1,3-bis(hydroxymethyl)urea

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 September 2016 to 28 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

2013

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis(hydroxymethyl)urea

EC Number:

205-444-0

EC Name:

1,3-bis(hydroxymethyl)urea

Cas Number:

140-95-4

Molecular formula:

C3H8N2O3

IUPAC Name:

1,3-bis(hydroxymethyl)urea

Test material form:

solid

Method

Target gene:

All Salmonella typhimurium strains contained mutations in the histidine operon.
Escherichia coli WP2 uvrA carried a defect in one of the genes for tryptophan biosynthesis.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver metabolising system (S9 mix)

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system. A maximum concentration of 5000 µg/plate was selected, in order that initial treatments were performed up to the maximum recommended concentration according to current regulatory guidelines (OECD, 1997).

Vehicle / solvent:

- Solvent used: water
- Justification for choice of solvent/vehicle: The selection of the solvent for this assay was undertaken based on the available information from the preliminary solubility test. Ultra pure water showed best performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ultra pure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

cumene hydroperoxide

other: 2-Aminoanthracene (2-AA); Daunomycin (DAUN)

Details on test system and experimental conditions:

METHOD OF APPLICATION:plate incorporation

DURATION
- Exposure duration: 2-3 days

NUMBER OF REPLICATIONS: 3 for each test concentration and positive control, 6 for solvent control

DETERMINATION OF CYTOTOXICITY
- Method: reduction of background growth

- OTHER:
Counting of revertant colonies:
Revertant colonies were either scored automatically with the „Sorcerer" or manually with the validated „Ames Study Manager" from Perceptive Instruments, Haverhill, Suffolk, UK. Tables of individual and mean values were generated automatically.
The presence of a background lawn of non-revertant cells was checked for each plate.

Evaluation criteria:

The assessment of test material-induced effects was dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which showed the highest number of colonies.
A test material was to be defined as negative or non-mutagenic in this assay if the assay was to be considered valid, and "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)
For valid data, the test material was considered to be positive or mutagenic if a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2810 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2810 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2810 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2810 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2810 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test material on the agar plates occurred.

HISTORICAL CONTROL DATA
Observed colony number remained mostly within the historical control values. Values outside those ranges remained without any biological significance.

Any other information on results incl. tables

Table 1: Summary of Series 1

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA 98	TA 100	TA 102	TA1535	TA 1537	WP2 uvrA
	Results without S9
Water	30 ± 5	102 ± 9	221 ± 19	21 ± 4	29 ± 4	36 ± 8
5	35 ± 6	100 ± 22	239 ± 47	22 ± 6	24 ± 6	36 ± 7
15.8	30 ± 4	115 ± 9	251 ± 37	25 ± 5	32 ± 5	34 ± 8
50	30 ± 8	116 ± 5	252 ± 52	25 ± 3	36 ± 5	36 ± 10
158	30 ± 7	102 ± 12	234 ± 44	22 ± 1	30 ± 5	38 ± 8
500	26 ± 6	108 ± 7	245 ± 21	22 ± 2	28 ± 8	32 ± 7
1580	28 ± 4	119 ± 13	352 ± 19	32 ± 9	25 ± 6	44 ± 9
5000	1 ± 1	1 ± 2	3 ± 2	1 ± 0	0 ± 1	42 ± 9
DAUN (1.0)	117 ± 12
NaN3 (2.0)		1446 ± 137		903 ± 15
CUM (200)			757 ± 15
9-AA (50)					1056 ± 286
NQO (2.0)						1509 ± 148
	Results with S9
Water	44 ± 6	125 ± 11	382 ± 33	24 ± 8	29 ± 4	43 ± 6
5	30 ± 8	133 ± 40	397 ± 41	27 ± 6	23 ± 4	37 ± 3
15.8	29 ± 5	126 ± 8	389 ± 27	27 ± 4	31 ± 5	40 ± 2
50	38 ± 2	123 ± 18	420 ± 26	21 ± 7	27 ± 14	46 ± 13
158	34 ± 5	136 ± 10	417 ± 26	25 ± 5	29 ± 2	33 ± 4
500	32 ± 3	116 ± 23	430 ± 18	29 ± 5	32 ± 3	49 ± 7
1580	31 ± 12	127 ± 8	458 ± 6	25 ± 7	26 ± 8	42 ± 40
5000	4 ± 3	9 ± 4	49 ± 10	1 ± 1	8 ± 6	48 ± 4
2-AA (2.0)	763 ± 47	1669 ± 146
2-AA (5.0)				254 ± 17	556 ± 58
2-AA (10)						512 ± 20
B(a)p (10)			1897 ± 213

 

 

Table 2: Summary of Series 2

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA 98	TA 100	TA 102	TA1535	TA 1537	WP2 uvrA
	Results without S9
Water	33 ± 7	117 ± 18	277 ± 16	30 ± 7	30 ± 3	30 ± 7
50	31 ± 3	124 ± 2	300 ± 32	27 ± 4	30 ± 4	36 ± 8
158	29 ± 5	126 ± 11	303 ± 15	18 ± 3	35 ± 3	31 ± 6
500	34 ± 7	117 ± 6	361 ± 39	28 ± 2	29 ± 3	35 ± 11
889	41 ± 11	118 ± 13	368 ± 16	30 ± 2	29 ± 6	34 ± 6
1580	30 ± 7	125 ± 7	476 ± 33	20 ± 2	28 ± 6	30 ± 3
2810	29 ± 6	117 ± 16	316 ± 96	18 ± 8	19 ± 4	42 ± 12
DAUN (1.0)	296 ± 29
NaN3 (2.0)		1709 ± 62		945 ± 27
CUM (200)			894 ± 90
9-AA (50)					1639 ± 183
NQO (2.0)						1608 ± 45
	Results with S9
Water	39 ± 9	132 ± 13	250 ± 28	24 ± 4	32 ± 3	37 ± 7
50	40 ± 8	143 ± 12	282 ± 55	24 ± 4	40 ± 4	39 ± 2
158	37 ± 11	140 ± 13	290 ± 31	25 ± 2	25 ± 8	41 ± 3
500	34 ± 11	139 ± 11	315 ± 37	24 ± 1	36 ± 5	43 ± 3
889	37 ± 2	130 ± 5	297 ± 41	24 ± 3	40 ± 6	38 ± 2
1580	29 ± 7	125 ± 11	414 ± 49	21 ± 8	29 ± 1	42 ± 8
2810	19 ± 6	129 ± 18	571 ± 72	22 ± 3	47 ± 6	48 ± 7
2-AA (2.0)	193 ± 43
2-AA (5.0)		1499 ± 61
2-AA (10)				160 ± 1	341 ± 17	149 ± 12
B(a)p (10)			1176 ± 45

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions the test substance was considered mutagenic in the bacterial reverse mutation assay.

Executive summary:

A study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and in the presence of a rat liver metabolising system (S9 mix) according to OECD 471. The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following test item treatments of all the tester strains in the absence and presence of S9 mix, a relevant dose dependent clear increase in revertant numbers was observed in Salmonella typhimurium TA 102 in the presence of S9 mix. It was concluded that the test item was mutagenic under the experimental conditions described.