6-methyl-2-oxoperhydropyrimidin-4-ylurea

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:

2012-01-10 to 2012-03-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(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 FÜR GESUNDHEIT UND LEBENSMITTELSICHERHEIT, LANDESINSTITUT FÜR ARBEITSSCHUTZ UND PRODUKTSICHERHEIT, Munich, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

6-methyl-2-oxoperhydropyrimidin-4-ylurea

EC Number:

214-447-6

EC Name:

6-methyl-2-oxoperhydropyrimidin-4-ylurea

Cas Number:

1129-42-6

Molecular formula:

C6H12N4O2

IUPAC Name:

(6-methyl-2-oxo-1,3-diazinan-4-yl)urea

Test material form:

other: dilution

Details on test material:

- Name of test material (as cited in study report): 6-methyl-2-oxoperhydropyrimidin-4-yl urea, Crotodur (CDU)
- Physical state: grey solid, granulate
- Analytical purity: < 100% Crotonylidendiurea (31% N) active component
- Lot/batch No.: Crotodur (32157)
- Storage condition of test material: at room temperature, separate from nitrate salt

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphtoflavone

Test concentrations with justification for top dose:

- Pre-experiment for toxicity: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate with and without metabolic activation
- Main experiments for mutagenicity: 31.6, 100, 316, 1000, 2500, and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water (A. dest.)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of bacteria and the S9 activity.

Controls

Controls 1

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

Remarks:

-S9: sodium azide (NaN3) 10 µg/plate in A. dest. (TA 100, TA 1535); 4-Nitro-o-phenylene-diamine (4-NOPD) 10µg/plate in DMSO (TA 98) & 40 µg/plate in DMSO (TA 1537); methylmethaneslfonate (MMS) 1 µL/plate in A. dest. (TA 102)

Controls 2

Positive control substance:

other: 2-aminoanthracene

Remarks:

+S9: 2-aminoanthracene (2-AA) 2.5 µg/plate in DMSO (TA 98, TA100, TA 1535, TA 1537) & 10 µg/plate in DMSO (TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation); Experiment 2: preincubation

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Cytotoxicity:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤0.5 in relation to the solvent control.
Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
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 strain TA 98, TA 100, and TA 102 the number of reversions is at least twice high
- if in tester strain TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Criteria of validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the control plates with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range): TA 98: 16-46 (-S9) and 18-56 (+S9); TA 100: 77-174 (-S9) and 80-165 (+S9); TA 1535: 5-29 (-S9) and 5-27 (+S9); TA 1537: 5-28 (-S9) and 5-34 (+S9); TA 102: 164-399 (-S9) and 163-458 (+S9)
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plates.

Statistics:

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.

Results and discussion

Test results

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 examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in Experiment 1 or 2 with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
No clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤0.5 in relation to the solvent control was detected for TA 98 or TA 100 with and without metabolic activation up to the limit concentration of 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
The data obtained for the solvent controls are within the range of the historical control data given in the material and method section.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects of the test item were noted in any of the 5 tester strains used up to the limit concentration of 5000 µg/plate evaluated with and without metabolic activation in Experiment 1 and 2. The reduction in the number of revertants down to a mutation factor of 0.5 found in Experiment 2 in tester strain TA 1537 at 1000 and 2500 µg/plate with metabolic activation was regarded not biologically relevant due to lack of a dose-response relationship.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Tabl.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	18±2.5	1.0	113±3.1	1.0	11±5.6	1.0	7±1.5	1.0	191±8.7	1.0
31.6	18±4.0	1.0	107±15.0	1.0	12±3.5	1.1	10±2.5	1.5	192±6.5	1.0
100	20±5.7	1.1	112±4.2	1.0	12±4.7	1.1	9±3.5	1.4	192±26.7	1.0
316	22±7.5	1.2	125±2.3	1.1	11±3.1	1.0	7±1.5	1.1	176±7.5	0.9
1000	16±9.5	0.9	123±9.0	1.1	13±1.0	1.2	7±1.5	1.1	193±7.4	1.0
2500	19±6.0	1.0	115±12.5	1.0	15±2.1	1.4	7±3.5	1.0	184±32.5	1.0
5000	13±6.1	0.7	112±8.1	1.0	12±4.0	1.1	12±2.5	1.9	195±12.1	1.0
Positive control	333±11.1	18.1	672±185.5	6.0	1206±96.5	109.7	169±39.6	25.4	1426±93.6	7.5

Solvent control: A. dest.

Positive controls: 4-NOPD (4-Nitro-o-phenylene-diamine; TA 98, TA 1537); NaN3 (sodium azide; TA 100, TA 1535); MMS (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	30±4.9	1.0	112±2.5	1.0	8±1.5	1.0	6±2.3	1.0	264±18.8	1.0
31.6	27±7.8	0.9	104±30.6	0.9	7±2.0	0.9	8±2.9	1.4	238±7.9	0.9
100	26±11.7	0.9	120±14.5	1.1	8±1.2	1.0	6±1.0	1.1	246±3.6	0.9
316	20±2.9	0.6	129±9.6	1.1	12±8.1	1.5	8±3.8	1.4	231±10.3	0.9
1000	21±10.4	0.7	156±9.2	1.4	8±3.0	1.0	6±1.7	1.1	231±18.2	0.9
2500	28±5.2	0.9	118±9.0	1.0	10±2.5	1.3	5±0.6	0.8	231±15.9	0.9
5000	22±5.6	0.7	129±12.3	1.1	9±2.1	1.1	8±3.2	1.4	243±16.5	0.9
Positive control	2834±183.1	93.4	1730±359.5	15.4	161±19.7	21.0	283±41.0	49.9	583±52.4	2.2

Solvent control: A. dest.

Positive controls: 2-AA (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	18±0.6	1.0	80±7.5	1.0	10±7.9	1.0	8±5.9	1.0	210±8.5	1.0
31.6	25±4.4	1.4	97±19.8	1.2	11±1.5	1.1	14±1.5	1.9	216±6.0	1.0
100	21±1.5	1.2	95±10.3	1.2	12±1.7	1.2	12±3.5	1.6	209±24.0	1.0
316	21±1.7	1.2	97±9.3	1.2	11±3.8	1.1	7±3.1	0.9	248±22.3	1.2
1000	20±4.9	1.1	98±6.2	1.2	7±2.1	0.7	9±0.6	1.2	184±15.3	0.9
2500	22±7.5	1.2	109±18.3	1.4	12±3.2	1.2	9±3.5	1.1	213±7.5	1.0
5000	18±1.5	1.0	85±8.7	1.1	12±1.7	1.2	7±3.6	0.9	199±37.8	0.9
Positive control	474±39.6	26.8	825±72.7	10.3	947±122.0	94.7	77±10.0	10.0	1478±164.8	7.0

Solvent control: A. dest.

Positive controls: 4-NOPD (4-Nitro-o-phenylene-diamine, TA 98, TA 1537); NaN3 (sodium azide; TA 100, TA 1535); MMS (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	35±4.0	1.0	130±5.5	1.0	20±4.9	1.0	14±5.0	1.0	307±20.0	1.0
31.6	35±14.6	1.0	122±12.9	0.9	17±1.5	0.9	11±2.6	0.8	317±29.3	1.0
100	32±5.0	0.9	115±11.8	0.9	15±2.5	0.8	9±5.0	0.6	320±49.7	1.0
316	28±2.5	0.8	110±2.9	0.8	15±4.5	0.8	9±2.6	0.6	321±9.8	1.0
1000	24±8.3	0.7	117±15.5	0.9	17±1.7	0.8	7±2.5	0.5	323±15.3	1.1
2500	25±4.7	0.7	129±17.0	1.0	17±3.5	0.8	7±2.6	0.5	318±29.5	1.0
5000	35±9.6	1.0	131±18.9	1.0	19±4.0	0.9	11±1.2	0.8	302±27.5	1.0
Positive control	3111±233.2	88.0	2070±388.7	15.9	85±10.7	4.2	227±69.6	16.2	647±55.2	2.1

Solvent control: A. dest.

Positive controls: 2-AA (2-aminoanthracene; all tester strains)

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative