Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In two in vitro genotoxicity studies (Ames test according to OECD TG 471 (Hüls AG, 1996) and HPRT test according to OECD TG 476 (LPT, 2015)) the test substance showed negative results.

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:
1996-03-14 to 1996-04-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1983)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(1992)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
mutated gene loci responsible for histidine auxotrophy
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Test concentrations with justification for top dose:
30 to 5000 µg/plate
Vehicle / solvent:
The solvent controls quantify the spontaneous mutation frequency of each bacterial strain in the presence of acetone.
Test concentration: 2.5 µg/plate (plate incorporation and preincubation test)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Aceton
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: with metabolic activation, 2-aminoanthracene, 2.5 µg/plate
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Metabolic activation system:   Aroclor 1254-induced rat liver S9 fraction, obtained from Cytotest Cell  Research GmbH & Co. KG (Rossdorf, Germany), lots No. 170795 and  050296,  prepared from male Wistar rats

ADMINISTRATION: 
- Dosing:   
 Plate incorporation test: 50/160/500/1600/5000 µg/plate (+/- metabolic  activation) repeated for TA 1537 (- metabolic activation) due to absence of 
 background lawn in all plates   Pre-incubation test: same concentrations for TA 100 and TA 1537; for TA  98 and TA 1535 only 30/100/300/1000/3000 µg/plate (+/- metabolic  activation)   repeated for TA 1535 (- metabolic activation) due to exceptionally low  revertant frequency in control (2 +/- 2; published background level 3 to  37) - Number of replicates: 3
- Application: solvent acetone (CAS No. 67-64-1)
- Positive and negative control groups and treatment:   
 positive without metabolic activation:   
TA 98: 2-nitrofluorene (2.5 µg/plate in dimethyl sulfoxide)   
TA 100: sodium azide (5.0 µg/plate in twice distilled water)   
TA 1535: sodium azide (2.5 µg/plate in twice distilled water)   
TA 1537: 9-aminoacridine (25 µg/plate in dimethyl sulfoxide)   
positive with metabolic activation and activity of metabolic system:   
all strains: 2-aminoanthracene (2.5 µg/plate in dimethyl sulfoxide)   
negative: solvent control, 25 µl/plate
- Pre-incubation: 30 minutes at 30 °C   incubation 72 hours at 37 °C

CRITERIA FOR EVALUATING RESULTS:    ratio of revertant rates treated/control >= 2 with generally positive  dose-response relationship in any strain in two independent experiments
Rationale for test conditions:
OECD Guideline 471
Evaluation criteria:
For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. A test article that does not meet these criteria will be called non-mutagenic in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.
Statistics:
The stated means, standard deviations and factors were calculated through a computer program written by Messrs BIOSYS. The usual statistical methods were used for the calculations.
Key result
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 5000 µg/plate in some cases, only with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS: 
- With metabolic activation: None
- Without metabolic activation: None   
The positive controls were functional.
PRECIPITATION CONCENTRATION: No precipitation was observed.
CYTOTOXIC CONCENTRATION (including effects on background lawn): 
- With metabolic activation only:   
 TA 98, TA 1535 in plate incorporation assay  
 TA 1537 in pre-incubation assay

no other results

Conclusions:
The test item did not induce a mutagenic effect in S. typhimurium TA 98, TA 100, TA 1535 and TA 1537, both in the presence and in the absence of Arochlor-induced liver microsomes . It is therefore not considered to be a bacterial mutagen.
Executive summary:

The test item was tested in the Ames Salmonella mutagenicity test for any mutagenic activity.

Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 were treated with the test compound by the Ames test plate incorporation ss well as the preincubation method.

Dose levels covering the range between 30 and 5000 fLg/plate, in triplicate both with and without the addition of a metabolising system

( Aroclor 1254 ioduced rat liver S9 mix) were employed. All four bacterial strains exhibited mutagenic responses to the appropriate positive control substances.

Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in an acceptable range.

Mutagenic activity of the test compound to one or more of the tester strains was not observed in either experiment with and without metabolic activation. It is therefore concluded, that the test item is not a bacterial mutagen.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-05-27 to 2015-07-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
OPPTS changed its name to OCSPP. The name change does not affect the guideline.
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):
V792 cells were maintained in Dulbecco's modified Eagle-Medium3 supplemented with 10% foetal calf serum4, penicillin (100 U/mL)3 and
streptomycin (100 μg/mL)3 called DMEM-FCS. Cultures were incubated at 37°C in a humidified atmosphere 90%) containing 10% CO2. For
subculturing, a trypsin4 (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used. Exposure to the
test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS)5 which additionally contained
20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid)4 pH 7.4 (PBSHEPES).
The cells were periodically checked for the absence of mycoplasma contamination by using the HOECHST stain 33258. The 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 and an NADPH-generating system (S9 mix) have been successfully used in eukaryotic in vitro systems for the activation of various compounds
Test concentrations with justification for top dose:
156.3, 312.5, 625, 1250 or 2500 µg/mL test item were selected for the experiments without and with metabolic activation (4-hour exposure)
78.1, 156.3, 312.5, 625 or 1250 µg/mL test item for the second experiment without metabolic activation (24-h exposure).
Vehicle / solvent:
Dimethyl sulfoxide (DMSO), a correction factor of 1.06 was used to correct for impurities
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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 (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).


DURATION
- 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 DMSO (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 4 or 5
* Afterwards cells were harvested by trypsinisation and replated at a density of 1 000 000 per 150mm 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).
* Plates were fixed and stained after about 8 days (plating efficiency plates) or 12 days (6-thioguanine plates).
- pH and osmolality measurements
* pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment
pH values: using a digital pH meter type WTW pH 540,
Osmolality: with a semi-micro osmometer
- 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 this preliminary experiment without and with metabolic activation pronounced to complete cytotoxicity and test item precipitation were noted at
concentrations of 1000 µg test item/mL and higher. Hence, 625 or 1250 µg test item per mL were employed as the top concentrations for the
mutagenicity tests without and with metabolic activation, respectively.
Evaluation criteria:
The following pre-determined 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.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
decreased plating efficiency were noted in the absence of metabolic activation: 4 hour exposure: 2500 and 1250 µg/mL; 24-hour exposure: 1250 µg/mL. Slightly decreased plating efficiency were noted with metabolic activation at 2500 µg/mL
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):
The concentrations employed were chosen based on the results of a cytotoxicity study with concentrations of 78.1, 156.3, 312.5, 625, 1250, 2500
and 5000 µg/mL. In this preliminary study cytotoxicity in form of decreased plating efficiency was noted at concentrations starting at 1250 µg/mL in the absence or at 2500 and 5000 µg/mL in the presence of metabolic activation (24-h or 4-h exposure, respectively). No changes in the pH and
osmolality were noted up to concentrations of 5000 µg/mL and higher in both experiments. Hence, the highest concentrations employed in the main study were 2500 µg test iteml/mL medium for a 4-h exposure in the absence and presence of metabolic activation and 1250 µg/mL for a
24-h exposure in the absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 cloneable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6 cloneable cells forand from 130.0 to 2693.3 x 10‑6 cloneable cells for DMBA. Hence, all
the mutation frequencies obtained for test item are within the vehicle control ranges. The test item is therefore negative in the HPRT-V79 mammalian cell mutagenicity test in the absence or presence of metabolic activation.


Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Mutagenicity

Experiments without metabolic activation

The mutation frequency of the vehicle control DMSO was 14.60 and 8.86 x 10‑6 cloneable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 156.3, 312.5, 625, 1250 or 2500 or 78.1, 156.3, 312.5, 625 or 1250 µg test item/mL culture medium (4-h or 24-h exposure, respectively) ranged from 4.38 to 19.46 x 10‑6 cloneable cells. These results are within the normal range of the vehicle controls. In the second experiment (24-h exposure) the mutation frequencies of 19.46 or 18.48 x 10‑6 cloneable cells were noted which were about 2-fold above the mean of the solvent control of 8.86 x 10‑6 cloneable cells. These findings are considered to be coincidental as the mutation frequencies were always lower than 40 x 10-6 , within the background data for the vehicle controls and no concentration response relationship was noted.

Experiments with metabolic activation

The mutation frequency of the vehicle control DMSO was 12.37 and 12.83 x 10-6 cloneable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 156.3, 312.5, 625, 1250 or 2500 µg test item/mL culture medium ranged from 3.33 to 18.33 x 10‑6 cloneable cells. These results are within the normal range of the vehicle controls.

The positive controls in the direct test EMS (ethyl methanesulfonate) and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 443.45 to 1107.78 x 10-6 cloneable cells in the case of EMS and ranging from 436.61 to 1383.08 x 10-6 cloneable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 cloneable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6 cloneable cells forand from 130.0 to 2693.3 x 10‑6 cloneable cells for DMBA. Hence, all the mutation frequencies obtained for test item are within the vehicle control ranges. The test item is therefore negative in the HPRT-V79 mammalian cell mutagenicity test in the absence or presence of metabolic activation.

The followingpH and osmolalitydata of the vehicle control and of all test item formulations in the medium were determined:

Concentration of test item
[µg/mL medium]

pH value

osmolality [mOsmol/kg]

Negative control:exposure medium with 1% DMSO

8.02

460

78.1

8.02

470

156.3

8.12

470

312.5

7.94

470

625

7.96

450

1250

7.94

450

2500

8.01

450

5000

8.07

440

Conclusions:
Under the present test conditions, the test item tested up to cytotoxic concentrations of 1250 or 2500 µg/mL medium (2500 µg/mL is equivalent to 15.6 mM) in the experiments without and with metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.
Executive summary:

The test item was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix.

The test item was completely dissolved in dimethylsulfoxide (DMSO). A correction factor of 1.06 was used to correct for the content of the sum of active ingredients of 94.4%. The vehicle DMSO served as the negative control.

Preliminary cytotoxicity test

The concentrations employed were chosen based on the results of a cytotoxicity study with concentrations of 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg tets item/mL. In this preliminary study cytotoxicity in form of decreased plating efficiency was noted at concentrations starting at 1250 µg/mL in the absence or at 2500 and 5000 µg/mL in the presence of metabolic activation (24-h or 4-h exposure, respectively). Hence, the highest concentrations employed in the main study were 2500 µg test item/mL medium for a 4-h exposure in the absence and presence of metabolic activation and 1250 µg/mL for a 24-h exposure in the absence of metabolic activation.

Main study

In the main study concentrations of 156.3, 312.5, 625, 1250or 2500 µg test item/mL were selected for the experiments without and with metabolic activation (4-hour exposure)and concentrations of 78.1,156.3, 312.5, 625 or 1250µg test item/mL for the second experiment without metabolic activation (24-h exposure).

Cytotoxicity

Cytotoxicityin form of decreased plating efficiencywas noted at the top concentrations in the absence of metabolic activation: 4‑hour exposure: 2500 and 1250 µg/mL; 24-hour exposure: 1250 µg/mL.Only slight signs of cytotoxicityin form of slightly decreased plating efficiency were noted in the first and second experiments withmetabolic activation at 2500 µg/mL of PE1= 0.44 or PE1= 0.34, respectively. In case of no cytotoxicity the highest concentration required by the guidelines is 5 mg/mL or 0.01 M. For relatively non-cytotoxic compounds the maximum concentration should be 5 mg/mL or 0.01 M, whichever is the lowest (OECD Guideline for Testing of Chemicals No. 476, paragraph 16). 0.01 M test item is equivalent to 1602.5µg test item/mL medium. Hence, no testing of concentrations higher than 2500 µg test item/mLwas needed.

Mutagenicity

Experiments without metabolic activation

The mutation frequency of the vehicle control DMSO was 14.60 and 8.86 x 10‑6cloneable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 156.3, 312.5, 625, 1250 or 2500 or 78.1, 156.3, 312.5, 625 or 1250 µg test item/mL culture medium (4-h or 24-h exposure, respectively) ranged from 4.38 to 19.46 x 10‑6cloneable cells. These results are within the normal range of the vehicle controls. In the second experiment (24-h exposure) the mutation frequencies of 19.46 or 18.48 x 10‑6cloneable cells were noted which were about 2-fold above the mean of the solvent control of 8.86 x 10‑6cloneable cells. These findings are considered to be coincidental as the mutation frequencies were always lower than 40 x 10-6, within the background data for the vehicle controls and no concentration response relationship was noted.

Experiments with metabolic activation

The mutation frequency of the vehicle control DMSO was 12.37 and 12.83 x 10-6cloneable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 156.3, 312.5, 625, 1250 or 2500 µg test item/mL culture medium ranged from 3.33 to 18.33 x 10‑6cloneable cells. These results are within the normal range of the vehicle controls.

The positive controls in the direct test EMS (ethyl methanesulfonate) and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 443.45 to 1107.78 x 10-6cloneable cells in the case of EMS and ranging from 436.61 to 1383.08 x 10-6cloneable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6cloneable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6cloneable cells forand from 130.0 to 2693.3 x 10‑6cloneable cells for DMBA. Hence, all the mutation frequencies obtained for test item are within the vehicle control ranges. The test item is therefore negative in the HPRT-V79 mammalian cell mutagenicity test in the absence or presence of metabolic activation.

Conclusion

Under the present test conditions, the test item tested up to cytotoxic concentrations of 1250 or 2500 µg/mL medium (2500 µg/mL is equivalent to 15.6 mM) in the experiments without and with metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

 

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

Genetic toxicity in vivo

Description of key information

The test item shows no clastogenic activity in the in vivo mouse micronucleus assay (Hüls AG, 1996).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-06-24 to 1996-09-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(1992)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Source: Harlan Winkelmann, Borchen (Germany)
- Age: young adults
- Weight at study initiation:   27.8 +/- 5.6 g (males); 25.9 +/- 5.2 g (females)
- No. of animals per dose: 5 males + 5 females per test duration
- Housing: max 5 animals per sex per cage
- Diet (e.g. ad libitum): Ssniff R 10, complete feed for rats, Ssniff Spezialfutter GmbH, 59494 Soest, Germany
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark rhythm
Route of administration:
oral: gavage
Vehicle:
- Vehicle: corn oil
Details on exposure:
ADMINISTRATION: 
- Vehicle: corn oil
- Control groups and treatment:    
negative: vehicle    
positive: 100 mg cyclophosphamide (CPA)/kg bw in 0.9 % aqueous NaCl   additional treated satellite group to replace mortalities
- Total volume applied: 10 ml/kg bw
- Duration of test: 24 hours; 48 hours
- Sampling times and number of samples: 24 hours; 48 hours
Duration of treatment / exposure:
Duration of test: 24 hours; 48 hours
Frequency of treatment:
single dose
Post exposure period:
Sampling times and number of samples: 24 hours; 48 hours
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males + 5 females 
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
positive: 100 mg cyclophosphamide (CPA)/kg bw in 0.9 % aqueous NaCl, oral gavage
Tissues and cell types examined:
EXAMINATIONS: 
- Organs examined at necropsy: femur bone marrow; others not specified in  report approx. 2000 PCE (polychromatic erythrocytes) per animal were analysed  for micronuclei  (5000 in re-evaluation of 48 hour vehicle and test  compound slides from males)
- Clinical observations: yes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- Criteria for selection of M.T.D.: maximum dose <= 2000 mg/kg bw without  mortalities within 48 hours

TREATMENT AND SAMPLING TIMES:
- Animals were killed by cervical dislocation 24 and 48 hours after test compound administration.
- Both femurs were dissected out from each animal, cleared of tissue and one epiphysis removed from each bone.
- The bone marrow was suspended in fetal calf serum (FCS) and erythrocytes were purified by means of a cellulose chromatography column.
- The eluate (3 x 1.5 ml) was centrifuged (5 min, 750 x g) and the pellet was again suspended in FCS/EDTA.

DETAILS OF SLIDE PREPARATION:
- The pure erythrocyte suspension was used to prepare "flat" cells on glass slides by means of a Shandon Cytospin 3 (2 slides per animal).
- Slides were air dried and stained with May­Grunwald/Giemsa.

METHOD OF ANALYSIS:
- The MIAMED image analyzer equipped with the "Micronucleus Test V 4.00" software was used for fully automated slide scoring.
- Where possible, a total of approximately 2000 PCE (i.e. 10,000 PCE per treatment group) were analyzed for micronuclei.
- The corresponding NCE were also scored for micronuclei. In addition, as an indicator for chemical-induced bone marrow toxicity, for each animal the PCE/NCE ratio was
determined on the basis of 2000 PCE scored.

Evaluation criteria:
- Criteria for evaluating results: Statistically significant and  biologically relevant increase in frequency of micronucleated  polychromatic erythrocytes of at least one test group as 
compared to the  negative control group of the same sampling time
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MORTALITY: 
- Phase 1 of dose finding = 2000 mg/kg bw: No mortalities within 48 hours  among 5 males + 5 females. No further phases were required.
- Main test: No mortalities
CLINICAL SIGNS: Predominant signs were hunched posture (only males),  slight sedation (only males), piloerection.
NECROPSY FINDINGS: No necropsy reported.
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: 
- The average PCE/NCE ratio of the positive control groups was  significantly lower than that of the corresponding vehicle controls 
(0.27  +- 0.09 vs 0.83 +- 0.20 for males, 0.32 +- 0.13 vs 0.96 +- 0.29 for  females).   
- The PCE/NCE ratio of the male 48 hour dosed group (0.45 +- 0.14) was  significantly lower than that of the corresponding vehicle control (0.93  +- 0.30).

GENOTOXIC EFFECTS:    
- The micronucleus frequencies of the negative controls were within the  range of historical control data of the performing laboratory.  - For the positive control a significant increase in the frequency of  micronucleated polychromatic erythrocytes was observed 
(4.78 +- 0.88 vs  0.22 +- 0.09 for males; 5.90 +- 1.03 vs 0.06 +- 0.07 for females). - No significant increase in the frequency of micronucleated  polychromatic erythrocytes over the control was found with the females  treated with the test substance. - In males of the 48 hour sampling time a weak but statistically  significant increase was observed (0.33 +- 0.12 vs 0.12 +- 0.10; 0.001 <  p < 0.01).
- Statistical significance was reduced to 0.01 < p < 0.05 and  the increase in micronucleus frequency was reduced to 0.26 +- 0.08 vs  0.16 +- 0.07 by scoring 5000 instead of 2000 PCE. According to Richold et  al. (1990), the "statistical significance should increase with increased  sample size ... if the response is real and not due to sampling error".  Since an opposite trend was observed, and since in addition the final  micronucleus frequency of 0.26 % is well within the range of historical  negative controls of the performing laboratory (0.00 - 0.40 %; 0.20 +-  0.10), the present observation was not considered of being indicative of  a clastogenic activity of the test compound. -The clinical symptoms indicate that the test substance or its metabolites  had reached the blood and hence the target organ, i.e. the bone marrow.  
Additional evidence for that comes from the reduction of the PCE/NCE  ratio in male animals of the 48 hours sampling time, which is indicative  of of bone marrow toxicity.

Treatment Sex Time % Micron PCE/NCE
in PCE
2000 T.S.   m    24 h    0.25 +- 0.07     0.60 +- 0.26
2000 T.S.   m    48 h    0.33 +- 0.12 **  0.45 +- 0.14 **
2000 T.S.   m    48 h    0.26 +- 0.08 * (1)
2000 T.S.   f    24 h    0.16 +- 0.19     0.76 +- 0.21
2000 T.S.   f    48 h    0.14 +- 0.10     1.21 +- 0.23  
Vehicle    m    24 h    0.22 +- 0.09     0.83 +- 0.20  
Vehicle    m    48 h    0.12 +- 0.10     0.93 +- 0.30  
Vehicle    m    48 h    0.16 +- 0.07   (1)  
Vehicle    f    24 h    0.06 +- 0.07     0.96 +- 0.29  
Vehicle    f    48 h    0.13 +- 0.08     1.47 +- 0.68  
100 CPA    m    24 h    4.78 +- 0.88 *** 0.27 +- 0.09 ***  
100 CPA    f    24 h    5.90 +- 1.03 *** 0.32 +- 0.13 ***
--------------------------------------------------------
T.S. = test substance (trimethylhexanediol; mg/kg bw)
CPA = cyclophosphamide (mg/kg bw)
* p < 0.05; ** p < 0.01; *** p < 0.001
(1) based on 5000 PCE/animal; other data based on 2000 PCE/animal

no other information

Conclusions:
From the results obtained, it is concluded, that the test item shows no clastogenic activity in this in vivo mouse micronucleus assay.
Executive summary:

In the in vivo mouse micronucleus assay, test item was tested for its potential to induce micronuclei in polychromatic erythrocytes (PCE) of NMRI mice.

In a preliminary toxicity test, 2000 mg/kg of test item were determined as the maximum tolerable dose.

In the main study, 2000 mg of test item/kg bodyweight were administered to male and female mice as a single oral dose (gavage). The negative control group received the vehicle, corn oil.

Animals of the positive control group were administered cyclophosphamide, at 100 mg/kg bodyweight.

Administration of each control substance was done by oral gavage, too. Five animals/sex/dose/sampling time were employed. Erythrocyte preparations were obtained from the negative and test compound groups at two sampling times, 24 and 48 hours after dosing. Erythrocytes from the positive control group were prepared 24 hours after dosing only. Slides were screened with an automatic image analyzer (LEITZ Miamed). One slide per animal was examined for the presence of micronuclei in at least 2000 PCEs (5000 PCEs in a re-evaluation of vehicle and test compound slides from the 48 hrs sampling time).

The ratio of PCE to normochromatic erythrocytes (NCE) as well as the incidence of micronucleated NCE was determined in parallel. At both sampling times, male and female mice treated with test item did not reveal biologically significant increases in the frequencies of micronucleated PCE.

The positive control compound, cyclophosphamide, induced highly significant increases in the frequency of micronucleated PCE, demonstrating the sensitivity of the test system.

From the results obtained, it is concluded, that the test item shows no clastogenic activity in this in vivo test system.

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

Additional information

Justification for classification or non-classification

According to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008 and based on the results of the in vitro and in vivo genotoxicity studies the test substance is not classified.