Reaction mass of 3-[3-(9-butyl-1,1,3,3,5,5,7,7,9,9-decamethylpentasiloxanyl)propoxy]-2-hydroxypropyl 2methylprop-2-enoate and 1-[3-(9-butyl-1,1,3,3,5,5,7,7,9,9-decamethylpentasiloxanyl)propoxy]-3-hydroxypropan-2-yl 2-methylprop-2-enoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro mutagenticity studies were conducted on the substance.

Bacterial reverse mutation assay (Ames test): Based on the study results, it was concluded that the test article was not mutagenic under the conditions of the study.

In vitro mammalian chromosome aberration test: The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Mammalian cell gene mutation assay (mouse lymphoma assay): The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Link to relevant study records

Reference

Reference 1

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:

April 6-27, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results. Conducted to method equivalent to OECD and to Japanese GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

To set the dose levels in this study, a dose-setting study was conducted to examine the number of reverse mutant colonies, antimicrobial activity and precipitation. In the dose-setting study, six dose levels were set up with 5000 μg/plate as the highest level and common ratio of 4.

The test article neither showed antimicrobial activity to any of the microbial strains regardless of the presence or absence of a metabolic activator at all dose levels nor increased the number of reverse mutant colonies twice or more than that in the negative vehicle. The test article precipitated at concentrations of 1250 μg/plate or higher regardless of the presence or absence of a metabolic activator but this had no effect on the counting of reverse mutant colonies.
Based on the above results, it was decided to use 5 dose levels with 5000 μg/plate as the highest dose level and common ratio of 2 for all microbial strains regardless of the presence or absence of a metabolic activator.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Genes involved in histidine synthesis
Genes involved in tryptophan synthesis

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

5, 20, 78, 313, 625, 1250, 2500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The vehicle for the test article was decided based on the result of the dissolution test, which was conducted in distilled water, DMSO, and acetone. The concentration of the dissolution test was set at 50 mg/mL in distilled water and DMSO and 100 mg/mL in acetone. The test article was added to each of the vehicles to give 50 or 100 mg/mL and solubility and reactions with the vehicle such as heat and fume generation were observed macroscopically. As a result, the test article dissolved in acetone. It also gave a uniformly dispersed suspension in DMSO by ultrasonication. Dispersion by ultrasonication was also attempted in distilled water but a uniform dispersion was not observed. The reactivity of the test article with vehicles such as heat and fume generation was examined. No reaction was observed with any of the vehicles. Based on the above results, acetone, which gave a solution of the test article without reacting and in which the test article was stable as a solution, was selected as the vehicle. The acetone used in the preparation was dehydrated by molecular sieving.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

benzo(a)pyrene

other: 2-(-2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Expression time (cells in growth medium): 48 hours

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
Revertant colonies and checking background lawn.

Evaluation criteria:

Dose-dependent increase in the reverse mutant colonies of any of the microbial strains regardless of the presence or absence of metabolic activation and a twice or more increase in comparison to the negative (vechicle) control.

Statistics:

Standard deviation.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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:

RANGE-FINDING/SCREENING STUDIES:
Six dose levels in total were prepared with the highest dose level of 5000 μg/plate, which was diluted by a common ratio of 4.

COMPARISON WITH HISTORICAL CONTROL DATA:
The reverse mutant colony counts in the negative (vehicle) control and the positive controls were within the normal ranges based on the respective background data. 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9—aminoacridine, 2-aminoanthracene and benzo[α]pyrene, used as positive controls, increased the reverse mutant colonies twice or more in comparison to the negative (vehicle) control. These results demonstrate appropriate conduct of the study. Neither environmental factor that might have affected the study reliability adversely nor deviation from the protocol was observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test article did not show a dose-dependent increase in the reverse mutant colonies of any of the microbial strains regardless of the presence or absence of metabolic activation and a twice or more increase in comparison to the negative (vehicle) control. The result was reproduced in the dose-setting study and the main study. In the sterility test performed in Nutrient broth No. 2 used in the pre-culture, in the test article solution at the highest concentration used in the study, S9 Mix and 0.1 M phosphate buffer, no microbial contamination was confirmed.

The test article did not show antimicrobial activity to any of the microbial strains regardless of the presence or absence of a metabolic activator. It was precipitated at doses of 625 μg/plate or higher in the absence of a metabolic activator and 1250 μg/plate or higher in the presence of a metabolic activator.

Remarks on result:

other: not mutagenic under the conditions of this study.

Please refer to the attached background material for the results of the dose-setting study (Table 1) and the result of the main study (Table 2).

Conclusions:

The test article did not show a dose-dependent increase in the reverse mutant colonies of any of the microbial strains regardless of the presence or absence of metabolic activation and a twice or more increase in comparison to the negative (vehicle) control. The result was reproduced in the dose-setting study and the main study. It was concluded that the test article was not mutagenic under the conditions of this study.

Executive summary:

In a reverse gene mutation assay in bacteria strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and strain WP2 uvr A of E. coli  were exposed to the test substance in acetone, at concentrations of 5, 20, 78, 313, 625, 1250, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation pre-incubation. 

 

The test substance was tested up to cytotoxic the limit concentration (5000 µg/plate). The test article precipitated at doses of 625 µg/plate or higher in the absence of a metabolic activator and 1250 µg/plate or higher in the presence of a metabolic activator.

No growth inhibition was detected.

The number of reverse mutant colonies was within the range of the historical data both in the negative and positive controls. Absence of contamination in the study system was also confirmed validating proper conduct of the study.

Based on the results, it was concluded that the test article was not mutagenic under the conditions of the study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 13 June 2008 and 28 August 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

See princilpes of method if other than guideline section for full information on deviation.

Principles of method if other than guideline:

Deviation: In the OECD 473 Guideline it is recommended that a repeat of the 4(20)-hour exposure with metabolic activation is performed if a negative response is seen in the first experiment, unless there is scientific justification for its omission. This study was run in parallel with a Mouse Lymphoma Assay (MLA) using L5178Y cells (project No. 2551/0027) which has the capability of detecting clastogenic activity. The study was performed to meet the requirements of the OECD 476 Guideline. It was therefore considered that the results of the MLA study gave adequate scientific justification for the omission of the repeat of the with metabolic activation exposure group.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: huma

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and β-naphthoflavone induced rat liver (S9 mix)

Test concentrations with justification for top dose:

Preliminary Toxicity Test: The dose range of test material used was 19.53 to 5000 µg/ml.

Experiment 1:
Without S9 mix: 0*, 10, 20, 40, 80*, 120*, 160*, MMC 0.4*
With S9 mix: 0*, 10, 20, 40, 80*, 120*, 160*, CP 4.5*

Experiment 2 (without S9 mix): 0*, 10, 20, 40, 80*, 120*, 160* MMC 0.2*

* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

Vehicle / solvent:

The test material was accurately weighed, dissolved in acetone and serial dilutions prepared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9, mitomycin C (MMC) 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.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9, cyclophosphamide (CP) was used at 4.5 µg/ml. It was dissolved in dimethyl sulphoxide.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.


DURATION
- Preincubation period: 48 hours.
- Exposure duration: Experiment 1; 4 hour exposure with and without S9 mix. Experiment 2; 24 hour continuous exposure without S9 mix.
- Expression time (cells in growth medium): 20 hours for 4 hour exposure period.
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours.


SELECTION AGENT (mutation assays): No selection agent.
SPINDLE INHIBITOR (cytogenetic assays): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/ml) two hours before the required harvest time.
STAIN (for cytogenetic assays): 5% Gurrs Giemsa


NUMBER OF REPLICATIONS: Duplicate cultures.


NUMBER OF CELLS EVALUATED: 100 per culture.



DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
- Scoring of Chromosome Damage; Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.


OTHER EXAMINATIONS:
- Determination of polyploidy: The frequency of polyploid cells were recorded.

-Qualitative Slide Assessment:
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.



RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 19.53 to 5000 µg/ml. The maximum dose was the maximum recommended dose level. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 156.25 µg/ml, in the 4(20)-hour exposure groups, and at and above 78.13 µg/ml in the 24-hour continuous exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 µg/ml in all three exposure groups. The mitotic index data are presented in Table 1. The test material did not demonstrate any clear evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level was based on the lowest precipitating dose level in the 4(20)-hour and 24 hour exposure groups, and was 160 µg/ml throughout the main experiments.



COMPARISON WITH HISTORICAL CONTROL DATA:
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials 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.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
EXPERIMENT 1:
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 at the maximum dose level of test material tested, 160 µg/ml in both the absence and presence of metabolic activation (S9).

The mitotic index data are given in Table 2. They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed, and that 20% mitotic inhibition was achieved at 160 µg/ml in the absence of S9, and 12% mitotic inhibition achieved at 80 µg/ml in the presence of S9. These observations were similar to those seen in the preliminary toxicity test.

The maximum dose level selected for metaphase analysis was the maximum dose level tested (160 µg/ml) in both the absence and presence of metabolic activation.

The chromosome aberration data are given in Table 4 and Table 5.

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

The polyploid cell frequency data are given in Table 7. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

EXPERIMENT 2:
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 at the maximum dose level of test material tested, 160 µg/ml.

The mitotic index data are given in Table 3. They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed, and that 20% mitotic inhibition was achieved at 120 µg/ml.

The maximum dose level selected for metaphase analysis was the same as Experiment 1, and was the maximum dose level tested (160 µg/ml).

The chromosome aberration data are given in Table 6.

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

The polyploid cell frequency data are given in Table 7. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in the absence of metabolic activation.


Please see attached background material for all results tables.

Remarks on result:

other: non-clastogenic to human lymphocytes in vitro.

There was no evidence of a response in the presence of metabolic activation in this study or in the MLA test performed on the test material (Project No. 2551/0027). This was taken as scientific justification to confirm that the repeat of the exposure group with metabolic action was not required.

Conclusions:

The test material 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 metabolising system in either of three separate exposure conditions. The test material 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 in so far as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 2000/32/EC. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. 

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising 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 in the absence of metabolic activation the exposure time was increased to 24 hours.

Results. 

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

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was non-toxic 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 was limited by the lowest precipitating dose level.

Conclusion.

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 24 June 2008 and 26 August 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

In the OECD 476 Guideline it is recommended that a repeat of the 4-hour exposure with metabolic activation is performed if a negative response is seen in the first experiment, unless there is scientific justification for its omission.

Principles of method if other than guideline:

Explanation of deviation: In the OECD 476 Guideline it is recommended that a repeat of the 4-hour exposure with metabolic activation is performed if a negative response is seen in the first experiment, unless there is scientific justification for its omission. This study was run in parallel with a Chromosome Aberration Test using Human Lymphocytes (project No. 2551/0026) which detects genotoxic (clastogenicity) activity. The study was performed to meet the requirements of the OECD 473 Guideline. There was no evidence of a response in the presence of metabolic activation in this study or in the Chromosome Aberration Test performed on the same test material. This was taken as scientific justification to confirm that the repeat of the exposure group with metabolic action was not required.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

This study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The stocks of cells are stored in liquid nitrogen at -196°C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/β-naphthoflavone induced rat liver (S9 mix).

Test concentrations with justification for top dose:

Preliminary Toxicity Test (4 hour exposure with and without S9 and 24 hour exposure without S9): The dose range used in the preliminary toxicity test was 19.53 to 5000 μg/ml.
Mutagenicity Test:
4-Hour exposure with and without Metabolic Activation: six dose levels of the test material (39.06 to 625 μg/ml)
24-Hour exposure without Metabolic Activation: The dose range of the test material was 39.06 to 625 μg/ml

The 6 dose levels covered are: 39.06, 78.13, 156.25, 312.5, 468.75, 625 μg/ml

Vehicle / solvent:

Solvent (acetone) treatment groups were used as the vehicle controls.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate (EMS) at 400 μg/ml and 150 μg/ml for the 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide (CP) at 2 μg/ml was used as the positive control in the presence of metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: Not stated.
- Exposure duration: 4 hour exposure and 24 hour exposure.
- Expression time (cells in growth medium): 48 hours.
- Selection time (if incubation with a selection agent): 10 - 14 days.



SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): MTT


NUMBER OF REPLICATIONS: Duplicate.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: Daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation.
The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate).

Evaluation criteria:

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.

Statistics:

The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test material was dosed into media.
- Effects of osmolality: The osmolaality did not increase more than 50 mOsm in the Chromosome Aberration Test performed on the same test material.
- Precipitation: Preliminary Toxicity Test - A cloudy precipitate of the test material was observed, overall, at and above 78.13 μg/ml, and a greasy / oily precipitate was formed at and above 625 μg/ml in the 4-hour exposure group in the presence of metabolic activation, and at and above 1250 μg/ml in both the 4-hour and 24-hour exposure groups in the absence of metabolic activation.



RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The results for the Relative Suspension Growth (%RSG) are shown in the remarks on results section.
Based on the %RSG values for all three of the exposure groups, maximum exposure of the test material to the cells appeared to occur at approximately 312.5 to 625 μg/ml. Therefore, the onset of the greasy/oily precipitate combined with the RSG values resulted in the maximum dose level in the mutagenicity test being set at 625 μg/ml. The elevated %RSG values observed at and above 2500 μg/ml in the 24-hour exposure group were considered to be due to the toxic nature of the solvent (acetone) during the extended exposure period i.e. the higher the dose level the lower the volume of acetone and, therefore, the higher the %RSG value when compared to the vehicle control culture.

COMPARISON WITH HISTORICAL CONTROL DATA:
Neither of the vehicle control mutant frequency values were outside the range of 50 to 200 x 10E-6 viable cells that is acceptable for L5178Y cells at Safepharm Laboratories. 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 (Tables 3 and 6).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutagenicity Test:
A summary of the results from the test is presented in Table 1.

4-Hour Exposure With and Without Metabolic Activation:
The results of the microtitre plate counts and their analysis are presented in Tables 2 to 10.
There was no evidence of any significant toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was also no evidence of any reductions in (%V) viabilities, therefore indicating that no residual toxicity had occurred (Tables 3 and 6). Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). A cloudy precipitate of test material was observed at and above 156.25 μg/ml and a greasy / oily precipitate was formed at and above 468.75 μg/ml. Evidence of heterogeneity (poor correlation between A and B cultures) was observed at 625 μg/ml in the presence of metabolic activation. However, with no evidence of any toxicologically significant responses, in any of the exposure groups, the data was considered acceptable for the purpose of this study. The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

24-Hour Exposure Without Metabolic Activation:
The results of the microtitre plate counts and their analysis are presented in Tables 8 to 10.
There was no significant evidence of toxicity following exposure to the test material as indicated by the %RSG and RTG values and also (%V) viabilities. The elevated RTG values observed in the test material dose levels, and elevated %RSG and RTG values in the positive control, were once again attributable to the toxic nature of the solvent (acetone) during the extended exposure period, and the use of Dimethyl sulfoxide as the solvent for the positive control (Table 9).

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had no effect on the toxicity of the test material.

The test material did not induce any statistically significant or dose-related increases in the mutant frequency in the absence of metabolic activation (Table 9). A cloudy precipitate of test material was observed at and above 156.25 μg/ml and a greasy / oily precipitate was formed at and above
468.75 μg/ml. Evidence of heterogeneity (poor correlation between A and B cultures) was observed at 312.5 μg/ml. However, with no evidence of any toxicologically significant responses, in any of the exposure groups, the data was considered acceptable for the purpose of this study.
The numbers of small and large colonies and their analysis are presented in Table 10.

There was no evidence of a response in the presence of metabolic activation in this study or in the Chromosome Aberration Test performed on the same test material (SPL Project No. 2551/0026). This was taken as scientific justification to confirm that the repeat of the exposure group with
metabolic action was not required.

Remarks on result:

other: non-mutagenic under the conditions of the test.

Preliminary Toxicity Test:

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
19.53	107	104	86
39.06	108	95	99
78.13	109	100	86
156.25	111	104	105
312.5	97	90	104
625	102	91	89
1250	114	95	107
2500	104	105	164
5000	100	102	241

Mutagencity Tests:

For all tables referenced above please refer to the attached background material.

CONCLUSION:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Conclusions:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction.

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Methods.

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. The exposure groups used were as follows: 4-hour exposures with and without metabolic activation and 24 hours without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 39.06 to 625 μg/ml for all three of the exposure groups.

Results.

The maximum dose level used in the mutagenicity test was limited to 625 μg/ml. This was due to the onset of a greasy / oily precipitate effectively reducing the exposure of the test material to the cells in the preliminary toxicity test. Whilst precipitate was observed at the end of the exposure periods of the mutagenicity test, it was not carried over into the maintenance phases of the test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, in any of the three exposure groups.

Conclusion.

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay (Ames test):

In a reverse gene mutation assay in bacteria strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and strain WP2 uvr A of E. coli  were exposed to the test substance in acetone, at concentrations of 5, 20, 78, 313, 625, 1250, 2500 and 5000µg/plate in the presence and absence of mammalian metabolic activation pre-incubation. 

 

The test substance was tested up to cytotoxic the limit concentration (5000µg/plate). The test article precipitated at doses of 625 µg/plate or higher in the absence of a metabolic activator and 1250 µg/plate or higher in the presence of a metabolic activator.

No growth inhibition was detected.

The number of reverse mutant colonies was within the range of the historical data both in the negative and positive controls. Absence of contamination in the study system was also confirmed validating proper conduct of the study.

Based on the results, it was concluded that the test article was not mutagenic under the conditions of the study.

In vitro mammalian chromosome aberration test:

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 (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 2000/32/EC. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising 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 in the absence of metabolic activation the exposure time was increased to 24 hours.

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

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was non-toxic 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 was limited by the lowest precipitating dose level.

Conclusion. The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Mammalian cell gene mutation assay (mouse lymphoma assay):

Introduction.

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Methods.

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. The exposure groups used were as follows: 4-hour exposures with and without metabolic activation and 24 hours without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 39.06 to 625 μg/ml for all three of the exposure groups.

Results.

The maximum dose level used in the mutagenicity test was limited to 625 μg/ml. This was due to the onset of a greasy / oily precipitate effectively reducing the exposure of the test material to the cells in the preliminary toxicity test. Whilst precipitate was observed at the end of the exposure periods of the mutagenicity test, it was not carried over into the maintenance phases of the test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, in any of the three exposure groups.

Conclusion.

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Justification for classification or non-classification

Based on negative results in three in-vitro studies, the substance is not classified for mutagencity.