Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from July 7 to August 26, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Source study has reliability 2; details on the read-across approach are attached in section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2000)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Standard plate test
1) TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA: 0, 60, 300, 1500, 7500, 15000 µg/plate.
2) TA 100: 0, 200, 400, 600, 800, 1000 µg/plate
3) TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA: 0, 1000, 2000, 3000, 4000 and 5000 µg/plate.

Preincubation test
4) TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA: 0, 30, 100, 300, 1000, 3000 µg/plate.
5) TA 100: 0, 500, 1000, 1500, 2000, 2500 µg/plate.
Vehicle / solvent:
- Vehicle: water.
- Justification for choice of solvent/vehicle: good solubility of test substance in water.
Controls
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene, 4-nitro-o-phenylendiamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation method

Test tubes containing 2-mI portions of soft agar (overlay agar, i.e. 100 ml agar (0.8 % agar + 0.6 % NaCI) and 10 ml amino acid solution (minimal aminoacid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in order:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S9 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 ca. 30 seconds.

Composition of the minimal glucose agar for Salmonella Typhimurium:
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 - 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Composition of the minimal glucose agar for Escherichia coli:
300 ml solution B (agar)
100 ml solution A (saline solution)
8 ml solution C (glucose solution)
10 ml solution D (casein solution)
After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

METHOD OF APPLICATION: preincubation test
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S9 mix or phosphate buffer are incubated at 37 °C for the duration of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within ca. 30 seconds.
After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies are counted.
Evaluation criteria:
The test substance is considered positive in this assay if a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either with or without S9 mix, is noted.

A test substance is generally considered nonmutagenic in this test if the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in standard plate test (from 3000 µg/plate), in preincubation test (from 1000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- precipitation: no test substance precipitation was found. As long as precipitation does not interfere with the colony scoring, 5 mg/plate is selected and analysed (if not toxic) as the maximum dose.

COMPARISON WITH HISTORICAL CONTROL DATA: at non cytotoxic levels, test results are in the range of historical control data, except for TA 100 strain with metabolic activation. Further investigation on ths specific strain did not confirm this results, which was then considered as accidental.

ADDITIONAL INFORMATION ON CYTOTOXICITY
- bacteriotoxic effect (reduced his- and trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in titer) was observed in the standard plate test depending on the strain and test conditions from about 3000 to 5000 µg/plate onward.
- in preincubation assay, batteriotoxicity (reduced his- and trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in titer) was obserevd depending on the strain and test conditions at doses ≥ 1000 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Standard plate test

historical data TA 1535 TA 100 TA 1537 TA 98  WP2 uvrA
without S9-mix 11-25 82-155 6-16 17-44 25-51
with S9-mix 12-25 88-156 6-19 20-49 25-56

µg/plate TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix
0 18 17 111 112 12 13 28 43 34 40
60 18 16 106 168 13 8 28 45 36 46
300 18 16 105 212 11 15 26 44 40 43
1500 15 18 103 170 9 15 30 35 40 44
7500 2 2 13 11 0 2 4 13 14 18
15000 0 0 0 0 0 0 0 1 3 5

µg/plate TA 100
with S9-mix
0 112
200 152
400 138
600 150
800 149
1000 119

µg/plate TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix
0 18 19 103 124 10 12 31 38 36 38
1000 17 20 111 130 11 9 28 38 32 41
2000 17 21 105 147 11 8 26 36 34 39
3000 13 22 62 78 4 6 23 39 35 33
4000 7 19 14 30 1 2 17 22 29 26
5000 1 8 9 10 0 1 8 12 21 18

Preincubation test

historical data TA 1535 TA 100 TA 1537 TA 98  WP2 uvrA
without S9-mix 13 -25 90 -160 5 -19 18 -44 25-54
with S9-mix 11-25 92 -159 6 -20 17 -50 25-59

µg/plate TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix without S9-mix with S9-mix
0 17 17 118 111 10 13 29 36 35 43
30 18 17 110 145 12 10 24 30 47 36
100 16 17 100 144 8 9 24 41 40 35
300 15 14 118 137 9 11 31 28 42 35
1000 9 20 29 189 3 4 13 30 22 42
3000 0 10 0 56 0 3 0 20 7 23

µg/plate TA 100
with S9-mix
0 111
500 136
1000 131
1500 110
2000 99
2500 88

Applicant's summary and conclusion

Conclusions:
Negative.
Under these experimental conditions, the substance is not a mutagenic agent in bacterial reverse mutation assay with Salmonella typhimurium and Escherichia coli strains. The occasionally observed slight increase in the number of revertant colonies using TA 100 with S9 mix both in the standard plate test and in the preincubation assay could not be confirmed in additional studies and is therefore regarded as incidental and not as indication of a point mutagenic activity of test substance.
Executive summary:

Method

Bacterial reverse mutation assay (Ames test) with Salmonella typhimurium (TA 1535, TA 100, TA 1537 and TA 98) and Escherichia coli (WP2 uvrA) without or with metabolic activation (S9 mix). Two methods were used for the assays: standard plate and preincubation methods. Water was used as vehicle due to the high solubility of test substance in water. In standard plate test, doses ranged between 60 and 15000 µg/plate; in preincubation test, doses were between 30 and 3000 µg/plate. Solvent controls, sterility controls and positive controls were used.

Results

Based on negative responses under test conditions, the substance is considered as non mutagenic. Indeed the number of revertant colonies was found to be within historical control ranges. Depending on strain and test conditions, from ca. 3000 - 5000 µg/plate onward the substance was cytotoxic in the standard plate test, while at doses ≥ 1000 µg/plate the substance showed cytotoxicity in preincubation test. Slight increase in revertant colonies using TA 100 with S9 mix was not confirmed in further studies with this strain; therefore, it was considered as accidental.