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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in bacterial cells (Ames) according to OECD TG 471: Negative

Genetic toxicity in mamalian cells (MLA) according to OECD TG 476 using read-across from 4 -tert-butylcyclohexanol (CAS 98 -52 -2): Negative

Chromosomal aberration in vitro according to OECD TG 473 using read-across from 4 -tert-butylcyclohexanol (CAS 98 -52 -2): Negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Oct 2016 to 28 Oct 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo Research Limited., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD UK
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat S9
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat S9
Test concentrations with justification for top dose:
EXPERIMENT 1: PLATE INCORPORATION METHOD
- Dose selection
The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
- Justification of top dose
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.

EXPERIMENT 2; PRE-INCUBATION METHOD
- Dose selection:
In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. Consequently the maximum recommended dose level of the test item (5000 μg/plate) or the toxic limit was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix: Salmonella strain TA98 and E.coli strain WP2uvrA (with S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. All other bacterial strains (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate. The eight test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle:
dimethyl sulphoxide
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: preincubation

DURATION
- Preincubation period: 20 min (experiment 2)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

OTHER
- The plates were scored for the presence of revertant colonies using an automated colony counting system.
- The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
1. A dose-related increase in mutant frequency over the dose range tested.
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.
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.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. Consequently the maximum recommended dose level of the test item (5000 μg/plate) or the toxic limit was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. The test item induced a stronger toxic response in the second mutation test (pre-incubation method), with weakened bacterial background lawns noted in the absence of S9-mix from 150 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535, TA98 and WP2uvrA). In the presence of S9-mix weakened bacterial background lawns were noted from 500 μg/plate (all Salmonella strains) and 1500 μg/plate (WP2uvrA).

Small, statistically significant increases in TA100 revertant colony frequency were observed in the second mutation test at 15 and 50 μg/plate in the presence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.3 times the concurrent vehicle control.

Conclusions:
The substance is not mutagenic in the Salmonella Typhimurium and Escherichia Coli reverse mutation assay performed equivalent to OECD 471.
Executive summary:

The mutagenic activity of the test substance was evaluated in a study equivalent to OECD TG 471 (1997) and according to GLP principles. A plate incorporation assay (experiment 1) and a pre-incubation assay (experiment 2) were performed in the absence and presence of S9 mix. The dose levels were selected based on observed cytotoxicity in the plate incorporation dose range finding study. In the pre-incubation assay, the maximum dose level of the test item in the first experiment (plate-incorporation method) was selected as the maximum recommended dose level of 5000 μg/plate. The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. Consequently the maximum recommended dose level of the test item (5000 μg/plate) or the toxic limit was employed in pre-incubation test. Negative, solvent and positive controls were included and gave appropriate responses. The substance did not induce a significant dose related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 metabolic activation in both assay formats. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across from a guideline study
Justification for type of information:
For assessing the genetic toxicity of Orivone in mamalian cells, read-across is used from p-tert-butylcyclohexanol. In the file attached and in the endpoint summary the full read-across is documented.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across from a guideline study
Justification for type of information:
For assessing the clastogenicity of Orivone, read-across is used from p-tert-butylcyclohexanol. In the file attached and in the endpoint summary the full read-across is documented.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

For Orivone an Ames test according to OECD TG 471 is available. No data on genetic toxicity in mamalian cells or clastogenicity is avialable, therefore data is derived from 4 -tert-butylcyclohexanol (CAS 98-52-2), a close analogue. First the available studies are summarised below. Thereafter the read-across justification is presented. The accompanying files are attached in the study record.

Ames test: Genetic toxicity in bacterial cells according to OECD TG 471 with Orivone

The mutagenic activity of the test substance was evaluated in a study equivalent to OECD TG 471 (1997) and according to GLP principles. A plate incorporation assay (experiment 1) and a pre-incubation assay (experiment 2) were performed in the absence and presence of S9 mix. The dose levels were selected based on observed cytotoxicity in the plate incorporation dose range finding study. In the pre-incubation assay, the maximum dose level of the test item in the first experiment (plate-incorporation method) was selected as the maximum recommended dose level of 5000 μg/plate. The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. Consequently the maximum recommended dose level of the test item (5000 μg/plate) or the toxic limit was employed in pre-incubation test. Negative, solvent and positive controls were included and gave appropriate responses. The substance did not induce a significant dose related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 metabolic activation in both assay formats. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.

MLA test according to OECD TG 476 with 4 -tert-butylcyclohexanol

In a mammalian cell gene mutation assay conducted according to OECD guideline 476 and GLP, mouse lymphoma L5178Y cells cultured in vitro were exposed to the test substance. Based on a preliminary cytotoxicity study, concentrations of 15.63, 31.3, 62.5, 125 and 250 µg were selected for the main test, both in presence and absence of induced rat S9 metabolic activation. Incubations were performed, in two independent experiments with expression times of 3 hours (with metabolic activation) or 3 and 24 hours (without metabolic activation). After a two-day expression period cells were exposed to 3 µg/mL trifluorothymidine (TFT) for 11 to 14 days for mutant selection. In the main study, cytotoxicity was noted at the top concentration (250 µg/mL) in absence and presence of metabolic activation. The observed mutant frequencies were within the ranges of the negative (vehicle) control. Vehicle and positive controls gave results within the historical data-range, and hence no mutagenicity was observed. In addition, no changes in the ratio of small to large mutant colonies were observed, indicating that the test substance did not exhibit clastogenic potential at the concentrations tested. Therefore, under the test conditions, the test substance is negative in the L5178Y TK+/- mouse lymphoma mutagenesis assay.

Chromosomal aberration assay according to OECDTG473 with 4 -tert-butylcyclohexanol

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, Chinese hamster lung fibroblasts (V79) were exposed to the test substance with and without exogenous metabolic activation (Arochlor 1254 induced rat liver S9). For dose selection the substance was evaluated at 10 concentrations up to the highest soluble concentration (2000 µg/mL in 1% ethanol) both with and without metabolic activation. The test was performed in 2 independent experiments with exposure times of 18 or 18 and 28 hours. Positive control substances and a negative control were included. The dose level reducing the mitotic index to approximately 50% of the solvent control was used as highest dose for metaphase analysis. Both the negative and positive controls in the assays fulfilled the requirements of a valid test. The test substance did not induce biologically significant increases in the chromosomal aberration frequency and is therefore judged to be not clastogenic in vitro both in absence and presence of metabolic activation. 

Assessing the genetic toxicity of Orivone (CAS# 16587-71-6) using read-across from the multi-constituent p-tert-butylcyclohexanol (CAS# 98-52-2)

1. Introduction and hypothesis for the analogue approach

Orivone (CAS# 16587-71-6) has a cyclohexanone backbone substituted with a (1,1)dimethyl-propyl moiety on thepara-position. For this substance gene mutation data in bacteria (Ames test) is available. However, data on gene mutation in mammalian cells and information on clastogenicity is lacking. Therefore, additional information is used in accordance with Article 13 of REACH where it is stated thatlacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, SARs, grouping and read-across. The multi-constituentp-tert-butylcyclohexanol (CAS# 98-52-2), which shares similarity in molecular structure and physico chemical properties, is used as source substance (see data matrix).

Hypothesis:Orivone (target) has similar genotoxic properties compared top-tert-butylcyclohexanol (source). This hypothesis builds on similarity in molecular structure, physico-chemical properties and the absence of genetic toxicity of both substances in the Ames test.

Available information:For Orivone an Ames test is available, which is performed according to OECD TG 471 and GLP. This study shows that Orivone is not genotoxic in bacterial cells. For the source substance, an Ames test (OECD TG 471 and GLP), a mouse lymphoma assay (MLA) (OECD TG 476 and GLP) and a chromosomal aberration test in Chinese hamster lung fibroblasts (V79) (OECD TG 473 and GLP) are available. All these tests were negative. The described studies are all scored with reliability 1.

2. Target and Source chemical

The chemical structures, physico-chemical data and a summary of the available genetic toxicity data are presented in the data matrix.

3. Purity / Impurities

The reported purity of the Orivone andp-tert-butylcyclohexanolis > 97.5%. For Orivone the major impurity identified iscis‐4‐(2‐methylbutan‐2‐yl)cyclohexanol (the alcohol version of the ketone being the same as the alcohol analogue), which is present at 1.6 %.Therefore itis not expected that this impurity affects the read-across.

4. Analogue justification

According to REACH Annex XI an analogue approach and structural alert information can be used to replace testing when information from different sources provides sufficient evidence to conclude that this substance has or does not have a particular dangerous property. The result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.The current read-across is based on structural similarity of Orivone (target) andp-tert-butylcyclohexanol (source).

Analogue selection:Based on database searches (RIFM database and OECD QSAR Toolbox using similarity with a Tanimoto coefficient of 80% and expert judgement the substancep-tert-butylcyclohexanone(CAS# 98-53-3) was identified as potential candidate. This molecule only differs from the target substance in a missing methyl group in the alkyl tail, which is not expected to affect the reactivity. In addition, the current sourcesubstance,p-tert-butylcyclohexanol (CAS#98-52-2),was identified as good analogue. This substance has, besides the missing methyl group in the tail, an alcohol instead of a ketone. These functional groups are easily converted into each other by either reduction or oxidation reactions, resulting in similar toxicological behaviour.Other hits were notpara-substituted and therefore not considered for read-across. Forp-tert-butylcyclohexanone (CAS# 98-53-3) the data on genetic toxicity is limited to an Ames test, thereforep-tert-butylcyclohexanol was selected as the most appropriate analogue for read-across.

Structural similarities and differences:Both the source and target substance have a cyclohexane backbone, which is either substituted with a ketone and a(1,1)dimethyl-propyl tail inpara-configuration (target, Orivone) or an alcohol and atertiary-butyl moiety inpara-configuration (source,p-tert-butylcyclohexanol). The missing methyl group in the alkyl tail does not significantly change the reactivity of the molecule, and therefore the mutagenic properties of the source and target substances are expected to be similar. In addition, the ketone (as present in the target molecule) and alcohol (source substance) are easily converted in each other and can therefore be considered to poses similar genotoxic properties.

Uncertainty of the prediction:There are no remaining uncertainties becausep-tert-butylcyclohexanolis a structurally close analogue with similar reactivity, as described above. In addition, both substances are negative in a bacterial mutagenicity assay, indicating similar genotoxic properties.In the ECHA guidance (RAAF, 2017) terminology the read-across receives code 5 (acceptable with high confidence).

5. Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix.

6. Conclusions per endpoint for hazard and C&L

Both Orivone (target) andp-tert-butylcyclohexanol (source) are negative in well conducted Ames tests with and without metabolic activation. For p-tert-butylcyclohexanol a genetic toxicity study and a chromosomal aberration test is performed mammalian cells. Based on the obtained results for p-tert-butylcyclohexanol and consequently for Orivone, the latter is not considered genotoxic in mammalian cells and not considered clastogenic.Therefore, Orivone is not genotoxic which will be forwarded to classification and labelling and the risk assessment

Data matrix: Relevant information on Orivone and p-tert-butylcyclohexanol for assessment of genetic toxicity.

Source/target

Target

Source

Common names

Orivone

p-tert butylcyclohexanol (multi constituent)

Chemical structures

 

 

 

 

 

CAS No.

16587-71-6

98-52-2 (generic)

EINECS

240-642-0

202-676-4

Tanimoto

1

0.77 (trans-isomer)

Empirical formula

C11H20O

C10H20O

Molecular weight

168.28

156.267

Physical state

Liquid

Solid

Melting point °C

< -20 (IFF measured)

56.6- 58.6 (ECHA dissemination site)

Vapour pressure Pa

4.2 (IFF measured)

6 (ECHA dissemination site)

Water solubility mg/L

370.8 at 24 °C (IFF measured)

264 at 20°C (ECHA dissemination site)

Log Kow

3.9 at 25 °C (IFF measured)

3.23

Genetic toxicity

 

 

in vitro gene mutation study in bacteria

Negative

(OECD TG 471)

Negative

(OECD TG 471)

in vitro gene mutation study in mammalian cells

Read-across fromp-tert-butylcyclohexanol

Negative

(MLA test, OECDTG476)

in vitro cytogenicity study in mammalian cells

Read-across formp-tert-butylcyclohexanol

Negative

(CAB, OECDTG473)

 

Justification for classification or non-classification

Based on the negative results of the Ames test, the mouse lymphoma assay, and the chromosome aberration test, classification for genotoxicity is not warranted in accordance with the EU CLP(EC No. 1272/2008 and its updates).