5-methylheptan-3-one oxime

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No effects seen in 3 in vitro methods

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: L5178T TK +/- Mouse Lymphoma Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May 2014 to 14 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Batch: PE00083052
Purity: 96.3%

Target gene:

This study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0, 5.59, 11.19, 22.38, 44.75, 89.5, 179, 358, 716, 1432 µg/mL

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate without S9 activation Cyclophosphamide with S9 activation

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

RESULTS

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 5.59 to 1432 μg/mL in all three exposure groups. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (µg/mL)	%RSG(-S9) 4-Hour Exposure	%RSG(+S9) 4-Hour Exposure	%RSG(-S9) 24-Hour Exposure
0	100	100	100
5.59	94	93	97
11.19	86	101	108
22.38	84	94	96
44.75	80	101	93
89.5	78	9	73
179	54	72	25
358	0	0	0
716	0	0	0
1432	0	0	0

There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. The onset of test item-induced toxicity was very sharp in all three of the exposure groups. A precipitate of the test item was observed at 1432 μg/mL in all three exposure groups at dosing. Based on the %RSG values observed, the maximum dose level in the subsequent mutagenicity experiment was limited by test item-induced toxicity.

 

Mutagenicity Test

Experiment 1

There was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of reductions in viability (%V) in the absence of metabolic activation, therefore indicating that residual toxicity had not occurred in this exposure group. In the presence of metabolic activation a reduction in viability can be observed, but only at a single dose level with excessive cytotoxicity (268.5 μg/mL). Based on the %RSG and RTG values observed, optimum levels of toxicity were achieved in the absence of metabolic activation only. Optimum levels of toxicity were not achieved in the presence of metabolic activation, due to the very steep toxicity curve of the test item.

Whilst optimum levels of toxicity were not achieved, a dose level in the presence of metabolic activation, that exceeded the upper limit of toxicity was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating (268.5 μg/mL). The excessive toxicity observed at 358 μg/mL in both the absence and presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 268.5 μg/mL in the presence of metabolic activation, exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell, at any of the dose levels (including the dose levels that exceeded the upper limit of acceptable toxicity), in either the absence or presence of metabolic activation. With no evidence of any toxicologically significant increases in mutant frequency in either the absence or presence of metabolic activation in this Experiment, the test item was considered to have been adequately tested in the absence of metabolic activation. Precipitate of the test item was not observed at any of the dose levels.

As was seen previously, there was evidence of marked toxicity in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. In the presence of metabolic activation a reduction in viability can be observed, but only at a single dose level with excessive cytotoxicity (270 μg/mL). Based on the RTG and / or %RSG values observed, very near to optimum levels of toxicity were considered to have been achieved in both the absence and presence metabolic activation. In the presence of metabolic activation the highest test item dose level (270 μg/mL) had a RTG value of 0.08 and a %RSG of 11%, which was very close to optimum toxicity. However, marginally fell below the acceptable level of toxicity and was therefore excluded from statistical analysis. The excessive toxicity observed at and above 180 μg/mL in the absence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a modest effect on the toxicity of the test item.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell, at any of the dose levels (including the dose levels that exceeded the upper limit of acceptable toxicity), in either the absence or presence of metabolic activation. It should be noted that all mutant frequency values were within the acceptable range for a vehicle control culture. With very near to optimum levels of toxicity achieved and no evidence of any toxicologically significant increases in mutant frequency in Experiments 1 and 2 inclusive, it was considered that the test item was adequately exposed. Precipitate of test item was not observed at any of the dose levels.

 

CONCLUSION

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests" adopted 21 July 1997, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods…….

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle, dimethyl sulfoxide (DMSO), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows:

 

Experiment 1

Group	Concentration of Stemone (μg/mL) plated for mutant frequency
4-hour without S9	22.38, 44.75, 89.5, 134.25, 179, 268.5
4-hour with S9 (2%)	22.38, 44.75, 89.5, 134.25, 179, 268.5

Experiment 2

Group	Concentration of Stemone (μg/mL) plated for mutant frequency
24-hour without S9	15, 30, 60, 90, 120, 150
4-hour with S9 (2%)	60, 90, 180, 210, 240, 270

 

Results……..

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle controls (DMSO) had mutant frequency values that wereconsidered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

 

Conclusion

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: Chromosome Aberration Test in Human Lymphocytes in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 June 2014 to 10 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch: PE00083052
Purity: 96.3%

Target gene:

The purpose of the study was to assess the potential chromosomal mutagenicity of the test item, Stemone, on the metaphase chromosomes of normal human lymphocytes. Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test.

Species / strain / cell type:

lymphocytes: human

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Dose levels (μg/mL):
0, 5.6, 11.2, 22.4, 44.8, 89.5, 179, 358, 716, 1432

Vehicle / solvent:

dimethyl sulphoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9, mitomycin C (MMC) (Sigma, Batch No. SLBD1982V) was used at 0.4 and 0.2 μg/mL for cultures in Experiment 1 and 2 respectively. It was dissolved in Minimal Essential Medium.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9, cyclophosphamide (CP) (Acros, Batch No. A0302605) was used at 5 μg/mL in both experiments. It was dissolved in dimethyl sulphoxide.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 5.6 to 1432 μg/mL. The maximum dose was the maximum recommended 10 mM concentration.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 716 μg/mL, in the 4(20)-hour exposure group in the presence of S9 and at 1432 μg/mL in the 4(20)-hour exposure group in the absence of S9 and in the continuous exposure group.

Haemolysis was observed following exposure to the test item at and above 179 μg/mL in all three of the exposure groups. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 358 μg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 179 μg/mL. The mitotic index data are presented in Table 1. The test item induced evidence of toxicity in all of the exposure groups.

The selection of the maximum dose level was based on toxicity in all exposure groups in both experiments.

 

Chromosome Aberration Test - Experiment 1

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of Stemone (µg/ml)
4(20)-hour without S9	0*	44.8	89.5*	179*	358*	537	716	MMC 0.4*
4(20)-hour with S9 (1%)	0*	44.8	89.5*	179*	358*	537	716	CP 5*

 

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

 

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 358 μg/mL in the absence of metabolic activation, and up to 537 μg/mL in the presence of metabolic activation (S9).

Precipitate observations were made at the end of exposure and precipitate was noted at 716 μg/mL in both exposure groups. Haemolysis was observed following exposure to the test item at and above 179 μg/mL in both of the exposure groups.

The mitotic index data confirmed the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 23% mitotic inhibition was achieved at 358 μg/mL in the absence of S9 the next dose level up had no metaphases. In the presence of S9 a dose-related inhibition of mitotic index was observed with 88% mitotic inhibition at 537 μg/mL, though this markedly exceeds the 50% optimum level of toxicity.

The maximum dose level selected for metaphase analysis was limited by toxicity to 358 μg/mL in both exposure groups.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

 

Chromosome Aberration Test - Experiment 2

The dose levels of the controls and the test item are given in the table below:

 

Group	Final concentration of Stemone (µg/ml)
24-hour without S9	0*	11.2	22.4	44.8*	89.5*	179*	358	MMC 0.2*
4(20)-hour with S9 (1%)	0*	44.8	89.5*	179*	358*	447.5	537	CP 5*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphami

 

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at 179 μg/mL in the absence, and 358 μg/mL in the presence of S9.

No precipitate of the test item was observed at the end of exposure in either exposure group. Haemolysis was observed following exposure to the test item at and above 179 μg/mL in both of the exposure groups.

The mitotic index data confirmed the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 50% mitotic inhibition was achieved at 179 μg/mL in the absence of S9. In the presence of S9 a dose-related inhibition of mitotic index was observed with 49% inhibition achieved at 358 μg/mL.

The maximum dose level selected for metaphase analysis was based on toxicity as it was in Experiment 1. The dose levels were 179 μg/mL in the absence of S9 and 358 μg/mL in the presence of S9, both achieved optimum toxicity.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

 

CONCLUSION

The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytesin vitro.

Conclusions:

The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al., 1991).

 

Methods…….

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group	Final concentration of test item Stemone (μg/mL)
4(20)-hour without S9	44.8, 89.5, 179, 358, 537, 716
4(20)-hour with S9 (2%)	44.8, 89.5, 179, 358, 537, 716
24-hour without S9	11.2, 22.4, 44.8, 89.5, 179, 358
4(20)-hour with S9 (1%)	44.8, 89.5, 179, 358, 447.5, 537

 

 

Results…….

All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item was cytotoxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition in some of the exposure groups.

 

Conclusion

The test item, Stemone was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: November 07, 2002, Experimental Completion Date: November 14, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Change of source supplier for TA 102 strain - No detrimental impact on the outcome of the study.

Deviations:

yes

Remarks:

Change of source for TA 102 strain - No detrimental impact on the outcome of the study.

Principles of method if other than guideline:

Deviations to Study Plan
Test System
Present:
The bacterial strains TA 1535, TA 1537, TA 100, and TA 102 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was obtained from E. Merck (D-64293 Darmstadt).
New:
The bacterial strains TA 1535, TA 1537, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was obtained from E. Merck (D-64293 Darmstadt). The bacterial strain TA 102 was obtained from RCC Ltd. (CH-4332 Stein).
Reason for the Alteration: updating
This deviation had no detrimental impact on the outcome of the study.
There was no deviation to the study plan.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: 9000479895
Purity: 96.9%

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Dose Selection
in the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as part of experiment I. Due to toxic effects observed in this pre-experiment 2500 µg/plate were chosen as maximal concentration in the main experiments.
The concentration range included two logarithmic decades. The following concentrations were tested:
10; 33; 100; 333; 1000; and 2500 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item STEMONE was dissolved in ethanol (purity> 99%, MERK, D-64293 Darmstadt). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
No precipitation of the test item occurred up to the highest investigated dose.

Untreated negative controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed.

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

DISCUSSION OF RESULTS

The test item STEMONE was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment l) and the pre-incubation test (experiment ll) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:

10; 33; 100; 333; 1000; and 2500 pg/plate

Toxic effects were observed at the following concentrations (pg/plate):`

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	100-2500	1000, 2500	1000, 2500	1000, 2500
TA 1537	1000, 2500	1000, 2500	1000, 2500	333 - 2500
TA 98	2500	100, 2500	1000, 2500	33 - 2500
TA 100	1000-2500	2500	1000, 2500	2500
TA 102	2500	1000, 2500	333 - 2500	333 - 2500

 

irregular background growth was observed in strain TA 98 at 100 ug/piate (with S9 mix), and in strain TA 100 at 2500 iig/plate (with and without 89 mix) in experiment I. in experiment Ii, irregular background growth was observed in strain TA 98 from 33 1000 ug/piate with 89 mix and at 33 ug/plate without S9 mix. No visible reduction of the background growth was observed in the remaining strains.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with STEMONE at any concentration level, neither in the presence nor absence of metabolic activation (S9mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The historical range of positive controls was exceeded in strains TA 1535 (Exp. ll) without metabolic activation and in strain TA 98 (exp. l) with metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay.

ln experiment l, the number of colonies did not quite reach the lower limit of our historical control data in strains TA 1535 and TA 1537 in the negative control with metabolic activation. ln addition the number of colonies exceed the upper limit of our historical control data in strain TA 98 in the negative and solvent control with metabolic activation. Since these deviations are rather small, these effect are judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, STEMONE is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of STEMONE to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

10; 33; 100; 333; 1000; and 2500 µg/plate

Toxic effects were observed at higher concentrations with and without metabolic activation in all strains used.

Irregular background growth was observed in strain TA 98 at 100 µg/plate (with S9 mix), and in strain TA 100 at 2500 µg/plate (with and without S9 mix) in experiment I. In experiment II, irregular background growth was observed in strain TA 98 from 33 - 1000 µg/plate with S9 mix and at 33 µg/plate without S9 mix. No visible reduction of the background growth was observed in the remaining strains.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with STEMONE at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in-· crease of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, STEMONE is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Chromosome Aberration Test in Human Lymphocytes in vitro

The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

L5178Y TK +/- Mouse Lymphoma Assay

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Salmonella typhimurium Reverse Mutation Assay with STEMONE

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, STEMONE is considered to be non-mutagenic.