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Genetic toxicity in vitro

Description of key information

Geranyl Isobutyrate MCS is negative in Ames based on read across from Citronellyl butyrate which was tested in an OECD TG 471.

Link to relevant study records

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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 justification is presented in the Endpoint summary Genetic toxicity. 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:
no 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
Remarks:
at 2500, and 5000 μg/plate with S9-mix only
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
Remarks:
at 5000 μg/plate in experiment 2 with S9-mix only.
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
Remarks:
at 333, 1000, 2500, and 5000 μg/plate with S9-mix only
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays, 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:
11 May 2016 - 01 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This information is used for Geranyl Isobutyrate MCS.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 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:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1, all 5 strains:
With and without S9-mix: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate
Experiment 2,
TA100 with and without S9-mix: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate
TA98, TA1535, TA1537 and WP2uvrA with and without S9-mix: 33, 100, 333, 1000, 2500, and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: The test substance was dissolved in DMSO (purity > 99 %). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine in DMSO for TA1537 and TA98
Remarks:
Without S9-mix
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO, 2.5 μg/plate for TA1535, TA1537, TA98 and TA100, and 10.0 μg/plate for WP2uvrA
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 replicates in both experiments.

NUMBER OF CELLS EVALUATED: 10^8 cells

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
Evaluation of Results:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Acceptability of the Assay:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce an increase above the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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
Remarks:
at 2500, and 5000 μg/plate with S9-mix only
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
Remarks:
at 5000 μg/plate in experiment 2 with S9-mix only.
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
Remarks:
at 333, 1000, 2500, and 5000 μg/plate with S9-mix only
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in the first experiment and at 5000 μg/plate in the second experiment. No precipitation of the test item in the overlay agar on the incubated agar plates was observed.

HISTORICAL CONTROL DATA:
These data represent the laboratory´s historical control data from January 2015 until December 2015 representing approx. 450 experiments (WP2 uvrA the historical data are based on approx. 200 experiments).

Strain
without S9 mix with S9 mix
Mean SD Min Max Mean SD Min Max
Solvent control 11 2.15 7 23 12 2.14 7 21
TA 1535
Untreated control 12 2.97 6 24 12 2.71 7 26
Positive control 1090 123.80 334 1372 392 62.85 176 549

Solvent control 10 1.83 6 18 13 3.27 7 27
TA1537
Untreated control 10 2.29 6 20 14 3.72 7 25
Positive control 83 12.28 55 131 175 44.44 82 327

Solvent control 24 3.75 16 36 33 5.55 18 51
TA 98
Untreated control 26 4.72 15 43 36 5.83 17 56
Positive control 344 51.13 211 599 3822 857.83 319 5048

Solvent control 155 24.19 84 194 145 31.81 81 204
TA 100
Untreated control 174 21.92 90 206 170 23.62 93 212
Positive control 1956 279.93 658 2528 3606 676.07 722 4940

Solvent control 41 5.72 27 63 51 6.91 37 72
WP2uvrA
Untreated control 42 6.01 31 63 53 7.05 38 88
Positive control 732 161.66 322 1066 362 72.26 212 858
Mean = mean value of revertants/plate SD = standard deviation Min = minimal value/Max = maximal value

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1537, TA 98 and, TA 100.
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent experiments, experiment 1 a plate incorporation test and experiment 2 a pre-incubation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Dose levels up to and including 5000 μg/plate were tested in the absence and presence of S9-mix. Adequate positive controls and solvent controls were included.

No precipitation of the test item in the overlay agar on the incubated agar plates was observed. The plates incubated with the test item showed reduced background growth in the strains TA 1537 and TA 100 in the presence of S9 mix in both experiments. Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1537, TA 98 and, TA 100.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Based on the results, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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

Additional information

Genetic toxicity in bacteria for Geranyl Isobutyrate MCS is based on read-across from Citronellyl butyrate. The executive summary of the Citronellyl butyrate is presented below followed by the read-across rationale.

Citronellyl butyrate Ames test information

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent experiments, experiment 1 a plate incorporation test and experiment 2 a pre-incubation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Dose levels up to and including 5000 μg/plate were tested in the absence and presence of S9-mix. Adequate positive controls and solvent controls were included. No precipitation of the test item in the overlay agar on the incubated agar plates was observed. The plates incubated with the test item showed reduced background growth in the strains TA 1537 and TA 100 in the presence of S9 mix in both experiments. Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1537, TA 98 and, TA 100. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Based on the results, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

The genetic toxicity (AMES study) of Geranyl Isobutyrate MCS based on read across from data available for Citronellyl butyrate (CAS #141-16-2)

 

Introduction and hypothesis for the analogue approach

Geranyl Isobutyrate MCS consists of 4 constituents which areIsobutyrate esters of a 3,7-dimethyloctanol chain and can be divided into two subgroups based on the number of unsaturated bonds. The Geranyl-type subgroup consists of two isomers of Geranyl Isobutyrate which have two double bonds in the chain and are present at a total of 70%. The Citronellyl-type subgroup consists of Citronellyl butyrate, which has one double bond in the chain and is present at ca. 25% and a Citronellyl like component without a double bond which is present at <5%.

For Geranyl Isobutyrate MCS no Ames data are available.In accordance with Article 13 of REACH, lacking information can be generated by means of QSARs, grouping and read-across.For assessing the mutagenic potential in bacteria of Geranyl Isobutyrate MCS, the analogue approach is selected because for structural analogue, Citronellyl butyrate, Ames data is available which can be used for read across.

Hypothesis: Geranyl Isobutyrate MCS has the samemutagenic potential as Citronellyl butyrate.

Available information:For Citronellyl butyrate a negative Ames study is available according to OECD TG 471 Rel. 1). The study was conducted in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA up to and including 5000 μg/plate with and without metabolic activation.

Target chemical and source chemical

Chemical structures of the target chemical and the source chemical(s) are shown in the data matrix, including physico-chemical properties and available toxicologicalinformation. Furthermore, a full list of constituents of Geranyl Isobutyrate MCS, including information relevant for read-across, is given in Appendix 1.

Purity / Impurities

The unidentified impurities of Geranyl Isobutyrate MCS are not considered to have a significant influence on the mutagenic potential..

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.

Analogue selection: For Geranyl Isobutyrate MCSCitronellyl butyrate was selected as an analogue because the constituents of Geranyl Isobutyrate MCS share a similar backbone and the same functional groups and Ames information is available for Citronellyl butyrate.

Structural similarities and differences: Geranyl Isobutyrate MCS has the same 3,7-dimethyloctanol chain and a butyrate ester as Citronellyl butyrate. They both have the same molecular weight. The difference is that Geranyl Isobutyrate MCS has constituents containing two double bonds in this chain compared to one for Citronellyl butyrate. The former also has an isobutyric ester while the latter has a butyric chain.

Toxico-kinetic: Geranyl Isobutyrate MCS and Citronellyl butyrate have similar absorption based on similar physico-chemical properties e.g. log Kows are around 5.5. Both substances have similar metabolic pathways. Both will enzymatically hydrolyse rapidly e.g. in the intestine before absorption and/or the liver (Longland, 1977) through carboxylesterases. Geranyl Isobutyrate MCS has some constituents, which have a double bond conjugated with the ester. This may result in a faster hydrolysis rate compared to non-conjugated esters because the ester bond is more electrophilic. In view of all esters will be cleaved by carboxylesterases (Toxicological Handbooks) the will not influence further the metabolic pathway. The terpene-type of alcohols formed, such as Geraniol and Citronellol, and their unsaturated analogues exhibit similar pathways of metabolism in animals resulting in participation in normal fatty acid metabolism and excretion in the urine, as such or as glucuronic acid conjugates (EFSA, 2008).

Toxico-dynamics: The reactive site in Geranyl Isobutyrate MCS is the conjugated ester group, while Citronellyl butyrate has not such a conjugated bond. In view of the kinetic profile this will not affect the mutagenic potential.

Uncertainty of the prediction: Besides the presented reasoning above, there are no other remaining uncertainties.

Data matrix

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

Conclusions per endpoint for hazard and risk assessment

For Geranyl Isobutyrate MCS no Ames mutagenicity is available. 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. The latter documentation is presented in the current document. For Citronellyl butyrate an OECD TG 471 (Reliability 1) with a negative result is available. Based on the reasoning presented Geranyl Isobutyrate MCS is considered not mutagenic in bacteria.

Final conclusion on hazard and risk assessment: Geranyl Isobutyrate is not mutagenic in the Ames test.

 

Data matrix for the read across from Citronellyl butyrate to Geranyl Isobutyrate MCS

Common name

Geranyl Isobutyrate MCS

Citronellyl butyrate

 

Target

Source

Chemical name

n.a.

3,7-dimethyloct-6-en-1-yl butanoate

Chemical structures

For a full list of constituents, see Appendix 1.

CAS #

--

141-16-2

EC #

905-357-5

205-463-4

REACH

2018

Registered

Empirical formula

n.a.

C14H26O2

SMILES

n.a.

CCCC(=O)OCCC(C)CCC=C(C)C

Physico-chemical data

 

 

Molecular weight

n.a.

226

Physical state

Liquid

Liquid

Log Kow

5.7 (exp.)

5.54 (est.)

Ws (mg/L)

17.4 (exp.)

1.63 (est.)

Vp (Pa)

1.2 (exp.)

6.2 (est.)

Human health endpoints

 

 

Ames

Based on read-across:

Not mutagenic

Not mutagenic

(OECD 471)

 

References:

EFSA, 2000, Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC) on a request from the Commission Flavouring Group Evaluation 6, Revision 1 (FGE.06Rev 1): Flavouring Group Evaluation 6, Revision 1 (FGE.06Rev1): Straight- and branched-chain aliphatic unsaturated primary alcohols, aldehydes, carboxylic acids, and esters from chemical groups 1 and 4 (Commission Regulation (EC) No 1565/2000 of 18 July 2000). The EFSA Journal (2008) 616, 1-74.

 

Longland, R.C., Shilling, W.H. & Gangolli, S.D. (1977) The hydrolysis of flavouring esters by artificial gastrointestinal juices and rat tissue preparations. Toxicology, 8, 197–204.

 

 

 


Appendix 1. Data matrix for the constituents of Geranyl isobutyrate MCS and Citronellyl butyrate

 

Geranyl isobutyrate MCS

 

 

 

Citronellyl butyrate

Constituent

1

2

3

4

 

Constituents

3,7-dimethyloctyl 2-methylpropanoate

3,7-dimethyloct-6-en-1-yl 2-methylpropanoate

(2Z)-3,7-dimethylocta-2,6-dien-1-yl 2-methylpropanoate

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

3,7-dimethyloct-6-en-1-yl butanoate

Chemical structures

 

CAS no

71662-25-4

97-89-2

2345-24-6

2345-26-8

141-16-2

Concentration range

3.7

25

14

56

 

Empirical formula

C14H28O2

C14H26O2

C14H24O2

C14H24O2

C14H26O2

Molecular weight

228

226

224

224

226

Physico-chemical data*

 

 

 

 

 

Water solubility, mg/l

0.56

0.68

0.82

0.82

0.59

Log Kow

5.6

5.5

5.4

5.4

5.5

* Episuite v4.11

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).