Urea, reaction products with formaldehyde, glyoxal and methanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In two reliable bacterial mutation assays the test item is considered to be non-mutagenic. A reliable gene mutation test in mammalian cells in vitro (HPRT test in V79 Chinese hamster cells) also revealed a negative result. A positive result in a reliable in vitro chromosome aberration test (induction of structural chromosome aberrations in V79 cells in the absence and presence of metabolic activation) was not confirmed by in vivo testing for chromosomal aberrations. The in vivo test for induction of micronuclei did not reveal clastogenic potential of the submission substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 000161MCA0

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from sprague Dawlay rat livers after Aroclor 1254 activation.

Test concentrations with justification for top dose:

0; 22; 110; 550; 2750; 5500 µg/plate (standard plate test)
0; 343.8, 687.5; 1375; 2750; 5500 µg/plate (preincubation test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see details

Details on test system and experimental conditions:

Standard plate test
Test tubes containing 2 mL portions of saft agar kept in a water bath at 45 °C, and the remaining companents are added in the following order:
0.1 mL test solution or vehicle
0.1 mL bacterial suspension
0.5 mL S-9 mix (in tests with metabolic activatian) or
0.5 mL phosphate buffer (in tests without metabalic activatian)
After mixing, the samples are poured onto Vogel-Bonner agar plates. After incubation at 37 °C for 48 -72 hours in the dark, the bacterial calonies (his+ revertants) are counted.

Preincubation test
0.1 mL test solution or vehicle, 0.1 mL bacterial Suspension and 0.5 ml S-9 mix are incubated at 37 °C for the duration of 20 minutes. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.

Both Tests
In each experiment 3 Test plates per dose per control used. After incubation at 37 °C for 48 -72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Posivite Control:
with S-9 mix: 2.5 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strains TA 100, TA 98, TA 1537and TA 1535
60 µg 2-aminoanthracene (2-AA)(dissolvecl in DMSO) for the strain E. coli WP2 uvrA
without S-9 mix: 5 µg N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (NOPD) (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine (AAC) chloride monohydrate (dissolved in DMSO) for the strain TA 1537
10 µg N-ethyl-N'-nitro-N-nitroso-guanidin (ENNG) (dissolved in DMSO) for the strain E. coli WP2 uvrA
The Titer was determined and in regularly measurements the strain characteristics were checked. Sterility control performed.

Evaluation criteria:

Positive results
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the result

Key result

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Solubilita: No precipitation of the test item was found.

Toxicity: No bacteriotoxic effect was observed under all test conditions

Mutagenicity: A 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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 22 JUN 2010 TO 30 SEP 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

(according to German Chemical Law and OECD Principles of GLP)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: OP1
- Expiration date of the lot/batch: October 16, 2014
- Certificate of analysis: N° 1873, 15/04/2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of solvent: Stable in water
- Storage: At room temperature

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: supplemented minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: checked for spontaneous mutant frequency

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

All test item concentrations were adjusted to solvent content (8 % water))
Range finding pre-experiment: 15.8 - 2022.0 µg/mL (ca. 10 mM),
Experiment 1: 101.1, 202.2, 404.4, 808.8, 1213.2, 1617.6 and 2022.0 µg/mL (4 h, with and without metabolic activation)
Experiment 1A: 505.0, 1011.0, 1263.8, 1444.3, 1685.0, 1838.2 and 2022.0 µg/mL (4 h, without metabolic activation)
Experiment 2: 3.8, 7.5, 15.0, 30.0, 60.0, 80.0 and 100.0 µg/mL (24 h, without metabolic activation)
200.0, 400.0, 800.0, 1200.0, 1600.0 and 2000.0 µg/mL (4 h, with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water (local tap water deionisised at Harlan CCR)
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 476

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: Experiment 1: 4 h (with and without metabolic activation); Experiment 2: 24 h (without metabolic activation), 4 h (with metabolic activation)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER: Two cultures were tested in each experiment

Evaluation criteria:

A test item is classified as positive if it induces either a reproducible concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent con-trols within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, bio-logical and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. To clarify the results obtained a confirmatory experiment IA was performed in the absence of S9 mix. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The cell cultures were evaluated at the following concentrations:

exposure
period S9
mix concentrations in µg/mL
Experiment I
4 hours - 404.4 808.8 1213.2 1617.6 2022.0
4 hours + 202.2 404.4 808.8 1213.2 1617.6
Experiment IA
4 hours - 1263.8 1444.3 1685.0 1838.2 2022.0
Experiment II
24 hours - 15.0 30.0 60.0 80.0 100.0
4 hours + 200.0 400.0 800.0 1200.0 1600.0 2000.0*

* mutagenicity evaluation was performed only in culture I

The maximum concentration of the test item in the first experiment equals a molar concentration of about 10 mM adjusted to solvent content (8 % water) according to the substance definition of REACH (REGULATION (EC) No 1907/2006). In experiment II the evaluation of dose groups was limited by test item induced cytotoxic effects. No precipitation of the test item was observed up to the maximum concentration in all experiments.
Cytotoxic effects occurred in the first experiment in the highest guideline required dose group (2022.0 µg/mL). In the presence and absence of S9 mix the cloning efficiency and/or the cell density were reduced to 10-20 % of the solvent controls. In the second experiment in the absence of S9 mix following 24 hours treatment the cloning efficiency I was reduced to approximately 10 % at the highest evaluated test item concentration of 100.0 µg/mL. In the presence of S9 relevant cytotoxic effects occurred at 1200.0 µg/mL and above. At 1200.0 µg/mL the relative cloning efficiency I was reduced to 17.9 and 14.2 % in culture I and II.
In experiment I following 4 hours treatment in the absence of S9 mix with 1617.6 and 2022.0 µg/mL three mutation frequencies (36.6, 39.5 and 56.6 mutant colonies/106 cells) exceeded the laboratory's historical data range of 0.6-32.4 mutant colonies/106 cells. The induction factor exceeded the threshold of three times the corresponding solvent control in the highest dose group in culture I and in the two highest dose groups in culture II. To verify this results a confirmatory experiment IA was performed in the absence of S9 mix under identical conditions but using narrower spacing of concentrations. In the highest dose group required by the OECD guideline relevant cytotoxicity was observed. The cloning efficiency I was reduced to 21.4 and 17.7 % of the solvent control in culture I and II. Following treatment with 1263.8 µg/mL the mutation frequency (35.6 mutant colonies/106 cells) exceeded the historical data range. The induction factor remained well within the threshold of three times the corresponding solvent control in all evaluated dose groups. Therefore, the effect observed in experiment I was not reproducible and is judged as not being biologically relevant.
In the second experiment three mutation frequencies (35.3, 43.5 and 50.1 mutant colo-nies/106 cells) exceeding the laboratory's historical data range (0.8 ¿ 33.2 mutant colo-nies/106 cells) were observed following treatment in the presence of S9 mix. The induction factor remained well within the threshold of three times the corresponding solvent control in all evaluated dose groups.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. In experiment I culture II (absence of S9 mix) and in experiment II in both cultures (presence of S9 mix) significant dose dependent trends of the mutation frequency indicated by a probability value of <0.05 were deter-mined. However, the trends were judged as biologically irrelevant since the trend was either not reproducible (experiment I) or the mutation frequency did not exceed the threshold described above (experiment II).
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 7.1 up to 28.3 mutants per 106 cells; the range of the groups treated with the test item was from 7.4 up to 56.6 mutants per 106 cells.
EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells with or without metabolic activation.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay with and without metabolic activation.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in three independent experiments, using two parallel cultures each. Experiment I was performed with and without S9 liver microsomal activation and a treatment period of 4 hours. A confirmatory experiment IA was performed with a treatment time of 4 hours without metabolic activation. Experiment II was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The following concentrations were tested:

Experiment 1: 101.1, 202.2, 404.4, 808.8, 1213.2, 1617.6 and 2022.0 µg/mL (4 h, with and without metabolic activation)

Experiment 1A: 505.0, 1011.0, 1263.8, 1444.3, 1685.0, 1838.2 and 2022.0 µg/mL (4 h, without metabolic activation)

Experiment 2: 3.8, 7.5, 15.0, 30.0, 60.0, 80.0 and 100.0 µg/mL (24 h, without metabolic activation); 200.0, 400.0, 800.0, 1200.0, 1600.0 and 2000.0 µg/mL (4 h, with metabolic activation)

The highest concentration in the main experiments (2022.0 µg/mL) was equal to a molar concentration of 10 mM adjusted to solvent content (8 % water) according to the substance definition of REACH (REGULATION (EC) No 1907/2006). In Experiment II the highest evaluated concentration was limited by strong test item induced cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells with or without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 JUN 2010 to 3 AUG 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemical Law and OECD Principles of GLP

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: OP1
- Expiration date of the lot/batch: October 16, 2014
- Certificate of analysis: N° 1873, 15/04/2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of solvent: Stable in water
- Storage: At room temperature

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and Escherichia coli WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

pre-experiment/experiment I (plate-incorporation test): 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
experiment II (pre-incubation test): 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: better than others

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (NaN3; strains TA1535, TA199); 4-nitro-o-phenylene-diamine (4-NOPD; strains: TA1537, TA98); methyl methane sulfonate (MMS; strain WP2uvrA)

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA; all strains)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (experiment I); preincubation (experiment II)

DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

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 WP2 uvrA) 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.

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation observed
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects observed

 Summary of Results Pre-Experiment and Experiment I

Study Name: 1342802	Study Code: Harlan CCR 1342802
Experiment: 1342802 VV Plate	Date Plated: 30/06/2010
Assay Conditions:	Date Counted: 05/07/2010

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			21 ± 7	11 ± 2	27 ± 6	119 ± 10	44 ± 3
	Untreated			19 ± 9	13 ± 4	32 ± 6	142 ± 37	56 ± 2
	test item	3 µg		15 ± 4	9 ± 3	30 ± 3	151 ± 21	57 ± 13
		10 µg		16 ± 4	14 ± 3	37 ± 9	123 ± 7	65 ± 5
		33 µg		18 ± 3	11 ± 2	28 ± 4	107 ± 12	64 ± 2
		100 µg		24 ± 3	11 ± 3	38 ± 9	95 ± 6	60 ± 7
		333 µg		22 ± 2	13 ± 3	34 ± 7	140 ± 13	48 ± 7
		1000 µg		21 ± 2	13 ± 3	40 ± 7	126 ± 9	51 ± 6
		2500 µg		14 ± 5	12 ± 4	37 ± 1	119 ± 22	61 ± 15
		5000 µg		24 ± 6	12 ± 5	37 ± 6	125 ± 13	47 ± 3
	NaN3	10 µg		1653 ± 459			910 ± 148
	4-NOPD	10 µg				371 ± 20
	4-NOPD	50 µg			76 ± 12
	MMS	3.0 µL						822 ± 16
With Activation	Deionised water			27 ± 1	21 ± 2	39 ± 3	124 ± 2	63 ± 9
	Untreated			22 ± 6	23 ± 7	93 ± 30	140 ± 4	65 ± 4
	ARKOFIX RCH	3 µg		24 ± 1	24 ± 7	52 ± 9	131 ± 5	68 ± 3
		10 µg		33 ± 4	24 ± 9	51 ± 9	121 ± 20	64 ± 3
		33 µg		23 ± 3	26 ± 16	43 ± 5	95 ± 20	88 ± 9
		100 µg		17 ± 5	20 ± 5	54 ± 10	103 ± 21	74 ± 3
		333 µg		24 ± 5	18 ± 2	50 ± 13	108 ± 22	58 ± 3
		1000 µg		23 ± 10	26 ± 1	47 ± 4	101 ± 10	74 ± 3
		2500 µg		26 ± 6	21 ± 8	56 ± 6	93 ± 7	78 ± 7
		5000 µg		23 ± 4	24 ± 2	45 ± 10	126 ± 3	83 ± 4
	2-AA	2.5 µg		277 ± 21	389 ± 23	2141 ± 218	1965 ± 88
	2-AA	10.0 µg						287 ± 33

Key to Positive Controls
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

 Summary of Results Experiment II

Study Name: 1342802	Study Code: Harlan CCR 1342802
Experiment: 1342802 HV2 Pre	Date Plated: 29/07/2010
Assay Conditions:	Date Counted: 03/08/2010

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			19 ± 3	23 ± 3	24 ± 3	147 ± 8	54 ± 11
	Untreated			12 ± 2	24 ± 4	27 ± 6	125 ± 8	52 ± 7
	test item	33 µg		18 ± 2	24 ± 4	22 ± 6	148 ± 2	40 ± 6
		100 µg		16 ± 1	29 ± 5	34 ± 3	134 ± 9	57 ± 6
		333 µg		16 ± 2	31 ± 4	23 ± 3	143 ± 14	41 ± 1
		1000 µg		15 ± 2	24 ± 5	24 ± 6	130 ± 17	56 ± 10
		2500 µg		16 ± 6	25 ± 7	28 ± 6	146 ± 2	42 ± 5
		5000 µg		20 ± 2	23 ± 10	35 ± 9	149 ± 10	59 ± 4
	NaN3	10 µg		1768 ± 52			1521 ± 45
	4-NOPD	10 µg				423 ± 55
	4-NOPD	50 µg			84 ± 13
	MMS	3.0 µL						456 ± 19
With Activation	Deionised water			26 ± 6	27 ± 7	45 ± 4	164 ± 15	70 ± 9
	Untreated			28 ± 1	33 ± 2	41 ± 10	178 ± 11	62 ± 4
	ARKOFIX RCH	33 µg		27 ± 3	31 ± 4	43 ± 3	165 ± 9	66 ± 11
		100 µg		27 ± 1	26 ± 2	45 ± 4	171 ± 10	73 ± 2
		333 µg		25 ± 11	32 ± 2	45 ± 5	168 ± 16	69 ± 3
		1000 µg		27 ± 3	34 ± 9	39 ± 2	142 ± 8	67 ± 6
		2500 µg		31 ± 3	28 ± 7	36 ± 2	158 ± 17	74 ± 18
		5000 µg		26 ± 1	36 ± 4	44 ± 12	160 ± 12	79 ± 13
	2-AA	2.5 µg		281 ± 23	394 ± 76	1824 ± 259	2469 ± 100
	2-AA	10.0 µg						363 ± 51

Key to Positive Controls
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

Conclusions:

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:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), 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 the test item at any dose 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.

In experiment II with S9 mix, the data in the untreated control of strain TA 1537 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.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 9 JUN 2010 to 22 JUN 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemical Law and OECD Principles of GLP

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: OP1
- Expiration date of the lot/batch: October 16, 2014
- Certificate of analysis: N° 1873, 15/04/2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of solvent: Stable in water
- Storage: At room temperature

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / beta-naphtoflavone induced rat liver S9

Test concentrations with justification for top dose:

The selction of the maximum dose was adjusted to a water content of 8 % (correction factor 1.09).
With and without metabolic activation:
Experiment I: 7.9, 15.9, 31.7, 63.4, 126.9, 253.8, 507.5, 1015.0 and 2030.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water (local tap water deionised at Harlan CCR)
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation (S9 mix)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (S9 mix)

Details on test system and experimental conditions:

According to the OECD Guideline only one experiment was performed. The exposure period was 4 hours with and without metabolic activation. The chromosomes were prepared 18 hours after start of treatment with the test item. Evaluation of two cultures per dose group was performed.

METHOD OF APPLICATION: in culture medium (minimal essential medium)

DURATION
- Exposure duration: 4 hours (+/- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: about 1.5

NUMBER OF CELLS EVALUATED: at least 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers

OTHER EXAMINATIONS:
- Determination of polyploidy: no evidence
- Determination of endoreplication: no evidence

Evaluation criteria:

Evaluation of the cultures was performed according to the OECD Guideline using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in the absence of S9 mix, where only 50 metaphases were scored.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) and relative cell numbers were determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix.
According to the OECD Guideline only one experiment was performed, since the test item was considered to be mutagenic after 4 hours treatment. The chromosomes were prepared 18 hours after start of treatment with the test item. The exposure period was 4 hours with and without metabolic activation.
In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control without metabolic activation and the highest test item concentration with and without metabolic activation, where only 50 metaphases were evaluated.
Neither precipitation nor relevant influence of the test item on pH value or osmolarity was observed (solvent control 387 mOsm, pH 7.4 versus 393 mOsm and pH 7.3 at 2030.0 µg/mL).
In the absence and presence of S9 mix cytotoxicity indicated by reduced mitotic indices (41.6 and 28.9 % of control, respectively) was observed at the highest evaluated concentration (2030.0 µg/mL). The observed cytotoxicity exceeded the cytotoxicity requested by the OECD guideline.
In the absence and presence of S9 mix statistically significant increases in the number of aberrant cells, excluding gaps (without S9 mix: 15.5, 76.0 %, with S9Mix: 16.5 and 30.0 %, respectively) were observed at the two highest evaluated concentrations (1015.0 and 2030.0 µg/mL). All values clearly exceeded the laboratory´s historical solvent control data range (0.0-4.0 % aberrant cells, excluding gaps).
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the controls. Either EMS (1000 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the absence and presence of S9 mix.

Remarks on result:

other: strain/cell type: V79 cells

Summary of results of the chromosome aberration study with the test item

Preparation	Test item	Cell numbers	Mitotic indices	Aberrant cells
interval	concentration	in %	in %	in %
	in µg/mL	of control	of control	incl. gaps*	excl. gaps*	with exchanges
Exposure period 4 hrs without S9 mix
18 hrs	Solvent control^1	100.0	100.0	3.0	2.5	0.5
	Positive control^2#	n.t.	105.9	42.0	39.0^S	25.0
	507.5	88.6	129.4	2.5	1.5	0.0
	1015.0	90.4	113.6	16.5	15.5^S	9.0
	2030.0^#	76.1	41.6	80.0	76.0^S	7.0
Exposure period 4 hrs with S9 mix
18 hrs	Solvent control^1	100.0	100.0	0.5	0.5	0.0
	Positive control^3	n.t.	52.6	10.0	9.5^S	3.0
	507.5	121.8	111.8	2.5	1.5	0.0
	1015.0	122.1	114.5	18.0	16.5^S	6.0
	2030.0^#	105.2	28.9	37.0	30.0^S	11.0

*     Inclusive cells carrying exchanges

^#     Evaluation of 50 metaphases per culture

n.t.  Not tested

^S     Aberration frequency statistically significant higher than corresponding control values

¹     Deionised water   10.0 % (v/v)

²     EMS                  1000.0 µg/mL

³    CPA                     1.4 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the absence and presence of metabolic activation.

Executive summary:

The test item, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V7 9cells of the Chinese hamster in vitro in one experiment. The following study design was performed:

	With and without S9 mix
Exposure period	4 hrs
Recovery	14 hrs
Preparation interval	18 hrs

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control without metabolic activation and the highest test item concentration with and without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentration (2030.0 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item, corrected by water content (correction factor 1.09) and with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data.

In the absence and presence of S9 mix cytotoxicity indicated by reduced mitotic indices was observed at the highest evaluated concentration. The observed cytotoxicity exceeded the cytotoxicity requested by the OECD guideline.

In the absence and presence of S9 mix statistically significant increases in the number of aberrant cells, excluding gaps (without S9 mix: 15.5 and 76.0%, with S9 mix: 16.5 and 30.0 %, respectively) were observed at the two highest evaluated concentrations (1015.0 and 2030.0 µg/mL). All values clearly exceeded the laboratory´s historical solvent control data range (0.0-4.0 % aberrant cells, excluding gaps).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mammalian Erythrocyte Micronucleus Test

Genetic toxicity of the test item has been investigated in an in vivo micronucleus assay in male NMRI mice (7 per dose group). The test item was administered once orally at the maximum recommended dose level of 2000 mg/kg bw, with 1000 and 500 mg/kg bw as the two lower dose levels. Animals were killed 24 or 48 hours later. Evaluation of polychromatic (PCE) or normochromatic (NCE) erythrocytes did not reveal any evidence of an increase in the incidence of micronucleated cells. No significant change in the PCE/NCE ratio was observed after dosing with the test item, and no signs of systemic toxicity were observed in animals dosed with the test item. The positive control material produced a marked increase in the frequency of micronucleated PCE. The test material was considered to be non-genotoxic under the conditions of the test (i.e. not clastogenic).

The result indicate that, under the conditions of the present study, there is no potential of the test item to induce chromosomal aberrations (i.e. not clastogenic). The positive control (cyclophosphamid) yielded positive results and therefore indicating the sensitivity of the system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Reverse mutation tests
The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain WP2 uvrA (Clariant, 1342802, 2010). The assay was performed in two independent experiments both with and without liver microsomal activation according to OECD guideline 471. Each concentration and the controls were tested in triplicate at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in any of the test groups.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose 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.
In experiment II with S9 mix, the data in the untreated control of strain TA 1537 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.
Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.
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 bacterial strains used.

In another Ames test according to OECD guideline 471 (BASF, 40 M0300/094292, 2010), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA were tested up to a concentration of 5500 µg/plate, both in the presence and in the absence of S-9 mix from Aroclor 1254 induced rats. No bacteriotoxic effect was observed under all test conditions.

In vitro Mammalian chromosomal Aberration Test
The test item was subject to an in vitro chromosome aberration test in chinese hamster cells (V79) according to OECD guideline 473 (Clariant, 1342803, 2010). The exposure period of the cells was 4 hours with and without metabolic activation with concentrations of 7.9, 15.9, 31.7, 63.4, 126.9, 253.8, 507.5, 1015.0 and 2030.0 µg/mL tested. The chromosomes were prepared 18 hours after start of treatment with the test item. Evaluation of two cultures per dose group was performed. In the absence and presence of S9 mix cytotoxicity indicated by reduced mitotic indices was observed at the highest evaluated concentration. The observed cytotoxicity exceeded the cytotoxicity requested by the OECD guideline. In the absence and presence of S9 mix statistically significant increases in the number of aberrant cells, excluding gaps (without S9 mix: 15.5 and 76.0%, with S9 mix: 16.5 and 30.0 %, respectively) were observed at the two highest evaluated concentrations (1015.0 and 2030.0 µg/mL). All values clearly exceeded the laboratory´s historical solvent control data range (0.0-4.0 % aberrant cells, excluding gaps). No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the absence and presence of metabolic activation.

In vitro Mammalian cell Gene Mutation Test
The test item was subject to a test on gene mutations at the HPRT locus in V79 Chinese hamster cells, according to OECD 476. 
The following concentrations were tested:
Experiment 1: 101.1, 202.2, 404.4, 808.8, 1213.2, 1617.6 and 2022.0 µg/mL (4 h, with and without metabolic activation)
Experiment 1A: 505.0, 1011.0, 1263.8, 1444.3, 1685.0, 1838.2 and 2022.0 µg/mL (4 h, without metabolic activation)
Experiment 2: 3.8, 7.5, 15.0, 30.0, 60.0, 80.0 and 100.0 µg/mL (24 h, without metabolic activation); 200.0, 400.0, 800.0, 1200.0, 1600.0 and 2000.0 µg/mL (4 h, with metabolic activation)
In Experiment II the highest evaluated concentration was limited by strong test item induced cytotoxicity. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells with or without metabolic activation.

Genetic toxicity of the test item has been investigated in an in vivo micronucleus assay in male NMRI mice (7 per dose group). The test item was administered once orally at the maximum recommended dose level of 2000 mg/kg bw, with 1000 and 500 mg/kg bw as the two lower dose levels. Animals were killed 24 or 48 hours later. Evaluation of polychromatic (PCE) or normochromatic (NCE) erythrocytes did not reveal any evidence of an increase in the incidence of micronucleated cells. No significant change in the PCE/NCE ratio was observed after dosing with the test item, and no signs of systemic toxicity were observed in animals dosed with the test item. The positive control material produced a marked increase in the frequency of micronucleated PCE. The test material was considered to be non-genotoxic under the conditions of the test (i.e. not clastogenic).
Moreover DMDHEU, a surrogate substance of the submission substance, was tested in an in vivo micronucleus test conducted according to OECD 474, too. The test compound was applied orally via gavage to male and female mice at concentrations of 500, 1000, and 2000 mg/kg bw (corresponding to 365, 730, and 1460 mg surrogate substance/kg bw).
The animals were treated once with the test compound and according to the test procedure the animals were killed 24 or 48 hours (only highest dose group) after administration of the test compound.
No signs of systemic toxicity or deaths were observed
The incidence of micronucleated polychromatic erythrocytes of the animals treated with test item was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased.The ratio of polychromatic/normochromatic erythrocytes in both male and femaleanimals remained unaffected by the treatment compared to control values.
The results indicate that, under the conditions of the present study, there is no potential of the test item (surrogate substance) to induce chromosomal aberrations (i.e. not clastogenic).
The positive control (cyclophosphamid) yielded positive results and therefore indicating the sensitivity of the system.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
In two reliable bacterial mutation assays and in a reliable gene mutation test in mammalian cells in vitro the test item is considered to be non-mutagenic. However, from the results of an in vitro CA study it can be concluded that the substance possesses clastogenic activity. In contrast, higher tier in vivo testing of chromosomal aberration (MN Test) revealed no clastogenic potential of the submission substance as well as of a surrogate substance (DMDHEU). Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.