2-hydroxy-1-(4-(4-(2-hydroxy-2-methylpropionyl)benzyl)phenyl)-2-methylpropan-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies conducted to recognised testing guidelines with GLP certification.

Link to relevant study records

Reference

Reference 1

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:

2012

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Hams F12 medium + supplements
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from phenobarbital and beta-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

first experiment +/- S9 mix: 7.8, 15.6, 31.3, 62.5, 125, 250 µg/ml
second experiment +/- S9 mix: 6.25, 12.5, 25, 50, 100, 150, 200 µg/ml

Vehicle / solvent:

Due to the limited solubility of the test substance in water, dimethyl sulfoxide (DMSO) was
selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT
assay and for which historical data are available.
The final concentration of the vehicle DMSO in the culture medium was 1% (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 3 days (4 hour treatment) or 2 days (24 hour treatment) at 37°C
- Selection time (if incubation with a selection agent): 6-7 days in TG-medium
- Fixation: At the end of the selection period, the medium will be removed and the remaining colanies will be fixed with methanol, stained with Giemsa and counted

SELECTION AGENT (mutation assays): TG-medium

NUMBER OF REPLICATIONS: two independent experiments, every sample in triplicate

NUMBER OF CELLS EVALUATED: all colonies are counted

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- pH
- osmolarity
- solubility
- cell morphology

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of
mutant frequencies. The number of mutant colonies obtained for the test substance treated
groups was compared with that of the respective vehicle control groups. A trend is judged as
statistically significant whenever the p-value (probability value) is below 0.10 and the slope is
greater than 0. However, both, biological and statistical significance will be considered
together.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 125 ug/ml onward

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: no
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: In the pretest for toxicity based on the purity and the molecular weight of the test substance 3 600 μg/mL (approx. 10 mM) of the test item was used as top concentration both with andwithout S9 mix at 4 hour exposure time and without S9 mix at 24 hour exposure time.

COMPARISON WITH HISTORICAL CONTROL DATA: historical negative control data from May 1994 - December 2011

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control
were observed in all experimental parts scored for gene mutations. In culture medium test substance precipitation occurred at 450 μg/mL and above at the beginning and at the end of treatment in the absence and presence of S9 mix. Cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% relative survival was observed at 112.5 μg/mL and above under all treatment conditions.

After 4 hours of treatment the morphology and attachment of the cells treated with at least the highest applied concentration was adversely influenced in all experimental parts scored for gene mutations. This occurred in samples regardless of the presence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test material was found not to be mutagenic under the conditions of the test.

Executive summary:

In this guideline (OECD 476) study conducted with GLP certification, the test material (EC: 444-860-9) was considered to be non-genotoxic. The HPRT test was conducted in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Based on the solubility properties of the test substance and according to an initial rangefinding cytotoxicity test doses up to 250 µg/ml were tested in presence and absence of a metabolic activation system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-11-06 until 2002-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted on 1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: "Kanpoan No. 287-Environmental Agency", "Eisei No. 127 ~ Ministry of Health & Welfare", "Heisei 09/10/31 Kikyoku No. 2-Ministry of International Trade & Industry"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No target gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate (doubling time of clone V79/T5 in stock cultures: 12 h) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study. The cells have a stable karyotype with a modal chromosome number of 22.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian microsomal fraction S9 mix

Test concentrations with justification for top dose:

With and without S9 mix: 20, 40, 80, 120, 160, 200 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive control substance without metabolic activation.

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Positive control substance with metabolic activation.

Details on test system and experimental conditions:

-
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 11.5 hours
- Selection time (if incubation with a selection agent): 2.5 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours


SPINDLE INHIBITOR (cytogenetic assays): colcemid


NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2x10E4-12x10E4 cells seeded per dish and 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- In addition, the number of polyploid cells in 500 metaphase
cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line
polyploid means a near tetraploid karyotype).

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in
the range of the historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical
control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural
chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and stafisfical significance were considered together. If the criteria mentioned above for the test item were not clearly met, the classificafion with regard to the historical data and the biological relevance was discussed and/or a confirmatory experiment is performed.

Although the inclusion of the structural chromosome aberrafions was the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the historical control data
(0.0 - 8.5 % polyploid cells).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

At concentration of 120 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 26.6 and 3400 µg/mL were applied. Clear toxic effects were observed after treatment with 106.3 µg/mL and above in the absence and presence of S9 mix. In addition, 24 hrs confinuous treatment with 26.6 µg/mL and above in the absence of S9 mix induced strong toxic effects
- In the pre-experiment, precipitation of the test item in culture medium was observed after 4 hrs treatment with 425 µg/mL and above in the absence and the presence of S9 mix. In addition, after 24 hrs confinuous treatment in the absence of S9 mix precipitafion occurred with 212.5 µg/mL and above at the end of treatment no relevant influence ofthe test item on the pH value or osmolarity was observed (solvent control 394 mOsm, pH 7.4 versus 349 mOsm and pH 7.4 at 3400 µg/mL)
- In the main experiment, no precipitafion in culture medium was observed 4 hrs after start of treatment up to the highest applied test item concentration (200 µg/mL)
- Toxic effects indicated by reduced cell numbers and/or mitotic indices of below 50 % of control were observed in all experimental parts of the
main experiment. In detail, strongly reduced cell numbers were observed after 4 hrs treatment with 120 µg/mL in the absence and the presence of S9 mix (11 % and 26 % of control, respecfively). In addition, the mitotic indices were cleariy reduced after 4 hrs treatment with 120 µg/mL in the absence and the presence of S9 mix (29 % and 27 % of control, respectively)
- In the absence and the presence of S9 mix the aberrafton rates (16.0 % and 22.5 %, respectively) were stafisfically significant and biologically relevant increased after treatment with 120 µg/mL as compared to the corresponding solvent controls (0.5 % and 2.0 %, respecfively). Also, the number of cells carrying exchanges was distinctiy increased (8.0 % and 12.5 %, respecfively) as compared to the solvent controls (0.0 %). Although these observations have been made, at highly cytotoxic concentrations they have to be regarded as biologically relevant
- In addifion, in the absence of S9 mix a single significant (p < 0.05) increase was observed in the absence of S9 mix after treatment with 40 µg/mL. Although this increase of 3.5 % aberrant cells, exclusive gaps, was statistically significant compared to the low response
(0.5 % aberrant cells, exclusive gaps) in the solvent control data, the response was within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Therefore, this statisfical significance was regarded as being biologically irrelevant. Furthermore, in the presence of S9 mix a dose-related increase in the number of aberrant cells (1.0, 2.0 and 22.5%) at the concentrations evaluated (40, 80, and 120 µg/mL, respectively)
was observed. However, the values at the two lower concentrations were within the historical control data range
- Occurrence of polyploid metaphases. No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.9 -3.3 %) as compared to the rates of the solvent controls (2.3 - 3.1 %)
- In this study, EMS (200 µg/mL) and CPA (0.7 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive

Executive summary:

In this guideline (OECD 473) study conducted with GLP certification, the test material (EC 444-860-9) was shown to not be genotoxic, but did exhibit a clastogenic response indicating cytotoxicity at 120 µg/L. The test was carried out over a range of concentrations (20 -200 µg/l) in Chinese hamster lung fibroblasts with and without metabolic activation. The results of the study indicate that the test material does not meet the criteria to be considered mutagenic under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Reference 3

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:

2002-10-10 until 2002-10-25

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:

"Ninth Addendum to OECD Guidelines for Testing of Chemicals", Section 4, No. 471: "Bacterial Reverse Mutation Test", adopted July 21,1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, Ll 362000, Annexe 4D", dated May 19, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his, trp

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Mammalian microsomal fraction S9 mix

Test concentrations with justification for top dose:

33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

- Experiment 1: The test item was assessed for its potential to induce gene mutations according to the plate incorporation test
- Experiment 2: pre-incubation test using Salmonella typhimurium strains TA 1535, TA 1537, TA98, and
TA 100, and the Escherichia coli strain WP2 uvrA
- The test item was tested in both experiments at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate
- Each concentration and the controls were tested in triplicate
- The following materials were mixed in a test tube and poured onto the minimal agar plates:
100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar
- In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

Evaluation criteria:

- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count ofthe corresponding solvent control is observed
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment
- A dose dependent increase in the'^number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxic effects, evident as a reduction in the number of revertants, were observed at 2500 µg/plate without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxic effects, evident as a reduction in the number of revertants, were observed at 5000 µg/plate with S9 mix in tester strain TA 100 at 5000 µg/plate in experiment II.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Irregular background growth was observed at 2500 and 5000 µg/plate in strain TA 1537 in experiment I. An irregularity in the background growth was also observed in strain TA 1535 at 33 µg/plate without S9 mix in experiment I. This effect was judged to be biologically irrelevant since it was only observed in one out of three plates and the number of revertant colonies remains well within the historical control range
- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance
- In experiment I, the data in the solvent control of strain TA 100 (with S9 mix) were slightly above the historical control range. In the presence of metabolic activation, the number of colonies did not quite reach the lower limit of the negative control of the historical control data in strains TA 1535 and TA 1537 (exp. II), and in strain WP2 uvrA (exp. I). Since these deviations were rather small, these effects were considered to be based upon biologically irrelevant fluctuations in the number of colonies
- The historical range of positive controls was exceeded in strains TA 1535 and TA 100 in experiment I without metabolic activation. This effect indicateed the sensitivity of the strains rather than compromising the assay
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies

Conclusions:

The test material was found not to be mutagenic under the conditions of the test.

Executive summary:

In this guideline (OECD 471) study conducted with GLP certification, the test material (EC 444-860-9) was determined not to be genetoxic but did exhibit cytotoxic effects. The reverse mutagenicity study was conducted in S. typhimurium (TA98, TA 100, TA 1535, TA 1537) and E. coli (WP2 uvr A) with and without metabolic acitvation. The results of the study indicate that the test material does not meet the criteria to be considered mutagenic under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

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:

2003-03-19 to 2003-02-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

, adopted on 1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration 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 Rossdorf, Switzerland
- Age at start of acclimization: 8-10 weeks
- Weight at study initiation: males mean value 33.39 g and females mean value 27.13 g
- Assigned to test groups randomly: yes
- Fasting period before study: Approximately 18 hours before treatment the animals received no food but water ad
libitum
- Housing: Individuelly in Makrolon type- 1 cages
- Diet: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe, Germany)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf, Germany)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 "C
- Humidity (%): 22 - 70 %
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: To: 2003-01-04 until 2003-02-07

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Not detailed

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Once

Post exposure period:

Sampling of the bone marrow was done 24 hours after treatment (to animals administered with 500, 1000, and 2000 mg/kg bw.of the test item) and 48 hours after treatment (to animals administered with 2000 mg/kg bw)
acute toxic symptoms were examined at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals (5 males, 5 females) per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide - 40 mg/kg b.w

Tissues and cell types examined:

Erythrocyres from the bone marrow

Details of tissue and slide preparation:

- 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-Grunwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample
- Evaluation of the slides was performed using NIKON microscopes with 100 x 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 item was classified as mutagenic if it induced 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 were used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Nonparametric Mann-Whitney test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- After treatment with the test item 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 item did not exert any cytotoxic effects in the bone marrow
- In comparison to the corresponding vehicle controls 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 item and with any dose level used
- Cyclophosphamide (positive control) administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

Conclusions:

Interpretation of results (migrated information): negative

Executive summary:

In this guideline (OECD 476) study conducted with GLP certification, the test material (EC 444-860-9) was determined not to be genotoxic. The test was conducted on mice (10 mice per sex per dose), with the substance administered via gavage in a single dose (nominal concentrations: 500, 1000 and 2000 mg/kg bw). The bone marrow of the test subjects was sampled at 24 and 48 hours. The results of the study indicate that the test material does not meet the criteria to be considered mutagenic under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Three in vitro assays were performed to evaluate the genotoxicity of the test substance. At first, the material was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e.Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The test item was applied at concentrations of 33 μg - 5000 μg/plate in presence and absence of a metabolic activation system (S9 mix). A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

The objective of the second assay was to evaluate the ability of the test substance to induce chromosomal aberrations in cultured Chinese hamster lung fibroblasts (V79) with and without exogenous metabolic activation. The test article was dissolved in DMSO. Replicate cultures of CHO cells were incubated with 20 to 200 µg/ml for 4.0 hours with and without S9 and harvested at 11.5 hours after the initiation of treatment. In the absence and the presence of S9 mix the aberration rates were statistically significant and biologically relevant increased after treatment with 120 µg/mL. Also, the number of cells carrying exchanges was distinctiy increased. Although these observations have been made at cytotoxic concentrations they have to be regarded as biologically relevant.

At last, the test material was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced. Based on the solubility properties of the test substance and according to an initial rangefinding cytotoxicity test doses up to 250 µg/ml were tested in presence and absence of a metabolic activation system. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies both without S9 mix.

At last an in vivo study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The material, dissolved in corn oil, was administered singly at concentrations of 500, 1000 and 2000 mg/kg bw. 24h and 48h after administration the bone marrow cells were collected for micronuclei analysis. To investigate a cytotoxic effect of the compound the ratio between polychromatic and normochromatic erythrocytes was determined. After treatment the number of PCEs was not substantially decreased as compared to the vehicle control thus indicating that the substance did not exert a cytotoxic effect in the bone marrow. In comparison to the corresponding vehicle controls 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 item and with any dose level used. In conclusion, the test item did not induce micronuclei and the test material is considered to be non-mutagenic in this micronucleus assay.

The test item did not induce mutations in bacteria or mammalian cells or chromosome aberrations in mammalian cells in vitro. An indiction of micronuclei in mice after oral treatment was also not observed. Therefore, the substance is not considered to be genotoxic under the conditions of these tests.

Short description of key information:
The test item was not mutagenic in a bacterial gene mutation assay but induced a positive (clastogenic) response in an in-vitro chromosome aberration test. The in-vivo micronucleus test in mice was negative and did not confirm the clastogenic effect from the in-vitro test.
No mutagenic potential was evident in an in-vitro gene mutation test in mammalian cells.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.