Reaction mass of Cobaltate(3-), bis[2-[[[3-[2-[1-[[(2-chlorophenyl)amino]carbonyl]-2-(oxo-κO)propyl]diazenyl-κN1]-4-(hydroxy-κO)phenyl]sulfonyl]amino]benzoato(3-)]-, sodium (1:3) and tetrasodium bis[2-[[[3-[[1-[(2-chloroanilino)carbonyl]-2-oxopropyl]azo]-4-hydroxyphenyl]sulphonyl]amino]benzoato(3-)]cobaltate(4-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test negative, in vitro mammalian cell gene mutation test negative; in vitro mammalian chromosome aberration test ambiguous

Link to relevant study records

Reference

Reference 1

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 29 April, 1993 to 20 July, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

May 26, 1983

Deviations:

yes

Remarks:

(statistical analysis was not performed. However at that time it was not recommended)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

September 19, 1984

Deviations:

yes

Remarks:

(statistical analysis was not performed. However at that time it was not recommended)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

July 13, 1987

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test material: FAT 92368/A
Batch No.: PLN 13
Purity: 80 %
Expiration date: March 1998
Vehicle: Bidistilled water

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Cytotoxicity test: 20.6 – 5000 µg/plate
Mutagenicity test: 61.7 – 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Bidistilled water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Untreated negative controls:

other: (same as solvent control)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(without metabolic activation for TA 100 and 1535)

Untreated negative controls:

other: (same as solvent control)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

(without metabolic activation for TA 98)

Untreated negative controls:

other: (same as solvent control)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

(without metabolic activation for TA 1537)

Untreated negative controls:

other: same as solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(Without metabolic activation with strain TA 102)

Untreated negative controls:

other: (same as solvent control)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(with metabolic activation for TA 100, TA 98, TA 1537 and TA 102)

Untreated negative controls:

other: (same as solvent control)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(with metabolic activation for TA 1535)

Details on test system and experimental conditions:

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 L-histidine and 0.5 mM (+)biotin dissolved in water.

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. In either case the final decision is based on the scientific judgment of the Study Director.
Criteria for a positive response:
The test substance is considered to be mutagenic in this test system if the following conditions are met: At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538. Generally a concentration-related effect should be demonstrable.

Statistics:

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Mutagenicity test, original experiment
In the original experiment carried out without metabolic activation, none of the tested concentrations of FAT 92368/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiment with activation performed on strain TA 102 a weak increase in the number of revertant counts was observed at the upper concentrations. No effects were observed with the other strains.

Mutagenicity test, confirmatory experiment
In the confirmatory experiment carried out without metabolic activation, again none of the tested concentrations of FAT 92368/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiment with activation performed on strain TA 102, a marginal increase in the number of revertant counts was observed at a single concentration only. No effects were observed with the other strains. In the mutagenicity tests without and with metabolic activation performed on strains TA 100, TA 102, TA 98 and TA 1537, due to a growth-inhibiting effect of the test material, a reduction in the number of revertant colonies was occasionally observed at the highest concentration.

Remarks on result:

other: all strains/cell types tested were negative

Toxicity test/range finding test:

In the experiment without and with metabolic activation due to an inhibiting effect of the test material on the growth of the bacteria, a slight reduction in the number of revertant colonies was observed at the highest concentration.

Conclusions:

FAT 92368/A was mutagenic in this reverse mutation assay when tested with S. typhimurium strain TA 102 with metabolic activation.

Executive summary:

An in vitro study was performed to investigate the potential of FAT 92368/A (of ca. 80 % purity) to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. The concentration range for the mutagenicity assay was determined in a preliminary test. 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. In order to confirm the results, the experiment was repeated twice. In preliminary toxicity test, In the experiment without and with metabolic activation due to an inhibiting effect of the test material on the growth of the bacteria, a slight reduction in the number of revertant colonies was observed at the highest concentration (5000 µg/plate). In the original as well as confirmatory experiment carried out without metabolic activation, none of the tested concentrations of FAT 92368/A led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the experiment with activation performed on strain TA 102 a weak increase in the number of revertant counts was observed at the upper concentrations. No effects were observed with the other strains. In the mutagenicity tests without and with metabolic activation performed on strains TA 100, TA 102, TA 98 and TA 1537, due to a growth-inhibiting effect of the test material, a reduction in the number of revertant colonies was occasionally observed at the highest concentration. Based on the above findings, it can be concluded that FAT 92368/A was mutagenic in this reverse mutation assay when tested with S. typhimurium strain TA 102 with metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 November, 2002 to 12 December, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Name: FAT 20049/H
Batch number: 004028F2
Date of receiving: 08-0ct-2002
Appearance: light brown powder

Target gene:

no data

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Source of cells: Cell bank of Genetic Toxicology, Aventis Pharma Deutschland GmbH, ProTox
Cell culture medium: RPMI-1640 medium containing approx. 13 % (v/v) HS (horse serum) and approx. 1.7 % sodium pyruvate
Experimental conditions in vitro: Approx. 37⁰C and approx. 4 % CO2 in microtiter plates

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

With metabolic activation: 60, 100, 200, 300 and 400 µg/mL
Without metabolic activation: 10, 25, 50, 100 and 125 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(wiith metabolic activation)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(wiithout metabolic activation)

Details on test system and experimental conditions:

Test organism: L5178Y mouse lymphoma cells
Experimental conditions in vitro: Approx. 37 °C and approx. 4 % CO2 in microtiter plates
Treatment time: With metabolic activation: 3 h; without metabolic activation: 24 h
Recovery time: With metabolic activation: 21 h; without metabolic activation: 24 h

Evaluation criteria:

Criteria for a positive response:
Both biological and statistical significances are considered together for evaluation purposes. The test substance is considered positive (clastogenic) if a dose-dependent or statistically significant increase (P <0.05, at least one concentration) in micronuclei formation outside of the historical control data range of the solvent control is observed.

Statistics:

Statistical analysis was performed with the Chi square-test. If mean micronucleus values in the concentration groups are below the mean micronucleus number of the solvent control no statistical analysis has to be performed.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

(this clastogenic effect correlated with heavy cytotoxicity (survival rate only 33.5 %) and is therefore considered to have only limited biological relevance)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The test substance was suspended in DMSO and diluted in the culture medium (final solvent concentration in the culture medium: 1 %) at concentrations of 1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL with and without metabolic activation to determine the cytotoxicity. Precipitation of the test substance in cell culture medium was not observed in concentrations below 2500 µg/mL. Cytotoxicity was assessed by cell counting. Cell survival rates decreased in this experiment dose-dependently reaching 55.4 % at a concentration of 100 µg/mL without S9 mix and 55 % at a concentration of 250 µg/mL with S9 mix. Cell cultures treated with 10, 25, 50 and 100 µg/mL in the absence of S9 mix were selected for evaluation. Higher concentrations were not evaluable because of heavy toxicity (lack of surviving cells). As a maximum concentration producing at least 50 % or more toxicity is required, an additional experiment had to be performed with a modified dose range (125-175 µg/mL). Similarly, in the presence of metabolic activation no concentration with a survival rate below 50 % was evaluable. Additionally, the cytotoxicity value of the positive control was not conclusive. Therefore, a complete second experiment with 12 concentrations ranged between 2.5 and 400 µg/mL was performed. Only cells treated with 125 µg/mL in the absence of S9 mix were evaluable for micronuclei formation, the other concentrations were not evaluable due to heavy toxicity. In the presence of S9 mix five concentration groups between 60 and 400 µg/mL were selected for evaluation. The highest concentration (400 µg/mL) results in a cell survival rate of 43.3 % of the solvent control. No increase in micronucleus formation was observed in the presence of metabolic activation in all concentration groups. In the absence of metabolic activation, a significant increase in micronucleus formation was observed at the highest concentration (125 µg/mL). However, this clastogenic effect correlated with heavy cytotoxicity (survival rate only 33.5 %) and is therefore considered to have only limited biological relevance. All other dose groups showed no increase in micronucleus formation. No dose-dependent effects were observed. The solvent and positive controls showed the expected increase in micronucleated cells, indicating the validity of the study.

Conclusions:

The test substance was clastogenic in the absence of metabolic activation correlated with heavy cytotoxicity, indicating limited biological relevance.

Executive summary:

The study was conducted to investigate the potential of the test substance (at ca. 80 % purity) to induce micronuclei in L5178Y cells with and without metabolic activation according to OECD Guideline 487. L5178Y cells were treated in 96-well microplates for 3 h with metabolic activation and 24 h without metabolic activation, followed by a recovery time of 21 and 24 h, respectively. Cells were treated with the test substance (suspended in DMSO) and evaluated for micronucleus formation up to concentrations producing distinct cytotoxicity (less than 50 % cell survival). Maximum concentrations evaluated were 400 µg/mL in the presence of S9 mix (43.3 % cell survival) and 125 µg/mL in the absence of S9 mix (33.5 % cell survival). The test substance did not precipitate in cell culture medium in concentrations below 2500 µg/mL. No increase in micronucleus formation was observed in the presence of metabolic activation in all concentration groups. In the absence of metabolic activation, a significant increase in micronucleus formation was observed at the highest concentration (125 µg/mL). However, this clastogenic effect correlated with heavy cytotoxicity (survival rate only 33.5 %) and is therefore considered to have only limited biological relevance. All other dose groups showed no increase in micronucleus formation. Hence, based on the findings of the study, it can be concluded, that the test substance was clastogenic in the absence of metabolic activation correlated with heavy cytotoxicity, indicating limited biological relevance.

Reference 3

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:

2014-02-04 to 2014-04-03

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)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

dated May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

EPA 712-C-98-221, August 1998

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 92368/Y
Batch No.: 1309001
Physical State / Colour: solid / orange brown
Storage Conditions: room temperature, protected from light
Expiry Date: 30.09.2018
Safety Precautions: gloves (butyl or neoprene), respiratory protection, safety glasses
Safety Precautions: The routine hygienic 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:

The S9 liver microsomal fraction induced in male wistar rats with Phenobarbital and ß-Naphthoflavone for 3 consecutive days

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation):
25, 50, 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
0.5, 1.0, 2.5, 5, 10, 25, 50, 100, 250 and 500 µg/mL
Experiment I
without metabolic activation: 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 7.5, 10 and 15 µg/mL
and with metabolic activation: 25, 50, 100, 150, 160, 180, 200, 225 and 250 µg/mL
Experiment II
without metabolic activation: 10, 25, 50, 100, 150, 200, 250 and 275 µg/mL
and with metabolic activation: 5, 15, 30, 70, 140, 170, 200, 210 and 220 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0 % FBS 4h treatment; MEM + 10% FBS 20h treatment). After processing the suspension with ultrasound for 5 min at 37 °C a solution was obtained.

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

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 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.

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: ≥ 7.5 μg/mL; experiment I with S9: ≥ 180 μg/mL; Experiment II without S9: ≥ 150 μg/mL; Experiment II with S9:≥ 170 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Precipitation:

No precipitation of the test item was noted in the experiments without and with metabolic activation.

 

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 6.9 % and 25.0 % for the highest concentrations (15 and 10 μg/mL) evaluated, respectively. The highest biologically relevant concentration evaluated with metabolic activation was 250 μg/mL with a relative growth of 11.5 %. In experiment II without metabolic activation the relative growth was 11.2 % for the highest concentration (275 μg/mL) evaluated. The highest concentration evaluated with metabolic activation was 220 μg/mL with a relative growth of 13.6 %.

 

Mutagenicity:

In experiment I without metabolic activation most 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 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 32.18 and 38.48, mutants/10E6 cells and in the range of 20.19 to 54.94 mutants/10E6 cells with the test item. The highest mutation rate (compared to the negative control values) of 1.55 was found at a concentration of 15 μg/mL with a relative growth of 6.9 %. In experiment I 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 30.06 and 34.32 mutants/106 cells and in the range of 8.06 to 38.10 mutants/10E6 cells with the test item. The highest mutation rate (compared to the negative control values) of 1.18 was found at a concentration of 200 μg/mL with a relative growth of 67.2 %. In experiment II without metabolic activation most 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 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 26.67 and 25.35 mutants/10E6 cells and in the range of 8.66 to 57.88 mutants/10E6 cells with the test item. The highest mutation rate (compared to the negative control values) of 2.23 was found at a concentration of 200 μg/mL with a relative growth of 46.6 %. 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 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 21.22 and 15.38 mutants/10E6 cells and in the range of 11.83 to 37.21 mutants/10E6 cells with the test item. The highest mutation rate (compared to the negative control values) of 2.03 was found at a concentration of 200 μg/mL with a relative growth of 43.2 %. According to OECD 476 the highest concentration tested should induce a reduced level of relative survival of approximately 10-20 %. In experiment I without metabolic activation this criterion was missed slightly. A concentration of 15 μg/mL resulted in a relative growth of 6.9 %, the next lower concentration (10 μg/mL) produced a relative growth of 25.0 %. Due to the fact that all stages of toxicity have been detected without any hint at mutagenicity, this deficiency is considered to be not biologically relevant. DMBA (0.8 and 1.0 μg/mL) and EMS (300 μg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Conclusions:

FAT 92368/Y 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 cultured in vitro were exposed to FAT 92368/Y at concentrations of

- 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 7.5, 10 and 15 µg/mL (without metabolic activation, Experiment I)

- 25, 50, 100, 150, 160, 180, 200, 225 and 250 µg/mL (with metabolic activation, Experiment I)

- 10, 25, 50, 100, 150, 200, 250 and 275 µg/mL (without metabolic activation, Experiment II)

- 5, 15, 30, 70, 140, 170, 200, 210 and 220 µg/mL (with metabolic activation, Experiment II).

FAT 92368/Y was tested up to cytotoxic concentrations. Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 6.9 % and 25.0 % for the highest concentrations (15 and 10 µg/mL) evaluated, respectively. The highest biologically relevant concentration evaluated with metabolic activation was 250 µg/mL with a relative growth of 11.5 %. In experiment II without metabolic activation the relative growth was 11.2 % for the highest concentration (275 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 220 µg/mL with a relative growth of 13.6 %. In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.55 was found at a concentration of 15 µg/mL with a relative growth of 6.9 %. In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.18 was found at a concentration of 200 µg/mL with a relative growth of 67.2 %. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.23 was found at a concentration of 200 µg/mL with a relative growth of 46.6 %. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.03 was found at a concentration of 200 µg/mL with a relative growth of 43.2 %. 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:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

in vivo micronucleus test is negative

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 23 September, 2002 to 17 January, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Age at study initiation: 8 -10 weeks
- Weight at study initiation: Males mean value 37.9 g (SD ±3.1 g); Females mean value 26.5 g (SD ±3.1 g)
- Assigned to test groups randomly: Yes
- Housing: Single
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): Tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-72 %
- Artificial light: 6.00 a.m-6.00 p.m

IN-LIFE DATES: From: 07 October, 2002 to 02 November, 2002

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Deionised water
- 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 deionised water. The vehicle was chosen for 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

Post exposure period:

no data

Dose / conc.:

437.5 mg/kg bw/day (nominal)

Dose / conc.:

875 mg/kg bw/day (nominal)

Dose / conc.:

1 750 mg/kg bw/day (nominal)

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: 40 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 (1750 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 2000 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 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

A test substance classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results. A test substance that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system

Statistics:

no data

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The mean number of polychromatic erythrocytes was not decreased after treatment with the test substance as compared to the mean value of PCEs of the vehicle control indicating that the test substance had no cytotoxic properties in the bone marrow. However, the urine of the treated animals was brown indicating the systemic distribution of the test substance and thus its bioavailibility. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant 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 with the test substance were below or near to the value of the vehicle control group. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Pre-experiment for toxicity:

In the first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg bw formulated in deionised water. The volume administered was 10 mL/kg bw. The animals treated with 2000 mg/kg bw expressed toxic reactions.

Toxic reactions	Hours post-treatment
	1 h	2-4 h	6 h	24 h	30 h	48 h
Reduction of spontaneous activity	2/2	2/2	2/2	1/-	1/-	1/-
Abdominal position	2/2	1/2	1/2	0/-	0/-	0/-
Eyelid closure	2/2	2/2	2/2	0/-	0/-	0/-
Ruffled fur	2/2	2/2	2/2	1/-	1/-	1/-
Apathy	2/2	1/1	1/2	0/-	0/-	0/-
Urine colour	-	brown	-	brown	-	-
Death	0/0	0/0	0/0	1/2	0/0	0/0

 

In the second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1500 mg/kg bw formulated in deionised water. The volume administered was 10 mL/kg bw. The animals treated with 1500 mg/kg bw expressed toxic reactions.

Toxic reactions	Hours post-treatment
	1 h	2-4 h	6 h	24 h	30 h	48 h
Reduction of spontaneous activity	2/2	2/2	2/2	2/2	2/2	2/2
Abdominal position	1/1	0/0	0/0	0/0	0/0	0/0
Eyelid closure	1/2	1/2	2/2	0/0	0/0	0/0
Ruffled fur	2/2	2/2	2/2	2/2	2/2	2/2
Apathy	1/1	0/0	1/1	0/0	0/0	0/0

 

In the third pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1750 mg/kg bw formulated in deionised water. The volume administered was 10 mL/kg bw. The animals treated with 1750 mg/kg bw also expressed toxic reactions.

Toxic reactions	Hours post-treatment
	1 h	2-4 h	6 h	24 h	30 h	48 h
Reduction of spontaneous activity	2/2	2/2	2/2	2/2	2/2	1/1
Abdominal position	1/2	1/2	2/1	0/0	0/0	0/0
Eyelid closure	1/2	1/2	2/2	0/0	1/1	1/1
Ruffled fur	2/2	2/2	2/2	1/1	1/1	1/1
Apathy	1/2	2/2	2/1	0/0	1/1	1/1

On the basis of these data 1,750 mg/kg bw were estimated to be suitable.

Toxic symptoms in the main experiment: In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 1750 mg /kg bw formulated in deionized water. The volume administered was 10 mL/kg bw. The animals treated with 2000 mg /kg bw expressed toxic reactions.

Toxic reactions	Hours post-treatment
	1 h	2-4 h	6 h	24 h
Reduction of spontaneous activity	12/10	12/11	12/12	3/6
Abdominal position	2/0	2/0	0/3	0/1
Eyelid closure	10/10	10/9	9/10	1/1
Ruffled fur	12/12	12/12	12/12	11/11
Brown urine colour	-/-	+/+	-/-	-/-
Death	0/0	0/0	0/0	1/1*

*1 female died after the 24 h observation interval

Conclusions:

The test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

The study was performed to investigate the potential of the test substance (at ca. 70-80 % purity) to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD Guideline 474 in compliance with GLP. The substance was formulated in deionised water, which was also used as vehicle control. 24 h and 48 h after a single administration, bone marrow cells were collected for micronuclei analysis. The highest dose was chosen based on a preliminary experiment. The following dose levels were investigated:

- 24 h preparation interval: 437.5, 875 and 1750 mg/kg bw

- 48 h preparation interval: 1750 mg/kg bw

 

In the main experiment, 3 animals died after treatment at this dose. After treatment with the test substance, the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control, thus indicating that the test substance did not exert cytotoxic effects in the bone marrow. However, the urine of the treated animals was brown, suggesting systemic distribution of the substance and thus bioavailability. Further, there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used. Hence, it can be concluded that the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test substance was tested in the standard Ames/ Salmonella gene mutation test and under the study conditions; the test substance was not mutagenic for S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 both in the absence and presence of metabolic activation. The test substance was also test in an in vitro cell gene mutagenicity test and under the experimental conditions reported, the test item was considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster. A study was conducted to investigate the potential of the test substance to induce micronuclei in L5178Y cells with and without metabolic activation according to OECD Guideline 487. Under the study conditions, the test substance was clastogenic in the absence of metabolic activation correlated with heavy cytotoxicity, indicating limited biological relevance. Finally a study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse in vivo according to OECD Guideline 474 in compliance with GLP. Under the study conditions, the test substance did not induce micronuclei. Therefore, the test substance is considered to be non-mutagenic in this in vivo mouse micronucleus assay.

Short description of key information:
The test substance was evaluated for genotoxic potential in the standard Ames/Salmonella gene mutation test, in the in vitro mammalian cell gene mutation test (HPRT-locus) and the in vitro micronucleus study and an in vivo bone marrow micronucleus test. The test substance was negative in the Ames test and negative in the in vitro mammalian gene mutation test. However, the results from the in vitro mammalian micronucleus test were ambiguous. A subsequent in vivo bone marrow micronucleus test was negative.

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential of the test substance (Ames test negative, in vitro mammalian cell gene mutation test negative; in vitro mammalian chromosome aberration test ambiguous, and an in vivo micronucleus test is negative) the substance is deemed non-genotoxic and accordingly does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council) as implementation of UN-GHS in the EU.