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Genetic toxicity: in vitro

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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The purpose of this study is to evaluate Oxypropazon for mutagenic activity (gene mutation) in bacteria with and without the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and AMES (1983).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(3-hydroxypropyl)oxazolidin-2-one
EC Number:
431-840-1
EC Name:
3-(3-hydroxypropyl)oxazolidin-2-one
Cas Number:
87010-29-5
Molecular formula:
C6 H11 N O3
IUPAC Name:
3-(3-hydroxypropyl)oxazolidin-2-one
Details on test material:
- Name of test material (as cited in study report): Oxypropazon
- Physical state: colourless to light yellowish, viscous liquid, clear
- Analytical purity: 97.2%
- Lot/batch No.: 708598
- Stability: August 1998
- Storage condition of test material: container tightly closed, stored at a cool, dry and well-ventilated place
- Density: 1.231 g/cm2 at 20°C
- pH-value: 7.0-7.2

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 (Aroclor 1254-induced)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
Principle
The Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures his- his+ reversion induced by chemicals which cause base changes or frameshift mutations in the genome of this organism. Upon a layer of histidine-free agar (minimum agar) a second layer, containing test organisms and test substance (top agar) was placed. A trace of histidine in the top agar allows the logarithmic division of the histidine-requiring bacteria in the presence of the test compound and any of its metabolites generated by the S9 mix. This period of several generations of auxotrophic cell division is essential for the fixation of pro-mutagenic lesions in the DNA, and results in the formation of a lawn of histidine-requiring bacteria whose further division is prevented by exhaustion of histidine. Only that small fraction of bacteria which has reverted to histidine-independence (either spontaneously or by the action of the test chemical) will continue to divide to form discrete, randomly distributed visible colonies each one of which consists of the progeny of a single mutant bacterium. The assay determines whether the addition of graded doses of the test substance to a series of such plates induces a dose-related increase in mutant colonies compared with plates treated only with the appropriate volume of the vehicle. Two independent experiments were carried out each with and without metabolic activation, each experiment consisting of 3 plates/concentration. The first test was carried out as a plate incorporation test and, because it was negative, a second experiment was conducted as a preincubation test.

Tester strains
The following Salmonella typhimurium strains were used in this study - obtained from Dr. Bruce N. AMES-: TA 1 537, TA 102 and TA 98 which primarily respond to frameshift mutagens and TA 1535 and TA 100 which respond to base-pair substitution mutagens. In addition to the mutation in the histidine operon these strains contain several other mutations that greatly increase their ability to detect mutagens. The growth requirements and the genetic identity of the strains, their sensitivity to UV-radiation and crystal violet and their resistance to ampicillin are regularly checked. The strains used yield spontaneous revertants within the frequency ranges expected.

Preparations
Procedure for growing cultures
Test strains were kept as frozen permanents in liquid nitrogen in nutrient broth containing 8% dimethyl sulfoxide (DMSO). For the mutagenicity experiments frozen permanent copies of the tester strains were thawed at room temperature and then used for inoculating the overnight: cultures. Overnight cultures were grown in a gyrorotary incubator (10h/37° C) in Oxoid 2 nutrient broth. The final cell density was approximately 10e8 – 10e9 cells/ml.

Metabolic Activation System
Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 was prepared according to MARON and AMES (1 983). 59 was collected from 20-30 rats. The pooled fraction was tested for:
.- protein content, according to LOWRY et al. (1951)
- Cytochrome P-450 content

The protein content of the S9 fraction was 34.12 mg/ml S9, cytochrome P-450 content: 0.19 nmol/mg protein. The S9 fraction was stored in liquid nitrogen. The S9 mix was freshly prepared on the day of the test according to MARON and AMES (1983): containing 5% S9 and the following components (per 100 ml):
- 5.0 ml rat liver S9 (Aroclor 1254-induced)
- 2.0 ml 1.65 M MgCI2 + 0.4 M KCI-salt solution (sterile stock solution)
- 141 mg glucose-6-phosphate
- 306.5 mg NADP
- 50.0 ml 0.2 M phosphate buffer, pH 7.4 (sterile stock solution)
- sterile aqua ad iniectabilia ad 100 ml

Afterwards the S9 mix was filter-sterilized by using a 0.45 pm filter and kept on ice.

Test substance preparation
The test substance was dissolved in aqua ad iniectabilia and several dilutions (1.00 to 100 µI/plate = 123 to 12300 µg/plate) ware prepared. The test solution was freshly prepared immediately before use. In line with normal practice in this type of in vitro study, the protocol did not require analysis of the dose form.

Concentrations employed
In a preliminary experiment (plate incorporation method) using strain TA 100 only slight cytotoxicity was observed at the highest tested concentrations of 31.6 and 100 µl Oxypropazon/plate. Hence, it was decided to use 100 µI Oxypropazon per plate as the top concentration for the two independent experiments of the main study.

Five concentrations ranging from 1 .0 to 100 µl of Oxypropazon per plate were employed far the two independent experiments with and without metabolic activation.

Negative and positive control substances
Aqua ad iniectabilia served as negative control.
The following chemicals served as positive control substances:

without metabolic activation:
1. sodium azide in H20 (10 µg/plate): TA 1535, TA 100 0
2. 2-nitro-9H-fluorene in DMS0 (10 µg/plate):TA 98
3. 9-amino-acridine in ethanol (100 µg/plate): TA 1537
4. methyl methane sulfonate: TA 102

with metabolic activation:
2-aminoanthracene (2 µg/plate) in DMSO: all strains

Main test procedure
1st independent experiment - Plate Incorporation Method
Sterile top agar containing 0.6% agar and 0.5% NaCl was molten an the day of the test. 10 ml of a sterile solution af 0.5 mM L-histidine HCI/0.5 mM biotin were added to 100 ml af molten agar. 2 ml of tap agar were distributed into culture tubes held at 45°C in a heating block. 0.1 ml of salmonella cell suspension, 0.1 ml of test substance solution (or 0.1 ml solvent/or 0.1 ml mutagen) and 0.5 ml of S9 mix were added. In the assay without metabolic activation the S9 mix was substituted by 0.5 ml phosphate buffer mentioned above. The test components were mixed by vortexing the soft agar far 3 sec at low speed and then poured onto a coded minimal glucose agar plate (Vogel-Bonner medium E). To achieve a uniform distribution of the top agar on the surface of the plate, the uncovered plate was quickly tilted and rotated and then placed, covered, on a level surface to harden. Within an hour the plates were inverted and placed in a dark 37°C incubator for 48 hours (in case of a cell cycle delay after 72 hours). The revertant colonies on the test plates and on the control plates were counted, and the presence of the background lawn on all plates was confirmed. A lawn that was thin compared to the lawn on the negative control plate was evidence of bacterial toxicity. The results of the first experiment were negative, therefore a second independent
experiment was conducted employing the preincubation method.

2nd independent experiment - Preincubation Method
0.5 ml phosphate buffer or 0.5 ml S9-mix for the study with metabolic activation were delivered to sterile capped culture tubes in an ice bath. 0.1 ml of bacterial culture and 0.1 ml of the test substance solution (or solvent/or mutagen) were added. The tubes were gently vortexed and incubated at 37° C for 60 minutes. The tubes were shaken at a moderate speed during the incubation. Then 2 ml of the top agar were added to the tube. The remaining steps were the same as described for the plate incorporation method.

Quality criteria
The genotypes of the tester strains are regularly confirmed in the following way:
a) Histidine and biotin requirement (his) (bio)):
Each of the four strains is streaked onto two Vogel-Bonner medium E plates in the following
1) with 0.1 mM L-histidine and 0.5 mM biotin (100 µl/each)
2) with 0.5 mM biotin (100 µl)
None of the strains should grow on plate 2); all strains should show excessive growth on plate 1).

b) (rfa) deep rough character:
10 µl of 0.1% crystal violet applied with a paper disc should give zones of inhibition in all tester strains.

c) UV-sensitivity (uvr B):
Plates are covered partly with black paper and placed under germicidal UV-irradiation.
All strains should only grow under the covered portion of each plate.

d) Ampicillin-resistance (pMK 101):
0.8 mg ampicillin/plate is placed onto plates seeded with bacteria: Absence of zones of inhibition around the discs indicate resistance to ampicillin (TA 98, TA 100 and TA 102), whereas strains TA 1535 and TA 1537 show zones of inhibition.

Evaluation
A test chemical is considered to show a positive response if
- the number of revertants is significantly increased (p < 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA 98. TA 100 and TA 102 and 3-fold of the solvent control for TA 1 535 and TA 1 537 in both independent experiments.
- in addition, a significant (p < 0.05) dose (lag value)-related effect (Spearman's rank correlation coefficient) is observed.
- positive results have to be reproducible end the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.

The range of spontaneous reversion frequencies in this laboratory are generally
TA 98: 20 - 60
TA 100: 100 - 200
TA 102: 240 - 320
TA 1535: 10 - 35
TA 1537: 3 - 20

The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment.

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary test
Ten (10) concentrations of Oxypropazon were examined in the preliminary test using strain TA 100 ranging from 0.00316 to 100 µl/plate. The test substance Oxypropazon was dissolved in aqua ad iniectabilia. Only slight cytotoxicity was observed at the highest tested concentrations of 31.6 and 100 µI Oxypropazon/plate. Hence, 100 µl/plate was chosen as top concentration for the main study.


Main study
Cytotoxicity
Five (5) concentrations ranging from 1.0 to 100 µl of Oxypropazon per plate ( 123 to 12300 µg/plate) were employed for the 1st independent experiment (plate incorporation test) and for the 2nd independent experiment (preincubation test) with and without metabolic activation.

Plate incorporation test
In the experiments without and with metabolic activation slight cytotoxic effects were noted at 31.6 and 100 µl/plate at all strains.

Preincubation test
In the experiments without and with metabolic activation slight cytotoxic effects were noted at 31.6 and 100 µl/plate at all strains.

Mutagenicity
No mutagenic effect was observed for Oxypropazon up to the cytotoxic concentration of 100 µl/plate (12300 µg/plate) in any of the 5 tester strains in the two independent experiments with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion