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Genetic toxicity in vitro

Description of key information

The test article was not mutagenic in two Ames tests and in an HPRT study and was not clastogenic in an in vitro chromosome aberration and an in vitro micronucleus study.

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Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Apr 2015 - 11 Sep 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL) and 1% (v/v) amphotericine B (250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitaland β-naphthoflavone induces rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment
4 hours exposure, 24 hours harvest time, without S9 mix 0; (3.13); 6.25; 12.50; 25.00; (50.00); (100.00) μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix 0; (3.13); 6.25; 12.50; 25.00; 50.00; (100.00) μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix 0; (3.13); 6.25; 12.50; 25.00; (50.00); (100.00) μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix 0; 3.13; 6.25; 12.50; 25.00; (50.00); (100.00) μg/mL
Concentratons in parantheses were not evaluated.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which had been demonstrated to be suitable in the in vitro micronucleus test and for which historical control data are available. The final concentration of the vehicle DMSO in culture medium was 1% (v/v).
Untreated negative controls:
yes
Remarks:
only for 24/24 hrs; culture medium w/o DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
without S9: EMS; with S9: CPP
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 hrs
- Expression time (cells in growth medium): 20 and 40 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 44 hrs

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) and propidium iodide

NUMBER OF REPLICATIONS:
Two independent experiments were carried out and at least 2 cultures were prepared per test group.

NUMBER OF CELLS EVALUATED:
At least 1000 cells per culture were evaluated for the occurrence of micronucleated cells.

DETERMINATION OF CYTOTOXICITY
- Method: cell count, proliferation index

Evaluation criteria:
A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions. If the results of this test were statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01 or S) have been printed in the tables.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance treatment
- Effects of osmolality: not influenced by test substance treatment
- Precipitation: No precipitation of the test substance in culture medium was observed.
- Other confounding effects: In the absence of metabolic activation cell attachment/morphology was adversely influenced (grade > 2) at 50 μg/mL and above in the 1st Experiment and at 25 μg/mL and above in the 2nd Experiment. Besides, in the presence of metabolic activation cell attachment/morphology was adversely influenced at 100 μg/mL in the 1st Experiment and at 50 μg/mL and above in the 2nd Experiment. No slides were prepared due to strongly reduced cell numbers at the highest applied test substance concentration of 100 μg/mL in all experimental parts. The slides were not scorable for cytogenetic damage due to strong cytotoxicity and/or poor quality in the 1st Experiment in the absence of S9 mix at 50 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
In the pretest the pH value was not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations tested. In addition, a solution of the test substance in the vehicle DMSO was obtained in the stock preparation (Test group: 2000 μg/mL). In culture medium test substance precipitation occurred at 250 μg/mL and above at the end of treatment under all experimental conditions. After 4 hours or 24 hours treatment in the absence and presence of S9 mix cytotoxicity indicated by reduced RPD of below 40 - 50% was observed at 62.5 μg/mL and above

COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.1 – 0.7% micronucleated cells) were close to the concurrent vehicle and negative control values (0.2 – 1.0% micronucleated cells) and clearly within the range of the 95% control limit of our historical negative control data (0.0 - 1.0% micronucleated cells).
The positive control substances EMS (without S9 mix; 400 or 500 μg/mL) and CPP (with S9 mix; 0.5 μg/mL) induced statistically significant increased micronucleus frequencies in both independently performed experiments. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (2.1 – 4.9% micronucleated cells) was clearly above the range of our historical negative control data (0.1 - 1.5% micronucleated cells) and close to our historical positive control data range (2.3 – 13.8% micronucleated cells).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
RPD (relative population doubling): In both main experiments in the absence and presence of S9 mix cytotoxicity indicated by reduced RPD of below 50% of control was observed at least at the highest applied test substance concentration. These values were calculated based on cell numbers determined at the end of each experiment. In detail, in the absence of S9 mix reduced RPD was obtained from 50 μg/mL onward after 4 and 24 hours exposure, respectively (-20.3% or -59.5%, respectively). Besides, in the presence of S9 mix RPD was decreased after 4 hours exposure with 100 μg/mL (-174.4%) at 24 hours preparation interval in the 1st Experiment and from 50 μg/mL onward (-46.2%) at 44 hours preparation interval in the 2nd Experiment.
CBPI (cytokinesis-block proliferation index): Distinctly reduced proliferative activity indicated by elevated cytostasis of above 50% was not observed in any test group scored for the occurrence of micronuclei. In detail, in the 1st Experiment in the absence of metabolic activation the highest scorable concentration of 50 μg/mL led to a cytostasis of 43.6%. Due to strong cytotoxicity indicated by severe cell loss no slides could be prepared at the next higher concentration of 100 μg/mL. In the 2nd Experiment in the absence of S9 mix after 24 hours continous exposure the highest scored concentration led to a slight reduction of CBPI indicated by 8.6% cytostasis. In addition, in the presence of metabolic activation in the 1st Experiment at 24 hours preparation interval no cytostasis was observed after treatment up to 25 μg/mL (-0.4%). But, due to strong cytotoxicity the slides of the next higher concentration of 50 μg/mL were not scorable. Besides, in the 2nd Experiment in the presence of S9 mix the highest scorable concentration of 25 μg/mL led to a cytostasis of 40.2%. Due to severe cytotoxicity indicated by RPD at the next higher concentration of 50 μg/mL the slides were not scored.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary of Results

            Cytotoxicity
Exp. Exposure/
Preparation interval
Test groups S9 mix Prec.* Genotoxicity Micro- nucleated
cells**
[%]
Proliferation index cytostasis (CBPI)
[%]
Relative population doubling (RPD)
[%]
1 4/24 hrs Vehicle control1 - n.d. 0.4 0 100
3.13 µg/mL - - n.d.  n.d. 98.6
6.25 µg/mL - - 0.7 -3.9 99.7
12.50 µg/mL - - 0.3 -1.3 93.1
25.00 µg/mL - - 0.2 -0.4 75.7
50.00 µg/mL - - n.s. n.s. -20.3
100.00 µg/mL  - - n.p. n.p. -219.2
Positive control2 - n.d. 2.1S 1.6 91
2 24/24 hrs Vehicle control1 - n.d. 0.3 0 100
Negative control - - 1 26.8 109.9
3.13 µg/mL - - n.d.  n.d.  108.4
6.25 µg/mL - - 0.2 5.3 106.4
12.50 µg/mL - - 0.2 14 95.6
25.00 µg/mL - - 0.1 40.2 75.6
50.00 µg/mL - - n.d. n.d. -59.5
100.00 µg/mL  - - n.p. n.p. -239.4
Positive control2  - n.d. 0.4 -26.2 122.6
Positive control3 - n.d. 2.8S 11.3 130.8
1 4/24 hrs Vehicle control1 + n.d. 0.2 0 100
3.13 µg/mL + - n.d. n.d.  89.9
6.25 µg/mL + - 0.5 1.4 100.4
12.50 µg/mL + - 0.6 5 99.3
25.00 µg/mL + - 0.4 7.7 102.9
50.00 µg/mL + - 0.3 43.6 75.4
100.00 µg/mL  + - n.p.  n.p. -174.4
Positive control4 + n.d. 4.0S 31.9 101.2
2 4/44 hrs Vehicle control1 + n.d. 0.2 0 100
3.13 µg/mL + - 0.4 1.1 94.3
6.25 µg/mL + - 0.4 -3.5 64.1
12.50 µg/mL + - 0.4 5 70.5
25.00 µg/mL + - 0.5 8.6 17.6
50.00 µg/mL + - n.d. n.d. -46.2
100.00 µg/mL + - n.p.  n.p. -308.7
Positive control4 + n.d. 4.9S -19 92.3

*       Precipitation in culture medium at the end of exposure period (macroscopic)

**    Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

S      Frequency statistically significant higher than corresponding control values

n.d. Not determined

n.p. No cytospin slides prepared due to strong cytotoxicity

n.s. Not scorable due to strong cytotoxicity

1      DMSO 1% (v/v)

2      EMS 400 μg/mL

3      EMS 500 μg/mL

4     CPP 0.5 μg/mL

Conclusions:
Under the experimental conditions described, the test article is not considered to have a chromosome-damaging (clastogenic) potency or to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells.
Executive summary:

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure, 24 hours harvest time, without S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL

A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. The vehicle/negative controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei. Cytotoxicity indicated by clearly reduced relative cell count or proliferation index (CBPI) was observed at least at the highest applied test substance concentration in all experimental parts of this study. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test article is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-10-09 to 1996-10-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1987
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix induced with phenobarbital and beta-naphtolflavone
Test concentrations with justification for top dose:
33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: strains TA1535 and TA100: sodium azide, TA1537 and TA98: 4-nitro-o-phenyle-diamine, WP2 uvrA: methyl methane sulfonate; with metabolic activation: alls trains: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation), preincubation

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A test article was considered positive if either a dose related and reproducible increase in the number of revertants or a reproducible increase for at least one test concentration was induced. A test article producing neither a dose related and reproducible increase in the number of revertants nor a reproducibly positive response at any one of the test points was considered non-mutagenic in this system. A test article was considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions was at least twice as high and in the strains TA 1535, and TA 1537 at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent and reproducible increase in the number of revertants was regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
not required
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test article precipitated in the overlay agar at concentrations of 2500 and 5000 µg/plate. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test article a pre-experiment was performed with strains TA98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Number of revertants per plate (mean from three plates), experiment I (Plate incorporation Test)

    strain TA1535       strain TA1537       strain TA98

      strain TA100

    E. coli WP2uvrA
 Conc. [µg/plate]  -MA  +MA   -MA   +MA   -MA   +MA   -MA   +MA   -MA   +MA
 negative control  15  18  9  10 18   19  115  136  38  45
 solvent control  20  16  12  7  16  19  94  120  32  38
 positive control*  706  522  315  261  427 967  521  750  585  187
 33.3  20  21  14  8  17  20  106  105  33  46
 100  17  18  13  9  17 16  90  97  32  40
 333.3  20  18  14  12  15  21  111  137 40  37
 1000  21  15  12  13  18  19  112  110  34  38
 2500  16  24  12  12  15  17  95  113  42  36
 5000  22  24  15  9  16  15  115  122  35  39

*without MA: Sodium azide (10 µg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strain TA 98 (10 µg/plate) strain TA 1537 (50 µg/plate)

with MA: 2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98, and TA 100; 2-aminoanthracene (10.0 µg/plate) strain WP2 uvrA

Table 2: Number of revertants per plate (mean from three plates), experiment II (Pre-Incubation Test)

      strain TA1535       strain TA1537       strain TA98

      strain TA100

    E. coli WP2uvrA
 Conc. [µg/plate]  -MA  +MA   -MA   +MA   -MA   +MA   -MA   +MA   -MA   +MA
 negative control  16  19  7  9 18   24  112  147  29  45
 solvent control 16  19  7  10  18  22  120  145  30  38
 positive control*  676  442 95 279  436 580  594  677  563  430
 33.3  15  18  6  11  16  23  107  131  28  32
 100 16  17  7  10  19 21  108  145  33  40
 333.3  14  17  7  11  18  23  113  141 35  35
 1000  14  17  6  8  16  21  101  140  24  28
 2500  15  15  6  11  15  22  131  131  23  31
 5000  12  16  5  8  13  18  122  129  23  37

*without MA: Sodium azide (10 µg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strain TA 98 (10 µg/plate) strain TA 1537 (50 µg/plate)

with MA: 2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98, and TA 100; 2-aminoanthracene (10.0 µg/plate) strain WP2 uvrA

Conclusions:
Under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-09-04 to 1996-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1987
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (phenobarbital / beta-Naphthoflavone - induced)
Test concentrations with justification for top dose:
without S9 mix: experiment I: 1, 3, 5, 10, 30, 50 µg/mL, experiment II: 3, 5, 10, 20, 30, 40 µg/mL
with S9 mix: experiment I: 3, 10, 30, 100, 300, 3500 µg/mL, experiment II: 5, 10, 30, 50, 100, 3500 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: ethanol, final concentration of ethanol in the culture medium did not exceed 1 % (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
without metabolic activation: ethylmethane sulfonate, with metabolic activation: cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h with metabolic activation, 18 h and 28 h without metabolic activation

NUMBER OF REPLICATIONS: 1

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosome aberrations or a significant and reproducible positive response for at least one of the test points. A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosome aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system. Both biological and statistical significance should be considered together.
Statistics:
Statistical significance was confirmed by means of the Fischer's exact test.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9-mix: cultures after treatment with 30 mg/mL showed a reduction of cell numbers in both experiments, with S9-mix: reduced cell number at both intervals in experiment II after treatment with 3500 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation occured in concentrations from 100 µg/mL onwards.

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. As recommended in this guideline 3500 µg/mL (~ 10 mM) of the test substance were applied as top concentration for treatment of the cultures in the pre-test. Test article concentrations between 1 and 3500 µg/mL (with and without S9-mix) were chosen for the evaluation of cytotoxicity using the determination of cell numbers 24 h after start of treatment. In the absence of S9 mix dose selection was influenced by strong induced cytotoxicity. Therefore, in the cytogenetic experiments, 50 µg/mL were chosen as top treatment concentration in the absence of S9 mix, whereas in the presence of S9 mix 3500 µg/mL were used as top treatment concentration.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix, in both experiments the mitotic, indices were reduced at each fixation interval after treatment with the highest evaluated concentration, 30 µg/mL (18 h interval: exp. I 35.4 %, exp. II 54.3 % of the corresponding control; 28 h interval: exp I 14 7 %, exp. II 59.8 % of the corresponding control). In additon, cultures after treatment with 30 µg/mL exhibited reduced cell numbers, except in experiment II at interval 28 h. Cytogenetic evaluation of cultures after treatment with 50 µg/mL (exp. I) and 40 µg/mL (exp, II) was not feasible, since no or to less metaphases were detected and the mitotic indices were strongly reduced.
In the presence of S9 mix, in both experiments at interval 18 h and 28 h the mitotic indices were not reduced in cultures after treatment with the test article, except in experiment I at interval 28 h after treatment wdth 100 µg/mL (54.5%).
In the presence of S9 mix, determination of cell numbers as indicator for cytotoxicity revealed a reduced number at both intervals in experiment II after treatment with 3500 µg/ml, a concentration with visible precipitation (18 h interval 46,1 %; 28 h interval 44,7 %), In addition, in experiment II at interval 18 h, cultures after treatment with 50 µg/ml (without precipitation) and 100 µg/ml (first concentration with precipitation) showed reduced cell numbers. However, qualitative assessment of the cultures 4 h after start of the treatment revealed precipitation of the test article at concentrations of 100 µg/ml
and above.

Table 1: Summary of results of the chromosome aberration study without metabolic activation

Exp.

 

preparation interval

conc. Test substance [µg/mL]

 

polyploid cells in %

mitotic index in % of solvent control

 

incl gaps

aberrant cells in % excl. gaps*

 

exchanges

I

18 h

negative control

3

92.1

2

2

0

solvent control

2.5

100

4

1.5

0

3

4.5

101.9

1

0

0

10

3

85

3

1

0-5

30

3

35.4

3

1.5

0

II

18 h

negative control

3

120.5

4

2.5

0.5

solvent control

1.5

100

2

1

0

10

3.5

101.1

1

1

0

20

2.5

86.7

2

1

0

30

3.5

54.3

1

1

0

I

28 h

solvent control

3

100

4

2

0.5

30

4

14.7

3.5

3

0.5

II

28 h

solvent control

5

100

4

2.5

0

30

4

59.8

1.5

1.5

0.5

*inclusive cells carrying exchanges

 

Table 2: Summary of results of the chromosome aberration study with metabolic activation

Exp.

 

preparation interval

conc. Test substance [µg/mL]

 

polyploid cells in %

mitotic index in % of solvent control

 

 

incl gaps

aberrant cells in % excl. gaps*

 

exchanges

I

18 h

negative control

4

102

3.5

2.5

0

solvent control

3.5

100

5

4

0

10

2.5

103.7

1.5

1

1

30

3.5

97.1

4

3.5

1.5

100p

4

101.4

3

3

0

3500p

2

106.3

4.5

3.5

2.5

II

18 h

negative control

3

105.6

0.5

0.5

0

solvent control

2.5

100

0

0

0

30

1.5

103.8

0

0

0

50

3.5

101.7

3

2.5s

0

100

2.5

86.4

2.5

2.5s

1

3500

1.5

99

4.5

3s

0.5

I

28 h

solvent control

2.5

100

2.5

1.5

0.5

100p

5

54.5

6

5s

2.5

3500p

3

93

8.5

5.5s

0

II

28 h

solvent control

2.5

100

1.5

1.5

0

50

1.5

89.4

4

2.5

1

3500p

3.5

82.9

5

3.5

1

*inclusive cells carrying exchanges

SAberration frequency statistically signiflcant higher than corresponding solvent control values

pPrecipitation of (he test article 4 h alter start of treatment was observed.Aberrant cells in the positive control groups: 9.0 % - 28,5 %

 

 

 

Conclusions:
Under the experimental conditions reported, the test article did not induce structural chromosome aberrations in V79 cells.
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:
1996-10-08 to 1996-11-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-and guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1987
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, phenobarbital and beta-naphtoflavone-induced
Test concentrations with justification for top dose:
experiment 1:
without S9 mix: 1.0; 10.0; 30.0, and 100.0 µg/mL
with S9 mix: 1.0; 10.0; 30.0, and 100.0 µg/mL

experiment 2:
without S9 mix: 3.0; 10 0; 30.0; 15.0; and 200.0 µg/mL
with S9 mix: 3,0; 30.0; 100.0; 150.0; and 200.0 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative non-toxicity to the cells. The final concentration of ethanol in the culture medium did not exceed 1%.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation: ethylmethane sulfonate; with metabolic activation: 7,12-dimethylbenz(a)anthracene
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Selection time (if incubation with a selection agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 6 days for determination of toxicity, 15 days for determination of cloning efficiancy, 16 days for selection of mutants

NUMBER OF REPLICATIONS: 5 flasks for mutant selection and 2 flasks for determination of cloning efficency (toxicity)

STAIN: methylene blue

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test article is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response for one of the test points. A test article producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test article is classified as mutagenic if it induces a reproducible mutation frequency that is at least three times higher than the spontaneous mutation frequency in the experiment at one or more of the concentrations. The test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (1-32 mutants per 10^6 cells) a concentration-related increase of the mutations within this range has to be discussed.
Statistics:
Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Strong toxic effects occurred in the precipitating range at concentrations of 100 µg/mL and above without metabolic activation. No relevant toxic effects occurred in the presence of metabolic activation up to the maximal concentration tested.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test article occurred at concentrations of 100 µg/mL and above in both experiments with and without metabolic activation

RANGE-FINDING/SCREENING STUDIES:
A pre-experiment was performed with eight concentrations ranging from 0.3 to 400.0 µg/mL with and without metabolic activation. Precipitation of the test article was observed at 100 µg/mL and above. In experiment I the maximal concentration (100 µg/mL) showed precipitation but strong toxic effects occurred solely at the maximal concentration in the absence of metabolic activation. To cover possible effects of the test substance in the range well above the limit of precipitation two concentrations above this limit were applied in the second experiment.

Table 1: Mutagenicity data , experiment I, without metabolic activation

 

Conc. [µg/mL]

Mutant colonies (mean of 5 flasks) *

Mutant colonies/10^6 cells

cell densitiy (% of control) as measure of toxicity

Negative control

0

2.4

8.2

100

Solvent control (ethanol)

0

2

7.1

100

Positive control (EMS)

600

129.2

603.8

79.4

Test substance

0.3

culture was not continued

Test substance

1

2.6

7.6

104.3

Test substance

3

culture was not continued

Test substance

10

1.6

4.6

108.2

Test substance

30

2.4

6.3

98.9

Test substance

100

1.2

3.7

77.6

*only colonies with more than 50 cells were scored

 

Table 2: Mutagenicity data , experiment I, with metabolic activation

 

Conc. [µg/mL]

Mutant colonies (mean of 5 flasks) *

Mutant colonies/10^6 cells

cell densitiy (% of control) as measure of toxicity

Negative control

0

0.4

1.3

100

Solvent control (ethanol)

0

2.4

7.3

100

Solvent control (DMSO)

 

6

22.5

100

Positive control (DMBA)

600

179

871.4

48.5

Test substance

0.3

culture was not continued

Test substance

1

7

17.3

93.6

Test substance

3

culture was not continued

Test substance

10

3

9.2

100.2

Test substance

30

2.2

8.5

102.8

Test substance

100

4.2

11.6

89.5

*only colonies with more than 50 cells were scored

 

Table 3: Mutagenicity data , experiment II, without metabolic activation

 

Conc. [µg/mL]

Mutant colonies (mean of 5 flasks) *

Mutant colonies/10^6 cells

cell densitiy (% of control) as measure of toxicity

Negative control

0

1.4

4.6

100

Solvent control (ethanol)

0

2

6.4

100

Positive control (EMS)

600

104.6

399.6

75.5

Test substance

3

2.6

8

77.5

Test substance

10

2

6.2

90.7

Test substance

30

5.2

15

81.7

Test substance

100

culture was not continued

31

Test substance

150

1.6

4.9

66.9

Test substance

200

3.6

10.7

52.3

*only colonies with more than 50 cells were scored

 

Table 4: Mutagenicity data , experiment II, with metabolic activation

 

Conc. [µg/mL]

Mutant colonies (mean of 5 flasks) *

Mutant colonies/10^6 cells

cell densitiy (% of control) as measure of toxicity

Negative control

0

1.4

3.2

100

Solvent control (ethanol)

0

1.6

4.9

100

Solvent control (DMSO)

0

2.4

8.9

100

Positive control (DMBA)

3.85

92.8

421.1

77

Test substance

3

4

13.7

102.6

Test substance

10

culture was not continued

90.2

Test substance

30

2.8

11.2

94.7

Test substance

100

1.6

4.4

84.7

Test substance

150

8.2

25.8

100.9

Test substance

200

5.8

19.5

92.9

*only colonies with more than 50 cells were scored

 

 

Conclusions:
Under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay

A GLP-compliant Ames test was performed following OECD guideline 471 to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without rat liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate. The plates incubated with the test article showed no reduction of the background growth up to 5000.0 µg/plate with and without metabolic activation in both independent experiments. No toxic effects evident as a reduction in the number of revertants were observed up to the highest investigated dose in both experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this reverse mutation assay (1996).

This result is supported by an older study investigating point mutations in Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537. The test substance dissolved in DMSO was tested at concentrations of 25, 75, 225, 675 and 2025 µg/ 0.1 mL both with and without metabolic activation. No differences in the number of histidine-prototrophic mutants in the controls and treated plates were recorded. The substance is therefore considered to be non-mutagenic in this assay (1980).

 

Chromosome aberration test in chinese hamster V79 cells

The test article, dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment period was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up and 100 metaphases were scored for structural chromosome aberrations for each culture. The concentrations used were determined in a pretest.

In both experiments, at both preparation intervals in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence of S9 mix, the aberration rates of the cells after treatment with the test article were near to the range of the solvent control values and within the range of our historical control data. However, in the presence of S9 mix, at both fixation intervals and both experiments, the test article did induce 5 unreproduced statistically significant increases in cells carrying structural chromosome aberrations. At interval 28 h in experiment I two significant increase were observed after treatment with 100 µg/ml (5.0 %) and 3500 µg/ml (5.5 %). These increases slightly beyond the historical range (0.0 - 4,0 %) were not reproduced in experiment II and occurred at concentrations with visible precipitation. With regard to the observed precipitation, physical or osmotic influences might be responsible for this observation produced by an evaluation of cytogentic damage in a limited sample size. In experiment II at interval 18 h three significant increases after treatment with 50 µg/ml, 100 µg/ml (2,5 % aberrant cells each) and 1000 µg/ml (3,0% aberrant cells) were observed. These significant increases due to a low solvent control value (0,0 %) were within the historical control data range (0 % - 4 %) and must be regarded as biologically irrelevant. In conclusion, no reproduced and concentration related increase was observed. Therefore, with regard to our evaluation criteria, the significant increases must be regarded as statistical anomalies without biological relevance (1996).

 

Gene mutation in mammalian cells (HPRT)

A GLP-compliant in vitro gene mutation assay in Chinese hamster V79 cells was conducted following OECD guideline 476. The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments with and without rat liver microsomal activation. The test article was tested at the following concentrations:

Experiment I:

Without S9 mix: 1.0; 10.0; 30.0; and 100.0 µg/mL

With S9 mix: 1.0, 10 0; 30.0; and 100.0 µg/mL

Experiment II:

Without S9 mix: 3.0; 10 0; 30.0, 150.0, and 200.0 µg/mL

With S9 mix: 3.0; 30 0; 100.0; 150.0, and 200.0 µg/mL.

Precipitation of the test article was observed at 100 µg/mL and above. Strong toxic effects occurred in the precipitating range at concentrations of 100 µg/mL and above without metabolic activation in both experiments. The mass cultures turned out to be less sensitive and the cell density was only moderately reduced in the precipitating concentration range without metabolic activation. No relevant toxic effects occurred in the presence of metabolic activation up to the maximal concentration tested. No substantial increase in the number of mutant colonies occurred at any of the evaluated concentrations neither in the presence nor absence of metabolic activation. There was no indication of a dose dependent increase in the number of colonies even below the threshold of biological relevance. The number of mutant colonies in any of the test groups with or without metabolic activation remained well within the range of the historical negative controls (1 - 32 colonies). In all experiments of this study (with and without S9 mix) the range of the negative and solvent controls was from 1.3 up to 22.5 mutant colonies per 106 cells; the range of the groups treated with the test article was from 3.7 up to 25.8 mutant colonies per 106 cells. At two concentrations (150 and 200 µg/mL) in the second experiment with metabolic activation the number of colonies exceeded the threshold of three times the corresponding control slightly. This effect was judged as biologically irrelevant since it is based upon the low values of the corresponding control caused by statistical fluctuations and does not indicate a mutagenic response. EMS (0.6 mg/mL) and DMBA (3.85 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells (1996).

 

In vitro micronucleus

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) following OECD guideline 487 and in compliance with GLP. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to the results of the present in vitro micronucleus assay, the test substance did not lead to a biologically relevant increase in the number of micronucleated cells either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The frequencies of micronuclei after test substance treatment were close to the range of the concurrent vehicle control values at both exposure times and clearly within the range of the 95% control limit of our historical negative control data. The number of micronucleated cells in all vehicle/negative control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the micronucleus test in V79 cells was demonstrated either by the laboratory’s historical control database on vehicle and positive controls or by X-bar chart to identify the variability of the vehicle control data. The increase in the frequencies of micronuclei induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and/or the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen here, the conclusion is drawn that Irgamod 295 has not the potential to induce micronuclei (no clastogenic and/or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation (2015).

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the substance is not classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not classified for genetic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.