4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch Number: 9000479770
Appearance: light yellow liquid
Storage: 1-10°C in the dark
Purity: 86.9%
Expiry Date: 4 Feb 2003

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post--mitochondrial fraction (S-9) - prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Range finding: 1.6, 8, 40, 200, 1000 and 5000 μg/plate
Experiment 1: 1.6, 8, 40, 200, 1000 and 5000 μg/plate
Experiment 2: perfromed up to an estimate of the lower limit of toxicity 1250 μg/plate for stain TA 1537 in the absence of S-9 and strain TA102 in the absence or presence of S-9) or up to 5000 μg/plate (all other treatments). Dose ranges of 78.13 to 5000 μg/plate and 19.52 to 1250 μg/plate were employed)

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: Glutaraldehyde (for TA102 without S-9), 2-aminoanthracene (for TA100, TA1535, TA1537, TA102 with S-9)

Evaluation criteria:

The test article was considered to be mutagenic if:
1. The assay was valid (see below)
2. Dunnett's test gave a significant response (p=<0.01) and the data set(s) showed a significant dose correlation
3. The positive responses described above were reproducible

Acceptance criteria
The assay was considered valid if the following criteria were met:
1. the mean negative control counts fell within the normal ranges as deinfined in Appendix 3
2. the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
3. no more than 5% of the plates ere lost through contamination or some other unforeseen event

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

It was concluded that Kephalis did not induce mutation in five histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1537 and TA102) when tested under the conditions of this study. The conditions included treatments at concentrations up to either 5000 μg/plate, or the lower limit of toxicity, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Qualifier:

according to guideline

Guideline:

other: European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.49 “InVitro Mammalian Cell Micronucleus Test". Official Journal of the European Union No. L142

Version / remarks:

Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identification: Kephalis
Chemical Name (IUPAC): 4-(1-ethoxyvinyl)-3,3,5,5,-tetramethylcyclohexanone (main component)
Molecular formula: C14H24O2
Molecular weight: 224.4
CAS Number: 36306-87-3
EC Number: 252-961-2
Description: Clear colourless to slightly yellow liquid (determined at WIL Research Europe B.V.)
Batch: PE00089447
Purity/Composition: 86.1% (sum of two peaks, 78.8% and 7.3%)
Test substance storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 22 November 2014 (expiry date)

Target gene:

none

Species / strain / cell type:

lymphocytes: cultured peripheral human

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

In the first cytogenetic assay, Kephalis was tested up to 100 and 125 μg/ml - Toxicity was reached at these dose levels.
In the second cytogenetic assay, Kephalis was tested up to 60 μg/ml - Appropriate toxicity was reached at this dose level.

Vehicle / solvent:

Kephalis was dissolved in dimethyl sulfoxide of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany). Kephalis concentrations were used within 2 hours after preparation.
The final concentration of the solvent in the culture medium was 1.0% (v/v).
No correction was made for the purity/composition of the test substance.

Untreated negative controls:

yes

Remarks:

vehicle: DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchine

Details on test system and experimental conditions:

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline.

Blood was collected from healthy adult, non-smoking, male volunteers (aged < 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2013) are presented below:

Dose range finding study: age 25, AGT = 12.9 h
First cytogenetic assay: age 22, AGT = 12.8 h
Second cytogenetic assay: age 31, AGT = 13.5 h

Cell Culture

Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Invitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ltd., United Kingdom) was added.

Environmental conditions
All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 52 - 90%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.9 - 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage occurred that were caused by opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.


Metabolic activation system
Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox); 4 μmol HEPES (Invitrogen Corporation). The above solution was filter (0.22 μm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v)
S9-fraction) to complete the S9-mix.

Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Evaluation criteria:

Acceptability of assay
An in vitro micronucleus test was considered acceptable if it meets the following criteria:
a) The positive control substance colchicine produced at least a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number mononucleated cells with micronuclei and the positive control substances MMC-C and CP produced at least a statistically significant increase in the number of binucleated cells with micronuclei.
b) A homogeneous response between the duplicate cultures is observed.

a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Species / strain:

lymphocytes: cultured peripheral human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

It is concluded that this test is valid and that Kephalis is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

Principles of method if other than guideline:

1. In the first experiment in the absence of S9-mix, cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Evaluation: Mistake in the exposure medium. The use of R10 medium instead of R5 medium is of no influence on the results of the study.
The study integrity was not adversely affected by the deviation

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

Specific details on test material used for the study:

Test substance information: TS 205540/A
Identification: Kephalis
Chemical Name (IUPAC): 4-(1-ethoxyvinyl)-3,3,5,5,-tetramethylcyclohexanone (main component)
Molecular formula: C14H24O2
Molecular weight: 224.4
CAS Number: 36306-87-3
EC Number: 252-961-2
Description: Clear colourless to slightly yellow liquid (determined at WIL Research Europe B.V.)
Batch: PE00089447
Purity/Composition: 86.1% (sum of two peaks, 78.8% and 7.3%)
Test substance storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 22 November 2014 (expiry date)

Test substance information: TS 205540/B
Identification: Kephalis
Chemical Name (IUPAC): 4-(1-ethoxyvinyl)-3,3,5,5,-tetramethylcyclohexanone (main component)
Molecular formula: C14H24O2
Molecular weight: 224.4
CAS Number: 36306-87-3
EC Number: 252-961-2
Description: Pale yellow liquid
Batch: PE00097828
Purity/Composition: 85.2% (sum of two peaks)
Test substance storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 14 March 2015 (expiry date)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells -Recommended test system in international guidelines (e.g. OECD, EC) and literature (see chapter 9 for references).
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

In the first experiment, Kephalis was tested up to concentrations of 90 μg/ml in the absence and presence of S9-mix
In the second experiment, Kephalis was tested up to concentrations of 65 μg/ml in the absence of S9-mix.
Since Kephalis was poorly soluble in the exposure medium, the highest tested concentration was 512 μg/ml exposure medium.

Vehicle / solvent:

The test substance was dissolved in dimethyl sulfoxide (SeccoSolv, Merck Darmstadt, Germany). Amber-coloured glassware or tubes wrapped in tin-foil were used when preparing the test solutions.
Kephalis concentrations were used within 2 hours after preparation. The final concentration of the solvent in the exposure medium was 1.0% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale: Recommended test system in international guidelines (e.g. OECD, EC) and literature (see chapter 9 for references).
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).

Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 106 cells/ml.

Cell culture
Horse serum
Horse serum (Invitrogen Corporation) was inactivated by incubation at 56°C for at least 30 minutes.

Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Invitrogen), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Invitrogen Corporation).

Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).

Exposure medium

For 3 hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium), except in the first mutation test in the absence of S9-mix. In this part of the study, the cells were exposed to the test substance in R10-medium (see protocol deviation 1).

For 24 hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).

Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).

Environmental conditions
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 48 – 89%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.3 – 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

Metabolic activation system
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix components contains per ml: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES. The above solution was filter (0.22 μm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 (ref. 12).

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Acceptability of the assay
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 106 survivors, and for CP not below 700 per 106 survivors.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Kephalis is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range (See APPENDIX 3, Table 13). The growth rate over the two-day expression period for cultures treated with DMSO was between 13 and 19 (3 hours treatment) and 98 and 109 (24 hours treatment) (See APPENDIX 2). Mutation frequencies in cultures treated with positive control chemicals were increased by 10- and 9.9-fold for MMS in the absence of S9-mix, and by 14-fold for CP in the presence of S9-mix (See Table 3 and Table 4). It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay (See APPENDIX 3, Table 14). In the absence of S9-mix, Kephalis did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Kephalis did not induce a significant increase in the mutation frequency. In conclusion, Kephalis is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)