N-2-hydroxyethyllactamide

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:

03 - 16 Aug 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:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-2-hydroxyethyllactamide

EC Number:

226-546-1

EC Name:

N-2-hydroxyethyllactamide

Cas Number:

5422-34-4

Molecular formula:

C5H11NO3

IUPAC Name:

N-2-hydroxyethyllactamide

Method

Target gene:

his/trp operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of California, Berkely, USA; British Industrial Biological Research Association, UK

Additional strain / cell type characteristics:

other: All TA strains carry a mutation of the uvr B gene coding for the DNA excision repair system (uvr B) and the deep rough mutation (rfa), TA 100 and TA 98 contain the R-factor plasmid (pkM101). E.coli contains an uvr A DNA repair deficiency

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (10% S9-mix), prepared from the livers of rats treated with phenobarbital/ β-naphtolflavone

Test concentrations with justification for top dose:

Experiment I:
- 1.5, 5, 15, 50, 150, 500, 1500 and 500 µg/plate (with and without metabolic activation)
Experiment II:
- 15, 50, 150, 500, 1500 and 500 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: The test item was fully miscible in sterile distilled water at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9-mix: N-ethyl-N´-nitro-N-nitrosoguanidine (ENNG), 9-Aminoacridine (9AA), 4-Nitroquinoline-1-oxide (4NQO); +S9-mix: 2-Aminoanthracene (2AA), Benzo(a)pyrene (BP)

Remarks:

ENNG: 2, 3, 5 μg/plate (E. coli, TA 100, TA 1535); 9AA: 80 μg/plate (TA 1537); 4NQO: 0.2 μg/plate (TA 98); 2AA: 1, 2, 10 μg/plate (TA 100, TA 1535 + TA 1537, E.coli); BP: 5 μg/plate (TA 98)

Details on test system and experimental conditions:

METHOD OF APPLICATION:; in agar (plate incorporation); pre-incubation

DURATION
- Pre-incubation period: 20 min (pre-incubation test)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicate cultures for each test concentration, negative (untreated), vehicle and positive control

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

The test item was tested under the experimental test conditions recommended in the appropriate OECD guideline 471.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study: a dose-related increase in mutant frequency over the dose range tested; a reproducible increase at one or more concentrations; biological relevance against in-house historical control ranges; statistical analysis of data as determined by UKEMS; fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response). A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p <0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed in any experiment either in the presence or absence of metabolic activation.

HISTORICAL CONTROL DATA
- Positive historical control data: The results were within the range of historical control data.
- Negative/solvent historical control data: The results were within the range of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No cytotoxicity was observed in any experiment either in the presence or absence of metabolic activation.

Any other information on results incl. tables

Table 1: Test results - Experiment I

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	TA 1535	E. coli WP2	TA 98	TA 1537
-	Solvent	68 ± 4.6	11 ± 2.5	15 ± 1.2	22 ± 3.0	14 ± 6.7
-	1.5	74 ± 7.6	12 ± 3.5	15 ± 5.1	23 ± 6.1	9 ± 1.2
-	5	76 ± 14.2	13 ± 1.5	12 ± 0.6	19 ± 2.5	12 ± 2.5
-	15	67 ± 8.3	12 ± 4.0	17 ± 5.5	20 ± 6.7	15 ± 2.3
-	50	64 ± 5.8	12 ± 1.5	19 ± 6.7	22 ± 3.1	12 ± 7.5
-	150	65 ± 2.3	14 ± 3.5	15 ± 4.6	25 ± 6.7	13 ± 6.0
-	500	70 ± 4.2	10 ± 4.0	11 ± 3.8	23 ± 1.2	6 ± 2.3
-	1500	66 ± 4.7	12 ± 3.5	14 ± 1.0	20 ± 3.6	9 ± 4.6
-	5000	72 ± 11.9	11 ± 3.0	14 ± 7.4	22 ± 4.0	9 ± 4.9
	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 plates ± SD)	319 ± 31.6	306 ± 99.1	525 ± 23.6	225 ± 9.3	226 ± 46.5
+	Solvent	72 ± 6.4	12 ± 2.6	26 ± 2.3	22 ± 5.5	13 ± 0.6
+	1.5	74 ± 9.6	9 ± 1.7	25 ± 10.5	28 ± 3.1	11 ± 2.9
+	5	76 ± 0.0	9 ± 1.2	20 ± 7.4	17 ± 1.7	19 ± 4.2
+	15	82 ± 12.7	9 ± 1.5	17 ± 2.1	29 ± 4.7	18 ± 5.2
+	50	77 ± 4.2	12 ± 3.2	23 ± 5.5	30 ± 4.7	13 ± 3.0
+	150	72 ± 3.5	11 ± 3.2	28 ± 2.1	21 ± 4.6	14 ± 0.6
+	500	66 ± 3.1	10 ± 0.6	18 ± 3.2	20 ± 4.5	11 ± 4.0
+	1500	70 ± 11.9	11 ± 1.7	23 ± 4.4	22 ± 2.6	10 ± 3.5
+	5000	65 ± 4.4	7 ± 1.0	18 ± 4.9	18 ± 0.6	12 ± 7.8
	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 plates ± SD)	1176 ± 45.1	137 ± 10.3	461 ± 27.5	114 ± 1.5	243 ± 32.1

ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine

9AA: 9-Aminoacridine

4NQO: 4-Nitroquinoline-1-oxide

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

Table 2: Test results - Experiment II

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	TA 1535	E. coli WP2	TA 98	TA 1537
-	Solvent	90 ± 3.2	15 ± 4.9	30 ± 14.0	30 ± 3.5	14 ± 3.0
-	15	91 ± 1.5	20 ± 5.0	23 ± 4.9	29 ± 2.5	19 ± 6.6
-	50	77 ± 2.5	22 ± 3.1	24 ± 5.1	27 ± 11.4	15 ± 1.0
-	150	90 ± 3.5	18 ± 7.9	23 ± 7.1	33 ± 3.6	19 ± 4.0
-	500	99 ± 7.8	15 ± 2.1	18 ± 5.0	33 ± 6.0	20 ± 7.0
-	1500	98 ± 10.7	14 ± 3.2	15 ± 5.8	26 ± 4.5	16 ± 1.0
-	5000	79 ± 10.4	10 ± 3.1	23 ± 1.7	25 ± 2.5	18 ± 5.2
	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 plates ± SD)	654 ± 73.5	1661 ± 349.5	1003 ± 60.6	272 ± 14.2	618 ± 292.7
+	Solvent	84 ± 10.8	22 ± 6.2	23 ± 3.8	28 ± 11.5	15 ± 4.6
+	15	94 ± 7.6	22 ± 2.6	30 ± 6.7	35 ± 9.1	20 ± 3.5
+	50	93 ± 8.5	24 ± 4.7	27 ± 5.1	31 ± 8.9	16 ± 3.5
+	150	102 ± 6.1	17 ± 11.5	24 ± 3.0	28 ± 5.3	14 ± 2.1
+	500	98 ± 8.7	15 ± 4.0	26 ± 1.2	24 ± 4.9	19 ± 2.1
+	1500	101 ± 12.5	16 ± 6.1	28 ± 2.5	22 ± 3.1	12 ± 5.2
+	5000	90 ± 3.0	21 ± 9.5	26 ± 4.9	30 ± 9.0	18 ± 0.6
	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 plates ± SD)	964 ± 71.8	241 ± 14.5	193 ± 7.8	146 ± 51.1	268 ± 9.0

ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine

9AA: 9-Aminoacridine

4NQO: 4-Nitroquinoline-1-oxide

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.