Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No substantial increase in revertant colony numbers of any of the five tester strains in an Ames test was observed following treatment with the test item, neither in the presence nor absence of metabolic activation (S9 mix).

During the mutagenicity test described the test item did not induce mutations in the HGPRT locus in V79 cells of the Chinese hamster in the absence and presence of metabolic activation.

Treatment with test item in the absence and presence of S9 mix in a chromosomal aberration assey in vitro, cused no reduction in mitotic index was observed at the tested concentrations. The observed mean mitotic index in the absence of metabolic activation were 9.52, 9.76, 9.70, 9.61 and 7.48, in the presence of metabolic activation were 9.31, 9.69, 9.54, 9.32 and 7.16 for NC, VC, T1, T2 and T3 concentrations respectively. The material was not clastogenic

Therefore, the test item is considered “non-mutagenic”.

Link to relevant study records

Referenceopen allclose all

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:
2015 -04-01 till 2015-05-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline-conform study under GLP without deviations
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dissolved in deionised water[
- Justification for choice of solvent/vehicle: best suitable solvent
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
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:
TEST-SPECIFIC CONFOUNDING FACTORS
Water solubility: soluble
Precipitation:
The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate with and without S9 mix in experiment I and from 2500 to 5000 µg/plate with S9 mix in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol¬ic activation.
Remarks on result:
other: other: reverse mutation assay
Remarks:
Migrated from field 'Test system'.

Summary Tabellen

Table1     Summary of Experiment I

Study Name: 1684702

Study Code: Harlan CCR 1684702

Experiment: 1684702 VV Plate

Date Plated: 01/04/2015

Assay Conditions:

Date Counted: 08/04/2015

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

Deionised water

 

 

9 ± 1

9 ± 3

27 ± 6

156 ± 19

41 ± 6

Untreated

 

 

8 ± 3

9 ± 1

23 ± 7

176 ± 21

37 ± 4

3 µg

 

9 ± 3

10 ± 1

25 ± 7

158 ± 14

40 ± 8

4N trocken

10 µg

 

9 ± 3

10 ± 2

30 ± 6

176 ± 9

49 ± 1

 

33 µg

 

9 ± 2

10 ± 2

25 ± 8

173 ± 10

46 ± 10

 

100 µg

 

10 ± 2

9 ± 3

25 ± 4

184 ± 9

41 ± 10

 

333 µg

 

11 ± 3

10 ± 3

27 ± 4

178 ± 19

45 ± 11

 

1000 µg

 

9 ± 3

6 ± 2

24 ± 6

161 ± 6

46 ± 7

 

2500 µg

 

8 ± 2P

8 ± 3P

24 ± 6P

166 ± 4P

44 ± 2P

 

5000 µg

 

8 ± 2P

9 ± 1P

23 ± 2P

169 ± 18P

42 ± 5P

NaN3

10 µg

 

1175 ± 97

 

 

2116 ± 124

 

4-NOPD

10 µg

 

 

 

325 ± 21

 

 

4-NOPD

50 µg

 

 

68 ± 9

 

 

 

MMS

2.0 µL

 

 

 

 

 

850 ± 137

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

11 ± 3

14 ± 3

38 ± 1

121 ± 16

53 ± 5

Untreated

 

 

9 ± 4

14 ± 2

39 ± 10

122 ± 3

50 ± 7

Sanodal Gold

3 µg

 

11 ± 2

13 ± 1

38 ± 10

122 ± 8

45 ± 5

4N trocken

10 µg

 

10 ± 3

12 ± 3

35 ± 7

121 ± 6

50 ± 6

 

33 µg

 

13 ± 1

13 ± 2

32 ± 9

130 ± 26

54 ± 5

 

100 µg

 

10 ± 3

15 ± 5

35 ± 11

122 ± 2

47 ± 7

 

333 µg

 

9 ± 2

14 ± 5

37 ± 5

140 ± 17

66 ± 4

 

1000 µg

 

11 ± 2

12 ± 3

33 ± 6

151 ± 7

52 ± 10

 

2500 µg

 

10 ± 3P

13 ± 2M P

35 ± 2P

136 ± 5P

43 ± 8P

 

5000 µg

 

10 ± 3P

14 ± 2M P

38 ± 15P

165 ± 15P

54 ± 16P

2-AA

2.5 µg

 

454 ± 30

165 ± 20

4724 ± 512

3255 ± 160

 

2-AA

10.0 µg

 

 

 

 

 

379 ± 34

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

 

 


Table2     Summary of Experiment II

Study Name: 1684702

Study Code: Harlan CCR 1684702

Experiment: 1684702 HV2 pre

Date Plated: 28/04/2015

Assay Conditions:

Date Counted: 04/05/2015

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

Deionised water

 

 

8 ± 1

11 ± 3

23 ± 8

179 ± 11

41 ± 8

Untreated

 

 

10 ± 2

8 ± 2

32 ± 9

189 ± 17

39 ± 3

Sanodal Gold

33 µg

 

8 ± 2

13 ± 2

24 ± 3

202 ± 24

43 ± 7

4N trocken

100 µg

 

6 ± 5

8 ± 5

23 ± 7

197 ± 19

45 ± 9

 

333 µg

 

9 ± 3

8 ± 1

25 ± 10

158 ± 0

38 ± 9

 

1000 µg

 

13 ± 4

11 ± 5

29 ± 6

204 ± 15

39 ± 11

 

2500 µg

 

12 ± 2

9 ± 4

22 ± 2

183 ± 7

41 ± 5

 

5000 µg

 

14 ± 1

8 ± 2

27 ± 8

182 ± 13

36 ± 1

NaN3

10 µg

 

890 ± 39

 

 

1670 ± 149

 

4-NOPD

10 µg

 

 

 

334 ± 38

 

 

4-NOPD

50 µg

 

 

74 ± 3

 

 

 

MMS

2.0 µL

 

 

 

 

 

661 ± 69

 

 

 

 

 

 

 

 

 

With Activation

Deionised water

 

 

11 ± 2

17 ± 0

30 ± 7

204 ± 6

42 ± 7

Untreated

 

 

10 ± 3

21 ± 4

30 ± 6

189 ± 7

48 ± 7

Sanodal Gold

33 µg

 

6 ± 1

12 ± 3

32 ± 5

202 ± 19

40 ± 8

4N trocken

100 µg

 

18 ± 4

17 ± 6

38 ± 2

193 ± 29

55 ± 8

 

333 µg

 

9 ± 1

10 ± 3

39 ± 9

144 ± 7

50 ± 9

 

1000 µg

 

12 ± 3

14 ± 1

34 ± 6

192 ± 23

53 ± 3

 

2500 µg

 

10 ± 5

14 ± 3

33 ± 3

228 ± 129

40 ± 11

 

5000 µg

 

9 ± 2

16 ± 4

36 ± 4

154 ± 18

39 ± 11

2-AA

2.5 µg

 

176 ± 14

155 ± 29

3415 ± 381

3108 ± 509

 

2-AA

10.0 µg

 

 

 

 

 

333 ± 121

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

 

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

 

 

 

Conclusions:
Interpretation of results (migrated information): negative

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, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test item 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 the Escherichia coli strain WP2 uvrA.

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:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate with and without S9 mix in experiment I and from 2500 to 5000 µg/plate with S9 mix in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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 in­crease of induced revertant colonies.

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:
November 28, 2015 to May 03, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD and GLP
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
absence and presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.063 (T1), 0.125 (T2) and 0.25 (T3) mg/mL
Nc- negative control
VC - Vehicle control
T1- Test item concnetration 1
T2- Test item concnetration 2
T3- Test item concnetration 3
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
Phosphate Buffer Saline
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
Without metabolic activation : EMS; Ethyl methanesulfonate ____________With metabolic activation: CPA; Cyclophosphamide Monohydrate
Key result
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The maximum concentration 0.25 mg/mL was selected as the highest concentration for the treatment based on the cytotoxicity experiment
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: human lymphocytes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Treatment with test item in the absence and presence of S9 mix, there was no reduction in mitotic index was observed at the tested concentrations. The observed mean mitotic index in the absence of metabolic activation were 9.52, 9.76, 9.70, 9.61 and 7.48,in the presence ofmetabolic activation were 9.31, 9.69, 9.54, 9.32 and 7.16 for NC, VC, T1, T2 and T3 concentrations respectively.
Executive summary:

The clastogenic potential of the test item was determined in a chromosomal abberration test according to OECD TG 473.

CYTOTOXIC EXPERIMENT

The cytotoxicity due to treatment with test item was assessed based on the mitotic index. The cytotoxicity experiment was conducted at the concentrations of 0.125 (T1), 0.25 (T2), 0.5 (T3) and 1 (T4) mg/mL of culture media. A reduction in mitotic index was observed in the treated concentrations, both in the absence and in the presence of metabolic activation (1%), respectively.In the absence of S9 mix, the mean mitotic index observed was 9.57 (NC), 9.49 (VC), 9.30 (T1), 7.60 (T2), 4.15 (T3) and 2.13 (T4) and 9.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 9.63 (NC), 9.53 (VC), 9.02 (T1), 7.40 (T2), 4.29 (T3) and 2.32 (T4) and 9.73 (PC).

In the cytotoxicity experiment, the highest test concentration 0.5 (T3), 1 (T4) mg/ mLof culture mediashowed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence these concentrations were not selected for the main study.  

PHASE I

In the experiment, the cultures were exposed to Sanodal Gold 4N trocken for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%). The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 1.000 (T1), 1.000 (T2), 1.000 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.667 (T1), 0.667 (T2), 1.333 (T3) and 11.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.063 (T1), 0.125 (T2) and 0.25 (T3) mg/mL and positive controls, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamideat the concentration of30 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells.Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

Treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index was observed at the tested concentrations. The observedmeanmitotic indexin the absence of metabolic activation were 9.54, 9.74, 9.31, 9.45 and 7.43,in the presence ofmetabolic activation were 9.76, 9.84, 9.73, 9.66 and 7.67 for NC, VC, T1, T2 and T3 concentrations respecti

PHASE II

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were 0.063 (T1), 0.125 (T2) and 0.25 (T3) mg/mL culture both in presence and in absence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC), 0.667 (T1), 1.000 (T2), 1.000 (T3) and 9.333 (PC) in the of absence metabolic activation and 0.667 (NC), 0.333 (VC), 1.000 (T1), 1.000 (T2), 1.000 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.063 (T1), 0.125 (T2) and 0.25 (T3) mg/mL of culture and positive control, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamideat the concentration of30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index was observed at the tested concentrations. The observedmeanmitotic indexin the absence of metabolic activation were 9.52, 9.76, 9.70, 9.61 and 7.48,in the presence ofmetabolic activation were 9.31, 9.69, 9.54, 9.32 and 7.16 for NC, VC, T1, T2 and T3 concentrations respectively.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 19, 2016 to February 15, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD and GLP
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted 28th July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cell gene mutation assay (HGPRT)
Specific details on test material used for the study:
The dose concentrations for the study were calculated based on the content of the trisodium trioxalatoferrate and not on the sample as such. Sodium sulfate is considered as inactive compound
Target gene:
hypoxanthine guanine phosphoribosyl transferase Locus (HGPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Cell line V79
Tissue Lung (Chinese Hamster)
Cell type Adherent
Culture Medium DMEM with 10 % foetal bovine Serum
Culture Conditions Temperature : 37 ± 2oC, Carbon dioxide : 5 ± 1%
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix: AroclorTM 1254 induced liver homogenate s9 S9 supernatant is thawed and mixed with co-factor solution, to result in a final concentration in the S9 mix
Test concentrations with justification for top dose:
125 µg/mL is the highest cocentration both in the presence and absence of metabolic activation
Justification : The cytotoxic effect was observed at concentrations of 500 μg/ml and 250 μg/ml
Vehicle / solvent:
Distilled water
Untreated negative controls:
yes
Remarks:
DMEM medium
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Chinese Hamster V79 cell line
METHOD OF APPLICATION: DMEM medium
- Cell density at seeding (if applicable):1.5x10^6

DURATION
SEEDING
Three days old exponentially growing stock cultures (more than 50% confluent) were trypsinized at 37°C for 5 minutes. Then enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared
- Preincubation period:
The culture flasks were incubated at 37°C in humidified atmosphere with 5% CO2 for 24 hours.
- Exposure duration:
4 hours in DMEM medium containing various concentrations of the test item either in the presence or absence of metabolic activation.
Positive and solvent controls were performed in parallel.
After 4 h the medium was replaced with complete medium following two washing steps with DMEM0 for Phase I and Phase II was performed with treatment duration of 24 hours in the absence of metabolic activation system and 4 hours in the presence of metabolic activation system
- Expression time (cells in growth medium):
The phenotypic expression was for a period of 7 days
- Selection time (if incubation with a selection agent):
7 days

SELECTION AGENT (mutation assays):
6-thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency;
Evaluation criteria:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
• The increase is concentration-related
Any of the results are outside the distribution of the historical negative control data
Statistics:
The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control (vehicle) groups using Mann-Whitney test. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance was considered together
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic effect was observed at concentrations of 500 μg/ml and 250 μg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

SUMMARY RESULTS           

 

Conc., µg/mL

S9 mix

Culture I

Culture II

CE Relative (Survival)

CE Relative (viability)

Mutant colonies /106Cells

Induction factor

CE Relative (Survival)

CE Relative (viability)

Mutant colonies /106Cells

Induction factor

Phase I  

NC

0

-

100

100

4.1

0.95

100

100

4.8

1.04

VC

0

-

95

97

4.3

1.0

93

96

4.6

1.0

T1

15.63

-

96

94

7.6

1.77

93

93

10.2

2.22

T2

31.25

-

90

89

7.1

1.65

86

88

11.7

2.54

T3

62.50

-

84

85

9.3

2.16

80

80

14.2

3.08

T4

125

-

80

78

13.0

3.02

73

77

14.6

3.17

PC

150

-

66

75

153.3

35.65

65

69

184.9

40.19

Key: NC = Negative control, VC = Vehicle Control,, CE = Cloning Efficiency

 

 

 

Conc., µg/mL

S9 mix

Culture I

Culture II

CE Relative (Survival)

CE Relative (viability)

Mutant colonies /106Cells

Induction factor

CE Relative (Survival)

CE Relative (viability)

Mutant colonies /106Cells

Induction factor

Phase I

NC

0

+

100

100

8.1

0.93

100

100

12.6

0.78

VC

0

+

93

98

8.7

1.0

93

95

16.0

1.0

T1

15.63

+

95

94

11.0

1.26

94

90

27.1

1.69

T2

31.25

+

91

91

11.6

1.33

86

85

16.0

1.0

T3

62.50

+

84

82

17.1

1.96

80

75

17.4

1.08

T4

125

+

77

72

20.5

2.35

75

67

33.9

2.11

PC

1.3

+

71

62

929.3

106.8

69

58

1059.0

66.18

Key:NC = Negative control, VC = Vehicle Control,, CE = Cloning Efficiency

 

   SUMMARY OF RESULTS (Contd.)

 

Conc., µg/mL

S9 mix

Culture I

Culture II

CE Relative (Survival)

CE Relative (viability)

Mutant colonies /106Cells

Induction factor

CE Relative (Survival)

CE Relative (viability)

Mutant colonies /106Cells

Induction factor

Phase II  

NC

0

-

100

100

7.0

0.84

100

100

4.9

0.87

VC

0

-

96

94

8.3

1.0

97

99

5.6

1.0

T1

15.63

-

96

93

14.5

1.75

95

91

5.9

1.05

T2

31.25

-

93

88

17.1

2.06

77

89

6.3

1.12

T3

62.50

-

82

82

25.9

3.12

84

81

10.1

1.80

T4

125

-

79

77

28.2

3.40

74

75

12.6

2.25

PC

150

-

61

74

167.1

20.13

66

66

188.0

33.58

Key: NC = Negative control, VC = Vehicle Control,, CE = Cloning Efficiency

 

 

Conc., µg/mL

S9 mix

Culture I

Culture II

CE Relative (Survival)

CE Relative (viability)

Mutant colonies /106Cells

Induction factor

CE Relative (Survival)

CE Relative (viability)

Mutant colonies /106Cells

Induction factor

Phase II

NC

0

+

100

100

6.5

0.49

100

100

6.3

0.93

VC

0

+

95

98

13.4

1.0

96

99

6.8

1.0

T1

15.63

+

101

97

17.8

1.33

97

97

8.7

1.28

T2

31.25

+

94

88

22.4

1.67

89

88

11.7

1.72

T3

62.50

+

85

84

23.8

1.78

84

82

13.6

2.00

T4

125

+

80

79

30.2

2.25

80

74

17.2

2.53

PC

1.3

+

60

67

1097.9

81.93

67

64

1000.4

147.1

Key: NC = Negative control, VC = Vehicle Control,, CE = Cloning Efficiency

 

CYTOTOXICITY            

TABLE 1   CLONING EFFICIENCY (CE) AND RELATIVE SURVIVAL GROWTH DURING THE EXPRESSION PERIOD WITHOUT METABOLIC ACTIVATION (-S9)

Doseµg/ml

Replicate

Cells seeded

 

Number of cells Immediately after treatment

Adjusted on Day 0

Number of colonies per flask

CE

Adjusted CE

Mean Adjusted CE

RS

NC

R1

500

510

510

525

1.05

1.05

1.02

100.00

R2

500

505

510

502

1.00

0.99

VC

R1

500

460

510

486

0.97

0.88

0.90

88.54

R2

500

475

510

501

1.00

0.93

T1

(15.63 µg/ml)

R1

500

385

510

397

0.79

0.60

0.58

64.09

R2

500

396

510

361

0.72

0.56

T2

(31.26 µg/ml)

R1

500

391

510

379

0.76

0.58

0.57

62.88

R2

500

366

510

388

0.78

0.56

T3

(62.5 µg/ml)

R1

500

371

510

371

0.74

0.54

0.57

62.60

R2

500

369

510

410

0.82

0.59

T4

(125 µg/ml)

R1

500

425

510

315

0.63

0.53

0.53

58.65

R2

500

380

510

360

0.72

0.54

T5

(250 µg/ml)

R1

500

121

510

340

0.68

0.16

0.14

15.31

R2

500

82

510

360

0.72

0.12

T6

(500 µg/ml)

R1

500

14

510

410

0.82

0.02

0.03

3.00

R2

500

20

510

405

0.81

0.03

Key:    R = Replicate, NC = Negative control, VC = Vehicle Control, SD = Standard Deviation

 

CLONING EFFICIENCY (CE) AND RELATIVE SURVIVAL GROWTH DURING THE EXPRESSION PERIOD WITH METABOLIC ACTIVATION (+S9)

Dose µg/ml

Replicate

Cells seeded

Number of cells Immediately after treatment

Adjusted on Day 0

Number of colonies per flask

CE

Adjusted CE

Mean Adjusted CE

RS

NC

R1

500

489

489

509

1.02

1.02

0.99

100.00

R2

500

478

489

495

0.99

0.97

VC

R1

500

463

489

482

0.96

0.91

0.91

91.30

R2

500

452

489

487

0.97

0.90

T1

(15.63 µg/ml)

R1

500

477

489

474

0.95

0.92

0.92

101.57

R2

500

481

489

466

0.93

0.92

T2

(31.26 µg/ml)

R1

500

461

489

410

0.82

0.77

0.81

89.33

R2

500

487

489

425

0.85

0.85

T3

(62.5 µg/ml)

R1

500

471

489

398

0.80

0.77

0.76

83.84

R2

500

464

489

397

0.79

0.75

T4

(125 µg/ml)

R1

500

385

489

375

0.75

0.59

0.57

62.71

R2

500

366

489

365

0.73

0.55

T5

(250 µg/ml)

R1

500

125

489

345

0.69

0.18

0.16

17.89

R2

500

100

489

362

0.72

0.15

T6

(500 µg/ml)

R1

500

50

489

380

0.76

0.08

0.06

6.65

R2

500

28

489

375

0.75

0.04

Key: R = Replicate, NC = Negative control, VC = Vehicle Control, SD = Standard Deviation

 

Conclusions:
During the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the HPRT locus in V79 cells of the Chinese hamster in the absence and presence of metabolic activation.
Therefore, SANODAL GOLD 4N TROCKEN is considered “non-mutagenic” in this HPRT assay
Executive summary:

This study was conducted to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The methods followed were as per OECD guideline No. 476, adopted on28thJuly 2015.

The assay was performed in two independent experiment, using two parallel cultures each. The experiment I was performed with and without liver microsomal activation at a treatment period of 4 hours. Experiment II was performed for a treatment period of 4 hours with and 24 hours without metabolic activation

500 µg/mL concentration was chosen as the highest dose for the cytotoxicity experiment, based on the solubility and precipitation properties of the test item.

The following concentrations were selected for the main study based on cytotoxicity results.

15.63 µg/mL, 31.25 µg/mL , 62.50 µg/mL , 125 µg/mL both in the presence and absence of metabolic activation

The cytotoxic effect was indicated by the Relative survival (RS) i.e., cloning efficiency (CE) of cells plated immediately after treatment, adjusted by any loss of cells during treatment, based on cell count, as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%). The cytotoxic effect was observed at concentrations of 500 μg/ml and 250 μg/ml. Relative survival at this concentrations was 3.00 in the absence of metabolic activation system, 6.65 in the presence of metabolic activation system for 500 μg/ml, 15.31 in the absence of metabolic activation system, 17.89 in the presence of metabolic activation system for 250 μg/ml and were found to be less than 50% of vehicle control thus indicates the cytotoxicity. The other tested concentrations were found to show more than 50% relative survival and hence these doses were selected for the main experiment (Phase-I and Phase-II).

PHASE-I

In culture I, the numbers of mutant colonies of Negative control (NC), Vehicle control (VC), Test concentration 1(T1), Test concentration 2 (T2), Test concentration 3 (T3), Test concentration 4 (T4) and Positive Control (PC) (EMS) were 4.1, 4.3, 7.6, 7.1, 9.3, 13.0, and 153.3/106cells respectively in the absence of metabolic activation, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 8.1, 8.7, 11.0, 11.6, 17.1, 20.5 and 929.3 per 106cells in presence of metabolic activation.

In culture II, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 4.8, 4.6, 10.2, 11.7, 14.2, 14.6, and 184.9/106cells respectively and in the absence of metabolic activation, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 12.6, 16.0, 27.1, 16.0, 17.4, 33.9, and 1059.0 per 106cells in presence of metabolic activation.

PHASE-II

In culture I, the numbers of mutant colonies NC, VC, T1, T2, T3, T4 and PC (EMS) were 7.0, 8.3, 14.5, 17.1, 25.9, 28.2, and 167.1/106cells respectively in the absence of metabolic activation. The numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 6.5, 13.4, 17.8, 22.4, 23.8, 30.2, and 1097.9./106cells in presence of metabolic activation.

In culture II, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 4.9, 5.6, 5.9, 6.3, 10.1, 12.6, and 188.0/106cells respectively in the absence of metabolic activation. The numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 6.3, 6.8, 8.7, 11.7, 13.6, 17.2, and 1000.4 per 106cells respectively in presence of metabolic activation.

In both the cultures, there was no distinct increase in the mutant frequency caused bySanodal Gold 4N trockenwhen compared to vehicle control and the induction factor did not exceed three times the corresponding vehicle controls. The test item did not induce mutations at the HPRT locus of V79 cells.

The positive controls used, EMS in the absence of metabolic activation and DMBA in the presence of metabolic activation, revealed significant increase in mutant colonies and induction factor is more than three times of vehicle control indicating that the test system was sensitive and the results are valid.

Note: NC: Negative control; VC: Vehicle control; T1: Test concentration1; T2: Test concentration 2; T3: Test concentration 3; T4: Test concentration 4; PC: Positive Control.

Conclusion

During the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the HPRT locus in V79 cells of the Chinese hamster in the absence and presence of metabolic activation.

Therefore, the test item is considered “non-mutagenic”in this HPRT assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

not applicable

Additional information

Justification for classification or non-classification

No classification

No signs of mutagenic activity were detected in bacteria or mammalian cells.