Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Sept 2012 - 6 Nov 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 21 July 1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(adopted 30 May 2008)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate
EC Number:
250-151-3
EC Name:
Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate
Cas Number:
30364-51-3
Molecular formula:
C17H33NO3.Na
IUPAC Name:
sodium N-methyl-N-(1-oxotetradecyl)aminoacetate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Target gene:
his operon (S. typhimurium strains)
trp operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA98, and TA100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbitone/beta-Naphthoflavone
Test concentrations with justification for top dose:
- Preliminary Toxicity Test:
TA100 or WP2uvrA: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Mutation Test - Experiment 1
All Salmonella strains (with and without S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate
WP2uvrA (with and without S9-mix): 15, 50, 150, 500, 1500 and 5000 μg/plate
- Mutation Test - Experiment 2
All Salmonella strains (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate
WP2uvrA (with and without S9-mix): 15, 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: sterile, distilled water
- Justification for choice of vehicle: based on solubility checks performed in-house
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
- S9: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate (WP2uvrA), 3 μg/plate (TA100), 5 μg/plate (TA1535); 9-Aminoacridine (9AA): 80 μg/plate (TA1537), 4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate (TA98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
+ S9: 2-Aminoanthracene (2AA): 1 μg/plate (TA100), 2 μg/plate (TA1535 and TA1537), 10 μg/plate (WP2uvrA); Benzo(a)pyrene (BP): 5 μg/plate (TA98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (Experiment 1) and pre-incubation (Experiment 2)

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

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies, a clearing or diminution of the background lawn (Preliminary Toxicity Test)
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:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. 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 judgement about test item activity. Results of this type will be reported as equivocal.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Preliminary Toxicity Test: TA100 at 500 μg/plate and above (+ and - S9); plate incorporation Experiment 1: all strains at 150 μg/plate and above (+ and - S9); pre incubation Experiment 2: all strains at 50 μg/plate and above (+ and - S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES: RANGE-FINDING/SCREENING STUDIES: Range finding test: in order to select the appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test substance. Ten concentrations of the test item formulation (0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) and vehicle control (sterile distilled water) were tested.
The test item was toxic to TA 100 from 500 µg/plate and non-toxic to WP2 uvrA.

COMPARISON WITH HISTORICAL CONTROL DATA: The results of the vehicle and positive controls (+ and - S9) are within the historical control data of the last two years.

Any other information on results incl. tables

Table 1 Test Results: Experiment 1 (plate incorporation) – Without Metabolic Activation

Test group

Number of revertants (mean) ± SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent control

105±7.6

18±3.8

34±9.8

21±6.8

15±3.5

Test item

1.5 µg/plate

96±3.1

16±2.6

N/T

19±7.4

9±1.5

5 µg/plate

97±11.7

21±2.6

N/T

22±5.6

16±2.1

15 µg/plate

94±4.7

16±3.5

29±2.0

16±3.2

9±2.5

50 µg/plate

107±16.5

13±3.2

28±2.9

22±8.5

10±2.3

150 µg/plate

79±6.0

11±4.0

35±7.6

13±2.6

9±5.5

500 µg/plate

58±4.7

7±4.2

32±8.1

12±3..1

2±1.2

1500 µg/plate

0±0

2±1.0

25±7.0

0±0

0±0

5000 µg/plate

N/T

N/T

29±5.5

N/T

N/T

Positive controls

S9 mix (-)

Name

Dose Level

No. of revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

514±45.2

453±17.9

859±31.6

159±12.7

1122±74.5

Table 2 Test Results: Experiment 1 (plate incorporation) – With Metabolic Activation

Test group

Number of revertants (mean) ± SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent control

125±10.2

11±1.0

36±6.0

26±4.2

17±7.0

Test item

1.5 µg/plate

122±12.5

11±1.2

N/T

26±7.1

16±7.6

5 µg/plate

119±7.8

11±2.6

N/T

19±5.0

15±6.1

15 µg/plate

124±26.0

12±3.5

40±8.1

21±4.2

14±1.2

50 µg/plate

126±12.8

11±5.5

33±7.1

22±8.7

16±3.8

150 µg/plate

103±15.5

11±0.6

39±6.7

24±7.1

10±0.6

500 µg/plate

68±5.2

5±0.6

34±3.8

11±2.6

3±1.2

1500 µg/plate

0±0

0±0

35±3.5

3±2.6

0±0

5000 µg/plate

N/T

N/T

30±2.6

N/T

N/T

Positive controls

S9 mix (+)

Name

Dose Level

No. of revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µ

10 µg

5 µg

2 µg

1939±74.5

314±31.6

362±29.3

169±4.2

384±9.3

Table 3 Test Results: Experiment 2 (pre-incubation) – Without Metabolic Activation

Test group

Number of revertants (mean) ± SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent control

109±7.6

21±2.3

29±3.0

19±3.1

12±2.6

Test item

0.5 µg/plate

116±2.5

23±4.2

N/T

19±2.6

15±3.5

1.5 µg/plate

103±8.5

22±4.0

N/T

19±5.1

12±2.0

5 µg/plate

87±13.2

19±4.0

N/T

17±3.8

12±4.7

15 µg/plate

98±8.5

17±3.1

26±8.2

18±4.5

6±1.0

50 µg/plate

100±11.6

18±3.5

27±13.2

15±1.2

5±2.1(S)

150 µg/plate

82±2.9

17±1.5

23±2.6

22±5.8

0±0 (V)

500 µg/plate

0±0

0±0

21±6.5

10±2.1

0±0 (V)

1500 µg/plate

N/T

N/T

21±5.0

N/T

N/T

5000 µg/plate

N/T

N/T

27±1.5

N/T

N/T

Positive controls

S9 mix (-)

Name

Dose Level

No. of revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

635±35.8

335±87.5

775±38.9

121±30.0

953±53.4

 


Table 4 Test Results: Experiment 2 (pre-incubation) – With Metabolic Activation

Test group

Number of revertants (mean) ± SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent control

105±15.0

11±1.2

33±7.6

28±4.0

13±5.9

Test item

0.5 µg/plate

106±7.6

9±0.6

N/T

22±7.6

14±2.6

1.5 µg/plate

101±4.7

13±3.0

N/T

25±3.2

13±3.8

5 µg/plate

94±6.2

10±0.6

N/T

21±1.5

12±1.7

15 µg/plate

91±11.9

11±1.2

33±2.6

28±4.2

11±6.7

50 µg/plate

89ׅ10.6

12±1.7

31±12.4

18±3.2

10±5.2

150 µg/plate

85±7.1

10±3.6

34±2.0

21±6.0

6±2.3

500 µg/plate

54±8.3

0±0

29±5.8

18±3.5

0±0

1500 µg/plate

N/T

N/T

30±5.5

N/T

N/T

5000 µg/plate

N/T

N/T

32±1.5

N/T

N/T

Positive controls

S9 mix (+)

Name

Dose Level

No. of revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1161±93.3

162±16.7

292±14.0

138±20.6

169±38.7

 

N/T Not tested at this dose level

(S) = Sparse bacterial background lawn

(V) = Very weak bacterial background lawn

solvent control:sterile, destilled water

positive controls (- S9):

-N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate (WP2uvrA), 3 μg/plate (TA100), 5 μg/plate (TA1537)

- 9-Aminoacridine (9AA): 80 μg/plate (TA1537), 4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate (TA98)

positive controls (+ S9):

- 2-Aminoanthracene (2AA): 1 μg/plate (TA100), 2 μg/plate (TA1535 and TA1537), 10 μg/plate (WP2uvrA)

- Benzo(a)pyrene (BP): 5 μg/plate (TA98)

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

Categories Display