Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: negative
HPRT Test: negative
In vitro Micronucleus Test: negative

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:
2011-09-26 to 2011-11-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP study without deviations
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
2005
Qualifier:
according to
Guideline:
other: ICH Guideline S2A:'Genotoxicity: Specific Aspects of Regulatory gentoxocoty tests for Pharmaceuticals (CPMP/ICH/141/95)'
Qualifier:
according to
Guideline:
other: ICH Giudeline S2B: 'A Standard Battery for Genotoxicity Testing of Pharmaceuticals (CPMP/ICH/174/95)'
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
mutated gene locus responsible for histidine auxotrophy
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: histidine auxotroph
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced rat liver S9; male rats
Test concentrations with justification for top dose:
Experiment I and II:
10.0, 31.6, 100, 316, 1000 and 3160 µg per plate.
Vehicle / solvent:
Test item was completely dissolved in dimethyl sulfoxide (DMSO).
Untreated negative controls:
no
Remarks:
solvent test will be used as negative reference item
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
for details see below
Positive control substance:
other: without metabolic activation: sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 2-nitroflurene in DMSO for TA 98, 9-amino-acridine in ethanol abs. for TA 1537, Cumene hydroperoxide in DMSO for TA 102
Remarks:
with metabolic activation: 2-aminoanthracene for TA 98, TA 102, TA 1537, Cyclophosphamide fpr TA 100, TA 1535
Details on test system and experimental conditions:
Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
10 concentrations ranging from 0.316 to 5000 µg/plate were tested. Cytotoxicity and/or test item precipitation were noted in the plate
incorporation test without and with metabolic activation at concentrations of 3160 and 5000 µg/plate.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction, the protein content of the S9 fraction was 34.2 mg/mL S9, cytochrome
P-450: 0.36 nmol/mg protein
ADMINISTRATION
- Dosing: 10.0, 31.6, 100, 316, 1000 and 3160 µg per plate.
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 10 µg/plate
2-nitroflurene in DMSO for TA 98, 10 µg/plate
9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
Cumene hydroperoxide in DMSO for TA 102, 10 µg/plate
- with metabolic acivation
2-aminoanthracene in DMSO for TA 98, TA 102, TA 1537, 2 µg/plate
Cyclophosphamide in aqua ad iniectabilia for TA 100, TA 1535, 1500 µg/plate
- negative control: solvent control: DMSO for all strains
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

Evaluation criteria:
The test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0 .05, U-test according to MANN and WHITNEY) compared to the solvent control to at least
2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both independent
experiments.
- additionally, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient) is observed.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.
Statistics:
According to the OECD Guideline 471, a statistical analysis of the data is not mandatory
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity and/or test item precipitation were noted in the plate incorporation test without and with metabolic activation at concentrations of 3160 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ


Remarks on result:
other: all strains/cell types tested

see attchached document

Conclusions:
Interpretation of results: negative

Under the present test conditions 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction
products with 2-butanone oxime tested up to a concentration of 3160 µg/plate, that led to cytotoxicity and/or test item precipitation, caused no
mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in
the preincubation test each carried out without and with metabolic activation.
Executive summary:

The test item was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

Six concentrations ranging from 10.0 to 3160 µg 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 2-butanone oxime per plate were employed in two independent experiments each carried out without and with metabolic activation.

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, test item precipitation and/or cytotoxicity (reduction of the number of revertants by more than 50 %) were noted at the top concentration of 3160 µg/plate in all test strains.

No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 2-butanone oxime, tested up to a concentrationof 3160 µg/plate, that led to cytotoxicity and/or test item precipitation, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

In conclusion, under the present test conditions the test item tested up to a concentration of 3160 µg/plate, that led to cytotoxicity and/or test item precipitation, caused no mutagenic effects in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

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:
2011-10-26 to 2012-01-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10 % fetal calf serum, penicillin (100 U/mL) and streptomycin
(100 µg/mL) called DMEM-FCS; Cells were periodically checked for the absence of mycoplasma contamination by using the HOECHST stain 33258;
Spontaneous mutation rate was continuously monitored
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial supernatant fraction derived from livers of Aroclor 1254-treated rats (S9 mix)
Test concentrations with justification for top dose:
Five concentrations: 15.63, 31.3, 62.5, 125, 250 µg/mL with and without metabolic activation
Vehicle / solvent:
The test item was completely dissolved in acetone. Preparations of the test item made on the day of use were employed.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethyl methanesulphonate (EMS) in direct mutagenicity experiment; 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay; both EMS and DMBA were dissolved in DMSO. The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL
Details on test system and experimental conditions:
CELLS AND TISSUE CULTURE MEDIA
- V79 cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10 % fetal calf serum, penicillin 3 (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS
- Incubation of cultures: at 37 °C in a humidified atmosphere (90 %) containing 10 % CO2
- For subculturing, a trypsin (0.05 %)-EDTA (ethylenediaminetetraacetic acid, 0.02 %) solution in modified Puck's salt solution A was used.

METHOD OF APPLICATION:
- Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).
- Vehicle: test item was completely dissolved in acetone. Preparations of the test item made on the day of use were employed.

DURATION (see tables below)
- Preincubation period:
* 1 day (in 30 mL DMEM-FCS)
- Exposure duration:
* 4 hours (1st experiment) and 24 hours (2nd experiment) without S9 mix, respectively;
* in the experiments with S9 mix, the medium was replaced by 18 mL S9 mix and the exposure limited to 4 hours.
* the negative control was treated with acetone (the vehicle) in the same way
* After removal of the test item and washing of the plates with PBS cells were trypsinised and a relative plating efficiency was determined for each
dose to obtain an accurate measure of the toxic effect of the chemical
- Expression time (cells in growth medium):
* Three replicate plates (60 mm diameter) were used with a known number of cells.
* Remaining cells were replated and the culture incubation continued until day 8 with 30 mL normal DMEM-FCS with one subcultivation on day 5.
* Afterwards cells were harvested by trypsinisation and replated at a density of 1 000 000 per 150 mm diameter dish in DMEM-FCS containing
6-thioguanine (10 µg/mL) for selection of mutants (5 replicate plates), or at approx. 100 to 150 cells (exact number known) per 60 mm diameter
dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE 2), (3 replicate plates).
- Fixation time:
* Plates were fixed and stained after about 8 days (plating efficiency plates) or 12 days (6-thioguanine plates).
- Positive control:
* ethyl methanesulphonate (EMS) in direct mutagenicity experiment;
* 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay
both EMS and DMBA were dissolved in DMSO.
* The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL


NUMBER OF REPLICATIONS: three
NUMBER OF CELLS EVALUATED: 1 500 000

DETERMINATION OF CYTOTOXICITY (same procedure was used as employed for the mutagenicity experiments, except that no mutant selection was carried out)
- Method: survival
- A concentration of the test item which produces a low level of survival (10 to 20 %) would be used as highest concentration and the survival in the
lowest concentration being approximately the same as that in the negative control.
- Five adequately spaced concentrations are employed
- In the preliminary experiment without and with metabolic activation test item precipitation was noted at concentrations of 250 and 500 µg test
item/mL and higher. Hence, 250 µg test item/mL were employed as the top concentration for the mutagenicity tests without and with metabolic
activation.
Evaluation criteria:
The following predetermined descriptive criteria are used for interpretation of the results:
- If in both independent experiments solvent and positive controls show results within the norm and if the test item does not increase the mutation
frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always lower than 40 x 10^-6 and if at least 1 000 000 cells per condition have been evaluated, the item is considered as negative in the test.
- In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold
solvent control and at least 40 x 10^-6 both in the presence and/or absence of S9 mix, the item is considered as positive in the test.
Statistics:
No satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments. See evaluation criteria
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
precipitation was noted at concentrations of 250 and 500 µg test item/mL and higher in the experiment without and with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (Preliminary cytotoxicity test):
In the preliminary experiment without and with metabolic activation test item precipitation was noted at concentrations of 250 and 500 µg test
item/mL and higher. Hence, 250 µg test item/mL were employed as the top concentration for the mutagenicity tests without and with
metabolic activation.
COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 106 survivors in non-activation solvent
controls and 6 to 46 per 106 survivors in S9 activation solvent controls [1]. The background data obtained at LPT are given at the end of this
chapter. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls EMS (600 and 700 µg/mL) and DMBA (20 and 30 µg/mL) should cause a 10-fold or greater increase in mutation
frequency.
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10 6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA
(see table below).
Remarks on result:
other: all strains/cell types tested

Criteria for assay acceptance

Solvent control: As the total number of colonies is normally low and as a single mutation may cause several colonies due to cell division during the expression period, a relatively large variation of the mutation frequency may result. This is especially true, if a low spontaneous mutation frequency is forced by cloning (in order to achieve a high sensitivity of the test).

The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 106survivors in non-activation solvent controls and 6 to 46 per 106survivors in S9 activation solvent controls [1]. The background data obtained at LPT are given at the end of this chapter. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls (600 and 700 µg/mL) and DMBA (20 and 30 µg/mL) should cause a 10-fold or greater increase in mutation frequency.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6clonable cells forand 130.0 to 2693.3 x 10-6clonable cells for DMBA (see table below).


The mutation frequencies of the solvent controls and the positive controls without and with metabolic activation for the last 58 experiments (most recent background data, not audited by the QAU-department) are given as follows:

Mutation frequency per 106 clonable cells

 

Without metabolic activation

(24-h exposure)

With metabolic activation

(4-h exposure)

Solvent control (n = 58)

mean

14.11

14.88

SD

7.42

8.20

range

1.30 - 34.80

2.18 - 38.36

Positive control (µg/mL) (n = 58)

 

EMS

(600)

EMS

(700)

DMBA

(20)

DMBA

(30)

mean

449.1

468.4

347.1

563.8444.2

SD

444.2

268.6

241.8

700.1

range

112.1 – 1708.4

152.0 – 976.9

  130.0 – 844.8

151.3 – 2693.3

SD     = Standard deviation

EMS   = ethyl methanesulfonate

DMBA = 9,10-dimethyl-1,2-benzanthracene

Conclusions:
Interpretation of results: negative

Under the present test conditions, the test item tested up to a concentration of 250 µg/mL, that led to test item precipitation in the experiments
without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test.
Executive summary:

The test item was tested for mutagenic potential in a HPRT Test on V79 cells performed according to OECD 476 both in the presence and absence of metabolic activation. In the first main experiment the exposure duration was 4 hours with and without S9 mix. The second experiment was performed with treamtent times of 4 hours with and 24 hours without S9 mix. The test item was completely dissolved in acetone. Five concentrations ranging from 15.63 to 250 µg test item/mL were selected for the experiments without and with metabolic activation based on precipitation of the test item at concentrations of 250 µg/mL and above.

Under the present test conditions the test item showed no substantial and reproducible increase in the mutation frequency. Thus, the test item was negative in the HPRT-V79 mammalian cell mutagenicity test.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: chromosome breakage (structural chromosomal aberrations) and misdistribution of chromosomes
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-26 to 2011-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP study without deviations
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals: In Vitro Mammalian Cell Micronucleus Test (MNvit), No. 487, Guideline July 22, 2010
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
mammalian cell system( Chinese hamster Ovary cells)
Species / strain / cell type:
other: Chinese hamster ovary (CHO-K1) cells
Details on mammalian cell type (if applicable):
Species/cell type: CHO cells as originally derived from the ovary of Chinese hamster, obtained from ATCC
CHO-K1, modal chromosome number of 20
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix based on liver homogenate fraction from male rats, induced with Aroclor 1254 (i.p.)
Test concentrations with justification for top dose:
12.5, 25, 50, 100 µg/mL with and without metabolic activation
Vehicle / solvent:
Vehicle: test item was completely dissolved in acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
vehicle: acetone
True negative controls:
no
Positive controls:
yes
Remarks:
clastogen
Positive control substance:
cyclophosphamide
Remarks:
+S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
vehicle: acetone
True negative controls:
no
Positive controls:
yes
Remarks:
clastogen
Positive control substance:
mitomycin C
Remarks:
-S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
aneugen
Positive control substance:
other: colchicine
Remarks:
-S9-mix
Details on test system and experimental conditions:
SYSTEM OF TESTING:
- Species/cell type: CHO-K1 cell line, cell cycle length 12 hours
- Metabolic activation system: male rat liver S9 from  Aroclor 1254 induced animals
ADMINISTRATION: 
- Solubility: test item was completely dissolved in acetone
- Preliminary experiment: precipitation were noted starting at a concentration of 100 µg test item/mL. Hence, 100 µg test item/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.
- Dosing:  12.5, 25, 50, 100 µg/mL with and without metabolic activation, 4 h exposure
12.5, 25, 50, 100 µg/mL without metabolic activation, 20 h exposure
In the main study test item precipitation was noted at the top concentration of 100 µg test item/mL in the experiments without and with
metabolic activation.
- Positive and negative control groups and treatment:    
negative: acetone
positive (+S9): cyclophosphamide 
positive (-S9): mitomycin C 
positive (-S9): colchicine
DURATION:
- most aneugens and clastogens are detected by a short term treatment period of 4 hours in the presence and absence of S9, followed by removal of
the test item and a growth period of 1.5 – 2.0 cell cycles. Cells were sampled at a time equivalent to about 1.5 – 2.0 times the normal (i.e. untreated)
cell cycle length either after the beginning or at the end of treatment. Because of the potential cytotoxicity of S9 preparations for cultured
mammalian cells, an extended exposure treatment of 1.5 – 2.0 normal cell cycles was used only in the absence of S9.
Cell treatment and harvest times for the used CHO cell line see table below.
As both initial tests of the short 4 h treatment are negative or equivocal, a subsequent, extended exposure treatment without S9 was used.
- Harvesting time: harvesting time was 20 hours after the end of exposure

STAIN (for cytogenetic assays): Each culture was harvested and processed separately. High-quality cell preparations for scoring were obtained. Cell
cytoplasm were retained to allow the detection of micronuclei and (in the cytokinesis-block method) reliable identification of binucleate cells. The
slides were stained using Giemsa.

NUMBER OF REPLICATIONS: 2, duplicate cultures were used for each test item concentration and for the solvent control cultures.

NUMBER OF CELLS EVALUATED: The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000
binucleated cells per culture; two cultures per concentration).

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
The CBPI indicates the average number of cell cycles per cell during the period of exposure to cytoB, and is used to calculate cell proliferation.
The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis:

OTHER EXAMINATIONS: 1000 binucleated cells per duplicate cell culture were scored to assess the frequency of cells with one, two, or more than
two micronuclei. Additionally, the cells were classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a
measure of toxicity.

Evaluation criteria:
The assay demonstrates its ability to reliably and accurately detect substances of known aneugenic and clastogenic activity, with and without
metabolic activation.
Solvent/vehicle control and untreated cultures give reproducibly low and consistent micronuclei frequencies, typically 5 – 25 micronuclei per 1000
cells according to OECD 487. Data from negative and positive controls are used to establish historical control ranges. These values are used in
deciding the adequacy of the concurrent negative/positive controls for an experiment .
Statistics:
The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for
continuity according to YATES.
If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing
micronuclei, it is classified as a positive result.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was noted at the top concentration of 100 µg test item/mL in the experiments without and with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The pH and osmolality of the negative control and test item formulations in the medium were determined for each experiment employing the
methods given below:
pH values: using a digital pH meter type WTW pH 525 (series no. 51039051),
Osmolality: with a semi-micro osmometer .
No relevant changes in pH or osmolality of the formulations were noted.

Test item was completely dissolved in acetone. Precipitation were noted starting at a concentration of 100 µg test item/mL. Hence, 100 µg test
item/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

Data from negative and positive controls are used to establish historical control ranges. These values are used in deciding the adequacy of the
concurrent negative/positive controls for an experiment i.e. the negative/positive control data must be within the historical ranges

Remarks on result:
other:

see attached tables

Conclusions:
Interpretation of results: negative

Under the present test conditions, the test item tested up to a concentration of 100 µg/mL, that led to test item precipitation in the absence and in
the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of
genotoxic properties in the in vitro micronucleus test.
Executive summary:

The test item (vehicle: acetone) was investigated in an in vitro micronucleus test using CHO cell cultures both in the presence and absence of metabolic activation according to OECD TG 487. Test item precipitation was noted at concentrations of 100 µg/mL and above. Thus, concentrations of 12.5 to 100 µg/mL were tested. Under the present conditions, the test item revealed no indications of genotoxic properties in the in vitro micronucleus test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item, tested in an Ames Test up to a concentration of 3160 µg/plate, that led to cytotoxicity and/or test item precipitation, caused no mutagenic effects in the Salmonella typhimuriumstrains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

The test item was tested for mutagenic potential in a HPRT Test on V79 cells performed according to OECD 476 both in the presence and absence of metabolic activation. In the first main experiment the exposure duration was 4 hours with and without S9 mix. The second experiment was performed with treatment times of 4 hours with and 24 hours without S9 mix. The test item was completely dissolved in acetone. Five concentrations ranging from 15.63 to 250 µg test item/mL were selected for the experiments without and with metabolic activation based on precipitation of the test item at concentrations of 250 µg/mL and above.

Under the present test conditions the test item showed no substantial and reproducible increase in the mutation frequency. Thus, the test item was negative in the HPRT-V79 mammalian cell mutagenicity test.

The test item (vehicle: acetone) was investigated in an in vitro micronucleus test using CHO cell cultures both in the presence and absence of metabolic activation according to OECD TG 487. Test item precipitation was noted at concentrations of 100 µg/mL and above. Thus, concentrations of 12.5 to 100 µg/mL were tested. Under the present conditions, the test item revealed no indications of genotoxic properties in the in vitro micronucleus test.

Justification for classification or non-classification

Because all in vitro genotoxicity studies revealed clearly negative results, it can be concluded that the test substance is not genotoxic in vitro and therefore must not be classifed according to the criteria of EU Regulation 1272/2008.