Heptanal, oligomeric reaction products with aniline

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 2016 to 03 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study performed in accordance with OECD test guideline in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

1

Reference substance name:

Heptanal, oligomeric reaction products with aniline

EC Number:

500-007-3

EC Name:

Heptanal, oligomeric reaction products with aniline

Cas Number:

9003-50-3

Molecular formula:

(C7H14O)n.(C6H7N)n

IUPAC Name:

Heptanal, oligomeric reaction products with aniline

Test material form:

liquid

Details on test material:

Test item: 207368/A
Identification: Hepteen Base®
Appearance: Clear amber liquid (determined by Charles River Den Bosch)
Batch: LT5C30Y170
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2016 (retest date)

Method

Target gene:

thymidine-kinase locus (TK-locus)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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 kept below 1 x 106 cells/ml.
Cell culture
Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.

Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies, Bleiswijk, The Netherlands) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).

Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).

Exposure medium
For 3 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).

For 24 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).

Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mixture

Test concentrations with justification for top dose:

Hepteen Base® was poorly soluble in the exposure medium, the highest tested concentration was 164 μg/ml exposure medium.

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

yes

Remarks:

Ethanol (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 kept below 1 x 106 cells/ml.
Cell culture
Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.

Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies, Bleiswijk, The Netherlands) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).

Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).

Exposure medium
For 3 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).

Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).

Environmental conditions
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 57 – 91%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 – 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

Rationale for test conditions:

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

Evaluation criteria:

Data evaluation and statistical procedures
In addition to the criteria stated below, 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.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

Solubility
Hepteen Base® precipitated in the exposure medium at concentrations of 52 μg/ml and above. The concentration used as the highest test item concentration for the dose range finding test was 164 μg/ml.

Dose range finding test
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 1.7 to 164 μg/ml 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.

In the absence of S9-mix, the relative suspension growth was 49% at the test item concentration of 164 μg/ml compared to the relative suspension growth of the solvent control.
In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to test item concentrations of 164 μg/ml compared to the solvent control.

The relative suspension growth was 30% at the test item concentration of 17 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentration of 52 μg/ml and upwards after 24 hours treatment.

Mutation experiment
The percentages of cell survival and the mutation frequencies for various concentrations of Hepteen Base®. Individual colony counts of cloning and selective plates and cell counts during subculturing are tabulated and listed in “Any other information on results incl. tables).

First mutagenicity test
Based on the results of the dose range finding test, the following dose range was selected for the first mutagenicity test:

Without S9-mix: 0.17, 0.54, 1.7, 5.4, 17, 30, 52, 85 and 164 μg/ml exposure medium.
With S9-mix: 0.05, 0.17, 0.54, 1.7, 5.4, 17, 52 and 164 μg/ml exposure medium.

Evaluation of toxicity
In the absence of S9-mix, the dose level of 52 μg/ml was not used for mutation frequency measurement, since this dose level showed an inconsistent RSG.
In the presence of S9-mix, no toxicity was observed and all dose levels were evaluated.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.17, 0.54, 1.7, 5.4, 17, 30, 85 and 164 μg/ml exposure medium.
With S9-mix: 0.05, 0.17, 0.54, 1.7, 5.4, 17, 52 and 164 μg/ml exposure medium.
In the absence of S9-mix, the relative total growth of the highest test item concentration was 32% compared to the total growth of the solvent controls.
In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level tested. .

Evaluation of the mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with Hepteen Base® either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Hepteen Base® treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test
To obtain more information about the possible mutagenicity of Hepteen Base®, a second mutation experiment was performed in the absence of S9-mix with a 24-hour treatment period.
Based on the results of the dose range finding test, the following dose levels were selected for mutagenicity testing: 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40 and 45 μg/ml exposure medium.

Evaluation of toxicity
The dose levels of 25 to 45 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 0.1, 0.5, 1, 5, 10, 15 and 20 μg/ml exposure medium.
The relative total growth of the highest test item was 7% compared to the total growth of the solvent controls;

Evaluation of mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with Hepteen Base®. The numbers of small and large colonies in the Hepteen Base® treated

Applicant's summary and conclusion

Conclusions:

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 suspension growth (SG) over the two-day expression period for cultures treated with ethanol was between 16 and 22 (3 hours treatment) and 136 and 150 (24 hours treatment).

In the absence of S9-mix, Hepteen Base® 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, Hepteen Base® did not induce a significant increase in the mutation frequency.

In conclusion, Hepteen Base® is not mutagenic in the TK mutation mouse lymphoma test system under the experimental conditions described in this report.

Executive summary:

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

 

This report describes the effects of Hepteen Base® 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 hours 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 no. 490.

 

Batch LT5C30Y170 of Hepteen Base® was a clear amber liquid with a purity of 99.7%. The test item was dissolved in ethanol.

 

In the first experiment, Hepteen Base® was tested up to concentrations of 164 μg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. Hepteen Base® precipitated in the culture medium at this dose level.

 

In the second experiment, Hepteen Base® was tested up to concentrations of 20 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. 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, Hepteen Base® 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, Hepteen Base® did not induce a significant increase in the mutation frequency.

 

It is concluded that Hepteen Base® is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions.