Reaction mass of sodium sulphate and trisodium trioxalatoferrate

Endpoint summary

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

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:

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

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. QA statement)

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 ± 2^P	8 ± 3^P	24 ± 6^P	166 ± 4^P	44 ± 2^P
		5000 µg		8 ± 2^P	9 ± 1^P	23 ± 2^P	169 ± 18^P	42 ± 5^P
	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 ± 3^P	13 ± 2^M P	35 ± 2^P	136 ± 5^P	43 ± 8^P
		5000 µg		10 ± 3^P	14 ± 2^M P	38 ± 15^P	165 ± 15^P	54 ± 16^P
	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.

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:

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

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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.

Reference 3

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

Guideline:

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

Version / remarks:

Adopted 28th July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 /10^6Cells	Induction factor	CE Relative (Survival)	CE Relative (viability)	Mutant colonies /10^6Cells	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 /10^6Cells	Induction factor	CE Relative (Survival)	CE Relative (viability)	Mutant colonies /10^6Cells	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 /10^6Cells	Induction factor	CE Relative (Survival)	CE Relative (viability)	Mutant colonies /10^6Cells	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 /10^6Cells	Induction factor	CE Relative (Survival)	CE Relative (viability)	Mutant colonies /10^6Cells	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 on28^thJuly 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

Justification for classification or non-classification

No classification

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