Uronium hydrogen sulphate

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:

end on 22-SEP-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Uronium hydrogen sulphate

EC Number:

244-343-6

EC Name:

Uronium hydrogen sulphate

Cas Number:

21351-39-3

Molecular formula:

CH4N2O.H2O4S

IUPAC Name:

hydrogen [amino(hydroxy)methylidene]azanium sulfate

Details on test material:

- Name of test material (as cited in study report): Aduct Urea-Sulfuric
- Physical state: not indicated
- Stability under test conditions: no data
- Storage condition of test material: At room temperature

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

- pre-experiment (experiment I): 3 – 5000 μg/plate
- experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9-mix: sodium azide (TA1535, TA100: 10μg/pl); 4-nitro-o-phenylene-diamine (TA1537, TA98: 10-50µg/pl); methyl methane sulfonate (TA102: 3μL/pl) - With S9-mix: 2-aminoanthracene (all strains: 2.5-10µg/pl)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and pre-incubation test (experiment II)

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: at least 48 hours at 37 °C in the dark

NUMBER OF REPLICATES: for each strain and dose level, including the controls three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: background growth

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 TA 102) 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

Species / strain:

other: Salmonella 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:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Aduct Urea-Sulfuric at any concentration 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. They showed a distinct in-crease in induced revertant colonies.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH, effects of osmolality, evaporation from medium, water solubility: no data.
- Precipitation: no precipitation of the test item occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES: reported as experiment I

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment II, the data in the untreated control with metabolic activation of strain TA 102 were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
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, occurred in the test groups with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: result of pre-experiment (experiment I)

Metabolic activation	Test group	Dose level (per plate)	Revertant colony counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	water		14 ± 5	7 ± 1	32 ± 3	145 ± 9	437 ± 36
	untreated		17 ± 7	8 ± 2	35 ± 7	137 ± 3	420 ± 13
	Aduct Urea-Sulfuric	3 µg	15 ± 2	7 ± 1	30 ± 7	143 ± 6	416 ± 6
		10 µg	14 ± 4	7 ± 2	34 ± 3	136 ± 13	424 ± 14
		33 µg	12 ± 4	9 ± 3	32 ± 10	125 ± 6	425 ± 30
		100 µg	10 ± 2	6 ± 1	31 ± 5	135 ± 20	445 ± 6
		333 µg	13 ± 3	6 ± 1	29 ± 6	131 ± 19	396 ± 38
		1000 µg	15 ± 2	6 ± 1	29 ± 9	125 ± 7	410 ± 37
		2500 µg	13 ± 0	9 ± 1	29 ± 3	134 ± 6	407 ± 15
		5000 µg	14 ± 3	9 ± 1	32 ± 6	135 ± 13	415 ± 18
	NaN3	10 µg	1606 ± 83			1790 ± 162
	4-NOPD	10 µg			298 ± 8
	4-NOPD	50 µg		65 ± 8
	MMS	3 µL					2268 ± 290
With Activation	water		20 ± 1	16 ± 5	42 ± 12	166 ± 10	561 ± 38
	untreated		21 ± 5	12 ± 4	31 ± 3	161 ± 13	539 ± 61
	Aduct Urea-Sulfuric	3 µg	21 ± 5	13 ± 1	46 ± 5	152 ± 3	574 ± 26
		10 µg	21 ± 3	13 ± 3	46 ± 10	147 ± 14	571 ± 14
		33 µg	17 ± 2	14 ± 4	41 ± 6	158 ± 22	569 ± 96
		100 µg	23 ± 5	18 ± 2	36 ± 12	151 ± 7	624 ± 27
		333 µg	25 ± 2	15 ± 5	43 ± 5	141 ± 13	615 ± 34
		1000 µg	21 ± 6	13 ± 3	30 ± 4	150 ± 8	594 ± 25
		2500 µg	20 ± 1	18 ± 3	42 ± 1	155 ± 17	627 ± 8
		5000 µg	24 ± 2	16 ± 2	38 ± 3	155 ± 20	625 ± 11
	2-AA	2.5 µg	473 ± 43	511 ± 45	2491 ± 156	4053 ± 143
	2-AA	10 µg					2304 ± 92

Table 2: result of experiment II

Metabolic activation	Test group	Dose level (per plate)	Revertant colony counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	water		17 ± 4	16 ± 3	37 ± 13	168 ± 24	418 ± 37
	untreated		17 ± 4	15 ± 8	31 ± 5	157 ± 28	436 ± 24
	Aduct Urea-Sulfuric	33 µg	17 ± 5	24 ± 8	34 ± 3	148 ± 11	449 ± 29
		100 µg	16 ± 1	17 ± 4	34 ± 8	160 ± 23	450 ± 24
		333 µg	17 ± 4	16 ± 3	41 ± 3	161 ± 9	460 ± 10
		1000 µg	19 ± 4	20 ± 6	35 ± 9	145 ± 11	418 ± 56
		2500 µg	13 ± 4	13 ± 4	34 ± 3	143 ± 18	444 ± 4
		5000 µg	17 ± 8	19 ± 2	31 ± 3	145 ± 5	442 ± 39
	NaN3	10 µg	1780 ± 54			1244 ± 225
	4-NOPD	10 µg			369 ± 14
	4-NOPD	50 µg		78 ± 6
	MMS	3 µL					2472 ± 404
With Activation	water		24 ± 6	26 ± 3	37 ± 9	195 ± 8	585 ± 77
	untreated		34 ± 12	26 ± 6	44 ± 8	185 ± 4	659 ± 21
	Aduct Urea-Sulfuric	33 µg	26 ± 7	27 ± 3	36 ± 2	211 ± 17	686 ± 25
		100 µg	22 ± 2	28 ± 5	33 ± 6	193 ± 14	634 ± 15
		333 µg	25 ± 1	22 ± 7	35 ± 11	200 ± 8	686 ± 3
		1000 µg	19 ± 5	30 ± 1	45 ± 6	183 ± 8	644 ± 12
		2500 µg	28 ± 5	22 ± 5	42 ± 8	190 ± 5	593 ± 36
		5000 µg	25 ± 10	24 ± 9	39 ± 4	171 ± 16	554 ± 20
	2-AA	2.5 µg	400 ± 63	357 ± 13	1950 ± 96	2862 ± 128
	2-AA	10 µg					2361 ± 277

Applicant's summary and conclusion

Conclusions:

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

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.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471, GLP), strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 of S. typhimurium were exposed to Aduct Urea-Sulfuric (Uronium hydrogen sulphate) at concentrations of 33; 100; 333; 1000; 2500; and 5000 μg/plate (experiment II) in the presence and absence of mammalian metabolic activation (pre-incubation). 

 

Aduct Urea-Sulfuric was tested up to limit concentration (5000 µg/plate) and no precipitation was observed.

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, 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 Aduct Urea-Sulfuric at any concentration 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.

The positive controls induced the appropriate responses in the corresponding strains.

 

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.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.