3-(N-p-methoxy cinnamidopropyl-N,N-dimethyl ammonium)-2-hydroxypropane-1-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance has been found to be non-mutagenic in three separate Ames studies and non-clastogenic in a chromosomal aberration test.

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:

Experimental Starting Date: 25 September 2013; Experimental Completion Date: 24 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella
Tryptophan for E. Coli

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9 Microsomal fraction (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1 (Plate Incorporation Method): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Experiment 2 (Pre-Incubation Method): 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in-house.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Used in plates without S9-mix

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Used in plates with S9-mix

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation)
Experiment 2I: preincubation

DURATION
- Preincubation period: 20 minutes (Experiment 2)
- Exposure duration: 48 hours (Experiment 1 and 2)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
-All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
-All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per ml.
-Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
-There should be a minimum of four non-toxic test item dose levels.
-There should be no evidence of excessive contamination.

Evaluation Criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA

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:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (see attached background information) and were
considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2 (see attached background material for all tables).

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:

Galaxy SunBeat was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline 870.5100 - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard eo-factors).

The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 50 to 5000 µg/plate.

Results

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

Conclusion

Galaxy SunBeat was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental study conducted between 08-26 February 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for salmonella

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 97a, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: 5000, 1500, 500 and 150 µg/plate
Main test: 5000, 1500, 500, 150, 50, 15 µg/plate

Untreated negative controls:

yes

Remarks:

spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

used wihout S-9 Mix

Positive control substance:

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: ICR 191

Untreated negative controls:

yes

Remarks:

spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Remarks:

with S-9 Mix

Positive control substance:

other: 2-aminoanthracene, 2-Aminofluorene, danthron

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and Experiment 2.

DURATION:
- Preincubation for bacterial strains: 12 - 14 hours
- Exposure duration: 48 hrs.

NUMBER OF REPLICATIONS: Triplicate plaing

DETERMINATION OF CYTOTOXICITY:
-Method: plates were asessed for numbers of revertant colonies and examined for effects on the growth of the bacterial lawn.

Evaluation criteria:

The mean number of histidine revertant colonies for all the treatment groups was compared with the number of revertants in the respective control group. The mutagenic activity of the test article was assessed by applying the following criteria:

Criteria for Evaluation
1. A test article is considered as mutagenic if it produces a concentration related increase over the range tested and lor a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.

The biologically significant increase would be assumed if the average colony counts are: at least twice as compared to those in control group for strain TA 97a, TA 98, TA 100 and TA102 and at least thrice as compared to those in control group for strain TA 1535.


2. A test article is considered as non-mutagenic, if it does not induce any increase in the number of revertants and does not show any dose response relationship, in two separate experiments, with any bacterial strain, either with or without metabolic activation.

Species / strain:

other: S. typhimurium TA 1535, TA 97a, TA 98, TA 100, TA 102

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:

valid

Positive controls validity:

valid

Conclusions:

Under the conditions of this study, it is concluded that Galaxy-SunBeat (5.0%) is non-mutagenic in Salmonella typhimurium strains TA 1535, TA 97a, TA 98, TA 100 and TA 102.

Executive summary:

Mutagenicity Study of Galaxy-SunBeat (5.0%) by Salmonella typhimurium Reverse Mutation Test was carried out according to OECD 471 (adopted 21 July 1997).

Galaxy-SunBeat (5.0%) was evaluated in the Ames / Salmonella Plate Incorporation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium, TA 1535, TA 97a, TA 98, TA 100 and TA 102, in the presence and absence of metabolic activation system (S9). Based upon preliminary tests conducted to assess the solubility/precipitation and cytotoxicity of Galaxy-SunBeat (5.0%), the tester strains were exposed to the test article in triplicate cultures at the doses of 5000, 1500, 500, 150, 50 and 15 µg/plate with and without a metabolic activation system (S9).

Distilled water was used as a vehicle. The exposed bacteria were plated onto minimal glucose agar medium supplemented with L-histidine. The plates were incubated for 37 °C for 48 hours after which the histidine revertant colonies were counted and their frequency compared with that in the control group.

Results of this test indicated that the revertant frequencies at all concentrations in strains TA 1535, TA 97a, TA 98, TA 100 and TA 102, with and without metabolic activation, were comparable to those observed in the control groups.

Plate counts for the spontaneous histidine revertant colonies in the control groups were found to be within the frequency ranges expected from the laboratory historical control data. Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation.

Under the conditions of this study, it is concluded that Galaxy-SunBeat (5.0%) is non-mutagenic in Salmonella typhimurium strains TA 1535, TA 97a, TA 98, TA 100 and TA 102.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

September 11 to September 15 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Single strain used.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

not specified

Test concentrations with justification for top dose:

Tested in triplicate:
10 µg, 25 µg, 250 µg, 500 µg, 1000 µg, 2500 µg and 5000 µg

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

yes

Remarks:

0.0 µg of test material used.

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Details on test system and experimental conditions:

Salmonella typhimurium - TA 100 was procured as a gift from Rallis Research Station, Rallis India limited, Bangalore. The fresh culture was sub-cultured under standard conditions of temperature and humidity.

Medium:
In the present study, Minimal Agar Medium supplemented with 40% Glucose solution was used both to culture and test the microbial system with the test substance.

Performance of the Assay:
Fresh cultures of Salmonella typhimurium - TA 100 grown to about 10 8 – 10 9 cells per ml and with an incubation temperature of 37ºC were used for the study. Different concentrations of the test substance and 0.1 ml of the fresh bacterial culture were added to 2.0 ml of overlay agar along with trace amount of histidine. Subsequently, the contents of each tube was mixed and poured over the surface of the selected and marked agar plate. The overlay agar was allowed to solidify and later, the plates were incubated at 37ºC for 48 to 72 hours.
All tests/platings were done in triplicated to establish the significance or no significance of the assay. After an incubation period of 72 hours, the revertant colonies of the microbe per plate was counted to establish the genotoxic potential of the test substance in the in vitro system.

Evaluation criteria:

After completion of the incubation period of 72 hours, each plate was counted to know the mean number of revertant colonies per plate, if any. The results obtained in different concentrations of the test substance were compared with those of negative and positive control.

Statistics:

The data from the control and test groups were subjected to Analysis of Variance (ANOVA) test. When a statistically significant difference was observed by ANOVA, paired comparisons between test and control groups were made using Student's - 't' test. In all cases a level of probability less than 0.05 was taken to indicate statistical significance.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results have indicated that the exposure of salmonella typhimurium - TA 100 to different concentrations of the test substance demonstrated no significant presence of revertants as compared to negative control. This has suggested the absence of mutagenic potential of the test substance. In comparison, the positive control treatment caused highly significant number of revertants.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The findings of the study has indicated that the test substance viz.. GALAXY - SUNBETAINE - MM - 17 has not induced any reverse mutation in Salmonella typhimurium TA 100.

Executive summary:

Salmonella typhimurium TA 100 were exposed to different concentrations of the test substance viz., GALAXY - SUNBETAINE - MM-17 under standard conditions for a period of 72 hours. The test was conducted using both negative control and positive control (Sodium azide).

Observations were made to know reverse mutations at the end of 72 hours. The test failed to suggest the induction of any significant histidine⁺ reverse mutants in all the test concentrations. In contrast, the positive control demonstrated a very significant number of histidine⁺ mutants. The results of the test substance (spontaneous revertants) was comparable with those of the negative control.

The findings of the study has indicated that the test substance viz.. GALAXY - SUNBETAINE - MM - 17 has not induced any reverse mutation in Salmonella typhimurium TA 100.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April to 20 May 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

- Type and identity of media:
RPMI 1640-10
RPMI 1640 with 10% Foetal calf serum, Penicillin, streptomycin, L-glutamine. pH - 7.2 to 7.4
RPMI 1640-2
RPMI 1640 with 2% Foetal calf serum, Penicillin, streptomycin, L-glutamine. pH - 7.2 to 7.4

- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background:no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Liver Fraction (S9)

Test concentrations with justification for top dose:

5000 µg/ml, 1500 µg/ml and 500 µg/ml, with and without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

vehicle

Negative solvent / vehicle controls:

yes

Remarks:

DSMO 0.2 ml

True negative controls:

not specified

Positive controls:

yes

Remarks:

30 µg/ml

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Remarks:

vehicle

Negative solvent / vehicle controls:

yes

Remarks:

DSMO 0.2 ml

True negative controls:

not specified

Positive controls:

yes

Remarks:

30 µg/ml

Positive control substance:

cyclophosphamide

Remarks:

with metabolica activation

Details on test system and experimental conditions:

Exposure Concentrations
Test concentrations for this study were selected from the results obtained with a solubility precipitation test and a preliminary cytotoxicity test of the test article, conducted with and without metabolic activation.

Solubility/Precipitation Test
Galaxy-SunBeat was dissolved in DSMO in which it was found to be completely soluble and when added to the final reaction mixture containing medium, serum and S9, no precipitation was observed at the concentrations of 5000 µg/ml. Hence the highest concentration of 5000 µg/ml was employed for the preliminary cytotoxicity test. Solubility was assessed by using unaided eye.

Preliminary Cytotoxicity Test
The test article was assessed for cytotoxicity to the cultured human lymphocytes. Four concentrations viz. 5000 µg/ml, 1500 µg/ml, 500 µg/ml and 150 µg/ml of the test article along with a concurrent solvent control were tested with a treatment period of 3 h with and without metabolic activation. Cytotoxicity was assessed on the basis of percent reduction in the mitotic index as compared to the solvent control.
In this sutotoxicity study, the mitotic index for the test concentrations of 5000 µg/ml, 1500 µg/ml, 500 µg/ml and 150 µg/ml was found to be comparable to that of the solvent control group.
Based upon the above results the highest recommended concentration of 5000 µg/ml was selected for the main study, along with the lower test concentrations of 1500 µg/ml and 500 µg/ml.

Stimulation of Mitosis
Microcultures containing 0.4 ml heparinised whole blood and 9.6 ml medium (RPMI 1640-10 and Phytohaemagglutinin-M) were incubated for 48 hours at 37°C, 5% CO2 in air and 90 to 100% humidity.

Treatment of Lymphocytes
Proliferating cells were treated with the test article in the presence and absence of metabolic activation system. Duplicate cultures were used at each concentration.

Experiment without metabolic activation
After 48 hours incubation, 0.2 ml aliquots of various dilutions of the test article were added to duplicate culture without changing the growth medium.
In the first experiment, after 3 hours of incubation at 37°C, the cultures were centrifuged at about 1000 rpm for 5 minutes, washed twice, resuspended in 10 ml warm medium (RPMI 1640-10) and were incubated until sampling at a time equivalent to about 1.5 cell cycle length i.e. 24 hours.
In the second experiment without activation was performed with continuous treatment until sampling at a time equivalent to about 1.5 cell cycle length i.e. 24 hours.

Experiment with metabolic activation
After 48 hours incubation, cultures were centrifuged at about 1000 rpm for 5 minutes. After discarding the supernatant the pellet was resuspended in 10 ml warm medium (RPMI 1640-2 and Phytohaemagglutinin-M) with 0.5 ml S9 mix. In treatment groups, the test article at different concentrations was added to each culture. After 3 hours of incubation at 37°C, the cultures were washed twice, resuspended in 10 ml warm medium (RPMI 1640-10) and incubated until sampling at a time equivalent to about 1.5 cell cycle length i.e. 24 hours.

Arresting of Metaphase
Colchicine at a final concentration of 0.5 ml µg/ml was added to each culture 2 hours before harvesting of the lymphocytes.

Fixation of Lymphocytes and Preparation of Slides
At end of the incubation period each culture was transferred to a centrifuge tube. The cell suspension was centriuged at about 1000 rpm for 5 minutes and the supernatant was discarded. To each of these tubes 5 ml of 0.56% potassium chloride (KCl) was added for hypertonic treatment of the cells at room temperature for 20+/- 2 minutes. After hypotonic treatment, the suspension was once again centrifuged at about 1000 rpm for 5 minutes and supernatant discarded. The freshly obtained pellet was fixed by resuspending in 5 ml fresh cold methanol: acetic acid (3:1 v/v) and left at room temperature for 10 minutes, then centrifuged at about 1200 rpm for 5 minutes and the supernatant was discarded.
After tht third change of fixative the pellet was resuspended in small volume of fixative and the solution was dropped on to clean chilled glass microsope slides.
The slides were kept in the dust free chamber for air drying and later stained with 5% Giemsa in Phosphate buffer (pH 6.8). The slides were air dried, coded and examined under microscope with 100X objective for well spread metaphases.

Observations
Mitotic Index (MI)
The mitotic index (MI) (% cells in mitosis) was determined for slides fo preliminary cytotoxicity test to establish the cytotoxicity of the test article. It was calculated as below:

Mitotic index = (No. of cells in dividion (metaphases)/Total no. of cells including metaphases) x 100

Evaluation criteria:

Chromosomal Aberrations
Only well spread metaphases were evaluated for chromosomal aberration. The coded slides were analyzed using oil immersion objective (100X). At least 200 metaphases plates, referred to as "Cells" in the text, were evaluated per test concentration and for the negative and positive controls, divided amoung the duplicates.
Aberrations such as gaps, break or fragment and exchanges were scored and noted as chromatid and chromosome type. Other abnormalities such as multiple breaks, pulverization, deletion, rind chromosome, polyploidy and endoreduplication were scored and recorded.

Evaluation of Results
Total number of aberrations, number of aberrations per cell, and proportion (%) of cells with aberrations were computed both inclusive and exclusive of gaps. Data were evaluated for statistical significance using the chi-square test.

Criteria for Evaluation
1. A test article is considered as mutagenic if it produces a concentration related increase over the range tested and/or a reproducible increase in number of cells with chromosome aberrations with or without metabolic activation system.
2. A test article is considered non-mutagenic if it does not induce any increase in the number of cells with chromosome aberrations, and does not show any dose response relationship, either with or without metabolic activation.

Species / strain:

lymphocytes: human peripheral

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:

Mitotic Index
The average mitotic indices, determined for the preliminary cytotoxicity test, following 3 hours treatment in the absence of metabolic activation system, observed at the concentration levels of 5000 µg/ml, 1500 µg/ml, 500 µg/ml and 150 µg/ml were 6.3, 4.8, 5.0 and 5.0 respectively. The same was 5.8 in the vehicle control group.
The average mitotic indices, determined for te preliminary cytotoxicity test, following 3 hours exposure in the presence of metabolic activation system, were 5.3, 4.5, 4.8 and 4.8 respectively at the concentration levels of 5000 µg/ml, 1500 µg/ml, 500 µg/ml and 150 µg/ml. The same was 5.5 in the vehicle control group.
These results indicated that Galaxy-SunBeat had no cytotoxic effect on the cultured human lymphocytes at the maximum test concentration of 5000 µg/ml as compared to the vehicle control group.

Chromosomal Aberrations
The aberrations encountered during the evaluations included: chromatid and chromosomal type gap and break, acentric fragment, polyploidy and endoreduplication.
For the two experiments carried out in the absence of metabolic activation system, following 3 hours and 24 hours exposure respectively, there was no significant concentration related increase in the incidence of structural chromosomal aberrations at any of the concentration levels of Galaxy-SunBeat when compared to the incidence in the respective solvent control group.
Similarly for the experiment carried out in presence of the metabolic activation system, following 3 hours exposure, there was no significant and concentration related increase inthe incidence of structural chromosomal aberrations at any concentration levels of Galaxy-SunBeat when compared to the incidence in the colvent control group.
The incidence of chromosomal aberrations was found to be in agreement with available laboratory control data.
The concurrent positive controls, Methyl methane sulphonate (30 µg/ml) and Cyclophosphamide (30 µg/ml) when tested respectively in the absence and presence of metabolic activation, induced significant increases in the incidence of structural chromosomal aberrations over that in the concurrent solvent control groups, thereby validating the test system.
Under the experimental conditions described, the results indicate that Galaxy-SunBeat did not induce chromosome aberrations in cultured mammalian cells, either in presence or in absence of metabolic activation system. Hence it can be concluded that in the present experimental conditions, Galaxy-SunBeat was found to be non-clastogenic.

Conclusions:

The comparison of incidence of chromosomal aberrations in Human Peripheral Blood Lymphocytes between the Galaxy-SunBeat treated groups and the control group showed no significant differences. No evidence of induced chromosomal damage was observed at and up to the concentration of 5000 µg/ml of Galaxy-SunBeat, either in presence or in absence of metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance is not classified based on negative results in three separate Ames studies and a chromosomal aberration test.