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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
  • Bacterial reverse mutation assay

In a bacterial reverse mutation assay in Salmonella typhymurium strains TA98 and TA100, performed according a method similar to OECD Guideline 471, it was concluded that T001036 has mutagenic properties towards Salmonella typhimurium strain TA100 in the absence and in the presence of an Arochlor 1254 induced rat liver S9 metabolic activation system. The test item was negative towards TA98. Although only 2 bacterial strains were tested, due to a positive result in strain TA100 and a positive result in the chromosome aberration study, it is considered that additional testing is not necessary. Therefore, this well-documented study is used as key study for in vitro gene mutation in bacteria and considered to be reliable.

  • Chromosome aberration study

In an in vitro chromosome aberration study in human lymphocytes, performed according to a method equivalent to OECD Guideline 473, T001036 was considered to be clastogenic to human lymphocytes in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987-06-16 to 1987-06-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: Only two tester strains were used instead of five
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Stability of the test article: known
- Storage condition of test material: at room temperature in closed containers
- Other: The test substance was synthesized in the Janssen Pharmaceutica Research Laboratories and passed the specifications of the chemical quality department.
Target gene:
histidine locus
Species / strain / cell type:
other: TA98 and TA100
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced rat liver S9 at 20 and 50 µg/plate
Test concentrations with justification for top dose:
- Preliminary dose range finding study: 0, 10, 100, 250, 500, 1000, 2500 and 5000 µg/plate
- Initial and repeat experiments: 0, 10, 100, 250, 500, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: In a preliminary solubility assay, the test substance (100 mg) was introduced in a test tube, with 2 mL distilled water and mixed vigorously. The test substance was still not soluble upon warming up to 45 °C, so a new solution was prepared in absolute ethanol following the same procedure. Since the test substance was not more soluble in ethanol than water, DMSO was chosen as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation; TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation; TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene at 1.0 µg/plate dissolved in DMSO
Remarks:
with metabolic activation; for TA98 and TA100
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- in agar (plate incorporation)
The following procedure was used for the plate incorporation assay: 0.1 mL of an overnight culture in nutrient broth (Oxoid) is introduced into test tubes. This corresponds to an average of 1-6 x 10^8 viable bacteria per tube. The histidine-biotine (0.05 mM) or tryptophane (5 µg) supplemented top agar (2 mL) is then added, followed by the substrate dilution (0.1 mL). When specified S-9 mix is then added (0.5 mL) and the content of the test tube well mixed. This mixture is then layered on minimal glucose agar (Vogel Bonner E medium) containing petri dishes following the method of Ames et al. The minimal glucose containing petri dishes are used within the week that they were prepared.

DURATION
- Exposure duration: 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: Method: reduction of the background lawn

OTHER EXAMINATIONS:
Before use of any test culture, the growth requirements and the genetic identity of the strains, their sensitivity to UV radiation and crystal violet and their resistance to ampicillin were checked. Each strain must present a spontaneous reversion rate within the historical control data of the testing facility.
After incubation of the plates the number of revertant are counted manually. The sensitivity of the strains and the activity of the metabolizing system are confirmed by testing specific mutagens. Historical control data on spontaneous revertants and on revertants in the positive and solvent control grops of each strain used in the study were included.
Evaluation criteria:
Reversion values were considered as positive if:
1) there was a significant increase in the number of revertant colonies (i.e., at least a two-fold increase compared to the solvent control;
2) a dose effect relationship was observed; and
3) these effects could be reproduced.

When equivocal results are obtained, more assays may be required, in order to evaluate the mutagenic potential of the test substance.
Statistics:
Based on the evaluation criteria, no statistical analysis was required.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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: In the preliminary toxicity test, low precipitation of the test substance occurred at 5000 µg/plate both with and without S9. No precipitation was reported in the main experiment.


RANGE-FINDING/SCREENING STUDIES:
A preliminary plate incorporation test was performed to determine the non-inhibitory concentrations of the test substance. In this pre-test, strain TA100 was tested with 6 concentrations of the test substance ranging from 100 - 5000 µg/plate with and without S9 metabolic activation. Toxic effects were evident by a clear reduction of the background lawn. The highest concentration that does not precipitate and that is not toxic for the bacteria, or the lowest concentration that gives visible toxic effects, was used as the highest one in the main assay.


COMPARISON WITH HISTORICAL CONTROL DATA:
Vehicle and positive control values were within historical data control range. The positive control substances induced marked increases in revertant colony numbers with all strains.
The test substance was toxic to the strain TA100 at 5000 µg/plate as the number of revertant colonies was found to be strongly reduced (pin-point colonies). Toxic action of the test substance to TA100 was also detected by a thinning of the bacterial lawn at 5000 µg/plate. Considering the reversion rate of TA100 to prototrophy with the test substance, a significant increase in the number of revertant colonies could be evidenced from dose levels of 250 µg/plate in the absence or in the presence of rat metabolic activation system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the preliminary dose range finding study toxicity to the strain TA100 was observed at 5000 µg/plate as the number of revertant colonies was found to be stronglyl reduced (pin-point colonies). Toxic action was also detected by a thinning of the bacterial background lawn at 5000 µg/plate.
- In the initial test in the absence and presence of S9, moderate toxicity was observed at 5000 µg/plate for TA98. For TA100, moderate and low toxicity were observed at 5000 µg/plate in the absence and presence of S9, respectively.
- In the repeat test in the absence of S9, low and moderate toxicity was observed for TA98 at 2500 and 5000 µg/plate, respectively. For TA100, moderate toxicity was observed at 5000 µg/plate. In the presence of S9, low toxicity was observed at 5000 µg/plate for both TA98 and TA100.

TA100 showed increased reversion to prototrophy at doses of 250 µg/plate and higher both in the presence and absence of S9. These findings were confirmed in the repeat test. The increased amount of S9 used (20 µL/plate-initial test increased to 50 µL/plate-repeat test) resulted in an increased reversion to prototrophy.

Conclusions:
Interpretation of results:
positive in strain TA100

The test substance was evaluated for mutagenic potential using the Ames Assay. Based on the increase of the reversion rates, it can be concluded that the test substance has mutagenic properties towards Salmonella typhimurium TA100 in the absence and in the presence of rat metabolic activation system. The test substance was negative towards TA98.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-09-11 (date test substance was received) to 2004-09-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
1) No data on mitogenic stimulation or the metaphase-arresting substance, 2) Only 100 metaphase spreads scored per concentration, 3) the solvent is not known.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Analytical purity: 100 %
- Lot/batch No.: RT001036G4B411 T1036
- Storage condition of test material: at room temperature in the dark
- Other: received on 2003-09-11
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
2 % induced rat liver homogenate metabolizing system (S9)
Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 0, 8.23, 14.46, 32.92, 65.83, 131.66, 263.33, 526.65, 1053.3 and 2106.6 µg/mL
- Group 1 (4(20)-hour without S9): 0, 130, 260, 520, 650, 780 and 1040 µg/mL (130, 260 and 650 µg/mL were selected for metaphase analysis.)
- Group 2 (4(20)-hour with S9): 0, 130, 260, 520, 650, 780 and 1040 µg/mL (130, 260, 520 and 650 µg/mL were selected for metaphase analysis.)
- Group 3 (24-hour without S9): 0, 32.5, 65, 130, 195, 260 and 390 µg/mL (65, 130 and 195 µg/mL were selected for metaphase analysis.)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
The identity was not supplied.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide at 10 µg/mL
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
The identity was not supplied.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C at 0.4 µg/mL for the 4h exposure and 0.2 µg/mL for the 24 h exposure
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: no data

DURATION
- Exposure duration: 4 hours (Groups 1 and 2); 24 hours (Group 3)
- Expression time: Groups 1 and 2: 20 hours was stated as the expression period in the study report. However, the study report failed to specify the time of addition of a spindle inhibitor.
Group 3: 0 hours
- Fixation time: 24 hours (all groups)

SPINDLE INHIBITOR: no data
STAIN: no data

NUMBER OF REPLICATIONS:
Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test material. Except where there was the need to clarify an equivocal response, only one of the duplicate cultures was assessed for the incidence of cells with chromosome aberrations.

NUMBER OF CELLS EVALUATED:
100 metaphases per analyzed culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no data

Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
No data was provided on the statistical tests performed. However, p<0.001 was considered significant in the study report.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
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: Precipitation was observed in the preliminary toxicity test.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
There was a precipitate of test substance observed at and above 1053.3 µg/mL in Groups 1 and 2 and at and above 526.65 µg/mL in the continuous exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 526.65 µg/mL in the pulse exposure groups and 263.33 µg/mL in the continuous exposure group. The mitotic index data showed that there was evidence of dose-related, test substance-induced toxicity in all exposure groups. In Groups 1 and 2 there was a steep toxicity curve, whereas in Group 3 the response observed was shallower. Therefore, the selection of the dose range for the chromosome aberration test was based on toxicity for all exposure groups and intermediate dose levels were included accordingly.

COMPARISON WITH HISTORICAL CONTROL DATA:
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control substances induced statistically significant increase in the frequency of cells with aberrations. The positive control for the with-metabolic activation group was highly toxic but a positive response was observed and therefore it was considered that the metabolic activation system was shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A microscopic assessment of the slides showed that metaphase cells were present at up to 780 µg/mL in Groups 1 and 2 and at up to 650 µg/mL in Group 3. The test substance induced significant mitotic inhibition at 650 µg/mL in Groups 1 and 2. In Group 3, 50% mitotic inhibition was practically achieved at 195 µg/mL. Dose selection for metaphase analysis was based on toxicity for all exposure groups.
Remarks on result:
other: Group 2

The test substance induced statistically significant increases in the frequency of cells with aberrations at 650 µg/mL in Group 2. An additional 100 metaphases were scored from the second culture to confirm the response. The test substance did not induce any statistically significant increases in either the pulse or the continuous exposure without metabolic activation.

The test substance did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.

Conclusions:
Interpretation of results:
positive

The test substance was evaluated for the induction of primary human lymphocytes in the presence and absence of metabolic activation. The test substance induced statistically significant increases in the frequency of cells with chromosome aberrations in the presence of a liver enzyme metabolizing system only. The test substance was therefore considered to be clastogenic to human lymphocytes in vitro.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: gene mutation
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay

A key study (K2, de Meester, 1987) was performed according to a method similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay). Salmonella typhimurium strains TA98 and TA100 were treated with solutions of the test item using the Ames plate incorporation method at 7 dose levels (experiments 1 and 2), in triplicate, both with and without the addition of an Arochlor 1254 induced rat liver S9 metabolic activation system. The dose range was 10 to 5000 µg/plate in both experiments.

Vehicle and positive control values were within the laboratories historical data control range. The positive control substances induced marked increases in revertant colony numbers in all strains.

The test item was toxic to the strain TA100 at 5000 µg/plate as the number of revertant colonies was found to be strongly reduced (pin-point colonies). Toxic action of the test item to TA100 was also detected by a thinning of the bacterial lawn at 5000 µg/plate. In the initial test in the absence and presence of S9, moderate toxicity was observed at 5000 µg/plate for TA98. For TA100, moderate and low toxicity were observed at 5000 µg/plate in the absence and presence of S9, respectively. In the repeat test in the absence of S9, low and moderate toxicity was observed for TA98 at 2500 and 5000 µg/plate, respectively. For TA100, moderate toxicity was observed at 5000 µg/plate. In the presence of S9, low toxicity was observed at 5000 µg/plate for both TA98 and TA100.

Based on the increase of the reversion rates, it can be concluded that T001036 has mutagenic properties towards Salmonella typhimurium strain TA100 in the absence and in the presence of metabolic activation. The test item was negative towards TA98.

Although only 2 bacterial strains were tested, due to a positive result in strain TA100 and a positive result in the chromosome aberration study, it is considered that additional testing is not necessary. Therefore, this well-documented study is used as key study for in vitro gene mutation in bacteria and considered to be reliable.

   

Chromosome aberration:

SafePharm Laboratories (K2, 2004) performed an in vitro chromosome aberration study in human lymphocytes (method equivalent/similar to OECD Guideline 473).

The test item, dissolved in vehicle, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and the presence of metabolic activation by S9 mix.

Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test material, ranging from 130 to 1040 µg/mL in the first experiment and from 32.5 to 390 µg/mL in the second experiment. A minimum of three concentration levels and the concurrent vehicle and positive controls were evaluated for chromosome aberrations for each exposure group. Except where there was the need to clarify an equivocal response only one of the duplicate cultures was assessed for the presence chromosome aberrations.

Two independent experiments were performed. In the first experiment (Groups 1 and 2) the exposure periods were 4 hours with and without S9 mix, with a 20 hour recovery period. In the second experiment the exposure period was 24 hours without S9 mix.

The test item induced significant mitotic inhibition at 650 μg/mL in the 4(20) hours pulse exposure groups. In the 24-hour exposure without S9 the ideal of 50% mitotic inhibition was practically achieved at 195 μg/mL. Dose selection for metaphase analysis was based on toxicity for all exposure groups.

100 metaphases were scored per concentration for the chromosome aberrations.

The test item induced statistically significant increases in the frequency of cells with aberrations at 650 μg/ml in the with-metabolic activation exposure group. An additional 100 metaphases were scored to confirm the response. The test item did not induce any statistically significant increases in either the pulse or the continuous exposure without metabolic activation.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

T001036 was therefore considered to be clastogenic to human lymphocytes in vitro.

 

In vitro gene mutation study in mammalian cells:

An in vitro gene mutation study in mammalian cells should be performed if a negative result is observed in Annex VII, section 8.4.1 and Annex VIII, section 8.4.2 of the REACH Regulation. As a positive result has been observed in both the bacterial reverse mutation assay and the in vitro chromosome aberration test, an in vitro gene mutation study in mammalian cells should not be performed.

 





Justification for classification or non-classification

As a positive result has been observed in both the bacterial reverse mutation assay and the in vitro chromosome aberration test, the substance is classified as mutagenic category 2 (H341) based on the criteria of the CLP Regulation (EC) No 1272/2008.