Registration Dossier

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: Meets generally accepted scientific principles with acceptable deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
; only 4 bacterial strains were used for testing (5 are recommended)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): D-Pentolacton kristallin
- Analytical purity: >99 %
- Storage condition of test material: room temperature

Method

Target gene:
Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from the liver of male Sprague-Dawley rats (treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice) and mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH 7.4).
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg per plate, in standard plate test and preincubation test, with and without S9-mix
Vehicle:
Aqua dest.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see "Deatils on test system and conditions"
Details on test system and conditions:
Standard plate test:
The experimental procedure is based on the method of Ames et al.
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
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:
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al.
0.1 ml test solution, 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.

Controls:
- 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 (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 and 10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98; 100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537.
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 (control)
- 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:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
No increase in the number of his revertants was seen in the standard plate test and in the preincubation test, with or without the addition of S9-mix.
No bacteriotoxic effect (reduced his background growth) was observed. The test material was completely soluble in aqua dest.

Any other information on results incl. tables

Table 1: Maximum revertants/plate and corresponding test concentrations in the standard plate test:

    

Strain

Tested compound

Maximum revertants/plate [corresponding dose unit in µg/plate]

 

 

without S9-mix

with S9-mix

S. typhimurium TA1535

Aqua dest

16 ± 4

13 ± 1

Test substance

24 ± 5 [5000]

21 ± 1 [20]

Positive Control

1927 ± 64

114 ± 17

S. typhimurium TA100

Aqua dest

105 ± 6 

112 ± 13

Test substance

118 ± 21 [20]

126 ± 13 [500]

Positive Control

2083 ± 161

1913 ± 42

S. typhimurium TA1537

Aqua dest

± 3 

12 ± 2

Test substance

12 ± 6 [500]

17 ± 3 [500]

Positive Control

527 ± 34

185 ± 19

S. typhimurium TA98

Aqua dest

30 ± 3

44 ± 1

Test substance

38 ± 6 [2500]

57 ± 11 [100]

Positive Control

815 ± 19

1447 ± 160

Table 2: Maximum revertants/plate and corresponding test concentrations in the preincubation test:

    

Strain

Tested compound

Maximum revertants/plate [corresponding dose unit in µg/plate]

 

 

without S9-mix

with S9-mix

S. typhimurium TA1535

Aqua dest

12 ± 2

13 ± 1

Test substance

19 ± 1 [2500]

14 ± 4 [500]

Positive Control

1024 ± 29

142 ± 2

S. typhimurium TA100

Aqua dest

104 ± 7

118 ± 4

Test substance

123 ± 3 [20]

132 ± 19 [100]

Positive Control

1203 ± 45

1260 ± 70

S. typhimurium TA1537

Aqua dest

± 1

12 ± 1

Test substance

11 ± 9 [2500]

12 ± 2 [100]

Positive Control

495 ± 161

162 ± 9

S. typhimurium TA98

Aqua dest

26 ± 6

42 ± 3

Test substance

37 ± 5 [5000]

48 ± 11 [500]

Positive Control

1034 ± 106

995 ± 205

Applicant's summary and conclusion