Heptanal, oligomeric reaction products with aniline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells

Key values were obtained in a GLP accredited laboratory using the thymidine kinase gene in accordance with OECD test guidline 490.

HEPTEEN BASE® is not mutagenic in the Thymidine Kinase mutation mouse lymphoma test system under the experimental conditions described in the report.

In Vitro Mammalian Chromosome Aberration Test with human lymphocytes

Key values were obtained in a GLP accredited laboratory using human lymphocytes in accordances with OECD test guideline 473. Under the experimental conditions of the study, Hepteen Base® is not clastogenic in human lymphocytes.

In vitro Reverse Mutation Assay using Salmonella Typhimurium and Escherichia Coli

Key values were obtained in a GKO Accredited laboratory using human lymphocytes in accordance with OECD, EU and MITI test guidelines. Under the experimental conditions of the study, Hepteen Base® is not mutagenic in the salmonella typhimurium and escherichia coli reverse mutation assays.

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:

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.

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

Target gene:

thymidine-kinase locus (TK-locus)

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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2016 to 29 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not applicable

Remarks:

Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mammalian cell line, other: human

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 Mixture

Test concentrations with justification for top dose:

The highest tested concentration was 52 μg/ml exposure medium. The dose range finding test (first cytogenic assay) that Hepteen Base (R) precipitated in the culture medium at concentrations of 52 and 164 μg/ml.

Vehicle / solvent:

Solubility in vehicle Ethanol: Not indicated by Sponsor but established by WIL to be soluble for the test
Stability in vehicle Ethanol: Not indicated

Untreated negative controls:

yes

Remarks:

Ethanol

Negative solvent / vehicle controls:

yes

Remarks:

Hanks’ Balanced Salt Solution (HBSS) (Life technologies, Bleiswijk, The Netherlands), without calcium and magnesium. (For positive controls)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Reference item - Negative control
The vehicle for the test item was ethanol (Extra pure, Merck, Darmstadt, Germany).

Positive controls
Without metabolic activation (-S9-mix):
Mitomycin C (MMC-C; CAS no. 50-07-7, Sigma, Zwijndrecht, The Netherlands) was used as a direct acting mutagen at a final concentration of 0.5 and 0.75 μg/ml for a
3 h exposure period, 0.2 and 0.3 μg/ml for a 24 h exposure period and 0.1 and 0.15 μg/ml for a 48 h exposure period.

With metabolic activation (+S9-mix):
Cyclophosphamide (CP; CAS no. 50-18-0. Baxter B.V., Utrecht, The Netherlands) was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 10 μg/ml for a 3 h exposure period (24 h fixation time)

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

All reference stock solutions were stored in aliquots at ≤-15°C in the dark. These solutions were thawed immediately before use.

Test item preparation
No correction was made for the purity/composition of the test compound.
Hepteen Base® was dissolved in ethanol absolute (Merck).
Hepteen Base® concentrations were used within approximately 2 hours after preparation.
The final concentration of the solvent in the culture medium was 0.5% (v/v).

Test system
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (OECD).
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). 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 & First cytogenetic assay: age 27, AGT = 13.5 h
Second cytogenetic assay: age 24, AGT = 12.4 h

Cell culture
Blood samples
Blood samples were collected by venepuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Life technologies) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added.

Environmental conditions
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 56 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 - 37.1°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. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic activation system
Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life technologies).
The above solution was filter (0.22 μm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml)
phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Rationale for test conditions:

Although in the absence of S9-mix the response of MMC-C was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Evaluation criteria:

Acceptability of the assay
A chromosome aberration 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 induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) All results are inside the 95% control limits of the negative historical control data range.

Species / strain:

mammalian cell line, other: Human

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose range finding test / First cytogenetic assay
At a concentration of 52 μg/ml Hepteen Base® precipitated in the culture medium. At the 3 h exposure time, blood cultures were treated in duplicate with 5.4, 17 and 52 μg test item/ml culture medium with and without S9-mix (first cytogenetic assay).

At the 24 hour and 48 hour exposure time single blood cultures were treated with 1.7, 5.4, 17, 52 and 164 μg Hepteen Base® /ml culture medium without S9-mix (dose range finding test).

Both in the absence and presence of S9-mix, Hepteen Base® did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome
aberrations.

Both in the absence and presence of S9-mix, Hepteen Base® did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Second cytogenetic assay
To obtain more information about the possible clastogenicity of Hepteen Base®, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Hepteen Base® in the absence of S9-mix for 24 or 48 hours. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix : 10, 15, 20, 30, 40 and 52 μg/ml culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).

Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix : 10, 20 and 40 μg/ml culture medium (24 h exposure time, 24 h fixation time). 10, 20 and 30 μg/ml culture medium (48 h exposure time, 48 h fixation time).

Hepteen Base® did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
Hepteen Base® did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Evaluation of the results
The ability of Hepteen Base® to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on the solubility of the test item in the culture medium for the 3-hour treatment and on inhibition of the mitotic index of about 50% or greater for the 24 and 48-hour treatment.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. Although in the absence of S9-mix the response of MMC-C was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix Hepteen Base® did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of Hepteen Base® on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.
Therefore it can be concluded that Hepteen Base® does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in the report.

Remarks on result:

other:

Remarks:

clastogenic in human lymphocytes under the experimental conditions

Conclusions:

Hepteen Base® is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Executive summary:

Evaluation of the ability of Hepteen Base® to induce chromosome aberrations in cultured peripheral human lymphocytes.

The report describes the effect of Hepteen Base® on the number of chromosome aberrations 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 of Hepteen Base® was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline. Batch LT5C30Y170 of Hepteen Base® was a clear amber liquid. The test item was dissolved in ethanol.

In the first cytogenetic assay, Hepteen Base® was tested up to 52 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Hepteen Base® precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Hepteen Base® was tested up to 40 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 30 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. Although in the absence of S9-mix the response of MMC-C was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Hepteen Base® did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No effects of Hepteen Base® on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Hepteen Base® does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Hepteen Base® is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In Vitro Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells

Evaluation of the mutagenic activity of Hepteen Base® in anin vitromammalian cell gene mutation test with L5178Y mouse lymphoma cells.

 

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).

 

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.

 

In Vitro Mammalian Chromosome Aberration Test with human lymphocytes

Evaluation of the ability of Hepteen Base® to induce chromosome aberrations in cultured peripheral human lymphocytes.

 

The effect of Hepteen Base® on the number of chromosome aberrations 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 of Hepteen Base® was tested in two independent experiments.

 

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

 

In the first cytogenetic assay, Hepteen Base® was tested up to 52 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Hepteen Base® precipitated in the culture medium at this dose level.

 

In the second cytogenetic assay, Hepteen Base® was tested up to 40 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 30 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.

 

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.

 

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.

 

Although in the absence of S9-mix the response of MMC-C was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Hepteen Base® did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

 

No effects of Hepteen Base® on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

 

Therefore it can be concluded that Hepteen Base® does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions and therefore is not clastogenic in human lymphocytes.

 

In vitro Reverse Mutation Assay using Salmonella Typhimurium and Escherichia Coli

Evaluation of the mutagenic activity of Hepteen Base® in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

 

Hepteen Base® was tested in theSalmonella 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).

 

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 mutation assay, Hepteen Base® was tested up to concentrations of 5000 μg/plate in the absence and presence 5% (v/v) S9-mix. Hepteen Base® precipitated on the plates at dose levels of 1600 and 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix.

 

In the second mutation experiment, Hepteen Base® was tested in the absence and presence of 10% (v/v) S9-mix at a concentration range of 1.7 to 1600 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and at a range of 5.4 to 5000 μg/plate in WP2uvrA. Hepteen Base® precipitated on the plates at dose levels of 1600 and 5000 μg/plate. Toxicity was observed in all tester strains in the absence and presence of S9-mix.

 

Hepteen Base® 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 Hepteen Base® is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

In Vitro Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells

Based on the results HEPTEEN BASE® does not have to be classified and has no obligatory labelling required for mutagenicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 - classification, labelling and packaging of substances and mixtures

 

In Vitro Mammalian Chromosome Aberration Test with human lymphocytes

Based on the results HEPTEEN BASE® does not have to be classified and has no obligatory labelling required for mutagenicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including amendments and Regulation (EC) No 1272/2008 - classification, labelling and packaging of substances and mixtures.

In vitro Reverse Mutation Assay using Salmonella Typhimurium and Escherichia Coli

Based on the results HEPTEEN BASE® does not have to be classified and has no obligatory labelling required for mutagenicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including amendments and Regulation (EC) No 1272/2008 - classification, labelling and packaging of substances and mixtures.