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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative based on read across from Myrcenyl acetate tested in OECD TG 471.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across information.
Justification for type of information:
The read across rationale is presented in the related Endpoint summary, the accompanying files are also attached there.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
E. coli WP2 uvr A
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
Remarks on result:
other: No mutagenic potential / read-across from Myrcenyl acetate
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay, based on the results of the source substance.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 20 April 2016 and 12 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The information is used for read across to Pseudo linalyl acetate.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: Histidine gene
E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of Phenobarbital and β-Naphta flavone
Test concentrations with justification for top dose:
Experiment 1: Plate Incorporation 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.

Experiment 2: Pre-Incubation Method:
The initial dose range used for Experiment 2 was determined by the results of Experiment 1 and ranged between 1.5 and 5000 μg/plate. In the initial second experiment the toxicity of the test item yielded results that differed slightly from the Experiment 1 and consequently an insufficient number of non-toxic dose levels were attained for TA1537 (absence and presence of S9-mix) and TA100 and TA98 (absence of S9-mix only). Final test item dose ranges were employed as follows:
Salmonella strains TA100 and TA98 (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Salmonella strain TA1537 (absence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Salmonella strains TA100 and TA1537 (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
Salmonella strain TA98 (presence of S9-mix) and E.coli strain WP2uvrA (presence and absence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
Salmonella strain TA1535 (presence and absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

Up to eight test item dose levels were selected in Experiment 2 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 / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- 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 the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
Study Controls:
The solvent (vehicle) control used was dimethyl sulphoxide. The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.

The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.

The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

Microsomal Enzyme Fraction:
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 04 March 2016 was used in this study.

S9-Mix and Agar:
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl / 0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Media:
Top agar was prepared using 0.6% Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd.

Test System and Supporting Information:
Bacteria:
The five strains of bacteria used, and their mutations, are as follows:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitution
TA100 his G 46; rfa-; uvrB-;R-factor
Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.

All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1712138 07/20) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Experimental Design and Study Conduct:
Test Item Preparation and Analysis:
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No correction was made for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method:
Without Metabolic Activation:
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation:
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring:
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method:
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Without Metabolic Activation:
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation:
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring:
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Acceptability Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
- 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).

- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- 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, which are at least the minimum positive control value over the previous two years, both with or without metabolic activation.

- There should be a minimum of four non-toxic test item dose levels.

- 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 can be used to determine the overall result of the study:
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 (Cariello and Piegorsch, 1996)).

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 judgment about test item activity. Results of this type will be reported as equivocal.
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:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
E. coli WP2 uvr A
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:
Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method) the test item induced a weakening of the bacterial background lawns of TA100 from 500 μg/plate, TA1535 from 1500 μg/plate and TA98 at 5000 μg/plate in both the absence and presence of S9-mix. Weakened lawns were also noted to TA1537 at 5000 μg/plate dosed in the absence of S9-mix only. There was no visible reduction in the growth of the bacterial background lawns noted for any of the remaining tester strains at any dose level, either in the presence or absence of metabolic activation (S9-mix).

Consequently the same maximum dose level or toxic limit was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. The test item was noted to induce a much stronger toxic response in the second mutation test after employing the pre-incubation method with a visible reduction in the growth of the bacterial background lawns noted to all of the tester strains dosed in the absence of S9-mix from 50 μg/plate (TA100), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (TA1535 and WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted to all of the bacterial strains, initially from 150 μg/plate (TA1535), 500 μg/plate (TA100, TA98 and TA1537) and at 5000 μg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (globular in appearance) was noted under a low-power microscope at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Statistically significant increases in TA1535 revertant colony frequency were observed in the presence of S9-mix at 500, 1500 and 5000 μg/plate in the second mutation test. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted at the same dose levels. These responses are, therefore, considered to be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

The vehicle (dimethyl sulphoxide) 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 in excess of the minimum positive control values over the previous two years, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
Remarks on result:
other: No mutagenic potential
Conclusions:
The test substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD TG 471 (1997) and according to GLP principles. The test was performed in two experiments. S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA were treated with the test item using both the plate incorporation (Experiment 1) and pre-incubation methods (Experiment 2) at up to eight dose levels, in triplicate, both with and without S9-mix. The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The dose range for the second mutation test was amended following the results of Experiment 1 and ranged between 0.15 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. The test item was noted to induce a much stronger toxic response in the second mutation test after employing the pre-incubation method with a visible reduction in the growth of the bacterial background lawns noted to all of the tester strains dosed in the absence of S9-mix from 50 μg/plate (TA100), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (TA1535 and WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted to all of the bacterial strains, initially from 150 μg/plate (TA1535), 500 μg/plate (TA100, TA98 and TA1537) and at 5000 μg/plate (WP2uvrA). No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 or 2. Considered not biologically relevant, but statistically significant increases in TA1535 revertant colony frequency were observed in the presence of S9-mix at 500, 1500 and 5000 μg/plate in the second mutation test. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted at the same dose levels. These responses are, therefore, considered to be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies. Adequate negative and positive controls were included. Under the conditions of the test the substance was considered to be non-mutagenic.

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

Additional information

Genetic toxicity is assessed based on read-across from Myrcenyl acetate to Pseudo Linalyl Acetate. The executive summary of the source is presented below followed by the read-across rationale.

Myrcenyl acetate 'mono' information

The mutagenic activity of Myrcenyl acetate was evaluated in accordance with OECD TG 471 (1997) and according to GLP principles. The test was performed in two experiments. S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA were treated with the test item using both the plate incorporation (Experiment 1) and pre-incubation methods (Experiment 2) at up to eight dose levels, in triplicate, both with and without S9-mix. The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The dose range for the second mutation test was amended following the results of Experiment 1 and ranged between 0.15 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. The test item was noted to induce a much stronger toxic response in the second mutation test after employing the pre-incubation method with a visible reduction in the growth of the bacterial background lawns noted to all of the tester strains dosed in the absence of S9-mix from 50 μg/plate (TA100), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (TA1535 and WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted to all of the bacterial strains, initially from 150 μg/plate (TA1535), 500 μg/plate (TA100, TA98 and TA1537) and at 5000 μg/plate (WP2uvrA). No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 or 2. Considered not biologically relevant, but statistically significant increases in TA1535 revertant colony frequency were observed in the presence of S9-mix at 500, 1500 and 5000 μg/plate in the second mutation test. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted at the same dose levels. These responses are, therefore, considered to be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies. Adequate negative and positive controls were included. Under the conditions of the test the substance was considered to be non-mutagenic.

The genetic toxicity (Ames study) of Pseudo linalyl acetate using read across from Myrcenyl acetate ‘mono’ (CAS 1118-39-4)

 

Introduction and hypothesis for the analogue approach

Pseudo linalyl acetate has one major, two minor constituents and a number of impurities. Myrcenyl acetate ‘mono’ with 25-35% is the main constituent. Alpha and Gamma Terpinlyl acetate are the minor ones, each between 10-20%. For Pseudo linalyl acetate there is no Ames information available.In accordance with Article 13 of REACH, lacking information can be generated by means other means than experimental testing, it is applying alternative methods such as QSARs, grouping and read-across. For assessing the mutagenicity of Pseudo linalyl acetate the analogue approach is selected because for its main constituent Myrcenyl acetate ‘mono’ an Ames test is available which can be used for read across.

Hypothesis: Pseudo linalyl acetate has the same Ames mutagenicity as Myrcenyl acetate ‘mono’.

Available information: The mutagenic activity of Myrcenyl acetate ‘mono’ was tested in an Ames test (OECD TG 471, Rel. 1). No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in experiment 1 or 2. Based on the results of this experiment, Myrcenyl acetate ‘mono’ was considered to be non-mutagenic.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical(s) are shown in the data matrix, including physico-chemical properties and available toxicologicalinformation.

Purity / Impurities

Pseudo linalyl acetate’s key constituents are covered by Myrcenyl acetate ‘mono’. All impurities (< 10%) are shown in Appendix 1 and are not expected to impact the assessment.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation.

Analogue selection: For Pseudo linalyl acetate the major constituent Myrcenyl acetate ‘mono’ has been selected, because it is considered to be the most electrophilic constituent and expected to present a conservative Ames test result.

Structural similarities and differences: The constituents of Pseudo linalyl acetate are branched or cyclic, saturated and non-saturated hydrocarbons to which mainly esters are attached. Myrcenyl acetate ‘mono’ has a conjugated double bond at the end of its alkyl chain and has a tertiary ester at the other end. The conjugated double bond is the most electrophilic and reactive site when compared to singe unsaturated bonds.

Toxico-kinetics,Absorption: Pseudo linalyl acetate constituents all are readily bioavailable because of the similar molecular weights and log Kows and Myrcenyl acetate ‘mono’ being its key constituents represents this bioavailability.Metabolism: Most of the constituents of the target substance and the source substance Myrcenyl acetate ‘mono’ are aliphatic esters that are hydrolysed and/or metabolised into the corresponding alcohols and acetic acid by carboxylesterases (Toxicological hand books) found in tissues throughout the body.

Genotoxic reactivity: For mutagenicity the electrophilicity is a key parameter. The major constituent of Pseudo linalyl acetate, which is Myrcenyl acetate ‘mono’ has a conjugated double bond in a terminal position and is therefore considered more electrophilic compared to the other constituents. This means that a negative result of Myrcenyl acetate ‘mono’ can be used for all constituents of Pseudo linalyl acetate.

Uncertainty of the prediction: There are no other remaining uncertainties other than already discussed above.

Data matrix

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

Conclusions on mutagenicity

For Pseudo linalyl acetate no Ames test is available. For its major constituents Myrcenyl acetate ‘mono’, such data is available which can be used for read across, when using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation, which is presented in the current document. The Myrcenyl acetate ‘mono’ Ames test is negative (OECD TG 471, Reliability 1) and therefore also Pseudo linalyl acetate is considered negative for mutagenicity.

Final conclusion on hazard and risk assessment:Pseudo linalyl acetate is not mutagenic in the Ames test.

Data matrix for Pseudo linalyl acetate and its read-across source Myrcenyl Acetate ‘mono’

Common name

Pseudo linalyl acetate

Myrcenyl acetate ‘mono’

Alpha-Terpinyl acetate

Gamma-Terpinyl acetate

 

Target

Source

Supporting Source

Minor constituent

Structure

Reaction mass

CAS #

Not applicable

1118-39-4

80-26-2

10235-63-9

EC #

944-488-2

214-262-0

201-265-7

233-564-3

REACH registration

2018

2018

Registered

Not found

Empirical formula

Not applicable

C12H20O2

C12H20O2

C12H20O2

Molecular weight

n.a.

196.29

196

196.29

Physico-chemical data

 

 

EpiSuite

EpiSuite

Log Kow

4.4 (exp.)

4.4 (exp.)

4.3

4.5

Human health endpoints

 

 

 

 

Ames

 

Negative

(Read-across)

Negative

(OECDTG 471)

Negative

(OECD TG 471)

Negative

(Read-across)

 

 Appendix 1: Constituents and possible impurities of Pseudo linalyl acetate

CAS#

Type of constituent

Type and Name

 

 

Esters linear and alicyclic

1118-39-4

Major

2-methyl-6-methylideneoct-7-en-2-yl acetate (Myrcenyl acetate ‘mono’)

7643-61-0

Impurity

(5Z)-2,6-dimethylocta-5,7-dien-2-yl acetate

7643-62-1

Impurity

(5Z)-2,6-dimethylocta-5,7-dien-2-yl acetate

105-87-3 *

Impurity

(2E)-3,7-dimethylocta-2,6-dien-1-yl acetate (Geranyl acetate)

80-26-2 *

Minor

2-(4-methylcyclohex-3-en-1-yl)propan-2-yl acetate (Alpha-Terpinyl acetate)

150461-96-4

Impurity

1-(3,3-dimethylcyclohex-1-en-1-yl)ethyl acetate

150461-97-5

Impurity

1-(5,5-dimethylcyclohex-1-en-1-yl)ethyl acetate

10235-63-9

Minor

1-methyl-4-(propan-2-ylidene) cyclohexyl acetate (Gamma-Terpinyl acetate)

20777-47-3

Impurity

cis-1-methyl-4-(prop-1-en-2-yl)cyclohexyl acetate (Beta-terpinyl acetate)

97890-05-6

Impurity

(2Z)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

76-49-3

Impurity

(1R,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-ylrel-acetate (Bornyl acetate)

210648-12-7

Impurity

3,3,5-trimethylcyclohept-4-en-1-yl acetate

 

 

Alcohol linear and alicyclic

543-39-5

Impurity

2-methyl-6-methylideneoct-7-en-2-ol (Myrcenol)

98-55-5 *

Impurity

2-(4-methylcyclohex-3-en-1-yl)propan-2-ol (Alpha-Terpineol)

586-81-2

Impurity

1-methyl-4-(propan-2-ylidene)cyclohexanol (Gamma-terpineol)

 

 

Justification for classification or non-classification

Based on the results, the substance does not need to be classified for genetic toxicity according to EU CLP (EC No. 1272/2008 and its amendments).