Reaction mass of Ammonium bisethanedioato bisaquo niobate(V) (anhydrous form) and Ammonium hydrogen ethanedioate ethanedioic acid (anhydrous form)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Aug 2012 - 07 Sep 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted 21 Jul 1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted 30 May 2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Erlangen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dehydrate

IUPAC Name:

Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dehydrate

Test material form:

solid

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented liver S9 homogenate from phenobarbital and ß-naphthoflavone induced male Wistar rats

Test concentrations with justification for top dose:

- pre-test (toxicity): 3.16, 10.0, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate
- main test (mutagenicity): 31.6, 100, 316, 1000, 2500, and 5000 µg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene

Remarks:

+S9: 2-aminoanthracene 2.5 µg/plate (TA 98, TA 100, TA 1535, TA 1537) 10 µg/plate (TA 102); -S9: 4-nitro-o-phenylene-diamine 10 µg/plate (TA 98) 40 µg/plate (TA 1537), sodium azide 10 µg/plate (TA 100, TA 1535), methylmethanesulfonate 1 µL/plate (TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates each in 2 independent tests

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of background lawn, reduction in the number of revertants down to a mutation factor ≤0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control.
A test item is considered as mutagenic if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100, TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535, TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase nor a reproducible biologically relevant response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Key result

Species / strain:

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

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no
No further information given in the study report.

RANGE-FINDING/SCREENING STUDIES:
The test item was not toxic to the tester strains TA 98 and TA 100 up to limit concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the positive and solvent controls are within the range of the historical control data.

Any other information on results incl. tables

Tab.1: Experiment 1 (plate incorporation) without metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA 98	Mutation Factor	TA 100	Mutation Factor	TA 1535	Mutation Factor	TA 1537	Mutation Factor	TA 102	Mutation Factor
Solvent control	22±5.5	1.0	98±11.9	1.0	6±0.6	1.0	8±2.0	1.0	211±22.9	1.0
31.6	15±2.5	0.7	88±7.2	0.9	9±2.3	1.4	8±1.7	1.0	189±7.4	0.9
100	17±5.0	0.8	92±9.5	0.9	5±2.6	0.8	8±2.6	1.0	184±16.0	0.9
316	20±3.8	0.9	112±16.5	1.1	7±0.6	1.1	9±3.8	1.1	186±20.0	0.9
1000	23±5.5	1.1	95±15.2	1.0	4±1.2	0.7	8±1.7	1.0	176±6.7	0.8
2500	16±2.5	0.7	88±7.8	0.9	5±0.6	0.7	7±1.0	0.9	187±20.3	0.9
5000	20±3.6	0.9	87±15.1	0.9	6±4.0	1.0	5±1.2	0.7	190±8.7	0.9
Positive control	251±17.6	11.6	642±137.8	6.5	996±137.1	157.2	78±10.7	9.8	2110±84.9	10.0

Solvent control: A. dest.

Positive controls: 4-nitro-o-phenylene-diamine (TA 98, TA 1537); sodium azide (TA 100, TA 1535); methylmethanesulfonate (TA 102)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

 

Tab. 2: Experiment 1 (plate incorporation) with metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA 98	Mutation Factor	TA 100	Mutation Factor	TA 1535	Mutation Factor	TA 1537	Mutation Factor	TA 102	Mutation Factor
Solvent control	34±7.6	1.0	113±13.5	1.0	7±2.5	1.0	8±3.8	1.0	296±16.1	1.0
31.6	29±6.0	0.9	130±20.8	1.1	8±1.2	1.0	6±4.2	0.8	278±25.1	0.9
100	26±1.2	0.8	111±5.9	1.0	5±2.1	0.7	7±1.7	0.9	255±14.8	0.9
316	30±3.0	0.9	113±4.6	1.0	9±1.5	1.2	8±1.0	1.0	281±18.5	0.9
1000	20±7.2	0.6	110±2.6	1.0	6±2.3	0.8	9±2.3	1.2	250±28.2	0.8
2500	25±1.7	0.7	101±6.0	0.9	8±1.5	1.0	8±0.6	1.1	213±8.7	0.7
5000	16±2.5	0.5	108±4.0	1.0	10±3.1	1.4	6±4.6	0.7	216±20.2	0.7
Positive control	2247±270.3	66.8	1768±89.8	15.6	56±7.2	7.6	263±40.9	34.4	944±42.7	3.2

Solvent control: A. dest.

Positive controls: 2-aminoanthracene (all tester strains)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

 

Tab. 3: Experiment 2 (preincubation) without metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA 98	Mutation Factor	TA 100	Mutation Factor	TA 1535	Mutation Factor	TA 1537	Mutation Factor	TA 102	Mutation Factor
Solvent control	17±1.2	1.0	84±11.6	1.0	6±3.1	1.0	8±3.5	1.0	171±13.5	1.0
31.6	23±3.8	1.3	104±5.7	1.2	9±1.7	1.6	9±2.6	1.1	183±21.6	1.1
100	23±3.0	1.3	82±5.0	1.0	6±3.1	1.1	9±1.0	1.1	188±10.5	1.1
316	21±4.7	1.2	87±9.5	1.0	9±1.5	1.5	8±4.6	1.0	162±16.1	0.9
1000	22±8.4	1.3	81±4.0	1.0	6±1.5	1.1	10±3.6	1.2	163±19.6	1.0
2500	18±7.8	1.1	71±9.7	0.8	7±0.6	1.3	7±4.9	1.1	184±6.8	1.1
5000	22±4.0	1.3	73±20.0	0.9	7±0.0	1.2	6±3.2	0.7	180±20.1	1.1
Positive control	390±33.5	22.5	1256±99.0	15.0	932±74.1	164.4	103±14.3	12.4	1811±82.9	10.6

Solvent control: A. dest.

Positive controls: 4-nitro-o-phenylene-diamine (TA 98, TA 1537); sodium azide (TA 100, TA 1535); methylmethanesulfonate (TA 102)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

 

Tab. 4: Experiment 2 (preincubation) with metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]	TA 98	Mutation Factor	TA 100	Mutation Factor	TA 1535	Mutation Factor	TA 1537	Mutation Factor	TA 102	Mutation Factor
Solvent control	37±10.1	1.0	104±7.8	1.0	6±1.2	1.0	9±0.6	1.0	256±30.9	1.0
31.6	42±7.5	1.1	101±16.6	1.0	11±2.1	1.8	9۬.3	1.1	277±25.5	1.1
100	41±13.1	1.1	84±8.5	0.8	9±2.1	1.5	9±3.8	1.1	277±11.2	1.1
316	31±6.7	0.9	102±20.5	1.0	7±1.5	1.2	8±4.4	0.9	270±19.5	1.1
1000	38±11.4	1.0	101±8.1	1.0	7±2.6	1.1	8±4.0	0.9	262±15.8	1.0
2500	30±9.3	0.8	86±13.7	0.8	7±2.5	1.1	6±5.9	0.7	225±1.7	0.9
5000	27±2.1	0.7	78±4.6	0.8	7±3.6	1.1	8±2.5	1.0	227±16.2	0.9
Positive control	2095±255.2	79.2	1933±303.9	18.6	81±18.1	12.8	190±40.9	21.9	612±102.0	2.4

Solvent control: A. dest.

Positive controls: 2-aminoanthracene (all tester strains)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative