N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

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:

02 May - 25 May 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland-Pfalz, Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

EC Number:

701-177-3

Molecular formula:

C21H39NO3

IUPAC Name:

N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102, TA 1535

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.

Test concentrations with justification for top dose:

First experiment: 50, 150, 500, 1501 and 5004 µg/plate with and without metabolic activation
Second experiment: 1250, 2500 and 5001 µg/plate with and without metabolic activation

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine (NPD; 80 µg/plate, -S9, TA98, TA97a and TA102); sodium azide (6 µg/plate, -S9, TA100 and TA1535), benzo-a-pyrene (BaP; 40 µg/plate, +S9, TA98), 2-Amino-anthracene (2-AA; 3 µg/plate, +S9, TA97a, TA100, TA102 and TA1535 )

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, first experiment); preincubation (second experiment)

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

NUMBER OF REPLICATIONS: 4 replications each in one independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: titre determination; the titre control should reach a concentration of at least 10E9 bacteria/mL (100 colonies/plate)

Evaluation criteria:

The test material may be considered as mutagen if at least one of the used strain (with or without metabolic activation) shows an increase of the revertant colonies over 2 times of the negative control (induction rate I ≥ 2) and/or a concentration-dependent relationship is apparent.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: The mutation rates of the positive controls were lower than the stated rates of Prof. Ames. However, the rates were within the range of the historical control data of the Laus GmbH. The rates of the spontaneous revertants were partly lower than the stated rates of Prof. Ames. However, the rates were within the ranges of the historical control data of the Laus GmbH.

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 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA97a
	Water	154 ± 20.2	16  ±8.4	n.d.	n.d.	n.d.
–	DMSO	132 ± 25.4	16 ± 5.8	153 ± 18.2	12 ± 2.1	120 ± 18.1
–	50	126 ± 19	12 ± 6	165 ±15	8 ±1	108 ± 3
–	150	155 ± 9	10 ± 3	161 ±45	15 ± 4	89 ± 10
–	500	135± 22	6 ± 5	146 ± 21	9 ± 4	128 ±10
–	1501	140±12	9 ± 1	161 ± 13	7 ± 2	127 ±32
–	5004	158 ± 22	14 ± 4	82 ± 55	9 ± 5	121 ± 54
Positive controls, –S9	Name	SA	SA	4NPD	4NPD	4NPD
	Concentrations (μg/plate)	6	6	80	80	80
	Mean No. of colonies/plate (average of 3 ± SD)	1001	1001	1001	1001	1001
	Water	n.d.	n.d.	n.d.	n.d.	n.d.
+	DMSO	140 ± 17.4	13 ± 3.4	145 ± 37.3	15 ±1.4	120 ± 18.1
+	50	125 ± 7	12 ± 6	158 ± 42	10 ± 3	115 ± 12
+	150	146± 13	12 ± 4	105 ± 61	8 ± 1	143 ± 18
+	500	166 ± 30	13 ± 5	152 ± 21	8 ± 2	149 ± 36
+	1500	143 ± 43	11 ± 6	141 ± 27	8 ± 2	151 ± 24
+	5000	0 148 ± 28	11 ± 3	166 ± 28	8 ± 2	144 ± 28
Positive controls, +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	3	3	3	40	3
	Mean No. of colonies/plate (average of 3 ± SD)	1001	1001	1001	150 ± 7	1001

1001 = more than 1000 colonies

4NPD = 4-Nitro-1,2-phenylendiamin

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

Table 2. Results of experiment 2 (pre-incubation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA97a
	Water	137 ± 30	12 ± 1	n.d.	n.d.	n.d.
–	DMSO	180 ± 9.5	12 ± 2.4	147 ± 29.6	9 ± 2.9	161 ± 65.9
–	1250	122 ± 32	9 ± 2	126 ± 9	9 ± 1	125 ± 22
–	2500	95 ± 15	11 ± 3	132 ± 46	6 ± 1	138 ± 27
–	5001	113 ± 12	11 ± 4	106 ± 48	7 ± 2	129 ± 15
Positive controls. –S9	Name	SA	SA	4NPD	4NPD	4NPD
	Concentrations (μg/plate)	6	6	80	80	80
	Mean No. of colonies/plate (average of 3 ± SD)	1001	1001	1001	1001	1001
	Water	n.d.	n.d.	n.d.	n.d.	n.d.
+	DMSO	174 ± 31.7	13 ± 2.9	117 ± 36.8	11 ± 3.7	132 ± 35.3
+	1250	143 ± 18	9 ± 1	136 ± 17	7 ± 3	159 ± 11
+	2500	142 ± 27	9 ± 2	154 ± 12	7 ± 1	116 ± 6
+	5001	130 ± 13	10 ± 2	68 ± 56	7 ± 2	131 ± 13
Positive controls. +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	3	3	3	40	3
	Mean No. of colonies/plate (average of 3 ± SD)	1001	1001	1001	1001	1001

1001 = more than 1000 colonies

4NPD = 4-Nitro-1,2-phenylendiamin

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative