3-(p-cumenyl)-2-methylpropionaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The Evaluation of the Mutagenic Activity of SILVIAL in an in vitro Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells performed according to the OECD Guideline No. 490 shows no mutagenic potential in the mouse lymphoma L5178Y test system under the experimental conditions.

The in vitro Micronucleus Assay with SILVIAL in Cultured Peripheral Human Lymphocytes performed according to the OECD Guideline No. 487 shows no clastogenic or aneugenic potential in human lymphocytes under the experimental conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 2016 to 16 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Principles of method if other than guideline:

There were no deviations from standard operating procedures that affected the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Name (as stated in the report): SILVIAL
Batch No.: SC00017272
Expiration date: 21 January 2017

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

In the first experiment, the test item was tested up to concentrations of 25 and 50 μg/ml in the absence and presence of S9-mix, respectively. In the second experiment, the test item was tested up to concentrations of 25 μg/ml in the absence of S9-mix. The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide (SeccoSolv, Merck Darmstadt, Germany).

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 106 cells/ml.

Rationale for test conditions:

Rationale: Recommended test system in international guidelines (e.g. OECD).

Evaluation criteria:

Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126. A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically
relevant and will be compared with the historical control data range. A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "Any other information on results" field

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The growth rate over the two-day expression period for cultures treated with DMSO was between 10 and 16 (3 hours treatment) and between 104 and 107 (24 hours treatment)

1st experiment (toxicity evaluation)

In the absence of S9-mix the dose levels of 1 to 10 μg/ml showed similar cytotoxicity. Therefore, the dose level of 1 μg/ml was not regarded relevant for mutation frequency measurement. Due to severe toxicity, the dose level of 30 μg/ml was removed during the subculture period.

In the presence of S9 -mix the dose levels of 55 and 60 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic or showed an inconsistent RSG (60 μg/ml). The dose levels of 1 to 40 μg/ml showed no cytotoxicity. Therefore, the dose levels of 1 and 5 μg/ml were not regarded relevant for mutation frequency measurement.

2nd experiment (toxicity evaluation)

In the absence of S9-mix the dose levels of 27.5 to 35 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose level of 10 μg/ml showed an inconsistent RSG. Therefore, this dose level was not regarded relevant for mutation frequency measurement.

Conclusions:

In the absence of S9-mix, SILVIAL did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, SILVIAL did not induce a significant increase in the mutation frequency.
In conclusion, SILVIAL is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

Evaluation of the mutagenic activity of SILVIAL in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

This report describes the effects of SILVIAL on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The study procedures described in this report were based on the most recent OECD guideline. Batch SC00017272 of the test item was a colourless liquid. The test item was dissolved in

dimethyl sulfoxide.

In the first experiment, the test item was tested up to concentrations of 25 and 50 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total

growth (RTG) was 4 and 11% in the absence and presence of S9-mix, respectively.

In the second experiment, the test item was tested up to concentrations of 25 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 7%.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative

control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, SILVIAL did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, SILVIAL did not induce a significant increase in the mutation frequency.

It is concluded that SILVIAL is not mutagenic in the mouse lymphoma L5178Y test system^under the experimental conditions described in this report.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 April 2016 to 17 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Principles of method if other than guideline:

There were no deviations from standard operating procedures that affected the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Name (as stated in the report): SILVIAL
Batch No.: SC00017272
Expiration date: 23 October 2016

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

In the first cytogenetic assay, SILVIAL was tested up to 90 and 130 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. In the second cytogenetic assay, SILVIAL was tested up to 70 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany).

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Colchicine (Colch; CAS no. 64-86-8, Acros Organics, Geel, Belgium)

Details on test system and experimental conditions:

Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2015) are presented below:
Dose range finding study: age 34, AGT = 12.9 h
First cytogenetic assay: age 33, AGT = 14.2 h
Second cytogenetic assay: age 30, AGT = 12.9 h

Rationale for test conditions:

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline.

Evaluation criteria:

An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analysed by the Chi-square test (one-sided, p < 0.05).

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

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:

The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database.
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

SILVIAL did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Conclusions:

Finally, it is concluded that this test is valid and that SILVIAL is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

An in vitro micronucleus assay with SILVIAL in cultured peripheral human lymphocytes.

This report describes the effect of SILVIAL on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity of SILVIAL was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

Batch SC00017272 of SILVIAL was a colourless liquid. The test item was dissolved in dimethyl sulfoxide.

In the first cytogenetic assay, SILVIAL was tested up to 90 and 130 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, SILVIAL was tested up to 70 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

SILVIAL did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Finally, it is concluded that this test is valid and that SILVIAL is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)