Reaction mass of disodium [2,2'-(imino-kappaN)dibutanedioato-kappa2O1,O4(4-)]manganese(2-) and sodium sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1997-01-23 - 1997-07-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well documented GLP-guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

144538-83-0

EC Number:

604-420-0

Cas Number:

144538-83-0

IUPAC Name:

144538-83-0

Constituent 2

Reference substance name:

Sodium-iminodisuccinate

IUPAC Name:

Sodium-iminodisuccinate

Constituent 3

Reference substance name:

sodium;(2S)-2-(1,2-dicarboxylatoethylamino)butanedioate

IUPAC Name:

sodium;(2S)-2-(1,2-dicarboxylatoethylamino)butanedioate

Constituent 4

Reference substance name:

IDS, Na-Salz

IUPAC Name:

IDS, Na-Salz

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): IDS, Na-Salz = Iminodisuccinate, Na-salt or Sodium-iminodisuccinate
- Molecular formula (if other than submission substance): C8H7NO8Na4
- Molecular weight (if other than submission substance): 337.1 [g/mol]
- Structural formula attached as image file (if other than submission substance): see Fig.1
- Substance type: chelate
- Physical state: white powder
- Analytical purity: 67,2 %
- Composition of test material, percentage of components: 67.2% IDS, Na-Salz, 10.1% DL-Asparic acid, Na-salt, 8.3% H20, 7.8% Fumaric acid, Na-salt, 4.2% DL-Malic acid, Na-salt, 1.1% Sodium hydroxide, 0.9% Maleinic acid, Na-salt (analytical result dated February 27, 1997)
- Purity test date: February 27, 1997
- Lot/batch No.: SAV B 0004
- Expiration date of the lot/batch: January 14, 1998
- Storage condition of test material: at room temperature

Method

Target gene:

his-

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: normal nutrient broth
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

TA 1535 and TA 100 bear the base-pair substitution, his G 46, and TA 100 additionally contains the plasmid pKM 101.
This R factor, also contained in TA 98 and TA 102, codes for an ampicillin resistance and should raise the sensitivity of the strains.
TA 102 carries the ochre Mutation his G 428 on the multicopy plasmid pAQl, which codes in addition for tetracycline resistance.
TA 1537 and TA 98 bear frameshift markers.
TA 1537 exhibits the +1 mutant, his C 3076, while TA 98 bears the +2 type, his D 3052.
With the exception of TA 102, all strains have reduced capability to repair DNA-damage which increases the likelihood that such damage results in mutations.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

- Type and identity of media: normal nutrient broth
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

TA 1535 and TA 100 bear the base-pair substitution, his G 46, and TA 100 additionally contains the plasmid pKM 101.
This R factor, also contained in TA 98 and TA 102, codes for an ampicillin resistance and should raise the sensitivity of the strains.
TA 102 carries the ochre Mutation his G 428 on the multicopy plasmid pAQl, which codes in addition for tetracycline resistance.
TA 1537 and TA 98 bear frameshift markers.
TA 1537 exhibits the +1 mutant, his C 3076, while TA 98 bears the +2 type, his D 3052.
With the exception of TA 102, all strains have reduced capability to repair DNA-damage which increases the likelihood that such damage results in mutations.

Metabolic activation:

with and without

Metabolic activation system:

the 9000 g fraction of homogenized mammalian Livers together with co-factors

Test concentrations with justification for top dose:

0, 40, 158, 500, 1581 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water (formed a clear colourless solution
- the positive controls were dissolved in DMSO
- Justification for choice of solvent/vehicle: The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether, and DMF according to information given by the internal sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

sodium azide (10 µg/plate, TA1535), nitrofurantoin (0.2 µg, TA100), 4-nitro-1,2-phenylene diamine (10 µg, TA1537), 4-nitro-1,2-phenylene diamine (0.5 µg/plate TA98), Cumene hydroperoxide (50 µg/plate) and 2-aminoanthracene (3 µg/plate, TA102)

Positive control substance:

sodium azide

cumene hydroperoxide

other: nitrofurantoin, 4-nitro-1,2-phenylene diamine, 2 aminoantracene

Remarks:

No "untreated" negative control was set up for the used solvent, since sufficient evidence was available ia the literature and from our own experience , indicating that this solvent had no influence on the spontaneous mutant counts of the used strains.

Details on test system and experimental conditions:

METHOD OF APPLICATION:in agar (plate incorporation)

For the mutant count, three plates were used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained three plates per strain.
The amount of solvent for the test substance and for the controls was 0.1 mL/plate.
The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 µg or 5 µL per plate were used as the highest dose. At least four, additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.
The first repeat was performed as preincubation in a water bath at 37°C for 20 minutes. At the end of the preincubation period 2 mL of molten soft agar were added to the tubes, the content mixed and plated.
For the mutant count, three plates were used for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive-control also contained three plates per strain. In experiments without S9 mix buffer was used as replacement.
The dose of this trial were determined on the basis of the results of the plate incorporation assay. Doses are given as µg/tube for better separation of plate incorporation and preincubation trials, despite the fact that µg/plate and µg/tube could be used synonymously.
The toxicity of the substance was assessed in two ways. The first method was a gross appraisal of background growth on the plates for mutant determination. A reduction in background growth was indicated by the letter "b" after the mutant count and only a single "b", without any other values, is noted for a concentration, this "b" represents three plates with reduced background growth. Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.
The bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37°C and 90 rpm. These suspensions were used for the determination of mutant counts. No standardized procedure was employed to set the bacterial suspensions at a defined density of viable cells per milliliter, since the chosen method of incubation normally produces the desired density. However, the numbers of viable cells were established in a parallel procedure by determining the titers of the negative controls with S9 mix.
The dilution of bacterial suspensions used for the determination of titers was 1:1, 000, 000 . Titers were determined under the same conditions as were the mutations, except that the histidine concentration in the soft agar was increased fivefold to permit the complete growth of bacteria.
The tests were performed both with and without S9 mix.
The count was made after the plates had been incubated for 48 hours at 37°C. If no immediate count was possible, plates were temporarily stored in a refrigerator.

Evaluation criteria:

The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data and/or the laboratories' own historical data
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.
Only trials which complied with all three of the above criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.

Results and discussion

Test results

Test results 1

Species / strain:

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

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 examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium 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 examined

Positive controls validity:

valid

Additional information on results:

other: Due to the results of the first trial, doses ranging from 1000 µg to 5000 µg per tube were chosen for the repeat tests.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The colony number of each plate and mean values of the plate incorporation assay are listed for each dose in the tables 4 to 8, 13 and 14. As may be seen, there was no indication of a bacteriotoxic effect of IDS, Na-Salz at doses of up to and including 1581 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. 5000 µg/plate had only a weak, strain-specific bacteriotoxic effect. Therefore this dose could nevertheless be used for assessment purposes.

Salmonella typhimurium TA 102 revealed without S9 mix an in crease in mutant counts of about 80 as compared to the respective negative control (Table 5). This increase could not be confirmed (Tables 11 and 12), and is therefore to be regarded as a random result.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-amino-anthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

The colony number of each plate and mean values of the preincubation assay are listed for each dose in Tables 6 to 10. As may be seen, there was no indication of a bacteriotoxic effect of IDS, Na-Salz at doses of up to and including 5000 µg per tube. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well.

None of the five strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls (Tables 6 to 10) and thus confirmed the results of the plate incorporation method.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-amino-anthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Table 2: Tabulated Summary of Data
Summary of Mean Values Without S9 Mix From Tables 1-12
Table and group	Strain
	TA 1535	TA 100	TA 1537	TA 98	TA 102
1-5
µ/plate
0	6	97	9	22	151
50	7	92	8	20	160
158	6	90	9	17	149
500	5	87	6	17	165
1581	4	88	7	22	191
5000	7	92	6	24	231
Na-azide	924
NF		209
4-NPDA			64	129
Cumene					242
6-10
µg/tube
0	7	127	8	21	315
1000	7	125	9	28	325
2000	6	128	7	29	327
3000	6	98	6	31	304
4000	7	136	4	32	255
5000	6	122	5	18	265
Na-azide	824
NF		216
4-NPDA			69	171
Cumene	A..				599
11-12
µg/plate
0					145	113
1000					142	120
2000					156	141
3000					123	160
4000					133	165
5000					163	159
Cumene					223	234

Table 3: Summary of Mean Values With S9 Mix From Tables 1-12
Table and group	Strain
	TA 1535	TA 100	TA 1537	TA 98	TA 102
1-5
µg/plate
0	11	105	9	30	314
50	13	96	14	26	271
158	12	111	10	26	243
500	9	104	8	33	295
1581	7	122	8	25	265
5000	12	119	8	25	332
2-AA	74	1675	91	2083	623
6-10
µg/tube
0	6	104	6	26	353
1000	5	105	5	24	357
2000	7	98	6	26	384
3000	8	108	5	29	380
4000	5	108	6	22	381
5000	6	95	6	21	404
2-AA	66	1227	26	1625	719
11-12
µg/plate
0					196	159
1000					174	181
2000					161	183
3000					168	151
4000					188	154
5000					178	156
2-AA					361	337

Bayer A.G.				Table : 4			Study Number:		T 5053854
Department of Toxicology							Study Director:		Dr.Herbold
Pharma Research Center							Technician:		Düver
Wuppertal Elberfeld							Date:		Feb. 13.1997
AMES Test with IDS Na-salt							Strain:	S.typhimurium TA 1535
Dose/Plate (µg/Plate)	Revertants per plate						Titer		Quotient
				10%			Dilution per mL
	-S9	M	SD	+S9	M	SD	10^+6	10^+8	-S9	+S9
Water	6	6	1	12	11	2	61	6.9	1.0	1.0
	5			8			77
	7			12
50	8	7	1	11	13	2	%	/	1.2	1.3
	7			14
	7			15
158	8	6	2	10	12	2	%	/	1.1	1.2
	6			14
	5			13
500	7	5	2	9	9	2	%	/	0.9	0.8
	4			10
	5			7
1581	5	4	1	7	7	1	%	/	0.7	0.7
	4			7
	4			8
5000	8	7	1	13 B	12	1	%	/	1.2	1.2
	6			11 B
	7			13 B
Na-azide 10	918	924	23	%	/	/	%	/	154. 1*	/
	905
	950
2-AA 3	%	/	/	77	74	5	%	/	/	6.9*
				68
				77
*: mutagenic effect					B: Background lawn reduced
%: not tested					SD: Standard-Deviation
M: Mean					+S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G.				Table : 5			Study Number:		T 5053854
Department of Toxicology							Study Director:		Dr.Herbold
Pharma Research Center							Technician:		Düver
Wuppertal Elberfeld							Date:		Feb. 13.1997
AMES Test with IDS Na-salt							Strain:	S.typhimurium TA 100
Dose/Plate (µg/Plate)	Revertants per plate						Titer		Quotient
				10%			Dilution per mL
	-S9	M	SD	+S9	M	SD	10^+6	10^+8	-S9	+S9
Water	106	97	9	102	105	3	42	5.0	1.0	1.0
	89			106			58
	97			108
50	86	92	7	90	96	7	%		0.9	0.9
	91			104
	100			95
158	80	90	13	123	111	15	%	/	0.9	1.1
	105			94
	86			116
500	81	87	11	106	104	15	%	/	0.9	1.0
	100			117
	81			88
1581	99	88	10	126	122	4	%	/	0.9	1.2
	84			118
	80			122
5000	93	92	4	114	119	6	%	/	0.9	1.1
	88			117
	95			126
NF 0.2	208	209	20	%	/	/	%	/	2.1*	/
	190
	229
2-AA 3	%	/	/	1785	1675	238	%	/	/	15.9*
				1838
				1402
*: mutagenic effect					B: Background lawn reduced
%: not tested					SD: Standard-Deviation
M: Mean					+S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G.				Table : 6			Study Number:		T 5053854
Department of Toxicology							Study Director:		Dr.Herbold
Pharma Research Center							Technician:		Düver
Wuppertal Elberfeld							Date:		Feb. 13.1997
AMES Test with IDS Na-salt							Strain:	S.typhimurium TA 1537
Dose/Plate (µg/Plate)	Revertants per plate						Titer		Quotient
				10%			Dilution per mL
	-S9	M	SD	+S9	M	SD	10^+6	10^+8	-S9	+S9
Water	8	9	1	7	9	2	88	8.9	1.0	1.0
	9			9			89
	9			10
50	8	8	1	13	14	1	%	/	0.9	1.6
	7			14
	9			14
158	8	9	1	12	10	2	%	/	1.0	1.2
	8			9
	10			10
500	9	6	3	10	8	2	%	/	0.7	0.9
	6			7
	4			6
1581	8	7	1	10	8	2	%	/	0.8	0.9
	7			7
	6			6
5000	7	6	1	7	8	2	%	/	0.7	0.9
	6			10
	6			7
4-NPDA 10	54	64	8	%	/	/	%	/	7.3*	/
	69
	68
2-AA 3	%	/	/	105	91	12	%	/	/	10.5*
				81
				87
*: mutagenic effect					B: Background lawn reduced
%: not tested					SD: Standard-Deviation
M: Mean					+S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G.				Table : 7			Study Number:		T 5053854
Department of Toxicology							Study Director:		Dr.Herbold
Pharma Research Center							Technician:		Düver
Wuppertal Elberfeld							Date:		Feb. 13.1997
AMES Test with IDS Na-salt							Strain:	S.typhimurium TA 98
Dose/Plate (µg/Plate)	Revertants per plate						Titer		Quotient
				10%			Dilution per mL
	-S9	M	SD	+S9	M	SD	10^+6	10^+8	-S9	+S9
Water	27	22	5	31	30	3	209	21.2	1.0	1.0
	17			33			215
	23			27
50	22	20	8	27	26	4	%	/	0.9	0.9
	12			22
	27			29
158	24	17	6	26	26	1	%	/	0.8	0.9
	13			27
	14			26
500	14	17	5	34	33	3	%	/	0.8	1.1
	14			29
	23			35
1581	19	22	3	25	25	2	%	/	1.0	0.8
	24			27
	22			23
5000	22	24	3	31	25	6	%	/	1.1	0.8
	23			19
	28			24
4-NPDA 0.5	138	129	8	%	/	/	%	/	5.8*	/
	127
	122
2-AA 3	%	/	/	1970	2083	103	%	/	/	68.7*
				2108
				2172
*: mutagenic effect					B: Background lawn reduced
%: not tested					SD: Standard-Deviation
M: Mean					+S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G.				Table : 8			Study Number:		T 5053854
Department of Toxicology							Study Director:		Dr.Herbold
Pharma Research Center							Technician:		Düver
Wuppertal Elberfeld							Date:		Feb. 13.1997
AMES Test with IDS Na-salt							Strain:	S.typhimurium TA 102
Dose/Plate (µg/Plate)	Revertants per plate						Titer		Quotient
				10%			Dilution per mL
	-S9	M	SD	+S9	M	SD	10^+6	10^+8	-S9	+S9
Water	151	151	6	302	314	15	68	6.0	1.0	1.0
	145			309			52
	156			331
50	169	160	17	254	271	15	%	/	1.1	0.9
	140			278
	171			282
158	168	149	17	247	243	6	%	/	1.0	0.8
	141			245
	137			236
500	175	165	9	258	295	35	%	/	1.1	0.9
	164			301
	157			327
1581	180	191	14	307	265	40	%	/	1.3	0.8
	185			261
	207			228
5000	269	231	33	350	332	17	%	/	1.5²	1.1
	211			329
	213			317
Cumene 50	249	242	17	%	/	/	%	/	1.6*	/
	222
	254
2-AA 3	%	/	/	629	623	14	%	/	/	2.0*
				607
				634
² see Chapter 5.1					*: Mutagenic effect
%: not tested					SD: Standard-Deviation
M: Mean					+S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G.				Table : 9			Study Number:		T 5053854
Department of Toxicology							Study Director:		Dr.Herbold
Pharma Research Center							Technician:		Düver
Wuppertal Elberfeld							Date:		Feb. 20.1997
AMES Test with IDS Na-salt							Strain:	S.typhimurium TA 1535
Dose/Plate (µg/Plate)	Revertants per plate						Titer		Quotient
				10%			Dilution per mL
	-S9	M	SD	+S9	M	SD	10^+6	10^+8	-S9	+S9
Water	7	7	1	7	6	1	223	23.8	1.0	1.0
	7			6			252
	6			6
1000	7	7	2	5	5	1	%	/	1.0	0.8
	8			5
	5			6
2000	5	6	1	5	7	2	%	/	0.9	1.1
	5			7
	7			8
3000	6	6	2	7	8	1	%	/	1.0	1.2
	5			9
	8			7
4000	8	7	2	4	5	2	%	/	1.0	0.7
	5			7
	7			3
5000	6	6	0	5	6	2	%	/	0.9	0.9
	6			5
	6			8
Na-azide 10	841	824	59	%	/	/	%	/	123.6*	/
	758
	872
2-AA 3	%	/	/	73	66	7	%	/	/	10.4*
				60
				65
*: mutagenic effect					B: Background lawn reduced
%: not tested					SD: Standard-Deviation
M: Mean					+S9: with S9 Mix
-S9: without S9 Mix

Bayer A.G.				Table : 10			Study Number:		T 5053854
Department of Toxicology							Study Director:		Dr.Herbold
Pharma Research Center							Technician:		Düver
Wuppertal Elberfeld							Date:		Feb. 20.1997
AMES Test with : IDS Na-salt							Strain:	S.typhimurium TA 100
Dose/Plate (µg/Plate)	Revertants per plate						Titer		Quotient
				10%			Dilution per mL
	-S9	M	SD	+S9	M	SD	10^+6	10^+8	-S9	+S9
Water	135	127	7	105	104	2	197	19.7	1.0	1.0
	124			102			196
	121			105
1000	121	125	4	95	105	10	%	/	1.0	1.0
	126			107
	128			114
2000	138	128	9	108	98	12	%	/	1.0	0.9
	125			101
	122			85
3000	96	98	13	105	108	12	%	/	0.8	1.0
	86			121
	111			97
4000	126	136	17	118	108	12	%	/	1.1	1.0
	126			110
	155			95
5000	123	122	6	103	95	9	%	/	1.0	0.9
	127			85
	115			97
NF 0.2	218	216	11	%	/	/	%	/	1.7*	/
	204
	225
2-AA 3	%	/	/	1219	1227	18	%	/	/	11.8*
				1248
				1214
*: mutagenic effect					B: Background lawn reduced
%: not tested					SD: Standard-Deviation
M: Mean					+S9: with S9 Mix
-S9: without S9 Mix

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Therefore, IDS, Na-Salz was considered to be non-mutagenic with and without S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Executive summary:

IDS, Na-Salz was investigated using the Salmonella/ microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants (Herbold, B, 1997a, OECD 471). These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. In a first experiment, doses up to and including 1581 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had only a weak, strain-specific bacteriotoxic effect. Due to the weakness of this effect this range could nevertheless be used for assessment purposes. Moreover, IDS, Na-Salz was investigated in an independent repeat using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per tube after preincubation for 20 minutes at 37°C on the same Salmonella typhimurium LT2 mutants. In this experiment, doses up to and including 5000 µg per tube did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed.

In both experiment, evidence of mutagenic activity of IDS, Na-Salz was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-amino-anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls. Therefore, IDS, Na-Salz was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.