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 TA98, TA100 and TA1535 and TA1537, performed according to a method similar to OECD Guideline 471, it was concluded that T000749 has mutagenic properties towards the Salmonella typhimurium strains tested in the absence and in the presence of S9-mix under the test conditions described in the report.

- Chromosome aberration study

In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to a method similar to the OECD Guideline 473, T000749 was considered to be clastogenic to human lymphocytes in vitro, in the absence and presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-04-04 to 2016-04-13
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)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A15JB2997
- Expiration date of the lot/batch: 2016-10-21
- Purity test date: 2015-10-22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions:not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated
Target gene:
The Histidine locus (S. typhimurium strains TA98, TA100, TA1535 and TA1537) and the Tryptophan locus (E. coli).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Aroclor 1254 induced rat liver metabolic activation system)
Test concentrations with justification for top dose:
Dose-range finding test (part 1): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (top dose selected based on the solubility findings)
Mutation experiment (part 2): 52, 164, 512, 1600, 3000 and 5000 μg/plate (top dose selected based on the dose-range finding test results)


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In DMSO, the test item was soluble at 50 mg/ml. Based on these solubility findings, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.
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, 5 μg/plate (TA1535)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without metabolic activation, 2.5 μg/plate (TA1537)
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, 10 μg/plate (TA98), dissolved in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation, 650 μg/plate (TA100), dissolved in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation, 10 μg/plate (WP2uvrA), dissolved in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix; 2.5μg/plate (TA1535 with 5 and 10% S9-mix, TA1537 with 5% S9-mix), 5μg/plate (TA1537 with 10% S9-mix), 1μg/plate (TA98 with 5 and 10% S9-mix, TA100 with 5% S9-mix), 2μg/plate (TA100 with 10% S9-mix), 15μg/plate (WP2uvrA with 5 and 10% S9-mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

DURATION
- Exposure duration:48+-4h
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. Typhimurium strains), Tryptophan (E. coli strains)

NUMBER OF REPLICATIONS: All concentrations for all experiments were tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.
In addition to the criteria for a positive or negative response, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3000 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
up to 7.0-fold increases
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3000 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
up to 5.8-fold increases
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3000 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
up to 5.6-fold increases
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3000 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
up to 6.6-fold increases
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3000 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
up to 3.8-fold increases
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
up to 2.8-fold increases
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
up to 2.3-fold increases
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
up to 4.1-fold increases
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
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml (test item formed droplets in water)
- Precipitation: At 512 µg/plate and upwards (Dose-range finding test); at 512 µg/plate and upwards (Mutation experiment, start of the incubation period); at 1600 µg/plate and above (Mutation experiment; end of the incubation period)

RANGE-FINDING/SCREENING STUDIES: The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. Based on the results, the results of the dose range finding test are reported as a part of the mutation experiment. In the second part of the mutation experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Six concentrations, 52, 164, 512, 1600, 3000 and 5000 μg/plate were tested in triplicate. In compliance with the OECD guideline No 471, there is no requirement for verification of a clear positive response. Since the test results of the mutation experiment showed clear positive responses, the follow-up experiment was not performed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The strain-specific positive control values were within the laboratory historical control data ranges
- Negative (solvent/vehicle) historical control data: The negative control values were within the laboratory historical control data ranges

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
- Other observations when applicable: Cytotoxicity was observed in the tester strains TA100, TA1535, TA1537 and TA98 in the absence and presence of S9-mix at the concentrations of 3000 and 5000 μg/plate. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

Although some increases were only observed at cytotoxic or precipitating dose levels or the number of revertants were within the historical control data ranges, clear dose related increases were observed in tester strains TA98 and TA100 both in the absence and presence of S9-mix.

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

Based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
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:
2003-12-22 (date test substance 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; 4) results from experiment 1 are not readable in the tables.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Description : light yellow liquid
Label : T 749
Date received : 2003-12-22
Storage conditions : Room temperature in the dark
Species / strain / cell type:
other: human lymphocytes
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
induced rat liver S9 (2%)
Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 0, 5.96, 11.92, 23.84, 47.69, 95.38, 190.75, 381.5, 763 and 1526 μg/mL
The results of a preliminary toxicity test were used to set the concentration range for the chromosome aberration test:
- Group 1 (4-hour without S9 with 20 hour expression time): 0, 95.38, 190.75, 381.5, 763, 1144.5 and 1526 μg/mL (0, 381.5, 763 and 1144.5, μg/mL were selected for metaphase analysis)
- Group 2 (4-hour with S9 with 20 hour expression time): 0, 95.38, 190.75, 381.5, 763, 1144.5 and 1526 μg/mL (0, 763, 1144.5 and 1526 μg/mL were selected for metaphase analysis)
- Group 3 (24-hour without S9 with 0 hour expression time): 0, 95.38, 190.75, 381.5, 572.25, 763 and 1144.5 μg/mL (0, 190.75, 381.5 and 572.25 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
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation; at 0.4 μg/mL for the 4(20) hour exposure and 0.2 μg/mL for the 24 hour continuous exposure (Groups 1 and 3 respectively)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation; at 10 μg/mL for the 4(20) hour exposure (Group 2)
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: 20 hours (Groups 1 and 2) was provided in the study report as the expression period. However, no information was given on the time of addition of a spindle inhibitor ; 0 hours (Group 3)
- Fixation time: 24 hours (all groups)

SPINDLE INHIBITOR: no data
STAIN: no data

NUMBER OF REPLICATIONS: Two cultures were tested per group. 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 cells per evaluated culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A positive response was reported 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 was generally required and appropriate statistical test may have been applied in order to record a positive response.
Statistics:
Statistics were included in the study. However, no information was provided on the tests performed.
Species / strain:
other: human lympocytes
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: human lymphocytes
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: human lymphocytes
Metabolic activation:
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
There was no precipitate of test material at any dose level in any exposure group.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed in order to determine the dose levels for the main chromosome aberration assay. The dose range for the toxicity test was 5.96 to 1526 μg/ml, based on a 10mM maximum dose level. The cultures were treated for 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). Cultures were taken down at 24 hours after the initiation of treatment. There was no precipitate of the test substance at any dose level in any exposure group. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to the maximum dose level of 1526 μg/ml in both the 4(20) hours pulse exposure groups and at up to 763 μg/ml in the 24 hour continuous exposure group. The data show a slight reduction in mitotic index at the maximum dose level tested in both 4(20)-hour groups. In the 24 hours group there was a dose-related toxic response that showed approximately 70% mitotic inhibition at 763 μg/ml. Therefore the selection of the dose range for the main test was based on the 10mM maximum recommended dose level for the pulse exposure groups and on toxicity for the continuous exposure group.

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 materials induced statistically significant increases in the frequency of cells with aberrations. It was therefore 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 the maximum 10 mM dose level, 1526 µg/mL in Groups 1 and 2 and at up to the intermediate dose level of 572.25 µg/mL in Group 3. The test substance was more toxic than expected from the Preliminary Toxicity test data and induced mitotic inhibition in Groups 1 and 3. In Group 1, 66 % mitotic inhibition was achieved at 1144.5 µg/mL and 20 % inhibition was achieved at 1526 µg/mL Group 2. In Group 3, there was a steep toxicity curve and the ideal of 50 % mitotic inhibition was not achieved although 80 % inhibition was observed at 572.25 μg/ml.
Remarks on result:
other: Group 1: 4 (20) hour - without S9

The test material did not induce statistically significant increases in the frequency of cells with aberrations in either of the 4(20) hours pulse exposures. However, there were modest increases in the frequency of cells with aberrations at the maximum dose level in each case, which included a small number of chromatid exchange aberrations. In the 24 hour continuous exposure group there was a statistically significant dose-related increase in the frequency of cells with aberrations. The greatest increase in aberrant cell frequency was observed at 572.25 μg/ml, a dose level close to the toxic limit. Many of the aberrations seen were chromatid breaks and also chromatid gaps, although one cell with a chromatid exchange was also recorded. The toxicological significance of the types of aberrations seen was not clear because, although some exchange aberrations were seen in all exposure groups their frequency was lower than may be expected, particularly for the large increase in aberrant cell frequency seen at the maximum dose level of the 24-hour exposure group.

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

Conclusions:
Interpretation of results:
negative in the 4 hour exposure period with and without metabolic activation
positive in the 24 hour exposure period without metabolic activation

Under the conditions of the study, it was concluded that the test substance induced statistically significant increases in the frequency of cells with chromosome aberrations in the absence of S9 after 24 hours continuous exposure. 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

Description of key information

 The micronucleus assay was performed in bone marrow cells of male/female NMRI mouse with T000749 according to OECD Guideline 474 (Durward R, 2004). 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.

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2003-12-22 (date test substance was received) to 2004-11-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
No detailed documentation is provided on test substance, animals, methods and results evaluation
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
No detailed documentation is provided on test substance, animals, methods and results evaluation
Qualifier:
according to
Guideline:
other: USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines for testing of new chemical substances.
Deviations:
no
Remarks:
No detailed documentation is provided on test substance, animals, methods and results evaluation
Principles of method if other than guideline:
not applicable
GLP compliance:
no
Type of assay:
micronucleus assay
Specific details on test material used for the study:
Description: Light yellow liquid
Date received: 2003-12-02
Storage conditions: Room temperature, in the dark
Species:
mouse
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS: no data

ENVIRONMENTAL CONDITIONS: no data

IN-LIFE DATES: no data
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: not indicated
- Concentration of test material in vehicle: not indicated
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:no data

DIET PREPARATION: no data
Duration of treatment / exposure:
Animals were treated once.
Frequency of treatment:
single oral dose
Post exposure period:
Test substance animals and vehicle control animals were sacrificed at 24 and 48 hours after treatment at the high dose group and 24 hours for the two lower dose groups were sacrificed at 24 hours. Positive control animals were sacrificed at 24 hours.
Dose / conc.:
2 000 other: mg/kg
Remarks:
24-hour and 48-Hour Sampling Time
Dose / conc.:
1 000 other: mg/kg
Remarks:
24-hour Sampling Time
Dose / conc.:
500 other: mg/kg
Remarks:
24-hour Sampling Time
No. of animals per sex per dose:
7 animals per group and sacrifice time point for the test substance and vehicle control groups, and 5 animals for the positive control group were used.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control: no data
- Route of administration: oral unspecified
- Doses / concentrations: 50 mg/kg
Tissues and cell types examined:
bone marrow (erythrocytes): at least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: 2000 mg/kg is the maximum recommended dose

TREATMENT AND SAMPLING TIMES: single oral administration; sampling 24h and 48h after administration

DETAILS OF SLIDE PREPARATION: Animals were killed at appropriate times, the bone marrow was extracted, and smear preparations were made and stained.

METHOD OF ANALYSIS: Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.
Evaluation criteria:
no data
Statistics:
Statistics were performed, but no data were provided on the tests that were run. P<0.001 was considered in the study to be statistically significant.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs, including hunched posture and ptosis, were observed in animals dosed with the test material at 2000 mg/kg in both the 24 and 48-hour groups.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: The study indicated that 2000 mg/kg produced toxicity but no premautre deaths. No further data were provided on the other dose levels were used.
- Solubility: no data
- Clinical signs of toxicity in test animals: No premature deaths were observed in test animals dosed with the test substance. However, clinical signs were observed at 2000 mg/kg as follows: hunched posture and ptosis.
- Evidence of cytotoxicity in tissue analyzed: no data
- Rationale for exposure: The range-finding test was performed to find suitable dose levels of the test substance for the main study and to investigate to see if there was a marked difference in toxic responses between sexes. There was no marked difference in toxicity of the test substance between sexes; therefore the main test was performed using only male mice.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test substance dose groups when compared to their concurrent vehicle control groups. However, the observation of clinical observations was taken to indicate that systemic absorption had occurred.
- The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes, hence confirming the sensitivity of the system to the known mutagenic activity of the positive control under the conditions of the test.
- Ratio of PCE/NCE: There were no statistically significant decreases in PCE/NCE ratio in the 24- or 48-hour test substance groups when compared to their concurrent vehicle control groups.
- Appropriateness of dose levels and route: The dose levels were chosen based on the results of the range-finding study: 2000 mg/kg bw was the maximum recommended dose
- Statistical evaluation: The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.
- Evidence of cytotoxicity: There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at 2000 mg/kg in both the 24 and 48-hour groups, these were as follows: hunched posture and ptosis.
Conclusions:
Interpretation of results: negative
The test substance was evaluated for the potential to produce a significant increase in the frequency of micronuclei of orally treated mice. The test substance was found to be negative for the induction of micronuclei and was therefore considered to be non-genotoxic under the conditions of the test.
Endpoint:
in vivo mammalian somatic cell study: gene mutation
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

In a K1 bacterial reverse mutation assay performed according to OECD Guideline 471, Verspeek-Rip C evaluated the potential to induce reverse mutations in Salmonella typhimurium strains TA98, TA100 and TA1535 and TA1537 and Escherichia coli (WP2uvrA).

The test item was dissolved in dimethyl sulfoxide at a concentration of 50 mg/ml.

In a dose range finding test, the test item was tested at a concentration range of 1.7 to 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at the top dose of 5000 μg/plate. In tester strain TA100, toxicity was observed at the dose level of 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

Based on the results of the dose range finding test, the test item was tested in the mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at dose levels of 1600 μg/plate and above. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction in the bacterial background lawn, was observed in all three tester strains in the absence and presence of S9-mix at the concentrations of 3000 and 5000 μg/plate.

In the absence of S9-mix, in tester strain TA1537 up to 5.8-fold increases the concurrent vehicle control, in tester strain TA98 up to 6.6-fold increases the concurrent vehicle control, in tester strain TA100 up to 2.8-fold increases the concurrent vehicle control and in tester strain WP2uvrA up to 4.1-fold increases the concurrent vehicle control were observed.

In the presence of S9-mix, in tester strain TA1537 up to 7.0-fold increases the concurrent vehicle control, in tester strain TA98 up to 5.6 increases the concurrent vehicle control, TA100 up to 3.8-fold increases the concurrent vehicle control and in tester strain WP2uvrA up to 2.3-fold the concurrent vehicle control were observed. Although some increases were only observed at cytotoxic or precipitating dose levels or the number of revertants were within the historical control data ranges, clear dose related increases were observed in tester strains TA98 and TA100 both in the absence and presence of S9-mix.

The test item did not induce a significant dose-related increase in the number of revertant colonies in tester strain TA1535.

Since the test results of this mutation assay showed clear positive responses, a follow-up experiment was not performed.

Based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

A supporting study (K2, Vanparys P., 1986) was performed according to a method similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay). The Ames Salmonella/microsomal activation assay was conducted with T000749 in triplicate using the strains TA98 and TA100 in the presence and in the absence of a metabolic activation system. The following dose levels were tested: 31.1, 62.5, 125, 250, 500, 1000 and 2000 µg T749/plate. The plate incorporation test was used.

In a preliminary range finding study using the trains TA98 and TA100 in the asbsence of S9 -mix, a thinning of the bacterial background lawn was found at 1000 µg/plate onwards. At concentration of 2500 µg/plate, T000749 was highly toxic to the strains TA98 so that 2000 µg/plate was selected as the highest acceptable concentration for the main study.

In the main study, the Salmonella typhimurium strains TA98 and TA100 did not reveal any increase in mutation rate at concentrations up to 2000 µg/plate, either in the presence or absence of metabolic activation. These findings were confirmed by repeated test.

As expected the appropriate positive control chemicals induced marked increases in revertant colony numbers will all the strains.

In vitro mammalian chromosome aberration test:

Wright (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 item, ranging from 95.38 to 1526 µg/mL in the first experiment and from 95.38 to 1144.5 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.

A microscopic assessment of the slides showed that metaphase cells were present at up to the maximum 10 mM dose level, 1526 μg/ml in both the 4(20)-hour exposure groups and at up to the intermediate dose level of 572.25 μg/ml in the 24-hour continuous exposure group. The test material was more toxic than expected from the Preliminary Toxicity test data and induced mitotic inhibition in both of the without-metabolic activation exposure groups. In the 4(20) hours exposure groups 66% mitotic inhibition was achieved at 1144.5 μg/ml without S9 and 20% inhibition at 1526 μg/ml with S9. In the 24-hour exposure group there was a steep toxicity curve and the ideal of 50% mitotic inhibition was not achieved although 80% inhibition was observed at 572.25 μg/ml.

Dose selection for metaphase analysis was therefore limited by toxicity in both of the without-S9 exposure groups but the maximum 10 mM concentration was scored for the with-S9 exposure group.

The test material did not induce statistically significant increases in the frequency of cells with aberrations in either of the 4(20) hours pulse exposures. However, there were modest increases in the frequency of cells with aberrations at the maximum dose level in each case, which included a small number of chromatid exchange aberrations. In the 24 hours continuous exposure group there was a statistically significant dose-related increase in the frequency of cells with aberrations. The greatest increase in aberrant cell frequency was observed at 572.25 μg/ml, a dose level close to the toxic limit. Many of the aberrations seen were chromatid breaks and also chromatid gaps, although one cell with a chromatid exchange was also recorded. The toxicological significance of the types of aberrations seen was not clear because, although some exchange aberrations were seen in all exposure groups their frequency was lower than may be expected, particularly for the large increase in aberrant cell frequency seen at the maximum dose level of the 24-hour exposure group.

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

T000749 was therefore considered to be clastogenic to human lymphocytes in vitro.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. It was therefore considered that the metabolic activation system was shown to be functional and the test method itself was operating as expected.

Genetic toxicity: in vivo

Micronucleus assay:

A micronucleus assay study (K2, Durward, 2004) was performed to investigate the potential of T000749 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum recommended dose (2000 mg/kg) with 1000 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone was marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence 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: 500, 1000 and 2000 mg/kg b.w
- 48 h preparation interval: 2000 mg/kg b.w

There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups. However, the observation of clinical observations was taken to indicate that systemic absorption had occurred.

There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

T000749 was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

 

 

 

Justification for classification or non-classification

Positive results were observed in the Ames test and in the in vitro chromosome aberration tests, and negative results were obtained in 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 T000749 should be classified as Muta 2 - H341 Suspected of causing genetic defects