Potassium carbamoylcarbamate

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:

2011-12-20 through 2012-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, OECD study performed without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Remarks:

OECD GLP and UK GLP

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Potassium carbamoylcarbamate

EC Number:

247-728-7

EC Name:

Potassium carbamoylcarbamate

Cas Number:

26479-35-6

Molecular formula:

C2H4N2O3.K

IUPAC Name:

potassium N-carbamoylcarbamate

Constituent 2

Reference substance name:

potassium allophonate

IUPAC Name:

potassium allophonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Potassium Allophonate, CAS No 26479-35-6, Purity: 83.8%
- Storage condition of test material: 15-30 degrees C protected from light
- Synonyms: Potassium Carbamoylcarbamate
- Physical state: powder

Method

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

S-9 prepared from Sprague Dawley male rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

0.32, 1.6, 8, 40, 200, 1000 and 5000 μg/plate, plus negative (vehicle) and positive controls with or without S9 (preliminary study and initial
mutagenicity assay).

156.25, 312.5, 625, 1250, 2500, 5000 μg/plate, plus negative (vehicle) and positive controls with or without S9 (independent repeat/confirmatory
mutagenicity assay).

Vehicle / solvent:

purified water

Controls

Untreated negative controls:

yes

Remarks:

vehicle, H2O

Negative solvent / vehicle controls:

yes

Remarks:

H2O

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene and 4-nitroquinoline 1-oxide

Details on test system and experimental conditions:

The test article was tested for mutation (and toxicity) in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) in two separate experiments at the concentrations detailed previously, using triplicate plates with and without S-9.
Negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate in both assays without and with S-9. These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46±1 degrees C:
• 0.1 mL bacterial culture
• 0.1 mL test article solution or control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1 degrees C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony counting).

As the results of the first experiment were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article or control solution, bacteria and S-9 mix detailed above, were mixed together and incubated for 1 hour at 37±1 degrees C, with shaking, before the addition of 2.5 mL molten agar at 46±1 degrees C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. Dunnett's test gave a significant response (p ≤ 0.01) which was concentration related;
2. The positive trends/responses described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfy the above criteria are dealt with on a case-by-case basis. Biological relevance is taken into account, for example,
consistency of response within and between concentrations and (where applicable) between experiments. Further experimental work may be deemed necessary to aid evaluation of these data.

Statistics:

Dunnett's test

Results and discussion

Test results

Species / strain:

S. typhimurium, other: S.typhimurium TA98, TA100, TA1535 and TA1537 & E. coli WP2 uvrA

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

Positive controls validity:

valid

Additional information on results:

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Potassium Allophonate at 0.32, 1.6, 8, 40, 200, 1000 and 5000 μg/plate, plus negative (vehicle) and positive controls. Following these treatments no evidence of toxicity, as would usually be manifest as a diminution of the background bacterial lawn, was observed in any of the tester strains either in the absence or in the presence of S-9.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 μg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 156.25 – 5000 μg/plate, in order to
examine more closely those concentrations of Potassium Allophonate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments no evidence of toxicity was observed in any of the tester strains either in the absence or in the presence of S-9.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Potassium AllophonateMean number of Revertants per Plate―Experiment 1       Without S-9
Compound	Concentration (ug/plate)	T98		TA100		TA1535		TA1537		WP2 uvrA
		Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation
Purified Water		18.0	2.3	106.0	12.3	16.4	3.6	12.4	3.8	13.4	2.9
Potassium Allophonate	0.32	23.0	8.2	113.0	20.7	17.0	3.6	11.7	3.8	14.3	3.1
Potassium Allophonate	1.6	11.7	0.6	112.7	10.7	18.3	3.1	10.0	4.0	13.7	9.9
Potassium Allophonate	8	15.3	4.0	127.0	13.9	22.3	4.2	13.3	3.1	8.0	2.6
Potassium Allophonate	40	13.7	4.6	124.0	20.8	21.3	6.4	11.7	3.1	12.7	2.9
Potassium Allophonate	200	16.7	2.1	117.7	14.5	17.0	4.4	7.3	2.9	12.3	3.2
Potassium Allophonate	1000	14.7	2.5	139.7	6.4*	21.0	9.5	10.0	4.6	10.7	1.2
Potassium Allophonate	5000	15.0	3.6	112.3	19.5	14.0	5.0	12.0	2.0	14.3	4.9
Positive Control		1263.3	134.1	502.3	24.0	670.0	70.6	379.7	125.9	652.3	74.8

Dunnett’s t-test significance values

*	p<0.05
**	p<0.01

 

 

Potassium AllophonateMean number of Revertants per Plate ―Experiment 1         With S-9
Compound	Concentration (ug/plate)	T98		TA100		TA1535		TA1537		WP2 uvrA
		Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation
Purified Water		29.0	7.7	127.6	9.4	19.0	1.4	18.0	3.4	19.8	4.5
Potassium Allophonate	0.32	30.7	4.2	134.3	19.6	22.3	5.5	10.3	4.0	24.7	2.5
Potassium Allophonate	1.6	35.0	3.6	130.0	8.9	19.0	3.6	16.7	3.1	16.0	2.6
Potassium Allophonate	8	39.7	13.8	106.3	30.9	23.3	6.0	13.7	1.5	19.0	3.0
Potassium Allophonate	40	39.3	3.1	105.3	17.2	24.3	0.6	13.7	4.6	21.7	4.0
Potassium Allophonate	200	30.0	8.7	111.0	7.2	19.0	4.4	13.3	2.3	16.7	7.4
Potassium Allophonate	1000	28.0	7.9	136.7	12.6	14.0	6.2	8.0	1.7	17.7	5.1
Potassium Allophonate	5000	32.3	4.7	139.0	9.8	11.0	1.0	8.7	1.5	15.0	4.0
Positive Control		449.0	29.3	539.3	24.2	265.7	23.1	109.0	3.6	238.0	4.6

Dunnett’s t-test significance values

*	p<0.05
**	p<0.01

 

 

Potassium AllophonateMean number of Revertants per Plate―Experiment 2       Without S-9
Compound	Concentration (ug/plate)	T98		TA100		TA1535		TA1537		WP2 uvrA
		Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation
Purified Water		20.6	4.9	120.8	9.8	17.2	3.7	14.4	6.0	18.4	1.5
Potassium Allophonate	156.25	14.0	5.0	125.3	19.4	9.3	3.5	17.7	4.6	17.0	1.7
Potassium Allophonate	312.5	15.7	2.1	126.7	5.5	15.3	2.5	9.7	1.5	16.3	2.9
Potassium Allophonate	625	18.7	0.6	115.3	6.5	10.0	4.6	13.0	3.6	15.0	4.6
Potassium Allophonate	1250	15.3	5.0	124.7	10.7	20.0	5.6	13.0	3.6	14.3	1.2
Potassium Allophonate	2500	15.3	4.6	129.0	12.8	12.0	2.6	14.0	6.6	15.0	4.6
Potassium Allophonate	5000	12.0	3.5	121.0	9.6	10.7	2.5	15.7	4.9	18.7	7.8
Positive Control		836.3	111.0	831.7	57.2	767.7	41.6	230.0	91.3	891.0	8.7

Dunnett’s t-test significance values

*	p<0.05
**	p<0.01

 

 

Potassium AllophonateMean number of Revertants per Plate―Experiment 2       With S-9
Compound	Concentration (mg/plate)	T98		TA100		TA1535		TA1537		WP2 uvrA
		Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation	Mean	Standard Deviation
Purified Water		38.8	4.0	142.4	3.9	19.8	4.9	18.0	2.9	17.2	2.2
Potassium Allophonate	156.25	26.7	4.9	147.7	3.2	22.3	2.9	18.7	5.5	15.7	4.5
Potassium Allophonate	312.5	34.7	7.2	148.0	4.6	24.3	8.1	22.7	2.5	16.7	5.1
Potassium Allophonate	625	32.7	7.5	137.0	7.9	27.0	1.7	21.3	2.3	20.0	4.0
Potassium Allophonate	1250	37.7	5.8	143.3	9.5	24.3	2.9	18.0	6.2	21.7	3.1
Potassium Allophonate	2500	31.3	4.0	149.3	0.6	23.0	3.0	18.7	3.2	19.7	4.2
Potassium Allophonate	5000	30.7	6.4	139.0	17.1	20.3	5.1	17.7	2.9	15.7	4.0
Positive Control		482.0	41.9	671.0	60.7	163.0	9.5	55.7	8.0	48.3	2.5

 

Dunnett’s t-test significance values

*	p<0.05
**	p<0.01

Historical negative (vehicle) control values

			Revertant numbers for individual plates
Strain	S-9	No. of studies	No. of plates	Mean	99% reference range^(1)	99% confidence interval for group mean of:
						4 values^(2)	5 values^(2)	6 values^(2)
TA98	-	51	525	25	9.0-44.0	16.0-34.3	16.8-33.1	17.4-32.3
TA98	+	51	538	35	16.0-58.0	25.0-46.6	26.0-45.3	26.7-44.4
TA100	-	51	600	111	73.0-156.5	88.7-133.5	90.8-130.9	92.4-129.0
TA100	+	51	604	117	71.0-168.0	91.8-144.3	94.3-141.2	96.2-139.0
TA1535	-	50	520	17	5.0-30.0	10.8-24.8	11.4-23.9	11.8-23.3
TA1535	+	50	525	17	6.0-32.0	10.8-24.5	11.4-23.6	11.8-23.0
TA1537	-	51	530	13	3.0-30.0	6.3-20.1	6.9-19.2	7.3-18.5
TA1537	+	51	523	18	4.0-30.0	10.4-25.6	11.0-24.6	11.5-23.9
WP2 uvrA	-	8	80	14	5.0-24.0	8.8-20.0	9.3-19.3	9.6-18.8
WP2 uvrA	+	8	80	16	7.0-39.0	9.5-24.2	10.1-23.2	10.6-22.6

⁽¹⁾  Reference ranges are calculated from percentiles of the observed distributions.

⁽²⁾  Calculated from square-root transformed data.

Applicant's summary and conclusion

Conclusions:

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

All criteria for a valid study were met. It was concluded that Potassium Allophonate did not induce mutation in four histidine-requiring strains of
Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA) when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended
concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).