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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from publication

Data source

Reference
Reference Type:
publication
Title:
Gene mutation toxicity study of test chemical
Author:
Hidesuke SHIMIZU et. al.
Year:
1985
Bibliographic source:
Jpn. J. Ind. Health.,1985

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
To determine the gene mutation toxicity of test chemical.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
IUPAC name: N,N'-ethylenedi(stearamide)
Mol. formula: C38H76N2O2
Molecular Weight : 593.03 gm/mol
Smiles: CCCCCCCCCCCCCCCCCC(=O)NCCNC(=O)CCCCCCCCCCCCCCCCC
InChI: InChI=1S/C38H76N2O2/c1-3-5-7-9-11-13-15-17-19-21-23-25-27-29-31-33-37(41)39-35-36-40-38(42)34-32-30-28-26-24-22-20-18-16-14-12-10-8-6-4-2/h3-36H2,1-2H3,(H,39,41)(H,40,42)

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 1538 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : liver of male Sprague-Dawley rats
- method of preparation of S9 mix : The S9 fraction was prepared from the liver of male Sprague-Dawley rats, weighing about 220g (seven weeks old), pretreated with polychlorinated biphenyl (KC 500) at a dose of 500mg/kg body weight five days before sacrifice.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.1 ml
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
Test concentrations with justification for top dose:
1, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and 9-aminoacridine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : not specified

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not specified
- Test substance added in medium; in agar (plate incorporation): 0.1ml of bacterial strain and 0.5ml of S9 mix or sodium phosphate buffer (pH 7.4) were added to a sterile test tube containing 0.1ml of various concentrations of the chemicals. This mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2ml of molten top agar (45°C).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): not specified
Rationale for test conditions:
not specified
Evaluation criteria:
Number for revertants analysed
Statistics:
not specified

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 1538 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
DMSO
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Not specified
Remarks on result:
other: No mutagenic potential observed

Any other information on results incl. tables

Table 1: Mutagenic activity of test chemical

Compound

Dose

µg/ plate

Revertant colonies/ plate

TA 100

TA 1535

WP2 uvrA

TA98

TA 1537

TA 1538

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

DMSO (Control)

 

150±16.8

154±17.5

30±5.6

15±7.1

30±9.7

34±10.9

32±7.7

42±10.1

18±8.6

22±6.3

22±7.3

28±5.7

Positive control

AF-2

0.01

501±84.7

-b

-

-

-

-

-

-

-

-

-

-

 

0.05

-

-

-

-

1082±293.7

-

278±64.8

-

-

-

-

-

ENNG

0.5

-

-

1101±683.1

-

-

-

-

-

-

-

-

-

9AC

80.0

-

-

-

-

-

-

-

-

889±275.7

-

-

-

4NQO

0.25

-

-

-

-

-

-

-

-

-

-

270±66.2

-

B(a)P

5.0

-

1084±236.3

-

-

-

-

-

809±108.4

-

313±41.6

-

354±89.4

2AA

5.0

-

 

-

440±198.6

-

359±127.0

-

-

-

-

-

-

Test chemical

1

150

169

35

19

22

53

28

40

6

12

16

28

5

156

162

29

20

33

42

27

36

13

13

23

22

10

141

166

35

11

39

36

31

35

5

11

21

34

50

152

161

32

13

31

33

23

35

8

12

19

23

100

165

161

39

14

31

39

23

31

12

8

20

20

500

152

163

26

25

22

34

31

37

10

13

24

30

1000

176

168

31

20

28

39

34

56

10

15

18

27

5000

139

185

24

23

25

41

19

28

8

10

17

16

-b: Not tested

Applicant's summary and conclusion

Conclusions:
Test chemical did not induce mutation in Salmonella Typhimurium TA 1538, 1535, 1537, 98, 100 and E. coli WP2 and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation study was performed on Salmonella Typhimurium strains to determine the mutagenic nature of test chemical. Salmonella Typhimurium TA 1538, 1535, 1537, 98, 100 and E. coli WP2 were used during the study. The study was performed in the presence and absence of S9 metabolic activation. The S9 fraction was prepared from the liver of male Sprague-Dawley rats, weighing about 220g (seven weeks old), pretreated with polychlorinated biphenyl (KC 500) at a dose of 500mg/kg body weight five days before sacrifice. The positive controls used were 4-nitroquinoline-N-oxide, 9-aminoacridine, 2-nitrofluorene, N-ethyl-N-nitro-N-nitrosoguanidine, benzo(a)pyrene, 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and 9-aminoacridine. 0.1ml of bacterial strain and 0.5ml of S9 mix or sodium phosphate buffer (pH 7.4) were added to a sterile test tube containing 0.1ml of various concentrations of the chemical. The test concentrations used were 1, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate and the test chemical was dissolved in DMSO. This mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2ml of molten top agar (45°C). This mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2ml of molten top agar (45°C). The contents of each tube were mixed and poured onto a minimal glucose agar plate immediately. The plates were incubated at 37°C for 48 hours, and then the number of revertant colonies on each plate was scored with an automated colony counter. The background bacterial lawn was checked routinely by dissected microscope. Negleble amount of revertant colonies were observed as compared to positive control substances. Thus the test chemical can be considered as non-mutagenic when Salmonella Typhimurium TA 1538, 1535, 1537, 98, 100 and E. coli WP2 were treated in the presence and absence of S9 activation system.