Reaction mass of sodium sulphate and trisodium trioxalatoferrate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium sulphate and trisodium trioxalatoferrate

EC Number:

947-037-7

IUPAC Name:

Reaction mass of sodium sulphate and trisodium trioxalatoferrate

Method

Species / strain

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.