Pseudoephedrine

Genetic toxicity: in vitro

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 guideline

Test guideline 1

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.

Test guideline 2

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

Test material information

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