Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: non mutagenic (WoE)

- MLA: non mutagenic (read-across, OECD 476, GLP, K, rel. 2)

- CAT: non clastogenic (read-across, OECD 473, K, rel.2)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 1989-03-10 to 1989-04-04
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Principles of method if other than guideline:
Ames test
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
not applicable
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor induced male rats
Test concentrations with justification for top dose:
8 to 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: none
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Remarks:
2-NF 2.5 µg/plate for TA 98 & TA 1538. SA 2.5 µg/plate for TA 100 and TA 1535. 9-AA 50 µg/plate for TA 1537
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
no data
Statistics:
no data
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
above 200-400 µg/plate
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
none

None

Conclusions:
The test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA1538, TA98 & TA100.
Executive summary:

In a reverse gene mutation assay S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed the test material diluted in DMSO both in the presence and absence of metabolic activation system (S9 fraction from the livers of Aroclor 1254 treated rats) using the plate incorporation method. The dose range was determined was 8 to 5000 µg/plate.

No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. 

It is therefore concluded that, under the test conditions, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA1538, TA98 & TA100.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2012-11-03 to 2012-11-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 471 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspection date: 2012-07-10)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene for S. thyphimurium and tryptophan gene for E.coli
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):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (S9-fraction obtained from the livers of rats induced with phenobarbitone/B-naphtoflavone)
Test concentrations with justification for top dose:
- Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
- Experiment 1:
E. coli: 50, 150, 500, 1500 and 5000 μg/plate .
TA 1537: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
TA100, TA1535, TA98: 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
- Experiment 2:
E. coli: 15, 50, 150, 500, 1500 and 5000 μg/plate .
TA 1537: 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
TA100, TA1535, TA98: 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.2.1/1
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.2.1/1
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
WIth S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 in agar (plate incorporation), Experiment 2 preincubation

DURATION
- Preincubation period (Exp 2): 20 minutes at 37°C.
- Exposure duration: ca. 48 hours at 37°C.

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: growth assessment of the bacterial background lawn

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay may be considered valid if the following criteria are met:
1. All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
2. All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to the historical control for 2010 & 2011).
3. All tester strain cultures should be in the approximate range of 0.9 to 9 billion bacteria per mL.
4. Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation (according to historical positive controls for 2010 & 2011).
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one or all of the following may be used to determine the overall result of the study, weighing the results in terms of their biological significance. The criteria are listed in order of priority:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
As recommended by UKEMS (Mahnon et al (1989).
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See "Table of results" in Attached background material
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: the test material is not classified as a VOC.
- Water solubility: the test material was solubilised in DMSO to improve solubility.
- Precipitation: None observed.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: The test item was toxic to TA100 from 500 µg/plate and non-toxic to WP2uvrA. The
test item formulation and S9-mix used in this experiment were both shown to be sterile. See table 7.6.1/2.

COMPARISON WITH HISTORICAL CONTROL DATA:
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test item induced toxicity to all of the Salmonella strains as weakened bacterial background lawns, initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. No toxicity was noted to Escherichia coli strain WP2uvrA. The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Table 7.6.1/2: Test results: Preliminary toxicity test

Metabolic activation

Strain

Dose (µ/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

+

TA100

84

74

77

81

80

67

65

63

64T

57T

69T

-

82

65

81

82

73

87

74

97

81T

77T

61T

+

WP2

uvrA-

28

34

30

27

29

21

32

21

25

21

31

-

33

35

27

32

24

31

28

34

33

21

26

Conclusions:
The test item is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA were exposed the test item diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate. The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 0.5 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9 -mix. The experiment was repeated on a separate day (pre-incubation method) using an amended dose range (between 0.15 and 5000 μg/plate), fresh cultures of the bacterial strains and fresh test item formulations.

Additional dose levels and an expanded dose range were selected in both experiments (where applicable) in order to achieve four non-toxic dose levels and the toxic limit of the test item.

The vehicle (dimethtl sulfoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item induced toxicity to all of the Salmonella strains as weakened bacterial background lawns, initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. No toxicity was noted to Escherichia coli strain WP2uvrA. The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of Doysia, either with or without metabolic activation or exposure method

 

Under the test condition, the test item is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA98 & TA100, and in E.coli WP2 uvrA.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

The supporting substance and the registered substance only differ in the ratio of the isomers, therefore the analogue approach is justified (see §Toxicokinetics for read-across justification).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2008-10-02 to 2009-03-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 471 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon for S. typhimurium and tryptophan operon for E. coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction (S9-mix obtained from livers of Aroclor 1254-induced rats)
Test concentrations with justification for top dose:
- Range finding test: 5, 10, 50, 100, 500, 1000 and 5000 µg/plate.
- Definitive mutation assay: 5, 10, 20, 30 and 50 µg/plate for S. typhimurium -S9 / 250, 500, 1000, 2500 and 5000 µg/plate for E. coli -S9 / 10, 50, 100, 250 and 500 µg/plate for S. typhimurium +S9 / 250, 500, 1000, 2500 and 5000 µg/plate for E. coli +S9.
- Confirmatory mutation assay: 20, 30, 50, 100 and 500 µg/plate for S. typhimurium -S9 / 250, 500, 1000, 2500 and 5000 µg/plate for E. coli -S9 / 50 , 100, 250, 500 and 1000 µg/plate for S. typhimurium +S9 / 250, 500, 1000, 2500 and 5000 µg/plate for E. coli +S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Coniferan was not miscible in H2O. It was miscible in DMSO at 50 mg/mL and formed a clear solution.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
see Table 7.6.1/2
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
without S-9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
see Table 7.6.1/2
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S-9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48 to 72 hours at 37 +/- 1 °C

NUMBER OF REPLICATIONS: triplicate plates per dose level

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency
Evaluation criteria:
A response was considered to be positive if either strain TA98 or TA100 exhibited a mean reversion frequency that was a t least double that the mean reversion frequency of the corresponding solvent control in at least one concentrations, or if either strain TA1537, TA1537 or WP2 uvrA exhibited a three-fold increase in the mean reversion frequency compared to the solvent control in at least one concentration. In addition, the response must be concentration-dependent (i.e. increasing mean reversion frequencies observed over increasing test concentrations). In evaluating the results, consideration was given to the degree of toxicity exhibited by the concentration causing the 2 to 3-fold or greater increase in reversion frequency and the magnitude of the increase in reversion frequency.
A response was considered to be negative if all of the strains treated with the test article had mean reversion frequencies that were less than twice that of the mean reversion frequencies of the corresponding solvent control plates in TA98 and TA100 and less than three times in TA1535, TA1537 and WP2 uvrA, and there was no evidence of a concentration-dependent response.
A response was considered equivocal if it did not fulfil 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 data
Key result
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:
cytotoxicity
Remarks:
at 500 µg/plate -S9
Vehicle controls validity:
other: yes, except that the reversion frequency of the TA98 solvent control was slightly higher than SITEK historical range
Untreated negative controls validity:
not examined
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:
cytotoxicity
Remarks:
at 5000 µg/plate -S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: NA
- Effects of osmolality: NA
- Evaporation from medium: Test material vapour pressure is too low to expect a significant effect of evaporation on test results
- Water solubility: Test substance was solubilised in DMSO to improve solubility
- Precipitation: None observed
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: (See Table 1 & 2 in Attached background material)
TA100: the relative cloning efficiencies (RCEs) at the concentrations of 5.0 to 5000 µg/plate without activation ranged from 0% to 95%. Significantly decreased RCE values (< 50%) were observed at 50 µg/plate and above. Marked thinning of background lawn was also observed at 500 µg/plate and above but the revertant frequencies were not significantly decreased at any of dose levels. In the presence of the activation system, the RCEs at the concentrations of 5.0 to 5000 µg/plate range from 1% to 176%. Significant decreases in RCS (<50%) and the revertant frequency were observed at test concentration and above. Noticeable thinning of background lawn was also found at 1000 µg/plate and above. No precipitate was observed at any of the test concentrations.
WP2 uvrA: the RCEs at the concentrations of 5.0 to 5000 µg/plate without activation ranged from 206% to 362%. This may be the result of the low revertant frequency observed in the solvent control. No significant decreases in RCEs were observed at any dose level. A significant decrease revertant frequency and the absence of a background lawn were observed at 5000 µg/plate. In the presence of metabolic activation system, the RCEs at the concentrations of 5.0 to 5000 µg/plate ranged from 77% to 140%. No significant decreases in RCEs (<50%) or effects on the background lawn were found at any dose level. Compared to the solvent control the decreased number of revertant frequency that was observed at some dose levels was not the result of toxicity but rather due to the number of revertant frequency of the solvent control group being higher that SITEK historical range for solvent controls. No precipitate was observed at any of the tests concentrations.

None

Conclusions:
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of Coniferan, either with or without metabolic activation.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 & TA 100 and E.coli strain WP2 uvrA were exposed to Coniferan diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method.

The dose range for the definitive mutation assay was determined in a preliminary toxicity assay and was as follow: 5 to 50 µg/plate for S. typhimurium -S9; 10 to 500 µg/plate for S. typhimurium +S9; 250 to 5000 µg/plate for E. coli +/- S9.

A confirmatory mutation assay followed using the following dose range: 20 to 500 µg/plate for S. typhimurium -S9; 50 to1000 µg/plate for S. typhimurium +S; 9; 250 to 5000 µg/plate for E. coli +/- S9. With S. typhimurium, an additional dose level was included to allow for potential test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range, except that the number of revertant per plate for the solvent control for TA98 was slightly higher than the SITEK historical control data. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of Coniferan, either with or without metabolic activation.

 

Under the test condition, Coniferan is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA98 & TA100, and E.coli WP2 uvrA.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

The source substance is a mixture of isomers, including 15.4% of the target substance.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2008-10-30 to 2008-12-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 473 and in compliance with GLP. Some deviations to protocol were observed but they had no impact on the results or conclusion.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
: insufficient number of cells in metaphase was available to the analyse of chromosome aberrations in the Exp 2 at 35 µg/mL and for the positive control in one of the both culture. 200 metaphases were therefore observed in only culture.
Qualifier:
according to
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Deviations:
yes
Remarks:
: insufficient number of cells in metaphase was available to the analyse of chromosome aberrations in the Exp 2 at 35 µg/mL and for the positive control in one of the both culture. 200 metaphases were therefore observed in only culture.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A Medium (Gibco-BRL) supplemented or not with fetal bovine serum (Gibco-BRL)
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
- other: doubling time of approximately 12 hours
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction obtained from rat liver induced by phenobarbital-5,6-Benzoflavone (phenobarbital/beta-naphtaflavone) and the cofactor pool (Moltox, lot No. 2331)
Test concentrations with justification for top dose:
Range finding study (3+15 hrs): 0; 0.5; 5; 10; 50; 100; 500; 1000 and 5000 µg/mL with and without metabolic activation
Main study 1 (3+15 hrs): 0; 10; 20; 30; 35; 40 and 45 µg/mL without metabolic activation / 0; 25; 50; 60; 70; 80; 90 µg/mL with metabolic activation
Main study 2 (continuous exposure of 18 hrs): 0; 10; 20; 25; 30 and 35 µg/mL without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test article was not miscible with water. 50 mg of test article was miscible with DMSO in 0.1 mL final volume, resulting in a final concentration of 500 µg/mL. DMSO was selected as the solvent. Test article in DMSO and complete medium in a final test article concentration of 5000 µg/mL resulted in a clear solution.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 to 0.8 µg/mL
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
7.5 and 12.5 µg/mL
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours (in the range finding study and the main study 1) or 18 hours (in the Main study 2)
- Expression time (cells in growth medium): 18 hrs
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 18h

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/mL) added 2h before harvesting cells
STAIN (for cytogenetic assays): Methanol:glacial acetic acid (3:1)

NUMBER OF REPLICATIONS: 2 cultures in parallel

NUMBER OF CELLS EVALUATED: 100 cells of each culture (total of 200 cells/concentration)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
To consider that the assay is valid:
In the solvent control, the percentage of cells with aberrations should not exceed 4%.
At least 25% of cells scored in the positive control should show one or more chromosome aberrations.
At least one of the test concentrations scored should show approximately 50% reduction in the RCG and/or RMI. This requirement should not be applied to test articles where no apparent toxicity could be achieved at the maximum soluble concentration or the highest allowable concentration.

For the evaluation of the results:
The test article was considered to have caused a positive response in this assay if the test article showed a positive concentration-response trend and a statistically significant increase over that of the solvent controls in the percentage of cells with aberrations at one or more concentrations.
The test article was considered to have caused a negative response if none of the test concentrations showed a statistically significant increase in the percentage of aberrant cells.
The test article was considered to have caused an equivocal response if there was a statistically significant increase in the percentage of cells with aberrations without an accompanying positive concentration-response trend.
Statistics:
Chi-square tests were used to compare the percentage of cells with aberrations for each concentrations with the solvent control values (significant if p <= 0.05). Statistical analysis was not performed if the test concentration value was equal to or less than the concurrent or historical solvent control value.
If positive response with the Chi-square test, the Cochran-Armitage test (trend test) was performed for evidence of a concentration-related response (significant if p <= 0.05)
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH of the test item in the medium was checked and no adjustment of pH of the treatment medium was necessary.
- Effects of osmolality:not determined
- Evaporation from medium: not determined
- Water solubility: the test item solubilised in DMSO was then soluble in the complete medium, resulting in a clear solution.
- Precipitation: no precipitation was observed during the test

RANGE-FINDING/SCREENING STUDIES: significant toxicity was observed at 50 µg/mL and above without metabolic activation and at 100 µg/mL and above with metabolic activation as revealed by a decrease of the relative cell growth (RCG). Relative Mitotic index (RMI) are not available above 10 µg/mL without activation and above 50 µg/mL with activation because there were not enough cells to drop slides.

COMPARISON WITH HISTORICAL CONTROL DATA: the data obtained for the untreated (water) and the vehicle are consistent with the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in the confirmatory assay (Exp 2) the RCG (Relative cell growth) for the test article concentrations of 10-35 µg/mL ranged from 52-105% and the RMI (Relative Mitotic index) ranged from 65-205%. The reason for the relatively high RMI at certain concentration levels is a result of the low relative MI for the solvent control cultures. The reason for this low MI is unknown.

OTHER: The percentage of endoreduplicated cells was higher than the normal range (0-1%) and the historical data of the lab. However, CHO cells have a tendency to endoreduplicate even if not clearly visible in the historical data. Moreover they are a normal phenomenon, common in liver, salivary glands, etc. Endoreduplicated cells are not a marker of aneugenicity.

Table 7.6.1/2:Chromosomal aberration study results. The results are presented for the 2x100 evaluated cells.

Processing time (hrs)

S9 mix

Doses (µg/mL)

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other*

% cells with aberrations

Gap

% of polyploid cells

% of cells with endoredu

plication

Cell Proliferation

RCG (%)

RMI (%)

3-18

-

Untreated

0

0

0

0

0

0.0

0

0

0.5

87

128

DMSO

1

0

0

0

0

0.5

0

0

1.0

100

100

10

0

0

0

0

0

0.0

0

0

0.0

101

108

20

0

0

0

0

0

0.0

0

0

0.0

98

110

30

2

0

0

0

0

1.0

0

0.5

1.0

99

45

35

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

41

29

40

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

9

N/D

45

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

4

N/D

MMC (0.4)

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

95

19

MMC (0.8)

30

94

18

4

8

45.5

1

1.0

1.0

61

32

+

 

Untreated

1

0

0

0

0

0.5

0

0.5

0.5

96

113

DMSO

1

0

0

0

0

0.5

0

0.0

0.0

100

100

25

0

0

0

0

0

0.0

0

0.0

2.0

135

88

50

1

0

0

0

0

0.5

0

1.0

4.5

91

121

60

0

0

0

0

0

0.0

0

1.5

3.5

86

70

70

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

88

49

80

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

33

N/D

90

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

4

N/D

CP (7.5)

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

77

16

CP (12.5)

35

93

20

2

11

44.0

0

1.5

0.5

58

13

18-0

-

Untreated

0

0

1

0

0

0.5

0

1.0

0.0

113

168

DMSO

1

0

0

0

0

0.5

0

2.0

0.0

100

100

10

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

105

110

20

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

104

130

25

0

0

0

0

0

0.0

0

2.0

0.0

91

205

30

0

0

0

0

0

0.0

0

3.0

1.5

101

143

35

1

0

0

0

0

0.5

0

1.0

5.0

52

65

MMC (0.2)

20

23

9

1

1

26.0

0

0.0

0.0

83

37

MMC (0.4)

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

72

12

*:pulverized cells and severely damaged cells (i.e. cell with 10 or more aberrations)

N/D: No data

RCG : Relative cell growth = (Number of cells in test flask/Number of cells in solvent flask) x 100

RMI: Relative Mitotic index = (Test concentration MI / Solvent MI) x 100 (where MI = Number of dividing cells from 1000 cells / 10)


Conclusions:
Under the test conditions, Coniferan induced a negative response in the chromosomal aberration test using hamster cells.
Executive summary:

In a chromosomal aberration assay in mammalian cells, performed according to the OECD No.473,  Coniferan (99.3% purity) diluted in Dimethylsulphoxide (DMSO) was tested in Chinese Hamster Ovary (CHO) cells in the presence and the absence of mammalian metabolic activation (S9) for 3 or 18hrs at concentrations from 10 to 90 µg/mL.

A range-finding study was performed in order to evaluate the cytotoxicity of the test item and to determine the appropriate concentrations for the main test. Cytotoxicity was assessed by the calculation of the percentage of RCG (Relative cell growth) and of RMI (Relative mitotic index).

Coniferan was incubated with the cells in the first experiment for 3 hrs without S9 mix at concentrations of 10; 20; 30; 35; 40 and 45 µg/mL and with S9 mix at the concentrations of 25; 50; 60; 70; 80 and 90 µg/mL. After the exposure, the cells were washed and re-incubated in fresh culture medium for 15hrs. The cells were then harvest. Colcemid (0.1 µg/mL) was added 2hrs before harvesting cells in order to inhibit the spindle allowing therefore the observation of the chromosomes in the cells in metaphase. In a second experiment, the cells were incubated with Coniferan for 18hrs in the absence of metabolic activation at the concentrations of 10; 20; 25; 30 and 35 µg/mL. The analysis of the presence of chromosomal aberrations was in this case performed after the end of the exposure period.

Mitomycin C and Cyclophosphamide were used as positive controls and induced appropriate responses.

Coniferan was cytotoxic to CHO cells. No increase in the occurrence of chromatid or chromosome structural aberration was observed with and without metabolic activation and for all exposure periods tested. The number of polyploid cells was in the normal range.

The percentage of endoreduplicated cells was higher than the normal range (0-1%) and the historical data of the lab. However, CHO cells have a tendency to endoreduplicate even if not clearly visible in the historical data. Moreover they are a normal phenomenon, common in liver, salivary glands, etc. Endoreduplicated cells are not a marker of aneugenicity.

Under the test conditions, Coniferan induced a negative response in the chromosomal aberration test using hamster cells. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 473.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2009-02-23 to 2009-05-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 476 and in compliance with GLP with minor deviations: mycoplasma contamination checking and pH measurements not reported. These deviations had no impact on the results or conclusion.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
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 locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source: Jan Clarke and Richard Han, BioReliance, Rockville, Maryland
- Type and identity of media: 50% RPMI0, 50% RPMI10 (RPMI0 medium supplemented with 10% heat-inactivated horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: not applicable7
- Periodically "cleansed" against high spontaneous background: yes, by exposure to THMG for 24 hours and growth in THG for an additional 24hours .
Additional strain / cell type characteristics:
other: TK +/- 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor 1254-induced rat liver homogenate
Test concentrations with justification for top dose:
- Range finding test: 5, 10, 20, 30, 40, 50, 100 and 200 µg/mL
- Definitive mutation assay: 2*, 5*, 8*, 11*, 14*, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44 and 47 µg/mL without S9; 25, 40*, 55*, 70*, 85*, 100*, 115*, 130, 145, 160, 175, 190, 205, 220,235, 250 and 275 µg/mL with S9.
- Confirmatory mutation assay: 5*, 8*, 11*, 14*, 17*, 20*, 23*, 26*, 29, 32, 35, 38, 41, 44, 47 and 50 µg/mL without S9.
(* =cloned)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not miscible in water and readily miscible in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: no
- Exposure duration: 4 hours +/-S9 in the definitive mutation assay; 24 hours -S9 in the confirmatory mutation assay.
- Expression time (cells in growth medium): 44 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): Trifluorothymidine

NUMBER OF REPLICATIONS: 1

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth

OTHER: Colony sizes were determined in solvent and control groups
Evaluation criteria:
A response was considered positive if at least one culture had a MF 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 consider negative if all of the cultures exhibiting TG of approximately 10%and greater has 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 fulfil 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:
None
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 applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not tested above 10 mM
- Evaporation from medium: Test material vapour pressure is too low to expect a significant effect of evaporation on test results.
- Water solubility: Coniferan was diluted in DMSO to improve solubility.
- Precipitation: None observed
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES:
The RSG for cultures treated with Coniferan without activation indicated that Coniferan was completely toxic (0% RSG) at 30 µg/mL. The cultures treated with 20, 10, and 5 µg/mL have RSG of 69%, 69% and 85%, respectively. The RSG for cultures treated with Coniferan with S-9 activation indicated that Coniferan was completely toxic, i.e. 0% RSG, at 100 µg/mL. The cultures treated with 50, 40, 30, 20, 10 and 5 µg/mL had RSG of 21%, 24%, 35%, 59%, 71%, and 80%, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The positive control MMS and DMBA produced positive responses. The solvent controls’ 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:
- Definitive mutation assay: the RTG for the cloned cultures ranged from 47% to 67% for cultures treated without activation (it is ideal to have a treated culture with 10% to 20% RTG; however this test article had a very precipitous dose-response. The culture treated with 14 µg/mL had 46% RSG while the culture treated with 17 µg/mL had 0% RSG and was not clonable due to insufficient cells ) and for 24% to 88% for cultures treated in conjunction with exogenous activation.
- Confirmatory mutation assay: the RTG for the cloned cultures ranged from 14% to 73%.

Table 7.6.1/1: Definitive mutation assay

 

Treatment (µg/mL

-S9 (4-hour)

Treatment (µg/mL

+S9 (4-hour)

%RSG

%RTG

MF/1.0E+cells

%RSG

RTG

MF

Solvent

100

100

137

Solvent

100

100

102

2.0

82

63

186

40

80

88

111

5.0

81

63

206

55

74

65

150

8.0

63

63

182

70

68

63

121

11

82

67

189

85

72

50

167

14

46

47

150

100

43

35

143

17*

0

NA

-

115

17

14

173

* This concentration was highly toxic and therefore there were insufficient cells for cloning.

 

Table 7.6.1/2: Confirmatory mutation assay

 

Treatment (µg/mL

-S9 (24-hour)

%RSG

%RTG

MF/1.0E+cells

Solvent

100

100

98

5.0

74

73

74

8.0

60

43

108

11

65

49

138

14

41

46

108

17

36

40

92

20

34

39

84

23

35

36

102

26

11

14

105

Conclusions:
The test material did not induce any statistically significant or dose-related increases in the mutant frequency at the TK +/- locus in L5178Y cells and was therefore considered to be non-mutagenic under the conditions of the test.
Executive summary:

In a mammalian cell gene mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, L5178Y TK±mouse lymphoma cells were exposed to Coniferan diluted in DMSO, in the presence and absence of metabolic activation (S-9). Concentrations were selected from a preliminary cytotoxicity assay. The definitive mutation assay was performed using a 4-hour treatment period at concentrations ranging from 2.0 to 47 µg/mL without activation and from 25 to 100 µg/mL with S-9 activation. The confirmatory mutation assay was conducted without activation with a 24-hour exposure period with concentrations ranging from 5.0 to 50 µg/mL. The maximum dose level used was limited by cytotoxicity.

 

The vehicle (solvent) controls gave acceptable levels of mutant frequencies for the L5178Y cell line at the TK +/- locus.

 The positive control treatments, MMS and DMBA, both in the absence and presence of metabolic activation, induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

Coniferan did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. It was therefore considered to be non-mutagenic under the conditions of the test.

 

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

The supporting substance is a mixture of isomers, including 15.4% of 4-tert-butylcyclohexyl acetate.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substances have similar physico-chemical and toxicological properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances both contain the same two isomers, in a different ratio: the target substance is a multi-constituent, while the source substance is defined as a mono-constituent, the second constituent being an impurity.

3. ANALOGUE APPROACH JUSTIFICATION
No (reverse) gene mutations were detected in the Ames test performed on both the source and the target substances. The Ames test on the source substance provided in this dossier was performed according to OECD TG 471. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities
Therefore, based on the considerations above, it can be concluded that the result of the Ames test conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.1.

4. DATA MATRIX
Cf. Iuclid Section 13.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See "Table of results" in Attached background material
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: the test material is not classified as a VOC.
- Water solubility: the test material was solubilised in DMSO to improve solubility.
- Precipitation: None observed.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: The test item was toxic to TA100 from 500 µg/plate and non-toxic to WP2uvrA. The
test item formulation and S9-mix used in this experiment were both shown to be sterile. See table 7.6.1/2.

COMPARISON WITH HISTORICAL CONTROL DATA:
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test item induced toxicity to all of the Salmonella strains as weakened bacterial background lawns, initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. No toxicity was noted to Escherichia coli strain WP2uvrA. The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Table 7.6.1/2: Test results: Preliminary toxicity test

Metabolic activation

Strain

Dose (µ/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

+

TA100

84

74

77

81

80

67

65

63

64T

57T

69T

-

82

65

81

82

73

87

74

97

81T

77T

61T

+

WP2

uvrA-

28

34

30

27

29

21

32

21

25

21

31

-

33

35

27

32

24

31

28

34

33

21

26

Conclusions:
The source substance (and by analogy the target substance) is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA were exposed the test item diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate. The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 0.5 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9 -mix. The experiment was repeated on a separate day (pre-incubation method) using an amended dose range (between 0.15 and 5000 μg/plate), fresh cultures of the bacterial strains and fresh test item formulations.

Additional dose levels and an expanded dose range were selected in both experiments (where applicable) in order to achieve four non-toxic dose levels and the toxic limit of the test item.

The vehicle (dimethtl sulfoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item induced toxicity to all of the Salmonella strains as weakened bacterial background lawns, initially from 150 and 500 µg/plate in the absence and presence of S9-mix respectively. No toxicity was noted to Escherichia coli strain WP2uvrA. The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of Doysia, either with or without metabolic activation or exposure method

 

Under the test condition, the source substance (and by analogy the target substance) is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA98 & TA100, and in E.coli WP2 uvrA.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

The source and the supporting substance only differ in the ratio of the isomers, therefore the analogue approach is justified (see §Toxicokinetics for read-across justification).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substances have similar physico-chemical and toxicological properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is a multi-constituent. The source substance is defined as a multi-constituent, including the target substance as an impurity present at 15.4%.

3. ANALOGUE APPROACH JUSTIFICATION
 In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on both the source and the target substances.
 In vitro gene mutation study in mammalian cells
In the MLA test performed on the source substance, none of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
 In vitro cytogenicity study in mammalian cells
In the CAT performed on the source substance, no significant increases in the frequency of cells with aberrations was induced. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the MLA and CAT conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.2 & 8.4.3.

4. DATA MATRIX
Cf. Iuclid Section 13. 
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
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:
cytotoxicity
Remarks:
at 500 µg/plate -S9
Vehicle controls validity:
other: yes, except that the reversion frequency of the TA98 solvent control was slightly higher than SITEK historical range
Untreated negative controls validity:
not examined
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:
cytotoxicity
Remarks:
at 5000 µg/plate -S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: NA
- Effects of osmolality: NA
- Evaporation from medium: Test material vapour pressure is too low to expect a significant effect of evaporation on test results
- Water solubility: Test substance was solubilised in DMSO to improve solubility
- Precipitation: None observed
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: (See Table 1 & 2 in Attached background material)
TA100: the relative cloning efficiencies (RCEs) at the concentrations of 5.0 to 5000 µg/plate without activation ranged from 0% to 95%. Significantly decreased RCE values (< 50%) were observed at 50 µg/plate and above. Marked thinning of background lawn was also observed at 500 µg/plate and above but the revertant frequencies were not significantly decreased at any of dose levels. In the presence of the activation system, the RCEs at the concentrations of 5.0 to 5000 µg/plate range from 1% to 176%. Significant decreases in RCS (<50%) and the revertant frequency were observed at test concentration and above. Noticeable thinning of background lawn was also found at 1000 µg/plate and above. No precipitate was observed at any of the test concentrations.
WP2 uvrA: the RCEs at the concentrations of 5.0 to 5000 µg/plate without activation ranged from 206% to 362%. This may be the result of the low revertant frequency observed in the solvent control. No significant decreases in RCEs were observed at any dose level. A significant decrease revertant frequency and the absence of a background lawn were observed at 5000 µg/plate. In the presence of metabolic activation system, the RCEs at the concentrations of 5.0 to 5000 µg/plate ranged from 77% to 140%. No significant decreases in RCEs (<50%) or effects on the background lawn were found at any dose level. Compared to the solvent control the decreased number of revertant frequency that was observed at some dose levels was not the result of toxicity but rather due to the number of revertant frequency of the solvent control group being higher that SITEK historical range for solvent controls. No precipitate was observed at any of the tests concentrations.

None

Conclusions:
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of Coniferan, either with or without metabolic activation.
By analogy, the target substance is considered to be not mutagenic. The source substance is a mixture of isomers, including 15.4% of the target substance.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 & TA 100 and E.coli strain WP2 uvrA were exposed to Coniferan diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method.

The dose range for the definitive mutation assay was determined in a preliminary toxicity assay and was as follow: 5 to 50 µg/plate for S. typhimurium -S9; 10 to 500 µg/plate for S. typhimurium +S9; 250 to 5000 µg/plate for E. coli +/- S9.

A confirmatory mutation assay followed using the following dose range: 20 to 500 µg/plate for S. typhimurium -S9; 50 to1000 µg/plate for S. typhimurium +S; 9; 250 to 5000 µg/plate for E. coli +/- S9. With S. typhimurium, an additional dose level was included to allow for potential test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range, except that the number of revertant per plate for the solvent control for TA98 was slightly higher than the SITEK historical control data. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of Coniferan, either with or without metabolic activation.

 

Under the test condition, Coniferan is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA98 & TA100, and E.coli WP2 uvrA.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

By analogy, the target substance is considered to be not mutagenic. The source substance is a mixture of isomers, including 15.4% of the target substance.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substances have similar physico-chemical and toxicological properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is a multi-constituent. The source substance is defined as a multi-constituent, including the target substance as an impurity present at 15.4%.

3. ANALOGUE APPROACH JUSTIFICATION
 In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on both the source and the target substances.
 In vitro gene mutation study in mammalian cells
In the MLA test performed on the source substance, none of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
 In vitro cytogenicity study in mammalian cells
In the CAT performed on the source substance, no significant increases in the frequency of cells with aberrations was induced. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the MLA and CAT conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.2 & 8.4.3.

4. DATA MATRIX
Cf. Iuclid Section 13. 
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH of the test item in the medium was checked and no adjustment of pH of the treatment medium was necessary.
- Effects of osmolality:not determined
- Evaporation from medium: not determined
- Water solubility: the test item solubilised in DMSO was then soluble in the complete medium, resulting in a clear solution.
- Precipitation: no precipitation was observed during the test

RANGE-FINDING/SCREENING STUDIES: significant toxicity was observed at 50 µg/mL and above without metabolic activation and at 100 µg/mL and above with metabolic activation as revealed by a decrease of the relative cell growth (RCG). Relative Mitotic index (RMI) are not available above 10 µg/mL without activation and above 50 µg/mL with activation because there were not enough cells to drop slides.

COMPARISON WITH HISTORICAL CONTROL DATA: the data obtained for the untreated (water) and the vehicle are consistent with the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in the confirmatory assay (Exp 2) the RCG (Relative cell growth) for the test article concentrations of 10-35 µg/mL ranged from 52-105% and the RMI (Relative Mitotic index) ranged from 65-205%. The reason for the relatively high RMI at certain concentration levels is a result of the low relative MI for the solvent control cultures. The reason for this low MI is unknown.

OTHER: The percentage of endoreduplicated cells was higher than the normal range (0-1%) and the historical data of the lab. However, CHO cells have a tendency to endoreduplicate even if not clearly visible in the historical data. Moreover they are a normal phenomenon, common in liver, salivary glands, etc. Endoreduplicated cells are not a marker of aneugenicity.

Table 7.6.1/2:Chromosomal aberration study results. The results are presented for the 2x100 evaluated cells.

Processing time (hrs)

S9 mix

Doses (µg/mL)

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other*

% cells with aberrations

Gap

% of polyploid cells

% of cells with endoredu

plication

Cell Proliferation

RCG (%)

RMI (%)

3-18

-

Untreated

0

0

0

0

0

0.0

0

0

0.5

87

128

DMSO

1

0

0

0

0

0.5

0

0

1.0

100

100

10

0

0

0

0

0

0.0

0

0

0.0

101

108

20

0

0

0

0

0

0.0

0

0

0.0

98

110

30

2

0

0

0

0

1.0

0

0.5

1.0

99

45

35

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

41

29

40

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

9

N/D

45

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

4

N/D

MMC (0.4)

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

95

19

MMC (0.8)

30

94

18

4

8

45.5

1

1.0

1.0

61

32

+

 

Untreated

1

0

0

0

0

0.5

0

0.5

0.5

96

113

DMSO

1

0

0

0

0

0.5

0

0.0

0.0

100

100

25

0

0

0

0

0

0.0

0

0.0

2.0

135

88

50

1

0

0

0

0

0.5

0

1.0

4.5

91

121

60

0

0

0

0

0

0.0

0

1.5

3.5

86

70

70

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

88

49

80

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

33

N/D

90

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

4

N/D

CP (7.5)

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

77

16

CP (12.5)

35

93

20

2

11

44.0

0

1.5

0.5

58

13

18-0

-

Untreated

0

0

1

0

0

0.5

0

1.0

0.0

113

168

DMSO

1

0

0

0

0

0.5

0

2.0

0.0

100

100

10

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

105

110

20

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

104

130

25

0

0

0

0

0

0.0

0

2.0

0.0

91

205

30

0

0

0

0

0

0.0

0

3.0

1.5

101

143

35

1

0

0

0

0

0.5

0

1.0

5.0

52

65

MMC (0.2)

20

23

9

1

1

26.0

0

0.0

0.0

83

37

MMC (0.4)

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

N/D

72

12

*:pulverized cells and severely damaged cells (i.e. cell with 10 or more aberrations)

N/D: No data

RCG : Relative cell growth = (Number of cells in test flask/Number of cells in solvent flask) x 100

RMI: Relative Mitotic index = (Test concentration MI / Solvent MI) x 100 (where MI = Number of dividing cells from 1000 cells / 10)


Conclusions:
Under the test conditions, Coniferan induced a negative response in the chromosomal aberration test using hamster cells.
By analogy, the target substance is considered to be not clastogenic. The source substance is a mixture of isomers, including 15.4% of the target substance.
Executive summary:

In a chromosomal aberration assay in mammalian cells, performed according to the OECD No.473,  Coniferan (99.3% purity) diluted in Dimethylsulphoxide (DMSO) was tested in Chinese Hamster Ovary (CHO) cells in the presence and the absence of mammalian metabolic activation (S9) for 3 or 18hrs at concentrations from 10 to 90 µg/mL.

A range-finding study was performed in order to evaluate the cytotoxicity of the test item and to determine the appropriate concentrations for the main test. Cytotoxicity was assessed by the calculation of the percentage of RCG (Relative cell growth) and of RMI (Relative mitotic index).

Coniferan was incubated with the cells in the first experiment for 3 hrs without S9 mix at concentrations of 10; 20; 30; 35; 40 and 45 µg/mL and with S9 mix at the concentrations of 25; 50; 60; 70; 80 and 90 µg/mL. After the exposure, the cells were washed and re-incubated in fresh culture medium for 15hrs. The cells were then harvest. Colcemid (0.1 µg/mL) was added 2hrs before harvesting cells in order to inhibit the spindle allowing therefore the observation of the chromosomes in the cells in metaphase. In a second experiment, the cells were incubated with Coniferan for 18hrs in the absence of metabolic activation at the concentrations of 10; 20; 25; 30 and 35 µg/mL. The analysis of the presence of chromosomal aberrations was in this case performed after the end of the exposure period.

Mitomycin C and Cyclophosphamide were used as positive controls and induced appropriate responses.

Coniferan was cytotoxic to CHO cells. No increase in the occurrence of chromatid or chromosome structural aberration was observed with and without metabolic activation and for all exposure periods tested. The number of polyploid cells was in the normal range.

The percentage of endoreduplicated cells was higher than the normal range (0-1%) and the historical data of the lab. However, CHO cells have a tendency to endoreduplicate even if not clearly visible in the historical data. Moreover they are a normal phenomenon, common in liver, salivary glands, etc. Endoreduplicated cells are not a marker of aneugenicity.

Under the test conditions, Coniferan induced a negative response in the chromosomal aberration test using hamster cells. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 473.

By analogy, the target substance is considered to be not clastogenic. The source substance is a mixture of isomers, including 15.4% of the target substance.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substances have similar physico-chemical and toxicological properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is a multi-constituent. The source substance is defined as a multi-constituent, including the target substance as an impurity present at 15.4%.

3. ANALOGUE APPROACH JUSTIFICATION
 In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on both the source and the target substances.
 In vitro gene mutation study in mammalian cells
In the MLA test performed on the source substance, none of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
 In vitro cytogenicity study in mammalian cells
In the CAT performed on the source substance, no significant increases in the frequency of cells with aberrations was induced. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the MLA and CAT conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.2 & 8.4.3.

4. DATA MATRIX
Cf. Iuclid Section 13. 
Reason / purpose:
read-across source
Reason / purpose:
reference to other study
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 applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not tested above 10 mM
- Evaporation from medium: Test material vapour pressure is too low to expect a significant effect of evaporation on test results.
- Water solubility: Coniferan was diluted in DMSO to improve solubility.
- Precipitation: None observed
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES:
The RSG for cultures treated with Coniferan without activation indicated that Coniferan was completely toxic (0% RSG) at 30 µg/mL. The cultures treated with 20, 10, and 5 µg/mL have RSG of 69%, 69% and 85%, respectively. The RSG for cultures treated with Coniferan with S-9 activation indicated that Coniferan was completely toxic, i.e. 0% RSG, at 100 µg/mL. The cultures treated with 50, 40, 30, 20, 10 and 5 µg/mL had RSG of 21%, 24%, 35%, 59%, 71%, and 80%, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The positive control MMS and DMBA produced positive responses. The solvent controls’ 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:
- Definitive mutation assay: the RTG for the cloned cultures ranged from 47% to 67% for cultures treated without activation (it is ideal to have a treated culture with 10% to 20% RTG; however this test article had a very precipitous dose-response. The culture treated with 14 µg/mL had 46% RSG while the culture treated with 17 µg/mL had 0% RSG and was not clonable due to insufficient cells ) and for 24% to 88% for cultures treated in conjunction with exogenous activation.
- Confirmatory mutation assay: the RTG for the cloned cultures ranged from 14% to 73%.

Table 7.6.1/1: Definitive mutation assay

 

Treatment (µg/mL

-S9 (4-hour)

Treatment (µg/mL

+S9 (4-hour)

%RSG

%RTG

MF/1.0E+cells

%RSG

RTG

MF

Solvent

100

100

137

Solvent

100

100

102

2.0

82

63

186

40

80

88

111

5.0

81

63

206

55

74

65

150

8.0

63

63

182

70

68

63

121

11

82

67

189

85

72

50

167

14

46

47

150

100

43

35

143

17*

0

NA

-

115

17

14

173

* This concentration was highly toxic and therefore there were insufficient cells for cloning.

 

Table 7.6.1/2: Confirmatory mutation assay

 

Treatment (µg/mL

-S9 (24-hour)

%RSG

%RTG

MF/1.0E+cells

Solvent

100

100

98

5.0

74

73

74

8.0

60

43

108

11

65

49

138

14

41

46

108

17

36

40

92

20

34

39

84

23

35

36

102

26

11

14

105

Conclusions:
The test material did not induce any statistically significant or dose-related increases in the mutant frequency at the TK +/- locus in L5178Y cells and was therefore considered to be non-mutagenic under the conditions of the test.
By analogy, the target substance is considered to be not mutagenic. The source substance is a mixture of isomers, including 15.4% of the target substance.
Executive summary:

In a mammalian cell gene mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, L5178Y TK±mouse lymphoma cells were exposed to Coniferan diluted in DMSO, in the presence and absence of metabolic activation (S-9). Concentrations were selected from a preliminary cytotoxicity assay. The definitive mutation assay was performed using a 4-hour treatment period at concentrations ranging from 2.0 to 47 µg/mL without activation and from 25 to 100 µg/mL with S-9 activation. The confirmatory mutation assay was conducted without activation with a 24-hour exposure period with concentrations ranging from 5.0 to 50 µg/mL. The maximum dose level used was limited by cytotoxicity.

 

The vehicle (solvent) controls gave acceptable levels of mutant frequencies for the L5178Y cell line at the TK +/- locus.

 The positive control treatments, MMS and DMBA, both in the absence and presence of metabolic activation, induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

Coniferan did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. It was therefore considered to be non-mutagenic under the conditions of the test.

 

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

By analogy, the target substance is considered to be not mutagenic. The source substance is a mixture of isomers, including 15.4% of the target substance.

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

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°

Test substance

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Uhlenbrock, 1989

4-tert-butylcyclohexyl acetate

Ames Test

WoE, rel. 4

Gene mutation

TA 1535,

TA 1537,

TA 1538,

TA 98,

TA 100

-S9

+S9

Up to 5000 µg/plate

-S9 : non mutagenic

+S9 : non mutagenic

2

 

Envigo, 2013

Cis-4-tert-butylcyclohexyl acetate

Ames Test

(OECD 471)

WoE, rel. 1

Gene mutation

TA 1535,

TA 1537

TA 98

TA 100

WP2 uvrA

-S9

+S9

Up to 5000 µg/plate

-S9 : non mutagenic

+S9 : non mutagenic

3

 

SITEK, 2009a

2-tert-butylcyclohexylacetate

Ames Test

(OECD 471)

WoE, rel. 1

Gene mutation

TA 1535,

TA 1537

TA 98

TA 100

-S9

+S9

Up to 5000 µg/plate or up to cytotoxic concentration

-S9 : non mutagenic

+S9 : non mutagenic

4

 

SITEK, 2009

2-tert-butylcyclohexylacetate

ML/TK test (OECD 476)

K, rel. 2

Gene mutation

mouse lymphoma L5178Y cells

-S9

+S9

-S9: up to 26 µg/mL

+S9: up to 115 µg/mL

-S9 : non mutagenic

+S9 : non mutagenic

5

 

SITEK, 2009b

2-tert-butylcyclohexylacetate

CHO CAT

(OECD 473)

K, rel. 2

Chromosomal aberration

Chinese Hamster Ovary cells

 

-S9

+S9

-S9: up to 45 µg/mL

+S9: up to 90 µg/mL

-S9 : non clastogenic

+S9 : non clastogenic

Gene mutation Assays (Tests n° 1 -4)

A bacterial reverse mutation assay (Ames test) was performed with 4-tert-butylcyclohexyl acetate (Test n°1). Since only a short abstract was available, a weight-of-evidence approach using studies conducted on supporting substances (isomers) was used to conclude on the potential of 4-tert-butylcyclohexyl acetate to induce gene mutation in bacteria. The read-across was deemed reliable based on the structural similarities of the substances and of their respective composition (non-negligible amount of the isomers of interests, cf. section 13 for detailled read-across justification):

- 4-tert butylcyclohexyl acetate is a multiconstituent substance (mixture of cis and trans isomers).

- Cis-4-tert-butylcyclohexyl acetate is a monoconstituent substance, nevertheless having up to 20% of the trans isomer.

- 2-tert butylcyclohexyl acetate is a muticonstituent substance, having approximately 15% of 4-tert butylcyclohexyl acetate (cis and trans isomers).

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in any tests, with any dose of test materials, either in the presence or absence of metabolic activation. All tests indicate that none of the isomers induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. 4-tert butylcyclohexyl acetate is therefore considered as non-mutagenic according to the Ames test.

No mammalian gene mutation test was located on 4-tert-butylcylclohexyl acetate. However, inability to produce gene mutation was confirmed on the supporting substance, 2-tert-butylcyclohexyl acetate using an in vitro forward mutation assay in mouse lymphoma TK L5178Y cells (ML/TK test) (Test n°4). None of the dose levels up to the cytotoxicity limit with 2-tert-butylcyclohexyl acetate, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. 2-tert-butylcyclohexyl acetate does not induce forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. 2-tert-butylcyclohexyl acetate, and by analogy 4-tert butylcyclohexyl acetate, are therefore considered as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames tests and extends the non-mutagenic effect of 4-tert-butylcyclohexyl acetate to mammalian cells.

Chromosomal aberration (Test n°5)

No mammalian chromosome aberration test was located on 4-tert-butylcylclohexyl acetate. However, the clastogenic potential of the supporting substance, 2-tert-butylcylclohexyl acetate, was determined using an in vitro chromosome aberration test in Chinese hamster ovary cells, which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured Chinese hamster ovary cells. None of the dose levels up to the cytotoxicity limit with 2-tert-butylcylclohexyl acetate, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. 2-tert-butylcylclohexyl acetate does not induce structural aberrations in the chromosomes of Chinese hamster ovary cells under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. 2-tert-butylcylclohexyl acetate, and by analogy 4-tert-butylcylclohexyl acetate, are therefore considered as negative for inducing chromosomal mutations in Chinese hamster ovary cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Based on the available information, no additional classification is proposed according to the CLP and the GHS.