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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
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

Constituent 1
Chemical structure
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.