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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 November 2012 to 14 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate
EC Number:
402-170-5
EC Name:
Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate
Cas Number:
106359-91-5
Molecular formula:
Hill formula: C26 H23 F N9 O13 S4 Na3
IUPAC Name:
trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate
Details on test material:
Name: FAT 40224/H TE
Batch No.: TZ 2382-BB-503C81
Physical State: powder
Colour: red
Density: 1.46 g/cm3 (20°C)
pH: 6.5 to 7.5 conc.(% w/w): 1%
Melting Point: > 200°C
Purity / Active Components: sum of all coloured substances: 85.4%, main constituents: 50.6%
Date of Analysis: 23.07.2012
Storage Conditions: at room temperature
Expiry Date: 28.01.2017
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: TZ2382-BB-503C81
- Expiration date of the lot/batch: 28-01-2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Method

Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment for experiment I (without metabolic activation):
5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
50, 100, 250, 500, 1000, 2000, 3000 and 5000 µg/mL
Experiment I
without metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, 2000 and 2500 µg/mL
and with metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000 and 2500 µg/mL
Experiment II
without metabolic activation: 10, 25, 50, 100, 200, 400, 600 and 800 µg/mL
and with metabolic activation: 100, 316, 1000, 1250, 1500, 2000, 2400, 2800 and 3000 µg/mL
Vehicle / solvent:
Vehicle (Solvent) used: cell culture medium (MEM + 0 % FBS 4h treatment; MEM + 10 % FBS 20h treatment). The test item was dissolved under stirring in cell culture medium and diluted prior to treatment.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; 300 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation; 1 and 1.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth
Evaluation criteria:
A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I without S9: ≥ 500 μg/mL; experiment I with S9: ≥ 100 μg/mL; Experiment II without S9: ≥ 400 μg/mL; Experiment II with S9:≥ 1000 μg/mL
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
FAT 40224/H is considered to be non-mutagenic in the HPRT locus using V 79 cells of the Chinese Hamster.
Executive summary:

The test item FAT 40224/H was assessed for its potential to induce gene mutations at the HPRT locus using V 79 cells of the Chinese hamster. The main experiments were carried out without and with metabolic activation. The experiments with metabolic activation were performed by including liver microsomes and NADP for efficient detection of a wide variety of carcinogens requiring metabolic activation. The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to the OECD guideline 476. In experiment 1 2500 µg/mL (with and without metabolic activation) was selected as the highest concentration. In experiment II 800 µg/mL (without metabolic activation) and 3000 µg/mL (with metabolic activation) were selected as the highest concentrations. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II was performed as 20 h long time exposure assay (without metabolic activation). The pH-value detected with the test item was within the physiological range. The test item was investigated at the following concentrations:


Experiment I


without metabolic activation:


S, 10, 25, 50, 100, 250, 500, 1000, 2000 and 2500 µg/mL


and with metabolic activation:


5, 10, 25, 50, 100, 250, 500, 1000 and 2500 µg/mL


Experiment II


without metabolic activation:


10, 50, 100, 200, 400, 600 and 800 µg/mL


and with metabolic activation:


100, 316, 1000, 1250, 1 500, 2000, 2400, 2800 and 3000 µg/mL


Precipitation:


No precipitation of the test item was noted in the experiments.


Toxicity:


A biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after the treatment with the test item in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 16.5 % for the highest concentration (2500 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2500 µg/mL with a relative growth of 17.4 %.


In experiment II without metabolic activation the relative growth was 12.2 % for the highest concentration (800 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 3000 µg/mL with a relative growth of 21.8 %, which is slightly higher than the postulated 10-20 % survival. Due to the fact that in both experiments with metabolic activation no hint at mutagenicity was found, this deficiency is considered to be not biologically relevant.


 


Mutagenicity:


In experiment I without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.


Mutation frequencies with the negative control were found to be 41.47 and 32.60 mutants/106 cells and in the range of 20.69 to 40.74 mutants/ 10cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.10 was found at a concentration of 1000 µg/mL with a relative growth of 45.6 %.


With metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 29.75 and 36.36 mutants/ 10cells and in the range of 5.22 to 43.12 mutants/10cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.30 was found at a concentration of 25 µg/mL with a relative growth of 82.1 %.


In experiment II without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICF. No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the tcst item did not show a biologically relevant increase as compared to the negative controls.


Mutation frequencies with the negative control were found to be 38.18 and 19.92 mutants/106 cells and in the range of 13.16 to 41.84 mutants 10cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.44 was found at a concentration of 50 µg/mL with a relative growth of 100.2 %.


In experiment II with metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 544 mutants per 10cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.


Mutation frequencies with the negative control were found to be 36.45 and 24.39 mutants/106 cells and in the range of 9.26 to 36.89 mutants/10cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.21 was found at a concentration of 2000 µg/mL with a relative growth of 35.5%. DMBA (1.0 and 1.5 µg/mL) and EMS (300 µg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation.


Based on the study results, FAT 40224/H is considered to be non-mutagenic in the HPRT locus using V 79 cells of the Chinese Hamster.