Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
Fenuron
EC Number:
202-941-4
EC Name:
Fenuron
Cas Number:
101-42-8
Molecular formula:
C9H12N2O
IUPAC Name:
fenuron
Test material form:
other: white to off-white powder
Details on test material:
- Particle size distribution: 100 % particle size < 12 µm (method: Laser Diffraction; Certificate of Analysis)
Particle size parameter determined with a Malvern Mastersizer 2000 (Non-GLP determination)
D10% = 1.18 µm
D50% = 7.72 µm
D90% = 16.00 µm
D: Diameter; 10, 50, 90: percentage cumulative
- Mass median aerodynamic diameter (MMAD): 1.595 µm
- Geometric standard deviation (GSD): calculated as 2.09
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.l: 0010416
- Expiration date of the lot/batch: April 2019
- Purity test date: 05/oct /2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (+10°C to +25°C) in a tightly closed container in a dry, cool and well-ventilated place, avoiding exposure to sunlight and moisture.

FORM AS APPLIED IN THE TEST: IsoQure UR 300 was completely dissolved in dimethyl sulfoxide (DMSO)

OTHER SPECIFICS: Tradename: IsoQure UR 300

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254, prepared according to MARON and AMES (1983) was obtained from Trinova Biochem . S9 was collected from male rats.
Test concentrations with justification for top dose:
Preliminary test: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 μg/plate
Main test: 31.6, 100, 316, 1000, 3160 and 5000 µg of IsoQure UR 300 per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (Batch no. STBG7748; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany).
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 and TA100 (10 µg/plate): without metabolic ativation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 (10µg/plate): without metabolic ativation.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (10µg/plate): without metabolic ativation.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
TA 102 (10µg/plate): without metabolic ativation.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
TA98, TA102, TA1537 (10 µg/plate): with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA100, TA1535 (2 µg/plate): with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
1st independent experiment - Plate Incorporation Method
2nd independent experiment - Preincubation Method
- Cell density at seeding (if applicable): 0.1 mL of Salmonella cell suspension (containing approximately 10E8 viable cells in the late exponential or early stationary phase)

DURATION
- Preincubation period: 20 min (2nd independent experiment)
- Exposure duration: 48 h to 72 h (1st independent experiment and 2nd independent experiment)

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn


Evaluation criteria:
Bacteria colonies were counted employing the Biosys Biocount 5000 system. Print outs of the colony counts were filed with the raw data. Occurrence of test item precipitation would have been documented after visual inspection of the cultures with the unaided eye. Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a scarce background lawn.
The results of the negative and positive control cultures should be within the range of the historical data generated by LPT.
The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20
Where concurrent negative or positive control data fall outside the range, they may be acceptable and considered for the inclusion into the historical control distribution as long as these data are not extreme outliers.

Statistics:
A test item 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 TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- Biological relevance of the results should be considered first.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium. A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Results and discussion

Test results
Species / strain:
other: TA1535, TA100, TA98, TA1537 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Citotoxicity
The reductions of the number of revertants by more than 50% in test strain TA1537 in the plate incorporation test without metabolic activation at 31.6 and 3160 µg/plate are considered to be caused by the high variation in individual counts and not due to cytotoxicity above all as no concentration response relationship was noted.
Mutagenicity
No increase in revertant colony numbers as compared with control counts was observed for IsoQure UR 300, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).
The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the range of the historical data generated by LPT

Applicant's summary and conclusion

Conclusions:
Under the present test conditions, IsoQure UR 300 tested up to a concentration of 5000 µg/plate (cytotoxic in the preincubation test) caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
Executive summary:

The test item was examined in the 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was completely dissolved in DMSO. The vehicle DMSO was employed as the negative control.

Preliminary test

The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000μg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000μg/plate. Hence, 5000μg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations of 31.6, 100, 316, 1000, 3160 and 5000μg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were noted up to the top concentration of 5000 µg IsoQure UR 300/plate in both experiments.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000μg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system

 In conclusion, under the present test conditions, IsoQure UR 300 tested up to a concentration of 5000 µg/plate (cytotoxic in the preincubation test) caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

 

Categories Display