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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Veilex 1 Ames information (based on read across): Negative
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 April 2006 to 19 May 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Labor, Notifications No. 77 and 67
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium TA1535: hisG46 rfa uvrB; S. typhimurium TA1537: hisC3076 rfa uvrB; S. typhimurium TA 1538: hisD3052 rfa uvrB; S. typhimurium TA98: hisD3052 rfa uvrB pKMl01; S. typhimurium TA100: hisG46 rfa uvrB pKM101; E. coli WP2 uvrA: trp
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 S9
Test concentrations with justification for top dose:
Dose-range finding test 1: a total of 6 doses consisting of 5000 μg/plate as the highest dose and 5 lower doses diluted with a geometric progression of 4 were employed.
Dose-range finding test 2 and main test: the highest dose at 78.1 μg/plate without S9 and 313 μg/plate with S9 and each lower 5 doses diluted with a geometric progression of 2.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was insoluble in distilled water at 50 mg/mL but it was soluble in DMSO at 50 mg/mL. The test substance solution of 50 mg/mL prepared with DMSO was considered to be stable as there was no change in colour no heat generation at room temperature within 2 hours after preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide
Remarks:
TA98, TA 100, and WP2uvrA without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine x 2 HCl
Remarks:
TA1537 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 48 hours

NUMBER OF REPLICATIONS: triplicate for the negative control group and duplicate per dose for the test substance and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition
Evaluation criteria:
The test substance was judged positive when the number of revertant colonies increased to twice or more that in the negative control in a concentration-dependent manner and also the reproducibility of the test results was obtained. In all other cases it was judged negative.
Statistics:
No statistical methods were used.
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: In the dose-range finding test 1 the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The precipitation of the test substance was not observed at any doses. The bacterial growth inhibition was observed at > 78.1 μg/plate in all test strains without S9 and >313 μg/plate with S9. In the dose-range finding test 2 the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The precipitation of the test substance was not observed at any doses. The bacterial growth inhibition was observed at >78.1 μg/plate in all test strains without S9, >156 μg/plate in TA100, TA1535, TA98 and TA1537 and >313 μg/plate in WP2uvrA with S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main test the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The precipitation of the test substance was not observed at any doses. The bacterial growth inhibition was observed at >78.1 μg/plate in all test strains without S9, >156 μg/plate in TA100, TA1535, TA98 and TA1537 and >313 μg/plate in WP2uvrA with S9.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The substance was concluded to give negative results in the Ames test.
Executive summary:

Genotoxicity of CP Formate in prokaryotes was studied in a GLP-compliant study performed according to the protocol similar to OECD guidelines 471 and 472 in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in Escherichia coli strain WP2uvrA, with and without metabolic activation. The experiments were performed by preincubation method. The bacterial growth inhibition was observed at > 78.1 μg/plate in all test strains without S9, > 156 μg/plate in TA100, TA1535, TA98 and TA1537 and > 313 μg/plate in WP2uvrA with S9. The numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all test strains, both with and without metabolic activation. Therefore the substance was concluded to give negative results in the Ames test.

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

Additional information

For Veilex 1 genotoxicity information in bacteria information is available but without the strain T102 or E.coli. In addition to this information also read across is applied to show that the missing information for Veilex1 can be covered using CP Formate and CP Acetate. First the available information from Veilex1 will be presented, thereafter the information from key source substance CP Formate and supporting substance CP Acetate and finally the read across justification will be presented.

Veilex1

The mutagenic activity of the substance was evaluated in a study similar to OECD 471. A plate incorporation assay was performed with S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 with and without metabolic activation (rat liver microsomes). All strains were dosed with 0.001, 0.01, 0.1, 1, 5, and 10 µL/plate test substance with and without metabolic activation. Adequate solvent (acetone) and positive control (2-aminoanthracene) were included. The experiment was repeated for TA 98 with as positive control Benzo(a)pyrene. TA 98 was dosed with 0.01, 0.1, 1, 5, and 10 µL/plate test substance with metabolic activation. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the S. typhimurium tester strains both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Read across CP Formate

Genotoxicity of CP Formate in prokaryotes was studied in a GLP-compliant study performed according to the protocol similar to OECD guidelines 471 and 472 in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in Escherichia coli strain WP2uvrA, with and without metabolic activation. The experiments were performed by preincubation method. The bacterial growth inhibition was observed at > 78.1 μg/plate in all test strains without S9, > 156 μg/plate in TA100, TA1535, TA98 and TA1537 and > 313 μg/plate in WP2uvrA with S9. The numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all test strains, both with and without metabolic activation. Therefore the substance was concluded to give negative results in the Ames test.

Read across CP Acetate

The mutagenic activity of the substance was evaluated in accordance with OECD 471 and according to GLP principles. A preincubation assay was performed with S. typhimurium strains TA100, TA1535, TA98, TA1537, and E. coli WP2uvrA with and without metabolic activation (Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone). Adequate solvent (DMSO) and positive controls were included. Two dose range finding tests were performed and one main test. In the main test all stains were dosed with 2.44, 4.88, 9.77, 19.5, 39.1, and 78.1 µg/plate without metabolic activation and 9.77, 19.5, 39.1, 78.1, 156, and 313 µg/plate with metabolic activation. The experiment was performed in duplicate. The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control groups of treatment with and without S9 mix. Under the conditions of the test the substance is not mutagenic.

The mutagenicity of Veilex#1 (CAS#63449-88-7) using read across from CP Formate (CAS#25225-08-5) and CP Acetate (CAS#25225-10-9)

Introduction and hypothesis for the analogue approach

Veilex#1 has acyclohexylethyl backbonewith abutyrate estergroup attached. For this substance themutagenicitydata available are not sufficient to cover the endpoint. Genotoxicity data may be generated by other means, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing themutagenicityin bacterial cells of Veilex#1 the analogue approach is selected because for a closely related analogue, CP Formatemutagenicityinformation is available which can be used for read across.

Hypothesis: Veilex#1 has similar genotoxic potential compared to CP Formate as thetwo methyl groups attached to the meta-position of the cyclohexylethyl backbone and the butyrate ester versus the formate ester, respectively, are not expected to influence mutagenicity.

Available information: The target chemical Veilex#1 is tested in an Ames test however an E. coli strain or S. typhimurium TA102 were absent. Therefore this study receives a reliability of 2 but missing essential information. The source chemical CP Formate was tested in an Ames test similar to OECD TG 471 and receives a Klimisch 1 study. In addition, for CP Acetate also an Ames study according to OECD TG 471 is available receiving reliability Klimisch 1.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemicals are shown in the data matrix, including physico-chemical properties and toxicological information, thought relevant for genetic toxicity, of all substances.

Purity / Impurities

Veilex#1 is a mono-constituent presenting a purity close to 100%. CP Formate is a multi-constituent composed of 65-85% 1-(3,3-dimethylcyclohexyl) ethyl formate and 8-18% 2,6,6-trimethylcycloheptyl formate. The minor constituent has a similar functional group but a larger ring structure of 7- C atoms. The similarity in the functional group will present similar reactivity and therefore will not affect the read across. The supporting substance CP Acetate, being a mono-constituent, does not have impurities that affect the read across.

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.In accordance with ECHA guidance (2015, RAAF) the analogue was selected from a group of related cyclohexyl-esters all negative in the Ames test and CP Acetate being the closest structural analogue. For support also information from CP Formate is presented (see Data matrix below)

Structural similarities and differences:Veilex#1 as well as the structural analogues have a common cyclohexylethyl backbone. CP Formate and CP Acetate, have two methyl groups attached to the meta-position of the cyclohexylethyl backbone, while Veilex#1 does not have additional substituents. A formate ester and an acetate ester are attached to the cyclohexylethyl backbone of CP Formate and CP Acetate, respectively, while Veilex#1 has a butyrate ester attached.These differences between the target and source chemicals are not expected to significantly influence the mutagenicity of thesechemicals.

Toxico-kinetic similarities and differences are expressed here because mutagenicity need to be assessed for both local and systemic sites:

Absorption:Veilex#1, CP Formate and CP Acetate have similar molecular weight and physico-chemical properties indicating similar absorption characteristics. All are liquids, molecular weight (184.3 - 198.3 g/mol)and log Kow (Ca. 3.8 – 5.2) indicate that Veilex#1 and its source chemicals will be absorbed via all routes of exposure. 

Metabolism:It can be anticipated that Veilex#1 will metabolise by carboxylesterases to butanoic acid (CAS#107-92-6) and 1-cyclohexylethanol (CAS#1193-81-3) while CP formate and CP Acetate will metabolise to 1-(3,3-dimethylcyclohexyl)ethanol (CAS#25225-09-6) and Formic acid (CAS#64-18-6) or Acetic Acid (CAS#64-19-7), respectively, as is presented in Fig. 1.

 

Fig. 1 The metabolisation pathway of Veilex#1, CP Formate and CP Acetate.

Uncertainty of the prediction:There is no remaining uncertainty, in view of similarities in structure, toxico-kinetic profile (absorption and metabolism) and reactivity profile between the substances. Therefore, read across can be applied. There are no structural alerts regarding mutagenicity for the target chemical, source chemicals and their metabolites (Toxtree V2.6.13 and OECD toolbox V3.3.0.132, data not shown). Therefore the mutagenicity data of both CP Formate and CP Acetate can be used for read across to Veilex#1. In accordance with RAAF (ECHA, 2015) a quality score of 5 can be assigned.

Data matrix

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

Conclusion for the Ames test result

For Veilex#1 an Ames test was performed test. In this study no mutagenic effects were observed. However, mutagenicity information from E. coli strain or the S. typhimurium TA102 strain were missing. In view of the negative results in these strains for two analogues (OECD TG 471 according to current guidelines), CP Formate and CP Acetate, it can be concluded that Veilex#1 is not mutagenic in the Ames test either.

Data matrix for the read across to Veilex#1, CP Formate, and CP Acetate

Common names

Veilex#1 (CP Butyrate)

CP Formate

CP Acetate

Chemical structures

 

Target

Key Source

Supporting source

CAS no

63449-88-7

25225-08-5

25225-10-9

EC no

REACH (2018)

939-618-9

Not registered

Empirical formula

C12H22O2

C11H20O2

C12H22O2

Physico-chemical data

 

 

 

Molecular weight

198.304

184.277

198.304

Physical state

liquid

liquid

liquid

Melting point,oC

< -20

 -20

13.46 (EpiSuite)

Boiling point,oC

246.5

219.9

>204 (EpiSuite)

Vapour pressure, Pa

6.5 at 24 °C

13.4 at 23 °C

6.7(EpiSuite)

Water solubility, mg/L

23.8 at 24 °C

26.12 at 23 °C

7.462 (EpiSuite)

Log Kow

5.2 at 20 °C

3.8 at 35 °C

4.42 (EpiSuite)

Human health endpoints

 

 

 

Genotoxicity – Ames test

Not mutagenic (IFF, 1978) Ames without E. coli strain or TA102

Not mutagenic similar to OECD TG 471

Not mutagenic, OECD TG 471 (IFF, 2008)

 

Justification for classification or non-classification

Based on the results of the Ames tests performed with the test substance and read across substances, the test substance does not have to be classified for mutagenicity in accordance with Regulation (EC) No. 1272/2008.

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