Benzenepropanal, 2-methyl-4-(2-methylpropyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria

The evaluation of the mutagenic activity of GR-88-0778 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay was performed in 2015 according to the OECD guideline No. 471.

It is concluded that GR-88-0778 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In vitro gene mutation study in mammalian cells

The evaluation of the mutagenic activity of NYMPHEAL in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells was performed in 2016 according to the OECD guideline No. 490.

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

In vitro Micronucleus Assay with NYMPHEAL in Cultured Peripheral Human Lymphocytes

An in vitro micronucleus assay with NYMPHEAL in cultured peripheral human lymphocytes was performed in 2017 according to the OECD guideline No.: 487.

It is concluded that this test is valid and that NYMPHEAL is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March 2015 to 23 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name (as stated in the report): GR-88-0778
Lot No: Batch 6
Aspect: Colourless liquid
Expiration date: March 29, 2017

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

In the first mutation assay, GR-88-0778 was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. GR-88-0778 precipitated on the plates at dose levels of 1600 μg/plate and upwards. Toxicity was observed in all tester strains, except in tester strain WP2uvrA in the presence of S9-mix. However since GR-88-0778 precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined in tester strain WP2uvrA.In the second mutation assay, GR-88-0778 was tested at appropriate dose ranges in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Toxicity was observed in all tester strains, except in WP2uvrA in the presence of S9-mix. However since GR-88-0778 precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined in tester strain WP2uvrA.

Vehicle / solvent:

GR-88-0778 was dissolved in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

TA1537 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA100 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2uvrA without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains with S9

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

In the first mutation assay, toxicity was observed in all tester strains, except in tester strain WP2uvrA in the presence of S9-mix (tested up to concentrations of 5000 μg/plate).
In the second mutation assay, toxicity was observed in all tester strains, except in WP2uvrA in the presence of S9-mix.
In the third mutation experiment performed on strain TA98 without S9 mix toxicity was observed at the dose levels of 164 μg/plate and upwards.

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that GR-88-0778 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of GR-88-0778 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay. GR-88-0778 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by induced by Aroclor 1254). An additional experiment was performed with the tester strain TA98 in the absence of S9-mix. The study procedures described in this report were based on the most recent OECD, EC and MITI guidelines. Batch 6 of GR-88-0778 was a colourless to pale yellow liquid with a purity of 98.6%. The test substance was dissolved in dimethyl sulfoxide. In the first mutation assay, GR-88-0778 was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. GR-88-0778 precipitated on the plates at dose levels of 1600 μg/plate and upwards. Toxicity was observed in all tester strains, except in tester strain WP2uvrA in the presence of S9-mix. However since GR-88-0778 precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined in tester strain WP2uvrA. In the second mutation assay, GR-88-0778 was tested at appropriate dose ranges in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Toxicity was observed in all tester strains, except in WP2uvrA in the presence of S9-mix. However since GR-88-0778 precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined in tester strain WP2uvrA. Since in tester strain TA98 in the second experiment too many dose levels showed severe toxicity in the absence of S9-mix, a third mutation experiment was performed with this strain in the absence of S9-mix. GR-88-0778 was tested up to the dose level 1600 μg/plate. Toxicity was observed at the dose levels of 164 μg/plate and upwards. GR-88-0778 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that GR-88-0778 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October 2016 to 12 December 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)

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): NYMPHEAL
Batch No.: SC00018060
Aspect: Colourless to pale yellow liquid
Expiration date: 28 April 2017

Target gene:

thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

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 12.5 and 65 μg/ml in the absence and presence of S9-mix, respectively. In the absence of S9-mix, the dose levels of 15 to 30 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix the dose levels of 80, 90 and 100 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the second experiment, the test item was tested up to concentrations of 10 μg/ml in the absence of S9-mix. Indeed the the dose levels of 13, 16 and 20 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing or showed an inconsistent RSG (13 μg/ml).

Vehicle / solvent:

The solvent for the test item, i.e. dimethyl sulfoxide (Merck, Darmstadt, Germany).

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

The solvent for the test item, i.e. dimethyl sulfoxide (Merck, Darmstadt, Germany).

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation (-S9-mix)

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

The solvent for the test item, i.e. dimethyl sulfoxide (Merck, Darmstadt, Germany).

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation (+S9-mix):

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

The test item was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods. Six or eight doses of the test item were tested in the mutation assay with a 3 and 24 hour treatment period, respectively. 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.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "additional information on results" field

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

First mutagenicity test:
In the absence of S9-mix, the dose levels of 15 to 30 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, the dose levels of 1.25 to 10 μg/ml showed no cytotoxicity. Therefore, the dose level of 1.25 μg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 80, 90 and 100 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

Second mutagenicity test:
The dose levels of 13, 16 and 20 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing or showed an inconsistent RSG (13 μg/ml).

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.

Conclusions:

In the absence of S9-mix, the test item 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, the test item did not induce a significant increase in the mutation frequency.
In conclusion, NYMPHEAL 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 NYMPHEAL in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

This report describes the effects of NYMPHEAL 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 SC00018060 of the test item was a colourless to pale yellow liquid. The test item was dissolved in dimethyl sulfoxide.

In the first experiment, the test item was tested up to concentrations of 12.5 and 65 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 14 and 3% in the absence and presence of S9-mix, respectively.

In the second experiment, the test item was tested up to concentrations of 10 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 9%.

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.

In the absence of S9-mix, the test item 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, the test item did not induce a significant increase in the mutation frequency.

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

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 September 2016 to 11 November 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)

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): NYMPHEAL
Batch No.: SC00018060
Aspect: Colourless to pale yellow liquid
Expiration date: 28 April 2017

Species / strain / cell type:

lymphocytes: peripheral humal lymphocytes

Details on mammalian cell type (if applicable):

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 25, AGT = 13.3 h
Second cytogenetic assay: age 24, AGT = 13.7 h

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

In the first cytogenetic assay, the test item was tested up to 50 and 70 μg/ml .
In the second cytogenetic assay, the test item was tested up to 55 μg/ml.
Appropriate toxicity was seen at those levels.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle for the test item was dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany).

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Colchicine

Remarks:

Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle for the test item was dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany).

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

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

Conclusions:

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

Executive summary:

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

This report describes the effect of NYMPHEAL 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 NYMPHEAL was tested in two independent experiments.

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

Batch SC00018060 of the test item was a colourless to pale yellow liquid. The test item was dissolved in dimethyl sulfoxide.

In the first cytogenetic assay, the test item was tested up to 50 and 70 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, the test item was tested up to 55 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. NYMPHEAL did not induce a statistically significant and 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 NYMPHEAL 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)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available on Nympheal, no classification is necessary according to the (EC) No 1272/2008 Regulation (CLP).