Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

-       Bacterial reverse mutation assay 

In a K1 bacterial reverse mutation assay in Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA), performed according to the OECD Guideline 471, it was concluded that T002102 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay in the absence and presence of metabolic activation.

 

-   Chromosome aberration study

Two key studies are used in weight of evidence approach to cover the endpoint.

In a K1 in vitro chromosome aberration study in Chinese hamster V79 cells, performed according to the OECD Guideline 473, T002102 induced structural chromosome aberrations in V79 cells in the presence and absence of metabolic activation under the conditions of this study.

In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to the OECD Guideline 473, the test item T002102 induced structural chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation under the conditions of this study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001-07-03 to 2002-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Guideline adopted July 21, 1997
Deviations:
yes
Remarks:
The following deviations from the protocol were reported: a) Seeding of cultures, b) preparation of cultures, c) acceptability of the test, and d) the evaluation of results sections were all updated. Deviations had no detrimental impact on study.
Qualifier:
according to
Guideline:
other: Commission Directive 2000/321EC, L1362000, Annex 4A, dated May 19, 2000.
Deviations:
yes
Remarks:
The following deviations from the protocol were reported: a) Seeding of cultures, b) preparation of cultures, c) acceptability of the test, and d) the evaluation of results sections were all updated. Deviations had no detrimental impact on study.
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate from the Federal Republic of Germany
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were seeded in Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum. For treatment, culture medium was replaced with serum-free medium (for treatment with s9) or complete medium (for treatment without S9) with 10 % FCS (v/v).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Cells have a high proliferation rate (doubling time of clone V79/T5 in stock cultures: 12h), reasonable plating efficiency of untreated cells (as a rule more than 70 %), and a stable karyotype with a modal chromosome number of 22.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/B-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-test on toxicity:
With and without S9 mix: 30.5, 60.9, 12.9, 243.8, 487.5, 957.0, 1950.0 and 3900.0 µg/mL

Main experiment:
75, 150, 225, 300, 450, and 600 µg/mL for the 4 hour exposure period.
150, 300, and 450 ug/mL were selected for metaphase analysis in the absence of S9 and 150, 225 and 300 µg/mL were selected for metaphase analysis in the presence of S9. The cytogenetic evaluation of higher concentrations in the respective intervals (with and without S9) was impossible due to strong test substance induced toxic effects (reduced cell numbers and/or low metaphase numbers, partially paralleled by poor metaphase quality).

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell culture.
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate at 1000 µg/mL dissolved in nutrient medium
Remarks:
without S9
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide at 0.7 µg/mL dissolved in nutrient medium
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9) with 10 % FCS (v/v) containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium were added. Concurrent negative, solvent and positive controls were performed.
After 4 hours the cultures were whashed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
DURATION
- Exposure duration: 4 hours
- Expression time: 11.5 hours
- Fixation time: 18 hours

SPINDLE INHIBITOR: 15.5 h after the start of the treatment colcemid was added (0.2 µg.mL culture medium) to the cultures. 2.5 hrs after the addition of colcemid, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37°C. After incubation in the hypotonic solution, the cells were fixed with a mixture of methanol and glacial acetic acid (3:1). Per experiment two slides per group were prepared. After preparation the cells were stained.
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
-100 well spread metaphase plates were scored per culture (only metaphases with characteristic chromosome numbers of 22 +/-1 were included in the analysis).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index or cell numbers

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

OTHER:
Prior to treatment the cells were washed with Ca-Mg-free salt solution and treated for 5 minutes with trypsin. Additionally, two cultures per test substance and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The cell number of the treated groups was given in percentage compared to the respective solvent control.

Evaluation criteria:
A test substance was classified as non-clastogenic if: the number of induced structural chromosome aberration in all evaluated dose groups are in the range of the testing lab's historical control data (0.0-4.0% aberrant cell exclusive gaps), and/or no significant increase of the number of structural chromosome aberrations was observed.
A test substance was classified as clastogenic if: the numbers of induced chromosome aberrations are not in the range of the testing lab's historical control data (0.0-4.0% aberrant cells exclusive gaps), and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Both biological and statistical significance should be considered together.
Although the inclusion of the structural chromosome aberrations is the purpose of the study, it is important to include the polyploids and endoreduplications. The following criteria is valid: a test substance can be classified as aneugenic if the number of induced numerical aberrations were not in the range of the testing lab's historical control data (0.0%-8.5% polyploid cells).
Statistics:
Statistical significance was confirmed using the Fisher's exact test (p<0.05).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the pre-experiment, no relevant influence on pH occurred (solvent pH 7.3 versus pH 7.3 at 3900 µg/mL).
- Effects of osmolality: In the pre-experiment, no relevant influence on osmolarity occurred (solvent 322 mOsm versus 285 mOsm at 3900 µg/mL).
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: In the pre-experiment, no precipitation occurred.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: A pre-test on cell growth inhibition to determine the toxicity of the test substance was performed. The general experimental conditions were the same as describe above for the cytogenetic main experiment. The following method was used. In a quantitative assessment, exponentially growing cell cultures (seeding 51700 cells/slide, with regard to the culture time 48 h) were treated with the test substance at concentrations ranging from 30.5-3900 µg/mL. A qualitative evaluation of cell number and cell morphology was made 4 h and 24 h after start of treatment. 24 h after start of treatment the cells were stained.
The highest concentration in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. Using reduced cell numbers as an indicator of toxicity, clear toxic effects were observed after 4 hours treatment with 487.5 µg/mL and above in the absence and presence of S9 mix. Additionally, 24 hour continuous treatment with 60.9 µg/mL and above in the absence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-experiments, 600 µg/mL (with and without S9 mix) was chosen as top concentration for the main experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: All positive, negative and solvent control values for % aberrant cells (including gaps, excluding gaps and with exchanges) were all within historical data control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main experiment, no clear dose related toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed at the concentrations evaluated. Higher concentrations could not be evaluated due to the strongly reduced metaphase cell numbers.
Remarks on result:
other: all strains/cell types tested

In this study, in the absence of S9 mix the aberration rate (16.0 %) was biologically relevant and statistically significant increased after treatment with 450 µg/ml as compared to the corresponding solvent control (1.0 %). Also, the number of cells carrying exchanges was distinctly increased (10.0 %) as compared to the solvent control (0.0 %). In the presence of S9 mix (treatment with 150, 225, and 300 µg/ml), biologically relevant and statistically significant increases in the aberration rates (12.0, 9.5, and 13.0 %, respectively) were observed. Additionally, the cells carrying exchanges observed (8.5, 4.5, and 6.5 %, respectively) give additional evidence for a clastogenic potential of the test item. In the main experiment, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.6 % - 3.0 %) as compared to the rates of the solvent controls (3.6 % - 3.8 %). In the cytogenetic experiment, EMS (1000 µg/ml)and CPA (0.7 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:
Interpretation of results:
positive with metabolic activation
positive without metabolic activation

The test substance induced structural chromosome aberrations in V79 cells in the presence and absence of metabolic activation after a four hour exposure under the conditions of this study.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2001-10-24 to 2001-11-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate from the Federal Republic of Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus and tryptophan locus
Species / strain / cell type:
other: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and B-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-experiment: concentration ranged from 3 to 5000 µg/plate
Experiments I & II: 33, 100, 333, 1000, 2500, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; at 10 μg/plate with TA1535 and TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-ophenylene-diamine
Remarks:
without S9; at 10 µg/plate with TA98, 50 µg/plate with TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; at 5 µg/plate with WP2uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
with S9; at 2.5 μg/plate with TA 1535, TA 1537, TA 98 and TA 100, at 10 μg/plate with WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation) (experiment I) and preincubation (experiment II)

Experimental performance:
The following materials were mixed in a test tube and poured onto the minmal agar plates:
- 100 µL of test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
- 500 µL of S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
- 100 µL of bacteria suspension (c.f. test system, pre-culture of the strains)
- 2000 µL overlay agar
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 h at 37°C in the dark.

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: at least 48 hours
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT: tryptophan (E. coli) and histidine (S. typhimurium)

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
Method:reduction in the number of spontaneous revertants or a clearing of the bacteria background lawn.

Evaluation criteria:
A test substance is considered positive if a dose related increase in the number of revertants exceeding the threshold or twice the colony count of the corresponding solvent control is observed at more than one concentration. Furthermore, a biologically relevant and reproducible increase exceeding the threshold at one test concentration is considered as positive.
A test substance producing neither a dose-related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in this system.

A biologically relevant response is described as follows:
An increase is considered relevant if in strains TA98, TA100, and WP2 uvrA the number of reversions is at least twice as high and in strains TA1535 and TA1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number or revertants is regarded as an indication of possible existing mutagenic potential of the test substance regardless whether the highest dose will induce the above described enhancement factors or not.
Statistics:
No statistical evaluation was performed.
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: No precipitation of the test substance occurred up to the highest investigated dose.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test substance a pre-experiment was performed with all the strains. Eight concentrations (3-5000 µg/plate) were tested for toxicity and mutation induction with three plates each. This pre-experiment was reported as part of Experiment I based on the following criteria being met: evaluable plates (>0 colonies) at five concentrations or more in all strains used. This pre-experiment was used to select dose levels for Experiment II.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle and negative control values were within historical data control ranges in the absence of metabolic activation. No data was given for historical ranges in the presence of metabolic activation.
 The historical range of positive controls was exceeded without metabolic activation in strains TA1535, TA100 and WP2 uvrA. This effect indicates the sensitivity of the strains rather than compromising the assay. The positive controls showed a distinct increase of induced revertant colonies. Appropriate mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without S9 in both experiments. Toxic effects, evident as a reduction in the number of revertants (below the factor of 0.5) were observed at the follow concentrations:
In experiment I, toxic effects were observed at 5000 µg/plate with and without S9 in TA1535 and TA1537, with S9 in TA98, and without S9 in WP2 uvrA.
In experiment II, toxic effects were observed at 333-5000 ug/plate without S9 in TA1535, 2500-5000 µg/plate with S9 in TA1535, 2500-5000 µg/plate without S9 in TA1537, 5000 µg/plate with and without S9 in TA98, 5000 µg/plate without S9 in TA100, 2500-5000 µg/plate without S9 in WP2 uvrA and 5000 µg/plate with S9 in WP2 uvrA.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutation by base pair changes or frameshifts in the genome of the strains used in the Ames test in the presence or absence of metabolic activation. Based on these results, the test substance is not to be classified as mutagenic.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2007-07-12 to 2007-08-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Guideline adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: Annex 4A Directive 2000/32/EC: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test"
Deviations:
no
GLP compliance:
no
Type of assay:
other: chromosomal aberrations in human lymphocytes in vitro
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Cytotoxicity Test:
Without S9 mix: 23.4, 40.9, 71.6, 125.3, 219.3, 383.8, 671.7, 1175.5, 2057.1 and 3600.0 µg/mL
With S9 mix: 23.4, 40.9, 71.6, 125.3, 219.3, 383.8, 671.7, 1175.5, 2057.1 and 3600.0 µg/mL
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible. With respect to the solubilty properties of the test item, 3600 µg/mL of T2102 (approx. 9.3 mM) was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 23.4 and 3600 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, no precipitation of the test item was observed before treatment up to the highest applicable concentration. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Main Experiment. Due to a technical error, the experimental part with S9 mix was repeated with the same top test item concentration. A second experiment was not performed, since the test item was considered to be clastogenic after 4 hr treatment.
Vehicle / solvent:
Water was used as the solvent based on the information provided by the Sponsor and compatibility with the target cells. This solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
The final concentration of deionised water in the culture medium was 10 % (v/v).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 at 825 µg/mL dissolved in nutrient medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 at 30 µg/mL dissolved in saline (0.9 % NaCl [w/v])
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- About 50-80 hrs after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test item. For the treatment with metabolic activation 50 µL S9 mix/mL medium were used. Concurrent solvent and positive controls were performed. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test substance was discarded and the cells were resuspended in "saline G". The washing procedure was repeated once as described. After washing the cells were resuspended in complete culture medium and cultured until preparation.
- All cultures were incubated at 37°C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

DURATION
- Exposure duration: 4 hours
- Expression time: ~ 15 hours
- Fixation time: 22 hours

SPINDLE INHIBITOR: Three hours before harvesting, colcemid (Fluka, 89203 Neu-Ulm, Germany) was added to the cultures (final concentration 0.2 μg/mL).
STAIN: The cells for evaluation of cytogenetic damage were stained with Giemsa or according to the Fluorescent plus Giemsa technique, respectively.

NUMBER OF REPLICATES: 2/doses/experiment

NUMBER OF CELLS EVALUATED: 100 well spread metaphase plates per culture were scored for structural chromosomal aberrations. Only metaphases with 46 ± 1 centromer regions were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index : To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.

OTHER EXAMINATIONS:
- The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37° C for 20 to 25 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide.
- The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives.
- Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates.
Evaluation criteria:
A test item is classified as non-mutagenic if:
− the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory's historical control data (0.0 - 4.0 % aberrant cells, excluding gaps).
− no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
− the number of induced structural chromosome aberrations is not in the range of the laboratory's historical control data (0.0 - 4.0 % aberrant cells, excluding gaps).
and
− either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data
(0.0 - 0.8 % polyploid cells).
However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the presence of S9 mix, clear cytotoxic effects were observed, after 4 hrs treatment with 671.7 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Neither precipitation of the test item in the culture medium nor relevant increase in the osmolarity or pH value was observed (solvent control: 253 mOsm, pH 7.4 versus 299 mOsm and pH 7.1 at 3600 µg/mL).

COMPARISON WITH HISTORICAL CONTROL DATA:
The values of the two highest evaluated concentrations clearly exceeded the laboratory’s historical control data range (0.0 – 4.0 % aberrant cells, excluding gaps). Therefore, these observations have to be considered as being biologically relevant.

ADDITIONAL INFORMATION:
In the absence of S9 mix, concentrations showing clearly reduced mitotic indices, were not scorable for cytogenetic damage. In the presence of S9 mix, clear cytotoxic effects were observed, after 4 hrs treatment with 671.7 µg/mL (46.0 % of control).

In the absence of S9 mix, the number of aberrant cells, excluding gaps (1.0 %, 5.5 %, and 16.5 %, respectively), after treatment with 125.3, 219.3, and 383.8 µg/mL, respectively, was increased in a dose-related manner. The number of cells carrying exchanges (4.0 %) observed at 383.8 µg/mL, gives an additional evidence for a clastogenic potential.

In the presence of S9 mix, a dose-dependent increase in cells showing structural chromosomal aberrations was observed at the evaluated concentrations 219.3, 383.8, and 671.7 µg/mL (2.0 %, 5.5 %, and 8.0 %, respectively). In addition, the value of the highest evaluated concentration, was statistically significant compared to the corresponding solvent control (3.0 % aberrant cells, excluding gaps), and clearly exceeded the laboratory’s historical control data range (0.0 – 4.0 % aberrant cells, excluding gaps). Therefore, these observations have to be regarded as being biologically relevant.
Remarks on result:
other: all strains/cell types tested

Either EMS (825 μg/mL) or CPA (30.0 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

The proliferation index of the lymphocytes in solvent control cultures in the 22 hrs preparation interval with and without S9 mix (4 hrs treatment; 1.04 and 1.74, respectively), was checked by analysing the proportion of mitotic cells in the 1st, 2nd and 3rd metaphase (M1, M1+, M2 and M3) indicating that the lymphocytes divided about 1.5 times within the early preparation interval. This is also proven by the occurrence of sufficient numbers of mitotic cells and by a clear clastogenicity observed after treatment with the positive control substances.

Since the test substance was considered to be clastogenic after 4 h treatment, a second experiment was not performed.

Conclusions:
Interpretation of results:
positive

Under the experimental conditions reported, the test item T002102 induced structural chromosomal aberrations in human lymphocytes in vitro, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The micronucleus assay was performed in bone marrow cells of male/female NMRI mouse with T002102 according to OECD Guideline 474. Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

The negative results in the in vivo study were not considered to be sufficient to overrule the in vitro positive results. A second in vivo study is recommended to conclude on the absence of positive results in vivo. However, no additional in vivo study will be performed for the genetic toxicity endpoint as the substance is classified at mutagenic category 2 based on a worst case assumption as described in the waiver.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2007-07-23 to 2007-08-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented GLP study performed in accordance with the OECD guideline N°474, Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000, and ICH-Harmonised Tripartites guidelines S2A and S2B, with minor deviations which do not impact the validity of the study.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
but data lacking about: some endpoints not recorded for toxic reactions during pre-experiment
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000.
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH-Harmonised Tripartite Guideline S2A (CPMP/ICH/141/95), dated April 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH-Harmonised Tripartite Guideline S2B (CPMP/ICH/174/95), dated July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH D-33178 Borchen
- Age at study initiation: 7-8 weeks
- Weight at study initiation: males mean value 37.3 g (SD ± 1.4 g) ; females mean value 31.1 g (SD ± 1.7 g)
- Animals were distributed into the test groups at random and identified by cage number.
- Fasting period before study: no data
- Housing: single housing in Makrolon Type I cages, with wire mesh top
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-84%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: 0.9 % NaCl
- Justification for choice of solvent/vehicle: chosen to its relative non-toxicity for the animals
- Concentration of test material in vehicle: 10 mL/kg bw
Details on exposure:
On the day of the experiment, the test item was formulated in 0.9 % NaCl. All animals received a single standard volume of 10 mL/kg body weight orally.
Duration of treatment / exposure:
one single administration
Frequency of treatment:
one single administration
Post exposure period:
Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval for the female animals
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval for the male and female animals
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval for the male and female animals, and 48 h preparation interval for the female animals
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
24 h preparation interval for the male animals, and 48 h preparation interval for the male animals
No. of animals per sex per dose:
Twelve animals, six males and six females, were treated per dose group and sampling time (except highest dose: 12 animals per sex, 6 per time point).
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): no
- Route of administration: orally, once
- Doses / concentrations: dissolved in deionised water, administered at 10 mL/kg bw (dose 40 mg/kg bw)
Tissues and cell types examined:
Bone marrow -at least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The volume to be administered should be compatible with physiological space available.
Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): At the beginning of the treatment, the animals (including the controls) were weighed and the individual volume to be initiated was adjusted to the animal body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time (24 or 48h). The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1h, 2-4h, 6h and 24h after administration of the test item. Sampling of bone marrow was done 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, D-64293 Darmstadt)/ Giemsa (Merck, D-64293 Darmstadt). Cover slips were mounted with Eukitt (Kindler, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sampled and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Twelve animals (6F+6M) per test groups were evaluated as described.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
The non parametric Mann-Whitney test was used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that T2102 did not have any cytotoxic properties in the bone marrow.
The highest dose (500 mg/kg b.w. for the female animals and 1000 mg/kg b.w. for the male animals) was estimated by pre-experiments to be suitable.
40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Pre-experiment results:

In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 100 mg/kg b.w. T2102 formulated in 0.9 % NaCl. The volume administered was 10 mL/kg b.w.. The animals treated with 100 mg/kg b.w. expressed no toxic reactions.

In a second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1000 mg/kg b.w. T2102 formulated in 0.9 % NaCl. The volume administered was 10 mL/kg b.w. A female died 2 -4 hours after administration of the test item.

In a third pre-experiment 2 males received orally a single dose of 1500 mg/kg b.w and 2 females received orally a single dose of 750 mg/kg b.w. T2102 formulated in 0.9 % NaCl. The volume administered was 10 mL/kg b.w. One male and one female died during the hour following the oral administration.

In a fourth pre-experiment 2 males received orally a single dose of 1250 mg/kg b.w and 2 females received orally a single dose of 500 mg/kg b.w. T2102 formulated in 0.9 % NaCl. The volume administered was 10 mL/kg b.w.. One male died during the hour following the oral administration.

On the basis of these data 1000 mg/kg b.w. was estimated to be suitable as the highest dose for the male and 500 mg/kg b.w. for the female animals.

Main experiment:

The animals treated with 1000 mg/kg and 500 mg/kg b.w. respectively expressed toxic reactions as shown in the table (left number: males, rigth number:females):

 Toxic reactions following administration  1h  2 -4h  6h  24h  48h*
 Reduction of spontaneous activity  9/10  10/12 9/8  0/0   0/0
 ruffled fur  12/12  12/12  12/12  7/8  0/0
 tremor  8/9  7/9  1/4  0/0

 0/0

*data from only 6 animals

Summary of micronucleus test results (main experiment, males)

 Test group  dose mg/kg b.w. sampling time (h)  PCEs with micronuclei (%) range  PCE per 2000 erythrocytes
Vehicle  0  24 0.175 1 -6 1298
 Test item  250

 24

 0.142

 1 -4

 1138

 Test item

 500

 24

 0.167

 1 -6

1236

  Test item

 1000

 24

 0.092

 0 -4

 1204

 positive control

 40

 24

 3.492

 56 -97

 1066

 Test item

 1000

 48

 0.120

 1 -4

 1119

Summary of micronucleus test results (main experiment, females)

Test group  dose mg/kg b.w. sampling time (h)  PCEs with micronuclei (%) range  PCE per 2000 erythrocytes
Vehicle  0  24 0.108 1 -4 1249
 Test item  125

 24

 0.142

 0 -6

 1281

 Test item

 250

 24

 0.100

 1 -4

1224

  Test item

 500

 24

 0.108

 0 -3

 1265

 positive control

 40

 24

 2.758

 39 -78

 1113

 Test item

 500

 48

 0.150

 1 -6

 1139

Conclusions:
Interpretation of results: negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Based on these results and the criteria in the CLP Regulation, the test substance is considered not classified as mutagenic.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity: in vitro

Bacterial reverse mutation assay:

An Ames test was performed according to OECD Guideline 471 and in compliance with the principles of GLP with the following Salmonella typhimurium strains: TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli strainWP2 uvrA (Wollny, 2002).

T002102 was tested with and without a metabolic activation system, according to the plate incorporation method (experiment 1) and the pre-incubation test (experiment 2). Bacteria were exposed to T002102 at six dose-levels selected from a preliminary toxicity test: 33, 100, 333, 1000, 2500 and 5000 μg/plate. After at least 48 hours of incubation at 37°C, the revertant colonies were scored. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Neither precipitate nor appreciable toxicity was observed. The substance is considered not mutagenic based on these results.

 

In vitro mammalian chromosome aberration test:

An in vitro chromosome aberration test was conducted according to OECD Guideline 473 and EU Method B.10 (Wolf, 2002).

The test material was evaluated in Chinese hamster V79 cells with and without metabolic activation, at the following dose levels: 75, 150, 225, 300, 450, and 600 µg/mL.

According to the conditions of the experiment, the test item induced a statistically significant increase in the frequency of cells structural chromosome aberrations in either the absence or presence of a metabolic activation system. No cytotoxicity was observed.

The second chromosome aberration test was conducted according to OECD Guideline 473 and Annex 4A Directive 2000/32/EC: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test" (Bohnenberger, 2007).

The test material was evaluated in human lymphocytes with and without metabolic activation, at the following dose levels: 23.4, 40.9, 71.6, 125.3, 219.3, 383.8, 671.7, 1175.5, 2057.1 and 3600.0 µg/mL. Based on the findings of this study, T002102 was concluded to induce structural chromosomal aberrations in vitro, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Neither precipitation of the test item in the culture medium nor relevant increase in the osmolarity or pH value was observed.

 

Genetic toxicity: in vivo

Micronucleus assay:

A micronucleus assay study was performed to investigate the potential of T002102 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (OECD 474, Honarvar, 2007).

Twelve animals (6 males, 6 females) per test group (except for the highest concentration 12 animals/sex were evaluated) were evaluated for the occurrence of micronuclei.

At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

The following dose levels of the test item were investigated:

- 24 h preparation interval for the female animals: 125, 250, and 500 mg/kg bw

- 24 h preparation interval for the male animals: 250, 500, and 1000 mg/kg bw
- 48 h preparation interval for the female animals: 500 mg/kg bw
- 48 h preparation interval for the male animals: 1000 mg/kg bw

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that T002102 did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

According to the requirements of Annexes IX and X, if there is a positive result in any of the in vitro studies from Annex VII or VIII and there are no appropriate results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study should be proposed. In the case of T002102, an in vivo micronucleus assay was performed and concluded on negative results. Based on the available tests and the testing strategy proposed in the Chapter R.7a of the endpoint specific guidance (Reference: ECHA-17-G-18-EN; Vresion 6.0; July 2017), the negative in vivo MNT results overrule the conclusions of the two chromosome aberration tests performed in vitro.

However, in order to implement appropriate risk management measures for substances potentially meeting the criteria for the highest risk categories 1A, 1B or 2 for genotoxicity, it is proposed to classify the substances showing positive in vitro results according to a worst case scenario. The classification is based on the assumption that all the selected follow-up in vivo experiments would confirm the already identified in vitro hazards. The existing negative in vivo results are not considered to conclusively overrule the positive in vitro results. It is acknowledged that some of these substances are probably overclassified and consequently “overprotect” the workers that will potentially be exposed to these substances.

This proposal is in accordance with the ICH safety guidelines S2 (R1) (http://www.ich.org/products/guidelines/safety/article/safety-guidelines.html) and in concordance with the classification related to preclinical and clinical development. Furthermore it minimises the use of animals without compromising the safety of the exposed population.

 

Justification for classification or non-classification

Positive results were observed in the two in vitro chromosome aberration tests, and negative results were obtained in the in vitro gene mutation assay and the in vivo micronucleus assay.

In order to implement appropriate risk management measures for substances showing positive in vitro results, the negative results in the in vivo genetic toxicity study were not considered to be sufficient to overrule the in vitro positive results.

Based on the available data, the criteria laid down in the CLP Regulation and the precautionary principle, the test item T002102 should be classified as Muta 2 - H341 Suspected of causing genetic defects.