N,N”-hexane-1,6-diylbis(N’-alkylaryl)urea

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:

18 Aug - 12 Sep 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): trade name given
- Substance type: white, crystalline powder
- Analytical purity: 99.5%
- Solubility: water: < 0.1 g/L
- Lot/batch No.: 1592ZG-076
- Expiration date of the lot/batch: Aug 2015
- Storage condition of test material: at room temperature 20 ± 5 °C

Method

Target gene:

his operon (for S. typhimurium strains)

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw i.p.)

Test concentrations with justification for top dose:

First experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation.
Second experiment: 78, 156, 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine (4-NOPD); 2-Amino-Anthracene (2-AA)

Remarks:

+S9: 2-AA (1 µg/plate, TA100, TA102, TA1535, TA97a); B[a]P (20 µg/plate, TA98); -S9: sodium azide (1 µg/plate, TA100); 4-NOPD (20 µg/plate, TA102, TA97a, TA98, TA1535)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: quadruplicates each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.
A test item is considered as a mutagen if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: As the test item was tested as suspension precipitation were observed on all plates in all tested concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA: All positive control values were within the range of the historical data. The most values of the spontaneous revertants of the solvent control ethanol which lie outside the range of the historical data are in the range of the literature data. Therefore, the study is concidered valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9 mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 4 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA102	TA1535	TA97a	TA98
-	0 (EtOH)	121 ± 17.1	248 ± 22.3	12 ± 1.0	99 ± 11.3	14 ± 1.0
-	50	115 ± 22.7	209 ± 8.9	13 ± 2.5	105 ± 4.5	13 ± 0.8
-	150	107 ± 12.0	208 ± 28.3	13 ± 3.2	112 ± 5.0	14 ± 0.6
-	500	109 ± 9.2	247 ± 42.0	14 ± 1.9	133 ± 9.1	10 ± 2.1
-	1500	115 ± 14.4	244 ± 47.7	15 ± 1.0	132 ± 1.8	13 ± 2.2
-	5000	119 ± 18.6	304 ± 102.8	15 ± 2.1	141 ± 31.0	15 ± 1.5
Positive controls, -S9 mix	Name	NaN3	4-NOPD	NaN3	4-NOPD	4-NOPD
	Concentrations (μg/plate)	1	20	1	20	20
	Mean No. of colonies/plate (average of 4 ± SD)	412 ± 30.1	748 ± 13.1	103 ± 14.5	525 ± 45.8	384 ± 54.0
+	0 (EtOH)	103 ± 9.7	229 ± 32.1	15 ± 1.0	125 ± 4.8	17 ± 3.0
+	50	116 ± 22.5	235 ± 16.4	13 ± 1.7	117 ± 5.7	13 ± 2.2
+	150	136 ± 4.1	238 ± 40.6	15 ± 2.4	113 ± 4.1	13 ± 3.1
+	500	126 ± 7.5	218 ± 19.1	13 ± 2.1	130 ± 16.4	14 ± 0.8
+	1500	125 ± 22.6	219 ± 14.1	14 ± 3.4	145 ± 15.2	13 ± 4.1
+	5000	107 ± 21.4	268 ± 36.5	14 ± 3.2	133 ± 9.0	14 ± 4.0
Positive controls, +S9 mix	Name	2-AA	2-AA	2-AA	2-AA	B[a]P
	Concentrations (μg/plate)	1	1	1	1	20
	Mean No. of colonies/plate (average of 4 ± SD)	492 ± 93.9	689 ± 80.2	107 ± 3.8	578 ± 34.3	99 ± 8.7

EtOH: Ethanol

NaN3: Sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

2-AA: 2-aminoanthracene

B[a]P: Benzo-a-pyrene

Table 2. Test results of experiment 2 (preincubation).

With or without S9 mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 4 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA102	TA1535	TA97a	TA98
-	0 (EtOH)	112 ± 5.2	199 ± 11.3	10 ± 0.8	101 ± 13.0	10 ± 2.1
-	78	135 ± 4.8	299 ± 59.9	13 ± 3.3	106 ± 13.7	10 ± 0.0
-	156	134 ± 4.9	324 ± 22.9	12 ± 1.5	108 ± 14.1	11 ± 3.2
-	312	122 ± 20.0	279 ± 76.5	14 ± 2.9	104 ± 10.0	10 ± 2.6
-	625	109 ± 16.1	275 ± 22.0	11 ± 1.0	121 ± 3.6	13 ± 2.9
-	1250	107 ± 7.9	230 ± 23.2	11 ± 4.0	119 ± 3.1	12 ± 2.2
-	2500	104 ± 9.5	240 ± 35.0	9 ± 2.2	114 ± 2.1	19 ± 2.1
-	5000	110 ± 20.0	248 ± 40.9	9 ± 1.9	113 ± 4.4	12 ± 2.4
Positive controls, -S9 mix	Name	NaN3	4-NOPD	NaN3	4-NOPD	4-NOPD
	Concentrations (μg/plate)	1	20	1	20	20
	Mean No. of colonies/plate (average of 4 ± SD)	563 ± 69.2	949 ± 113.2	114 ± 15.6	547 ± 76.3	111 ± 13.6
-	0 (EtOH)	99 ± 16.9	187 ± 5.7	9 ± 2.2	115 ± 2.7	17 ± 2.6
-	78	126 ± 14.2	327 ± 34.8	15 ± 4.0	126.± 7.3	12 ± 3.3
-	156	114 ± 21.1	285 ± 41.1	15 ± 2.6	115 ± 1.8	14 ± 5.1
-	312	115 ± 21.2	160 ± 13.5	12 ± 2.2	117 ± 6.6	13 ± 2.5
-	625	134 ± 9.3	318 ± 50.8	15 ± 2.2	109 ± 14.0	12 ± 2.4
-	1250	97 ± 6.9	295 ± 53.8	12 ± 3.6	113 ± 14.4	10 ± 0.5
-	2500	120 ± 13.4	349 ± 30.2	9 ± 2.2	115 ± 1.9	14 ± 2.4
-	5000	106 ± 17.1	237 ± 18.0	11 ± 2.4	119 ± 2.8	12 ± 1.3
Positive controls, +S9 mix	Name	2-AA	2-AA	2-AA	2-AA	B[a]P
	Concentrations (μg/plate)	1	1	1	1	20
	Mean No. of colonies/plate (average of 4 ± SD)	539 ± 113.6	1018 ± 101.2	99 ± 11.8	556 ± 72.2	114 ± 16.8

EtOH: Ethanol

NaN3: Sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

2-AA: 2-aminoanthracene

B[a]P: Benzo-a-pyrene

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative