6 (5-yl)

54830-99-8 (generic)

1361017-07-3(5-yl)

113889-23-9

EC no.

911-369-0

441-420-8

REACH registration

Registered

Registered

Molecular weight

192

220

Physico-chemical properties

 

 

Appearance

Liquid

Liquid

Water solubility

186

11.5

Log Kow

3.9

4.48

Human health

 

 

Genemutations in bacterial cells (Ames)

Negative

(OECD TG 471)

Negative

(OECD TG 471)

Genemutations in mammalian cells

Negative

(OECD TG 476)

No data available

In vitro cytogenicity (chromosomal aberration)

Read across from Cyclobutanate

Negative

(OECD TG 473

 

Justification for classification or non-classification

Based on the information presented above the substance is not mutagenic and therefore does not need to be classified according to EU CLP (EC 1272/2008 and its amendments).