Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial mutagenicity test (Ames test according to OECD 471), an in vitro mammalian cell genotoxicity test (HPRT test according to OECD 476) and an in vitro micronucleus test (according to OECD 487) are available for the test item. Based on results of the studies the test substance showed no genotoxic properties.

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:
2016-03-04 to 2016-03-17
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:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
march 2005. OPPTS changed its name to OCSPP,
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
mutated gene loci resposible for histidine auxotropy
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- obtained from Trinova Biochem according to Dr. Bruce N. AMES,
Additional strain / cell type characteristics:
other: histidine auxotroph
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced rat liver S9; male rats, obtained from Trinova Biochem
Test concentrations with justification for top dose:
Plate incorporation test: 10.0, 31.6, 100, 316, 1000 or 3160 µg per plate;
Preincubation test: 10.0, 31.6, 100, 316, 1000 or 3160 µg per plate;
Vehicle / solvent:
The test item was completely dissolved in dimethylsulfoxide (DMSO) . The vehicle dimethylsulfoxide (DMSO) served as the negative control. Fresh
preparations of the test item were used for the treatment in all experimental parts.
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, Mitomycin C in DMSO for TA 102
Remarks:
with metabolic activation: 2-aminoanthracene in DMSO for TA 100, TA 1535, Benzo(a)pyrene in DMSO for TA 98, TA 102, TA 1537
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,
- The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100.- Ten concentrations ranging from 0.316 to 5000 µg/plate were tested.
- Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted at concentrations of
3160 and 5000 µg/plate. Hence, 3160 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction.
ADMINISTRATION
- Dosing:
* Plate incorporation test: 10.0, 31.6, 100, 316, 1000 or 3160 µg per plate;
* Preincubation test: 10.0, 31.6, 100, 316, 1000 or 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 highly purified water 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
* Mitomycin C in in highly purified water for TA 102, 10 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100 and TA 1535, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, TA 102 and 1537, 10 µg/plate
- negative control: the vehicle DMSO was used as negative reference item (all test strains).
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

NUMBER OF REPLICATIONS: 3 per concentration and experiment

NUMBER OF CELLS EVALUATED: approximately 10E8 viable cells in the late exponential or early stationary phase

DETERMINATION OF CYTOTOXICITY
- Method: In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce
background lawn and reduction of the number of revertants) was noted at the top concentration of 3160 µg test item/plate in all test strains.
Evaluation criteria:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent
experiments.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation
coefficient may be applied.
- 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.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Acceptance Criteria
The results of the negative and positive control cultures have to be within the range of the historical data generated by LPT.

Statistics:
According to the OECD Guideline 471, a statistical analysis of the data is not mandatory
Key result
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:
Ccytotoxicity was noted at the top concentration of 3160 µg test item/plate in all strains
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Remarks:
see Vehicle
Positive controls validity:
valid
Additional information on results:
GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ

CYTOTOXICITY EFFECTS:
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce background
lawn and reduction of the number of revertants) was noted at the top concentration of 3160 µg test item/plate in all test strains.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

see attchached document

Conclusions:
In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 3160 µg/plate, caused no mutagenic effect
in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
Executive summary:

The purpose of this study was to evaluate the test item for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and (1983).

The potential of the test item to induce gene mutations was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 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.

The test item was completely dissolved indimethylsulfoxide (DMSO).The vehicle DMSOserved as the negative control.

Preliminary test

Test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted at concentrations of 3160 and 5000 µg/plate. Hence, 3160 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 10.0 to 3160 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation

Cytotoxicity

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 3160 µg test item/plate in all test strains.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to acytotoxic

concentration of 3160 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system

In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 3160 µg/plate, caused no

mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 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:
2016-02-22 to 2016-05-26
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)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Qualifier:
according to guideline
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. QA statement)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
The test item was completely dissolved in dimethylsulfoxide (DMSO)
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 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 fraction (S9 fraction) from rats treated with Aroclor 1254, prepared according to MARON and AMES was obtained from Trinova Biochem . S9 was collected from male rats.
Test concentrations with justification for top dose:
12.5, 25, 50, 75 and 100 µg test item/mL meedium were selected for the experiments without metabolic activation
100, 200, 400, 600 and 800 µg test item/mL medium were selected for the experiments with metabolic activation.
Vehicle / solvent:
Test item was completely dissolved in dimethylsulfoxide (DMSO). The solvent DMSO served as the negative control. Fresh preparations of the test item were used for the treatment in all experimental parts.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
CELLS AND TISSUE CULTURE MEDIA
- V79 cells were maintained in Dulbecco's modified Eagle-Medium supplemented 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), on day 1 of the experiment, approximately 1500000 cells were seeded in 30 mL DMEM-FCS per 150 mm diameter dish.
- Exposure duration:
* 4 hours, in the absence and presence of S9 mix, the cells were exposed to the test item in DMEM-FCS for 4 hours.
* In the experiments with S9 mix, the medium was replaced by 18 mL S9 mix. The solvent control was treated with DMSO in the same way. In addition, positive controls were employed.
* the negative control was treated with DMSO (the vehicle) in the same way
* At the end of the exposure period, the cells were washed and trypsinised and the relative plating efficiency (PE1) was determined for each dose to obtain an accurate measure of the toxic effect of the chemical.
- Expression time (cells in growth medium):
* . For the determination of the plating efficiency (PE1), cells were seeded on three replicate plates (60 mm diameter) with a known number of cells.
* Remaining cells were replated and the culture incubation continued until day 8 with 30 mL normal DMEM-FCS including cell passage on day 5. This period was required for expression of the new genotype, i.e. for sufficient dilution and catabolism of the previously expressed HPRT.
* 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 (plating efficiency plates. PE2) or 12 days (6-thioguanine plates).
- pH and osmolality measurements
* The pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
pH values: using a digital pH meter ,
Osmolality: with a semi-micro osmometer .

- Positive control:
* ethyl methanesulphonate (EMS) in mutagenicity experiments in the absence of exogenous metabolic activation;
* 9,10-dimethyl-1,2-benzanthracene (DMBA) in the S9 mix supplemented assays. This compound is mutagenic in V79 cells in the presence, but not in the absence of S9 mix.
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
- The concentrations employed in the main experiment were chosen based on the results of a preliminary cytotoxicity test with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL.
- In this preliminary test pronounced cytotoxicity in form of decreased plating efficiency was noted at concentrations of 100 µg/mL and higher in the experiment without metabolic activation and at 1000 and 2000 µg/mL in the experiment with metabolic activation.
- Test item precipitation was noted at concentrations of 1000 and 2000 µg/mL medium in both experiments.
- No changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO.
- Hence, the highest concentrations employed in the main study were 100 µg test item/mL medium in the absence and 800 µg/mL medium in the presence of metabolic activation.
Rationale for test conditions:
For relatively non-cytotoxic items the maximum concentration should be 2 mg/mL, 2 µL/mL or 0.01 M, whichever is the lowest, when not limited by solubility in the solvent or culture medium, or cytotoxicity.
Where there is cytotoxicity (tested in the pre-test), these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean that the concentration levels should be separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it should result in approximately 10 - 20% (but not less than 10%) relative survival (relative cloning efficiency) or relative total growth. Relatively insoluble items will be tested up to or beyond their limit of solubility under culture conditions.
The concentrations employed in the main experiment were chosen based on the results of a preliminary cytotoxicity test with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL. In this preliminary test pronounced cytotoxicity in form of decreased plating efficiency was noted at concentrations of 100 µg/mL and higher in the experiment without metabolic activation and at 1000 and 2000 µg/mL in the experiment with metabolic activation. Test item precipitation was noted at concentrations of 1000 and 2000 µg/mL medium in both experiments.
Test item was completely dissolved in dimethylsulfoxide (DMSO)
Evaluation criteria:
EVALUATION OF RESULT
Individual plate counts for the test item and controls are presented for both mutation induction and survival. Mutation frequency is expressed as number of mutants per number of surviving cells. Plating efficiencies (PE1 and PE2) and relative survival (RS) are presented in the tables.
The reported data, analysed data and the methods of calculation are listed below:
Results of determination of survival following exposure to the test item, expressed as Plating Efficiency 1 (PE1), are calculated using the following equations:

Plating Efficiency step Mean of the number of clones/culture dish /
Mean of the number of cells plated/culture dish

Relative PE1 (treated culture) /
PE1 (control culture)

Results of determination of survival following incubation for selection of mutants, expressed as Plating Efficiency 2 (PE2), are calculated using the following equations:

Plating Efficiency step Mean of the number of clones/culture dish/
Mean of the number of cells plated/culture dish

Results of determination of mutant frequency per 106 cells (MF/106) are calculated with the following equation:

Mean of mutant cells /
Mutant Frequency PE2

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:
Cytotoxicity in form of decreased plating efficiency was noted at 75 and 100 μg/mL medium in the absence of metabolic activation and at 600 and 800 μg/mL medium in the presence of 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):
The concentrations employed in the main experiment were chosen based on the results of a preliminary cytotoxicity test with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL. In this preliminary test pronounced cytotoxicity in form of decreased plating efficiency was noted at concentrations of 100 µg/mL and higher in the experiment without metabolic activation and at 1000 and 2000 µg/mL in the experiment with metabolic activation. Test item precipitation was noted at concentrations of 1000 and 2000 µg/mL medium in both experiments. No changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO. Hence, the highest concentrations employed in the main study were 100 µg test item/mL medium in the absence and 800 µg/mL medium in the presence of metabolic activation.




Remarks on result:
other:

Criteria for assay acceptance

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 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 solvent controls without metabolic activation and 6 to 46 per 106survivors in solvent controls with metabolic activation. The background data obtained atLPTare given at the end of this chapter . The spontaneous mutation frequency may vary 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 (seeText table 4.8-1).The mutation frequencies of the solvent controls andthe positive controlswithout and with metabolic activation for the last 21 experiments (most recent background data, not audited by the QAU-department) are given as follows:

Mutation frequency per 106cloneable cells

 

Without metabolic activation

(4-h exposure)

With metabolic activation

(4-h exposure)

Solvent control (n = 58)

mean

15.7

15.3

SD

9.6

7.7

range

6.8 - 47.3

5.7 - 46.7

Positive control (µg/mL)

 

EMS

(600)

EMS

(700)

DMBA

(20)

DMBA

(30)

mean

775.2

746.2

769.8

737.9

SD

313.4

351.5

324.9

307.2

range

378.4 – 1537.4

341.8 – 1506.7

195.7 – 1610.7

144.3 – 1545.9

SD     = Standard deviation

EMS   = ethyl methanesulfonate

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

If in both independent experiments solvent and positive controls show results within the range of historical control data and if the test item induces mutation frequencies within the range of historical control data obtained for the solvent controls, and if at least1000000 cells per condition have been evaluated, the item is considered as negative in the test.

In case of a dose-dependent or reproducible increase in mutation frequency exceeding the range of historical control data, the test item is considered as positive in the test.

pH values and osmolality

Following pH and osmolality data of the negative control and of the test item formulations in the medium were determined:

Text table5-1pH values and osmolality

 

Concentration of TEST item
[µg/mL medium]

pH value

Osmolality [mOsmol/kg]

Medium

8.08

320.0

Negative control

8.03

381.0

3.16

8.12

460.0

10.0

8.14

461.0

31.6

8.12

470.0

100

8.08

461.0

316

8.08

460.0

1000

7.78

450.0

2000

7.71

450.0

 

No relevant changes in pH or osmolality compared to the solvent control were noted.


Conclusions:
Under the present test conditions,the test item tested up to cytotoxic concentrations (4 hour exposure, 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 its 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 in the experiments without and with S9 mix.

The test item was completely dissolved in dimethylsulfoxide (DMSO). The solvent DMSO served as the negative control.Fresh preparations of the test item were used for the treatment in all experimental parts.

Preliminary cytotoxicity test

The concentrations employed in the main experiment were chosen based on the results of a preliminary cytotoxicity test with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL. In this preliminary test pronounced cytotoxicity in form of decreased plating efficiency was noted at concentrations of 100 µg/mL and higher in the experiment without metabolic activation and at 1000 and 2000 µg/mL in the experiment with metabolic activation. Test item precipitation was noted atconcentrations of 1000 and 2000 µg/mL medium in both experiments. No changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO. Hence, the highest concentrations employed in the main study were 100 µg test item/mL medium in the absence and 800 µg/mL medium in the presence of metabolic activation.

Main study

In the main study concentrations of 12.5, 25, 50, 75 and 100 µg test item/mL were selected for the experiments without metabolic activation and concentrations of 100, 200, 400, 600 and 800 µg/mL medium for the experiments with metabolic activation.

Cytotoxicity

Cytotoxicity in form of decreased plating efficiencywas noted at 75 and 100 µg/mL mediumin the absence of metabolic activationandat 600 and 800 µg/mL mediumin the presence of metabolic activation.

 

Mutagenicity

Experiments without metabolic activation

The mutation frequency noted for the solvent control DMSO was 35.13 and 42.98 x 10-6 cloneable cells. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 12.5, 25, 50 and 75 µg test item/mL culture medium ranged from 18.75 to 41.85 x 10-6

cloneable cells. These results are within the normal range of the solvent controls. The top concentration of 100 µg/mL medium was almost completely cytotoxic, and, hence, not used for evaluation.

 

Experiments with metabolic activation

The mutation frequency noted for the solvent control DMSO was 14.81 and 39.23 x 10-6 cloneable cells. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 100, 200, 400 and 600 µg test item/mL culture medium ranged from 12.47 to 44.24 x 106cloneable cells. These results are within the normal range of the solvent controls. The top concentration of 800 µg/mL medium was almost completely cytotoxic, and, hence, not used for evaluation.

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 506.05 to 839.11 x 10-6 cloneable cells in the case of EMS and ranging from 446.03 to 744.91 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 5.7 to 47.3 x 10-6 cloneable cells for the solvent controls. The mutation frequency of the positive controls at LPT ranges from 341.8 to 1537.4 x 10-6 cloneable cells for EMS and from 144.3 to 1610.7 x 10-6 cloneable cells for DMBA.

 

Conclusion

Under the present test conditions,the test item tested up to cytotoxic concentrations (4 hour exposure, 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:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-01-28 to 2013-05-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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
Specific details on test material used for the study:
The test item was completely dissolved in dimethylsulfoxide (DMSO).
Target gene:
mammalian cell system( Chinese hamster Ovary cells)
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
Human peripheral blood was obtained by venipuncture from young (approximately 18 – 35 years of age), healthy, non-smoking male or female individuals with no known recent exposures to genotoxic chemicals or radiation.
Metabolic activation:
with and without
Metabolic activation system:
Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
without S9-mix: 6.25, 12.5, 25, 50 and 100 or 2.5, 5, 10 and 20 µg/mL
with S9-mix: 12.5, 25, 50 and 100 µg/mL
Vehicle / solvent:
The test item was completely dissolved in dimethylsulfoxide (DMSO) .
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
aneugen
Positive control substance:
cyclophosphamide
mitomycin C
other: colchicine
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Species/cell type: Human peripheral blood was obtained by venipuncture from young (approximately 18 – 35 years of age), healthy, non-smoking male or female individuals with no known recent exposures to genotoxic chemicals or radiation, and collected in heparinised vessels.
- Metabolic activation system: male rat liver S9 from  Aroclor 1254 induced animals
ADMINISTRATION: 
- Solubility: The test item was completely dissolved in dimethylsulfoxide (DMSO) . The vehicle DMSO served as the negative control. Fresh preparations of the test item were prepared on the day of the experiment and used for the treatment in all experimental parts.
- Preliminary experiment: without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg test item/mL medium were employed. Test item precipitation was noted starting at the concentration of 316 µg/mL in both experiments. In the experiment without S9 mix complete cytotoxicity was noted at concentrations of 31.6 µg/mL medium and higher (24-hour exposure). In the experiment with S9 mix cytotoxicity was noted at concentrations of 100 µg/mL medium and higher (4-hour exposure).
Hence, 100 µg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation for a 4-hour exposure and 20 µg/mL for the experiment without S9 mix with a 24-hour exposure.
- Dosing:  
Without metabolic activation:
4-h exposure 6.25, 12.5, 25, 50 and 100 µg/mL medium;
24-h exposure: 2.5, 5, 10 and 20 µg/mL medium;
With metabolic activation:
4-h exposure: 12.5, 25, 50 and 100 µg/mL medium.
- Positive and negative control groups and treatment:    
negative/solvent: DMSO
positive, clastogen (+S9): cyclophosphamide in aqua ad iniectabilia, c = 10 µg/mL and c = 20 µg/mL
positive, clastogen (-S9): mitomycin C in aqua ad iniectabilia, c = 0.1 µg/mL and c = 0.2 µg/mL
positive, aneugen (-S9): colchicine in aqua ad iniectabilia, c = 0.01 µg/mL and c = 0.020 µg/mL

TREATMENT SCHEDULE:
- 0.5 mL of freshly prepared blood lymphocytes were seeded with 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.
- After initiation appropriate concentration of the test item in the vehicle were added to the cell cultures for each target concentration of the test item in the test medium and each experiment.
- Precipitation of the test item was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment.
- Theoretical considerations, together with published data, indicate that 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 cell cycles.
- Cells were sampled at a time equivalent to about 1.5 times the normal (i.e. untreated) cell cycle length either after the beginning or at the end of treatment.
- Sampling or recovery times would have been extended if it is known or suspected that the test item affects the cell cycling time (e.g. when testing nucleoside analogues). Because of the potential cytotoxicity of S9 preparations for cultured mammalian cells, an extended exposure treatment was used only in the absence of S9.
- All treatments were conducted while the cells were growing exponentially.

DURATION:
Cell treatment and harvest times for the used human lymphocytes line:
Without S9 mix: 4-hour exposure and 24-hour exposure
- 0.5 mL of freshly prepared blood lymphocytes were seeded with 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.
- After 48 hours the cultures were centrifuged (10 minutes at 800 – 900 rpm) and the medium was replaced by 4.95 mL of fresh Ham’s F10 medium with fetal calf serum (FCS).
- Five concentrations of 6.25, 12.5, 25, 50 or 100 µg test item/mL were employed. The test item treatments and the controls were added at a volume of 50 µL to obtain the corresponding target concentrations.
- The cultures were then incubated for 4 hours and 24 hours at +37°C.
- Afterwards the medium was removed and the cultures were washed twice with Ham’s F10 medium.
- After addition of 5 mL Chromosome medium containing 5 µg/mL Cytochalasin B the cultures were incubated for further 20 hours at 37°C.

With S9 mix: 4-hour exposure
- 0.5 mL of freshly prepared blood lymphocytes were seeded with 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.
- After 48 hours the cultures were centrifuged (10 minutes at 800 – 900 rpm) and the medium was carefully removed and replaced by 4.45 mL Ham’s F10 medium with FCS and 0.5 mL S9 Mix.
- Four concentrations of 12.5, 25, 50 or 100 µg test item/mL were employed. The test item treatments and the controls were added at a volume of 50 µL to obtain the corresponding target concentrations.
- The cultures were then incubated for 4 hours at +37°C. Afterwards the medium was removed and the cultures were washed twice with Ham’s F10 medium.
- After addition of 5 mL Chromosome medium containing 5 µg/mL Cytochalasin B the cultures were incubated for further 20 hours at 37°C.

see tables below

STAIN (for cytogenetic assays):
- Each culture was harvested and processed separately.
- After the test item incubation, mitotic activity was arrested by the addition of CytoB to each culture at a final concentration of 5 µg/mL.
- After an additional incubation of 20 hours the cultures were centrifuged for 10 minutes at 800 rpm, the supernatant was discarded and the cells resuspended in KCl (0.56%).
- After incubation for 17 minutes at 37°C, the cell suspensions were centrifuged for 10 minutes at 800 rpm. The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added.
- The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm.
- The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette.
- Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide and left to air-dry at room temperature.
- The slides were then stained using 10% Giemsa.


NUMBER OF REPLICATIONS: Two replicate cultures were used for each test item concentration and for the vehicle and positive 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:
At least 500 cells per replicate cell culture (two cultures per concentration in the main study, one culture per concentration in the preliminary test) were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity.
Thus, an RI of 53% means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53% of this number divided in the treated culture, i.e. 47% cytostasis

OTHER EXAMINATIONS:
- The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration). If substantially fewer than 1000 binucleate cells per culture are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test would be repeated using more cells, or at less toxic concentrations, whichever is appropriate. Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.

- pH values and osmolality measurements
The pH and osmolality of the negative control and all test item formulations in the medium of the preliminary experiment were determined employing the methods given below:
pH values: using a digital pH meter type SevenCompact s’210 .
Osmolality: with a semi-micro osmometer13F .


Rationale for test conditions:
The concentrations employed were chosen based on the results of a cytotoxicity study (see above).
Evaluation criteria:
The assay demonstrates its ability to reliably and accurately detect substances of known aneugenic and clastogenic activity, with and without
metabolic activation.
Acceptance of a test is based on the following criteria:
• The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database (Poisson-based 95% control limits). Where concurrent negative control data fall outside the 95% control limits, they may be acceptable for inclusion in the historical control data as long these data are not extreme outliers.
• Concurrent positive controls induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
• Adequate number of cells, cell proliferation criteria and concentrations are analysable and are consistent with those described in Exosure Concentrations

Vehicle control and untreated cultures give reproducibly low and consistent micronucleus frequencies. Data from vehicle and positive controls are used to establish historical control ranges. These values are used in deciding the adequacy of the concurrent vehicle controls or positive controls for an experiment.
Statistics:
Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction. Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures were determined. Individual culture data were provided.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
• the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
• any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).
The test chemical is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Key result
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity was noted at the top concentration of 100 µg test item/mL medium in the experiments without and with metabolic activation (4-hour exposure) and at 20 µg/mL medium in the 24-hour exposure experiment in the absence of metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The results for the vehicle controls were within historical control range.

The following pH and osmolality data of the vehicle control and of all test item formulations in the medium were determined in a preliminary test:

Concentration of
Test item
 

[µg/mL medium]

pH value

osmolality [mOsmol/kg]

Medium

7.90

302.5

0, vehicle control

7.58

430.0

3.16

7.66

440.0

10

7.66

440.0

31.6

7.63

440.0

100

7.61

445.0

316

7.5

440.0

1000

7.34

430.0

2000

7.13

425.0

 

No relevant changes in pH or osmolality of the test item formulations at concentrations of 3.16 to 2000 µg/mL medium were noted.

Conclusions:
Under the present test conditions, the test item tested up tocytotoxic concentrations 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 chromosomal damage in the in vitro micronucleus test.
The results for the vehicle controls were within historical control range.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.
 
Executive summary:

The in vitro micronucleus assay is a genotoxic test system for the detection of chemicals which induce the formation of small membrane bound DNA fragments i.e. micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes, which are unable to migrate with the rest of the chromosomes during the anaphase of cell division.

The purpose of the micronucleus assay is to detect those agents, which modify chromosome structure and segregation in such a way as to lead to induction ofmicronuclei in interphase cells.

Test samples of the test item were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a ratliver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.

The test item was completely dissolved in dimethylsulfoxide (DMSO). The vehicle DMSO served as the negative control. No correction factor was used.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg test item/mL medium were employed. Test item precipitation was noted starting at the concentration of 316 µg/mL in both experiments.In the experiment without S9 mix complete cytotoxicity was noted at concentrations of 31.6 µg/mL medium and higher (24-hour exposure). In the experiment with S9 mix cytotoxicity was noted at concentrations of 100 µg/mL medium and higher (4-hour exposure).Hence, 100 µg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation for a 4-hour exposure and 20 µg/mL for the experiment without S9 mix with a 24-hour exposure.

In the main study cytotoxicity was noted at the top concentration of 100 µg test item/mL medium in the experiments without and with metabolic activation (4-hour exposure) and at 20 µg/mL medium in the 24-hour exposure experiment in the absence of metabolic activation.

Mitomycin C (at 0.2 µg/mL) and colchicine (at 0.02 µg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 µg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of 6.25, 12.5, 25, 50 and 100 or 2.5, 5, 10 and 20 µg test item/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 2.0 to 5.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentrations of 100 or 20 µg/mL medium, respectively. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies.In this test the following frequencies were observed: vehicle control: 3.5 or 4.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24-hour exposure, respectively. The vehicle result was within the historical control ranges.

In the positive control cultures the micronucleus frequencies were increased to 23.5 or 16.5 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively.This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of 12.5, 25, 50 and 100 µg test item/mL medium(4-h exposure) in the presence of metabolic activation ranged from 3.0 to 4.5 micronucleated cells per 1000 binucleated cells.There was no dose-related increase in micronuclei upto the top concentration of 100 µg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a mean frequency of 4.5 micronucleated cells per 1000 binucleated cells was observed. The vehicle result was within the historical control ranges.

In the positive control culture the micronucleus frequency was increased to 18.0 micronucleated cells per 1000 binucleate cells for the 4-hour exposure.This demonstrated that cyclophosphamide induced significant chromosomal damage.


Conclusion

Under the present test conditions, the test item tested up tocytotoxic concentrations 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 chromosomal damage in the in vitro micronucleus test. The results for the vehicle controls were within historical control range.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.  

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

Genetic toxicity in vivo

Description of key information

No studies with respect to this endpoint have been performed.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro studies

A bacterial mutagenicity test (Ames test according to OECD 471), an in vitro mammalian cell genotoxicity test (HPRT test according to OECD 476) and an in vitro micronucleus test (according to OECD 487) are available for the test item.

Under the present test conditions, the test item tested up to a cytotoxic concentration of 3160 µg/plate, caused no mutagenic effect

in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation (LPT, 2016).

Furthermore, the test item tested up to cytotoxic concentrations (4 hour exposure, without and with metabolic activation) was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects (LPT, 2016).

The test item tested up to cytotoxic concentrations 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 chromosomal damage in the in vitro micronucleus test.

The results for the vehicle controls were within historical control range. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid (LPT, 2016).

Conclusion:

Three studies (Ames-Test, in vitro MN test, HPRT-Test) were conducted to examine potential genotoxic effects of the test substance. These studies are guideline studies with Klimisch score 1 (reliable without restrictions). Hence all of these studies are relevant for the assessment of the genotoxic potential of the substance.

In vivo studies

No studies with respect to this endpoint have been performed.

Justification for classification or non-classification

The test item showed no genotoxic properties in three in vitro studies. Based on these results it is concluded that the test substance is not genotoxic and therefore must not be classified according to the criteria of EC Regulation 1272/2008.