Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Disperse 086 is concluded to be not mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 27 September, 1993 to 02 February, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test material: FAT 36034/E
Batch No.: 9300102
Expiry date: July, 1998
Purity: 93 %
Solubility: in water <0.1 g/L
Colour: red
Storage: at room temperature
Target gene:
Histidine gene

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
The histidine auxotrophic strains of Salmonella typhimurium (TA 98, TA 102, TA 1535 and TA 1537) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A. Strain TA 100 was obtained from Dr. M. Schüpbach, Hoffmann-La Roche Ltd., Basel, Switzerland. Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NBagar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment. The characteristics of the strains were checked monthly. Histidine auxotrophy of the strains was demonstrated by the requirement for 1-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene (strains TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Strain TA 102 additionally was checked for tetracycline resistance (presence of multicopy plasmid pAQ1). The presence of the uvr+ gene was demonstrated by the resistance of strain TA 102 against UV-light. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Cytotoxicity test: 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate
Mutagenicity tests: 61.7, 185.2, 555.55, 1666.7 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on solubility
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA 100 and TA 1535 without metabolic activation
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
for TA 102 without metabolic activation
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
for TA 98 without metabolic activation
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA 1537 without metabolic activation
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
for TA 100, TA 102, TA 98 and TA 1537 with metabolic activation
Untreated negative controls:
other: same as solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide*H20
Remarks:
for TA 1535 with metabolic activation
Details on test system and experimental conditions:
Method of application: in agar (plate incorporation)

Setting up of the test plates: 0.1 mL of the overnight cultures were mixed with 2 mL of top agar, either 0.5 mL of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 mL of the activation mixture (experiments with activation) and 0.1 mL of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 mL of minimal agar (1.5 % agar supplemented with 2 % salts of the Vogel-Bonner Medium E and 2 % glucose). The top agar was composed of 0.6% agar and 0.6% NaCl. It was supplemented with 10 % of 0.5 mM 1-histidine and 0.5 mM (+)biotin dissolved in water.

Preliminary toxicity/range-finding test: A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. The plates were inverted and incubated for about 48 h at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration, as well as each negative control was used.

Mutagenicity test: The mutagenicity test was performed with strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 without and with microsomal activation. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied was 5000 µg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3. The plates were inverted and incubated for about 48 h at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.
Evaluation criteria:
Assay acceptance criteria: A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.
Statistics:
In deviation to the OECD guideline, a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(only a weak/slight decrease in the number of back mutants were observed)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA102, TA1535 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity test/range finding test:
Six concentrations ranging from 20.6 to 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. The numbers of revertant colonies were not reduced. Normal back-ground growth was observed. The test substance exerted no toxic effect on the growth of the bacteria. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

Mutagenicity test, original experiment:
In the experiment performed with metabolic activation, treatment of strain TA 98 with the test substance led to a slight, concentration dependent increase in the number of revertant colonies at the concentrations of 185.2 and 555.6 µg/plate, followed by a weak decrease in the number of back mutants at the concentrations of 1666.7 and 5000 µg/plate. A marginal increase in the number of revertant colonies occurred on strain TA 1537 at the concentrations of 555.6 to 5000 µg/plate. No effects were observed with the other strains. In the experiment carried out without metabolic activation, a marginal increase was observed in the number of revertant colonies at the concentrations of 1666.7 and 5000 µg/plate. No effects were seen with the other strains.

Mutagenicity test, confirmatory experiment:
In the experiment carried out with activation on strain TA 98, a slight, concentration dependent increase in the number of revertant colonies occurred at the concentrations of 555.6 and 1666.7 µg/plate, followed by a slight decrease in the number of back-mutants at the concentration of 5000 µg/plate. A marginal increase in the number of revertant colonies occurred on strain TA 1537 at the concentrations of 555.6 to 5000 µg/plate. Again, no effects occurred on the other strains. In the experiment performed without metabolic activation, a marginal increase was observed in the number of revertant colonies at the concentration of 5000 µg/plate only . Again, no effect was seen with the other strains.
Conclusions:
The test substance exerted a marginal mutagenic action on strain TA 1537. The metabolites of the test substance were weakly mutagenic with strain TA 98 and marginally mutagenic with strain TA 1537.
Executive summary:

An in vitro study was performed to investigate the potential of the test substance (of ca. 93 % purity) to induce gene mutations according to OECD Guideline 471, EU Method B.14 and EPA OTS 798.5265 in compliance with GLP. The concentration range of the test substance to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. An independent repetition of the experiments was performed with the same concentrations. In the preliminary experiment without and with metabolic activation performed on strain TA 100, no toxic effects were observed. In the original experiment performed, a slight increase in the number of revertant colonies at the concentrations of 185.2 to 5000 µg/plate was observed with TA 98 with metabolic activation, followed by a weak decrease in the number of back mutants at the concentrations of 1666.7 and 5000 µg/plate. A marginal increase in the number of revertant colonies occurred on strain TA 1537 at the concentrations of 555.6 to 5000 µg/plate. In the experiment without metabolic activation, a marginal increase was observed in the number of revertant colonies at the concentrations of 1666.7 and 5000 µg/plate. In the confirmatory experiment, a slight, concentration dependent increase in the number of revertant colonies occurred at the concentrations of 555.6 and 1666.7 µg/plate with activation on strain TA 98, followed by a slight decrease in the number of back-mutants at the concentration of 5000 µg/plate. A marginal increase in the number of revertant colonies occurred on strain TA 1537 at the concentrations of 555.6 to 5000 µg/plate. In the experiment without metabolic activation, a marginal increase was observed in the number of revertant colonies at the concentration of 5000 µg/plate only. In both the mutagenicity tests with metabolic activation, normal background growth was observed with all strains. The number of revertant colonies was not reduced. The test substance exerted no toxic effect on the growth of the bacteria. Under the study conditions, the test substance exerted a marginal mutagenic action on strain TA 1537. The metabolites of the test substance were weakly mutagenic with strain TA 98 and marginally mutagenic with strain TA 1537.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-03-26 to 2014-06-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Specific details on test material used for the study:
Name: FAT 36034/W TE
Physical State: solid
Storage Conditions: room temperature, protected from light
Colour: red
pH (specify): 10.2 (2 % (w/w) aqu. Suspension), RT
Active Components: >90 %
Purity (qualitative and quantitative): 91.7 % (w/w)
Solubility in water: 3.26 mg/l
Date of Analysis: 07/08/2013
Expiry Date: 08/08/2018
Safety Precautions: The routine hygienic procedures procedures were sufficient to assure personnel health and safety
Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
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):
0.001, 0.0025, 0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
0.001, 0.0025, 0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM
Experiment I
without and with metabolic activation: 0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM
Experiment II
without metabolic activation: 0.0025, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.15 and 0.20 mM
and with metabolic activation: 0.025, 0.035, 0.045, 0.055, 0.065, 0.075, 0.085, 0.095, 0.12 and 0.25 mM
Vehicle / solvent:
Vehicle (Solvent) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation; 0.8 and 1.0 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in DMSO
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.
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: ≥0.05 mM; Experiment II without S9: ≥0.04 mM; Experiment II with S9: 0.25 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
FAT 36034/W is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

In a mammalian cell gene mutation assay (HPRT locus),V79 cells culturedin vitro were exposed to FAT 36034/W TE at concentrations of


- 0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM (without and with metabolic activation, Experiment I)


- 0.0025, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.15 and 0.20 mM (without metabolic activation, Experiment II)


- 0.025, 0.035, 0.045, 0.055, 0.065, 0.075, 0.085, 0.095, 0.12 and 0.25 mM (with metabolic activation, Experiment II). FAT 36034/W was tested up to cytotoxic concentrations. A biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after the treatment with the test item in experiment I without and in experiment II without and with metabolic activation. In experiment I without metabolic activation the relative growth was 36 % for the highest concentration (0.25 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 0.25 mM with a relative growth of 77.5 %. In experiment II without metabolic activation the relative growth was 12.2 % for the highest concentration (0.20 mM) evaluated. The highest concentration evaluated with metabolic activation was 0.25 mM with a relative growth of 59.8 %. In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.54 was found at a concentration of 0.09 mM with a relative growth of 59 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.03 was found at a concentration of 0.06 mM with a relative growth of 92.8 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.30 was found at a concentration of 0.01 mM with a relative growth of 75.7 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.28 was found at a concentration of 0.085 mM with a relative growth of 78.2 %. The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Disperse Red 086 was not clastogenic in a micronucleus assay.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 November, 1996 to 04 March, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test), 1983
Deviations:
yes
Remarks:
(such as lower relative humidity, solubility in water not indicated. however, these had no influence on the study results)
Qualifier:
according to guideline
Guideline:
other: EEC Directive 92/69, L 383, Annex V, B.12, December 29, 1992.
Deviations:
yes
Remarks:
(such as lower relative humidity, solubility in water not indicated. however, these had no influence on the study results)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
Test material: FAT 36034/D
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL; CH-4414 Füllinsdorf
- Age at study initiation: 8 -12 weeks
- Weight at study initiation: Males mean value 30.8 g (SD ±2.9 g); Females mean value 22.5 g (SD ±1.5 g)
- Assigned to test groups randomly: Yes
- Housing: Single
- Diet: Pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water: Tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 5 °C
- Humidity: 20-70 %
- Artificial light: 6.00 a.m-6.00 p.m
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: polyethylene glycol (PEG 400)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Volume administered: 10 mL/kg bw
Details on exposure:
On the day of the experiment, the test substance was formulated in PEG 400. The vehicle was chosen to its relative non-toxicity for the animals. All animals received a single standard volume of 10 mL/kg bw orally.

Duration of treatment / exposure:
Single administration

Frequency of treatment:
Once

Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
670 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5/sex/group

Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: Cyclophosphamide
Dissolved in: Deionized water
Dosing: 20 mg/kg bw
Route and frequency of administration: Orally, once
Volume Administered: 10 mL/kg bw
Solution prepared on day of administration. The stability of positive control at room temperature is sufficient. At 25 °C only 3.5 % of its potency is lost after 24 h.
Tissues and cell types examined:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The highest dose (2000 mg/kg bw) was estimated by a pre-experiment to be suitable.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 h and 48 h after a single administration of the test substance the bone marrow cells were collected for micronuclei analysis.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 1000 erythrocytes. The analysis was performed with coded slides.


Evaluation criteria:
A test substance is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one ofthe test points. A test substance producing neither a dose-related increase in the number of micronucleated polychromatic elythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system. This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
(only slight reduction of spontaneous activity which resolve within 48 h)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
As estimated by a pre-experiment 2000 mg/kg bw (highest guideline-recommended dose) of the test substance were administered as maximum dose and tolerated by the animals. The animals expressed slight toxic reactions. The mean number of normochromatic erythrocytes was not increased after treatment with the test substance as compared to the mean values of NCEs of the corresponding vehicle controls, indicating that the test substance had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test substance. The mean values of micronuclei observed after treatment were in the range of the vehicle control group. 20 mg/kg bw cyclophosphamide used as positive control showed a statistically significant increase of induced micronucleus frequency.

Pre-experiment for toxicity:


In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg bw, test substance formulated in PEG 400. The volume administered was10 mL/kg bw. The treated animals expressed toxic reactions as shown in the table:


 























Toxic


reactions



Hours post-treatment (male/female)



1 h



6 h



24 h



48 h



Reduction of spontaneous activity



2/2



2/2



1/1



0/0



 


6 h after treatment the urine was red. On the basis of these data 2000 mg/kg bw were estimated to be suitable.


 


Deviations to the protocol:


One female did not reach the lower limit of the body weight range given in the protocol (19.3 g instead of 20 g). In deviation to the protocol the relative humidity was sometimes lower than 30 % (lowest value 20 %) and the temperature was lower than 21 ± 30 ⁰C (lowest temperature 16 ⁰C). As requested by the sponsor the stability of the test substance in PEG 400 which was used as vehicle was indicated (instead of the stability in water). Due to technical reasons 20 mg/kg bw instead of 40 mg/kg bw cyclophosphamide were applied as positive control. As the induced micronucleus frequency was lower than expected for 40 mg/kg bw CPA a second additional scoring of the slides of the positive control was performed. Therefore, the number of evaluated polychromatic erythrocytes in this experimental group is 2000. These deviations had no influence on the integrity and validity of the study.

Conclusions:
The test substance did not induce micronuclei in bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD Guideline 474 and EEC Directive 92/69, L 383, Annex V, B.12 in compliance with GLP. The test substance was formulated in polyethylene glycol 400 (PEG 400). 24 and 48 h after a single administration, bone marrow cells were collected for micronuclei analysis. Ten animals (5/sex) per test group were evaluated. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose levels of the test substance were investigated:


- 24 h preparation interval: 200, 670 and 2000 mg/kg bw


- 48 h preparation interval: 2000 mg/kg bw


 


The highest dose (2000 mg/kg bw) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. After treatment with the test substance, the number of NCEs was not increased, indicating that the test substance had no cytotoxic effects in the bone marrow. Further, there was no substantial enhancement in the frequency of micronuclei at any preparation interval and with any dose level used. Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The test substance was evaluated for genotoxic potential in the standard Ames/Salmonella gene mutation test and was shown to be able to induce gene mutations by frameshifts in the genome of the strains TA 1537 (with and without S9 mix) and TA 98 (with S9 mix). Therefore, the test substance is considered to be mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay. A subsequent in vitro mammalian cell gene mutation test demonstrated no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation) and concluded that the test item to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster. Additionally a study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD Guideline 474 and EEC Directive 92/69, L 383, Annex V, B.12 in compliance with GLP. After treatment with the test substance, the number of normochromatic erythrocytes was not increased, indicating that the test substance had no cytotoxic effects in the bone marrow. Further, there was no substantial enhancement in the frequency of micronuclei at any preparation interval and with any dose level used. Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in the micronucleus assay.


 


Conclusion:


Disperse Red 086 should be considered not mutagenic as it failed to reproduce the mutagenic effect observed in the Ames assay in the subsequently conducted mammalian cell gene mutation assay. It was also not clastogenic in the micronucleus assay. Hence, Disperse Red 086 can be considered to be not genotoxic. However, taking the positive outcome observed with the Ames assay into account, a testing proposal for Comet assay aimed at investigating the mutagenic potential in vivo has been included with the dossier.

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential of Benzenesulfonamide, N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthracenyl)-4-methyl- (Ames test positive, in vitro mammalian cell gene mutation test negative; in vivo mammalian micronucleus test negative) the substance is deemed non-genotoxic and accordingly does not need to be classified according to CLP (Regulation (EC) No 1272/2008 of The European Parliament and of The Council) as implementation of UN-GHS in the EU.