Urea, N,N''-1,4-cyclohexanediylbis-, trans-

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD Guideline 471 and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[(1r,4r)-4-(carbamoylamino)cyclohexyl]urea

EC Number:

813-604-1

Cas Number:

68533-01-7

Molecular formula:

C8 H16 N4 O2

IUPAC Name:

[(1r,4r)-4-(carbamoylamino)cyclohexyl]urea

Test material form:

other: solid

Details on test material:

Name of test substance: NM01
Test substance No.: 13/0143-1
Batch identification: GM0416/27
CAS No.: 68533-01-7
Purity/composition: 98.6 g/100 g
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
Storage stability: The stability of the test substance under storage conditions throughout the study period was guaranteed until 13 Mar 2015 as indicated by the sponsor, and the sponsor holds this responsibility.
Date of production/supply: 13 Mar 2013
Physical state, appearance: Solid, white
Storage conditions: Room temperature

Method

Target gene:

his-, trp-

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from rats treated with phenobarbital (i.p.) and β-naphthoflavone (orally) each on 3 consecutive days

Test concentrations with justification for top dose:

0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate

Vehicle / solvent:

ethanol (due to insolubility of the test substance in ultrapure water)

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD); 2-aminoanthracene (2-AA)

Remarks:

All positive control substances were used without S9 mix, except for 2-AA (with S9 mix)

Details on test system and experimental conditions:

TEST SYSTEM:
Deep-frozen bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) are thawed at room temperature,
and 0.1 mL of this bacterial suspension is inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. As a rule, a germ density of ≥ 108 bacteria/mL is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

SALMONELLA TYPHIMURIUM:
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) is determined. The tester strains TA 1535, TA 1537, TA 98 and TA 100 are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide
layer (rfa), which leads to an increase in permeability to lipophilic substances.
The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98. The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 (4) and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system,
which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

ESCHERICHIA COLI:
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions. The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

SCOPE OF TESTS AND TEST CONDITIONS:
All experiments were conducted with and without S9 mix, with 3 test plates per dose or per control, and applying the test substance at the following concentrations: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate.
1. Experiment: Standard plate test in TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
2. Experiment: Preincubation test in TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA (Reason: No mutagenicity was observed in the standard plate test)
3. Experiment: Preincubation test in TA 1537 (Reason: Concerning the bacteriotoxicity inconclusive values were observed in the 2nd Experiment)
4. Experiment: Preincubation test in TA 1537 (Reason: Due to technical reason, an evaluation of the TA 1537 in the 3rd Experiment was not possible.)

STANDARD PLATE TEST:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution [minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin (S. typhimurium); or 0.5 mM tryptophan (E. coli)] are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto minimal glucose agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants or trp+ revertants) are counted.

PREINCUBATION TEST:
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

TITER DETERMINATION:
The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments. In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. Test tubes containing 2-mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL vehicle (without and with test substance)
0.1 mL fresh bacterial culture (dilution: 10-6)
0.5 mL S9 mix
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S9 mix are incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Evaluation criteria:

Experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 109 cells per mL were used. For approval the titer of viable bacteria was ≥ 108 colonies per mL.

The test substance is considered positive if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

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:

cytotoxicity

Remarks:

weak bacteriotoxicity in preincubation test at 5 000 μg/plate only

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY:
No relevant increase in the number of his+ or trp+ revertants was observed in any of the strains in either test system.

TOXICITY:
No bacteriotoxic effect (reduced his- or trp- background growth, slight decrease in the number of his+ or trp+ revertants, slight reduction in the titer) was observed in the standard plate test up to the highest required concentration.
In the preincubation assay a weak bacteriotoxicity (slight decrease in the number of his+ revertants) was observed depending on the strain and test conditions at 5 000 μg/plate. The inconclusive Data observed in the 2nd Experiment was not confirmed in a repeat experiment and has to be considered as not relevant.

SOLUBILITY:
No test substance precipitation was found with and without S9 mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions chosen here, it is concluded that NM01 is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance NM01 was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several strains of Salmonella typhimurium and Escherichia coli (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA), in a reverse mutation assay. A standard plate test and a preincubation test were conducted, both with and without metabolic activation (liver S9 mix from induced rats), and both covering a dose range of 33 μg - 5 000 μg/plate. No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5 000 μg/plate. A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.