Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
only 4 strains were tested

Data source

Reference
Reference Type:
publication
Title:
Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals
Author:
Mortelmans, K., et al.
Year:
1986
Bibliographic source:
Environmental Mutagenesis, 7: 1-119

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance was assayed for mutagenicity in the Salmonella/microsome preincubation assay using a standard protocol approved by the National Toxicology Program [see also Haworth et al, 1983].
To the 13 X 100-mm test tube maintained at 37°C the following substances were added:
0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution.

The mixture was mixed and allowed to incubate without shaking at 37°C for 20 min, at which time 2.0 ml of molten (45°C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 ml of minimal glucose bottom agar [Vogel and Bonner, 1956] in 15 X 100-mm plastic petri dishes (Falcon Muta-Assay, 1028). When the top agar had solidified, the plates were inverted and incubated at 37°C for 48 hr. Concurrent solvent and positive controls were tested with and without the metabolic activation systems. Five dose levels of the test substance was tested, with three plates per dose level in four strains.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylamine
EC Number:
200-834-7
EC Name:
Ethylamine
Cas Number:
75-04-7
Molecular formula:
C2H5NH2
IUPAC Name:
ethanamine
Details on test material:
- Name of test material (as cited in study report): Ethylamine
- Analytical purity: no data
Specific details on test material used for the study:
- Supplier: Fluka Chemical Co.
- Testing lab: SRI

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Male Sprague-Dawley rats and male syrian hamsters; liver S9
- method of preparation of S9 mix: Aroclor 1254 (200 mg/ml in corn oil) was administered ip at 500 mg/kg 5 days prior to decapitation (EGG, SRI) or cervical dislocation (CWR). The animals were deprived of food 12-24 hr immediately preceding death; otherwise food and water were provided ad libitum. The livers were removed aseptically, washed in ice-cold 0.15 M KCl, and minced and homogenized (3 ml of 0.15 M KC1 per gm of wet tissue) in a Potter-Elvehjem apparatus with a Teflon pestle. CWR initially used a Waring blender, but switched to a Potter-Elvehjem apparatus. The S-9 fraction was obtained by centrifugation of the liver homogenate for 10 min at 9,000 g at 4°C. The S-9 mix was prepared immediately prior to each assay.
- Volumes of S9 mix:
Per milliliter:
S-9 fraction 0.10 ml
0.04 M MgC12 0.02 ml
1.65 M KC1 0.02 ml
0.04 M NADP 0.10 ml
0.05 M glucose-6-phosphate 0.10 ml
1.0 M NaH2P04, (pH 7.4) 0.10 ml
Distilled water 0.56 ml
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 10000 ug/plate
Vehicle / solvent:
Water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Potassium chloride
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: -4-nitro-o-phenylenediamine for TA98; - with metabolic activation: 2-aminoanthracene (all strains).
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3

METHOD OF TREATMENT/ EXPOSURE:
- preincubation test

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration: 48 h

FOR GENE MUTATION:
- Ames test
Evaluation criteria:
1) Mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible;
or when the response was of insufficient magnitude to support a determination of mutagenicity

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 10000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS:
- With metabolic activation: negative
- Without metabolic activation: negative

PRECIPITATION CONCENTRATION:
- No precipitate observed

CYTOTOXIC CONCENTRATION:
- With metabolic activation: 10000 ug/plate
- Without metabolic activation: 10000 ug/plate

Any other information on results incl. tables

Strain: TA1535

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

33

2.3

11

2.6

15

0.3

100     

30

4.9

10

2.3

10

3

333     

25

3.5

9

1.7

11

3.8

1000     

30

2.7

11

2

10

0.9

3333     

7s

4.1

15

3.5

18

1.7

10000     

0s

0

0s

0

0s

0

Positive Control

342

42.1

245

10.7

178

24.1

 

Strain: TA100

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

146

10.4

121

2.7

121

6.5

100     

118

5.1

114

8.6

136

11.5

333     

129

9.4

132

5.7

116

10.3

1000     

108

5.2

143

9.4

108

9.3

3333     

118

9

121

16.9

94

11.1

10000     

0s

0

0s

0

41s

20.5

Positive Control

377

8.7

966

42.3

456

24.8

 

Strain: TA98

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

16

2

26

5.7

25

1.9

100     

17

1.7

32

2.3

27

3.5

333     

13

3.1

32

3.2

25

0.6

1000     

12

3.2

34

4.7

32

2.3

3333     

8s

4.3

0s

0

23

4.5

10000     

t

 

t

 

0s

0

Positive Control

475

108.7

764

21.3

326

10.7

 

Strain: TA1537

Dose

No Activation
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

5

1.5

5

1.5

6

1.7

100     

11

2.1

7

1.2

6

1

333     

5

1.3

4

1

7

1.2

1000     

6

3.2

7

1.2

4

0.7

3333     

4

1.7

0s

0

3

0.7

10000     

0s

0

t

 

0s

0

Positive Control

350

41.7

406

11.5

135

8.7

  

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination

Applicant's summary and conclusion

Conclusions:
Based on the results of this publication, the test substance is negative in the Salmonella/microsome preincubation assay with and without metabolic activation.
Executive summary:

The test substance was evaluated for mutagenicity in the Salmonella/microsome preincubation assay using a standard protocol approved by the National Toxicology Program. Doses of 0, 100, 333, 1000, 3333 and 10000 ug/plate were tested in four Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537) in the presence and absence of Aroclor-induced rat or hamster liver S9. The test substance was evaulated as negative in all experiments.

Categories Display