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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (only 4 tester strains used according to OECD TG 471 adopted 1981). Based on the requirements of the OECD TG 471 (adopted 1997) an additional tester strain (TA102 or E.coli) is lacking.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 1981)
Deviations:
yes
Remarks:
Based on the requirements of the OECD TG 471 (adopted 1997) an additional tester strain (TA102 or E.coli) and a second positive control substance in addition to 2-aminoacridine (with metabolic activation) is lacking.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
The study was conducted in accordance with GLP with one deviation: no analytical investigation of the stability of the test substance in the vehicle was carried out.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
(+)-Pseudoephedrin-HCl
IUPAC Name:
(+)-Pseudoephedrin-HCl
Details on test material:
Batch No.: 27550
name of the test substance: (+)-Pseudoephedrin-HCl
Date of manufactoring: September 1990
Test substance No.: 91/80
Degree of purity: 99.0 - 100.5% related to the dried substance
Appearance, consistency: white crystals or powder
storage: Room temperature (darkness)

Method

Target gene:
his operon
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:
Male S.D. rats liver S9, induced by Aroclor 1254
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
aqua dest.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
solvent control and sterility control
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9: 2-aminoanthracene; without S9: N-methyl-N´-nitro-N-nitrosoguanidine, nitro-o-phenylendiamine, 9-aminoacridine chloride monohydrate
Details on test system and experimental conditions:
Tester strains, doses, number of plates:

1st Experiment:
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 20, 100, 500, 2500 and 5000 ug/plate
Solvent: aqua dest.
Type of test, test conditions: standard plate test with and without S-9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment:
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 20, 100, 500, 2500 and 5000 ug/plate
Solvent: aqua dest.
Type of test, test conditions: preincubation test with and without S-9 mix
Number of plates: 3 test plates per dose or per control

Negative control:
Parallel with each experiment with and without S-9 mix, a negative control (solvent control, sterility control) is carried out for each tester strain in order to determine the spontaneous mutation rate.

Positive controls:
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:
with S-9 mix: 10 µg 2-aminoanthracene (2-AA) (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535;
without S-9 mix: 5 µg N-methyl-N'-nitro-N-nitrosoguanidine ( MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535; 10 µg 4-nitro-o-phenylendiamine (NPD) (dissolved in DMSO) for the strain TA 98; 100 µg 9-aminoacridine chloride monohydrate (AAC) (dissolved in DMSO) for the strain TA 1537.

Standard plate test:
Test tubes containing 2 mL portions of soft agar which consists of 100 mL agar (0.6% agar + 0.6% NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution or solvent
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation )
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test:
0.1 mL test solution or solvent, 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.
Composition of the minimal glucose agar: 980 mL aqua dest., 20 mL Vogel-Bonner E Medium, 15 g Difco bacto agar, 20 g D-glucose, monohydrate.
After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

S-9 mix:
The S-9 mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S-9 fraction is thawed at room temperature and 1 volume of S-9 fraction is mixed with 9 volumes of S-9 supplement (cofactors).
Evaluation criteria:
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (compared to control values)
- dose-response relationship
- reproducibility of the results

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments without microsomal activation in the standard plate test.

Concentration

(µg/plate)

TA98

TA100

TA1535

TA1537

0

25

126

18

15

20

27

115

20

14

100

24

109

15

13

500

21

98

17

13

2500

19

112

18

12

5000

22

103

20

10

NPD

 

 

 

 

10

783

 

 

 

MNNG

 

 

 

 

5

 

1460

1583

 

AAC

 

 

 

 

100

 

 

 

1097

 Table 2: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments with microsomal activation in the standard plate test.

Concentration

(µg/plate)

TA98

TA100

TA1535

TA1537

0

39

98

16

17

20

52

105

16

18

100

51

107

21

13

500

48

118

18

17

2500

50

107

15

18

5000

49

102

17

20

2-AA

 

 

 

 

10

658

1253

231

116

 

Table 3: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments without microsomal activation in the preincubation test.

Concentration

(µg/plate)

TA98

TA100

TA1535

TA1537

0

25

87

11

11

20

21

73

16

8

100

25

100

17

7

500

24

101

11

9

2500

29

100

11

11

5000

25

93

14

11

NPD

 

 

 

 

10

1206

 

 

 

MNNG

 

625

603

 

5

 

 

 

 

AAC

 

 

 

 

100

 

 

 

235

Table 4: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments with microsomal activation in the preincubation test.

Concentration

(µg/plate)

TA98

TA100

TA1535

TA1537

0

40

89

12

11

20

44

90

15

11

100

43

98

13

7

500

36

90

14

8

2500

30

88

15

10

5000

44

95

10

5

2-AA

 

 

 

 

10

834

483

69

60

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative