Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

-       Bacterial reverse mutation assay 

A K1 GLP-compliant bacterial reverse mutation assay was performed according to OECD Guideline 471 and EU Method B.13/14 in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli strain WP2 uvrA. T001141 was concluded to be non-mutagenic in the presence or absence of metabolic activation.

-       Chromosome aberration study

In a K2 in vitro chromosome aberration study in human lymphocytes, performed according to a method equivalent to OECD Guideline 473, T001141 induced statistically significant dose related increases in the frequency of cells with chromosome aberrations in both the presence and the absence of a liver enzyme metabolising system. The test material was therefore 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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-04-11 to 2016-05-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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: I15FB3131
- Expiration date of the lot/batch: 2017-06-11 (retest date)
- Date of Manufacture: 2015-06-12

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in DMSO. Preparation of test solutions started with solutions of 50 mg/ml applying treatment with ultrasonic waves resulting in a clear colourless solution. The lower test concentrations were prepared by subsequent dilutions in DMSO. Test item concentrations were used within 2 hours of preparation.
Target gene:
The Histidine locus in S. typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Tryptophan locus in E. coli strains (WP2uvrA)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
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: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate with and without 5% S9-mix (TA100 and WP2uvrA) (top dose selected based
on the solubility findings)
Mutation experiment I: 52, 164, 512, 1600 and 5000 μg/plate with and without 5% S9-mix (TA98, TA1535 and TA1537) (top dose selected based on the dose ran
ge finding test results)
Mutation experiment II: 492, 878, 1568, 2800 and 5000 μg/plate with and without 10% S9-mix (all tester strains) (top dose selected based on the
dose range finding test and mutation experiment I results)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml (the test item sank to the bottom). The test item was soluble at 50 mg/ml (= 5000 μg/plate) in DMSO after treatment with ultrasonic waves. Based on these solubility findings, DMSO was selected as vehicle.
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)
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)
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)
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 ± 4 h
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. Typhimurium histidine-dependent strains); Tryptophan (E. coli tryptophan-dependent strains)

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

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn; increase in the size of the microcolonies; reduction of the 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.
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility : The test item was observed to be insoluble in water at 50 mg/ml (the test item sank to the bottom)
- Precipitation: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any of the strains in both experiments

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. Normal bacterial background lawn and no precipitate were observed up to the highest dose. Based on these results, the dose ranges for mutation experiment I (TA98, TA1535 and TA1537) were selected.

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: There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutation experiment 1

Toxicity: In strain TA1537 (absence and presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely that these reductions are caused by incidental fluctuations in the number of revertant colonies.

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

Based on the results of this study it is concluded that the test item is not 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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-12-22 (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
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 was not known.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Description : White crystalline solid
Chemical name : 1-acetyl-4-(4-hydroxyphenyl) piperazine
Purity : 100%
Label : T 1141 CODE NO.:012536
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 applicable
Metabolic activation:
with and without
Metabolic activation system:
2 % rat liver homogenate metabolizing system (S9)
Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 0, 8.61, 17.21, 34.42, 68.84, 137.69, 275.38, 550.75, 1101.5 and 2203 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): 0,137.69, 275.38, 550.75, 826.13, 1101.5, 1652.25 μg/mL (0, 550.75, 826.13and 1101.5 μg/mL were selected for metaphase analysis)
- Group 2 (4-hour with S9 with 20 hour expression time): 0, 137.69, 275.38, 550.75, 1101.5, 1652.25, 2203 μg/mL (0, 275.38, 550.75 and 1101.5 μg/mL were selected for metaphase analysis)
- Group 3 (24-hour without S9 with 0 hour expression time): 0, 4.31, 8.61, 17.21, 25.82, 34.42, 51.63 μg/mL (0, 8.61, 17.21 and 25.82, 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:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with 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 treated 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 analyzed culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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

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 statistically significant.
Species / strain:
other: human lymphocytes
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
There was no precipitate of test material observed at any dose level in any exposure groups.

RANGE-FINDING/SCREENING STUDIES:
Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 2203 μg/ml in the 4 hour pulse exposure groups and at up to 68.84 μg/ml in the 24 hour continuous exposure group. The data show dose-related toxicity in all of the exposure groups. Furthermore, in both 4(20)-hour pulse exposure groups there was approximately 50% mitotic inhibition at 1101.5 μg/ml. Therefore the selection of the dose range for the main test was based toxicity.

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 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 1652.25 ug/mL in both Groups 1 and 2, and at up to 51.63 ug/mL in Group 3. The test substance induced mitotic inhibition in all exposure groups. In Groups 1 and 2, 50% mitotic inhibition was achieved at 1101.5 ug/mL, while in Group 3, the test substance was more toxic than expected and there was approximately 75% mitotic inhibition at 25.82 ug/mL. Dose selection for metaphase analysis was limited by toxicity in all exposure groups.
Remarks on result:
other: Groups 1, 2 and 3

The test material induced statistically significant dose related increases in the frequency of cells with aberrations in all exposure groups, both with and without metabolic activation. The types of aberrations observed were mainly chromatid gap and breaks with few exchange-type aberrations. It is considered unusual to have so few exchange aberrations with a dose related positive response, as seen in this experiment.

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:
positive with and without metabolic activation

The test substance was evaluated for induction of chromosome aberrations in primary human lymphocytes. The test substance induced statistically significant dose-related increases in the frequency of cells with chromosome aberrations in both the presence and the absence of a liver enzyme metabolising system. 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

-       Micronucleus test

In a K2 in vivo micronucleus test in the mice, performed according to the OECD Guideline 474, T001141 was found to produce a statistically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test. The test material was therefore considered to be genotoxic under the conditions of the test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-12-22 (date test substance was received) to 2005-09-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
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
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
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
GLP compliance:
no
Type of assay:
micronucleus assay
Specific details on test material used for the study:
Description: white crystalline solid
Date received: 2003-12-22
Storage conditions: Room temperature, in the dark
Species:
mouse
Strain:
not specified
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: mean weight of 23.70 g
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: no data
- Diet: no data
- Water: no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: no data To: 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.:
1 500 other: mg/kg bw
Remarks:
24-hour and 48-Hour Sampling Time
Dose / conc.:
750 other: mg/kg bw
Remarks:
24-hour Sampling Time
Dose / conc.:
375 other: mg/kg bw
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(s): no data
- Route of administration: oral (unspecified)
- Doses / concentrations: 50 mg/kg bw
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: Dose levels were selected on the basis of the results of a range-finding study

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): single oral administration; sampling 24h and 48h after administration

DETAILS OF SLIDE PREPARATION: The animals were killed at the specified time (24 or 48 hours after treatment), the bone marrow extracted, and smear preparations made and stained.

METHOD OF ANALYSIS: Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.
Evaluation criteria:
no data
Statistics:
No data was provided on the statistical tests performed. However, statistical significance was measured down to p<0.05.
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Remarks:
hunched posture, ptosis, ataxia and lethargy
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 1000 mg/kg produced toxicity and 2000 mg/kg bw produced premautre deaths. No further data were provided on the other dose levels were used.
- Solubility: no data
- Clinical signs of toxicity in test animals: Adequate evidence of toxicity was observed in animals dosed with the test substance via the oral route, with a premature death occurring at 2000 mg/kg. Clinical signs were also observed at and above 1000 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 statistically significant increases in the frequency of micronucleated PCEs in all of the test substance dose groups when compared to their concurrent vehicle control groups. The response in the 24-hour groups was inversely dose-related; it was considered that this was due to the high levels of toxicity observed at the upper two test substance dose levels.
- Ratio of PCE/NCE (for Micronucleus assay): There were statistically significant decreases in the PCE/NCE ratio in all of the 24- or 48-hour test substance groups when compared to their concurrent vehicle control groups. This accompanied by the presence of clinical signs was taken to indicate that systemic absorption had occurred, and exposure to the target tissue was achieved.
- 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.
- Appropriateness of dose levels and route: The dose levels were chosen based on the results of the range-finding study: 1500 mg/kg bw was the maximum tolerated dose (MTD)
- Statistical evaluation: See the table below.
- Evidence of cytotoxicity: There were no premature deaths seen in any of the dose groups in the main test. Clinical signs were observed in animals dosed with the test material at and above 750 mg/kg in both the 24- and 48-hour groups where applicable; these were as follows: hunched posture, ptosis, ataxia and lethargy.

The group mean results of the micronucleus assay and cytotoxicity are presented below:

 

Micronucleus Results - Summary of Group Mean Data

 

Micronuclei per 2000 PCE

PCE/NCE Ratio

Treatment Group

Group Mean

SD

Group Mean

SD

Vehicle Control (48 h)

1.3

1.3

0.99

0.35

Vehicle Control (24 h)

1.3

1.5

0.95

0.22

Positive Control (24 h)

42.8***

16.3

1.24

0.38

1500 mg/kg (48 h)

24.1***

16.6

0.13***

0.06

1500 mg/kg (24 h)

12.6**

9.9

0.34***

0.07

750 mg/kg (24 h)

14.0***

8.6

0.55***

0.07

375 mg/kg (24 h)

38.0***

12.3

0.64*

0.20

* p<0.05; **p<0.01; *** p<0.0001

 

The test substance was found to produce a statistically significant increase in the frequency of miconuclei in PCEs of mice under the conditions of the test.

Conclusions:
The test substance was evaluated for induction of micronuclei in the bone marrow of orally treated male mice. The test substance was considered to be genotoxic under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Bacterial reverse mutation assay
A key study (K1, Verspeeck-Rip, 2016) was performed according to the test guidelines OECD Guideline 471 (Bacterial Reverse Mutation Assay) and the EC Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. 
The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor 1254 induced rat liver metabolic activation system).
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 5% (v/v) S9-mix in the strains TA100 and WP2uvrA. The test item did not precipitate on the plates up to the concentration of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of the dose range finding test were reported as part of the first mutation assay.
Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates up to the top concentration of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
In a second mutation assay, the test item was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item did not precipitate on the plates up to the top concentration of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in any of the experiments.
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.

A supporting study is also used to cover the endpoint: the study followed a method equivalent to the test guideline OECD 471 but was only performed on two strains (Vanparys, 1986). The test substance was shown to have no mutagenic properties towards Salmonella typhimurium strains TA98 and TA100 either with or without S9 metabolic activation.

Chromosome aberration in human lymphocytes

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 item, ranging from 137.69 to 1652.25 µg/mL without metabolic activation and from 137.69 to 2203 µg/mL with metabolic activation in the first experiment (4 hour exposure period followed by a 20 hour recovery period), and from 4.31 to 51.63 µg/mL in the second experiment (24 hour exposure period). 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.

A microscopic assessment of the slides showed that metaphase cells were present at up to 1652.25μg/ml in both the 4(20)-hour exposure groups and at up to 51.63μg/ml in the 24-hour continuous exposure group. The test material induced mitotic inhibition in all exposure groups. In the 4 hours pulse exposures the ideal of 50% mitotic inhibition was achieved at 1101.5 μg/ml in both the with and without S9 exposure groups. In the 24-hour exposure group the test material was more toxic than expected and there was approximately 75% mitotic inhibition at 25.82 μg/ml. Dose selection for metaphase analysis was limited by toxicity in all exposure groups.The test item did not induce statistically significant increases in the frequency of cells with aberrations in any of the exposure groups.
The test material induced statistically significant dose related increases in the frequency of cells with aberrations in all exposure groups, both with and without metabolic activation. The types of aberrations observed were mainly chromatid gap and breaks with few exchange-type aberrations. It is considered unusual to have so few exchange aberrations with a dose related positive response, as seen in this experiment.
The test material did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.

T001141 was therefore considered to be clastogenic to human lymphocytes in vitro.

In vivo micronucleus test in the mouse

SafePharm Laboratories (K2, 2005) performed an in vivo micronucleus test in the mouse (OECD Guideline 474, method B12 of the EEC Commission Directive 2000/32/EC).
The micronucleus test was conducted using the oral route in groups of 7 male mice at the maximum tolerated dose 1500 mg/kg and with 750 and 375 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained.
There were statistically significant decreases in the PCE/NCE ratio in all of the 24 or 48-hour test material group when compared to their concurrent vehicle control group. This accompanied by the presence of clinical signs was taken to indicate that systemic absorption had occurred, and exposure to the target tissue achieved.
There were statistically significant increases in the frequency of micronucleated PCEs in all of the test material dose groups when compared to their concurrent vehicle groups. The response in the 24-hour groups was inversely dose-related; it was considered that this was due to the high level of toxicity observed at the upper two test material dose levels.
The test material was found to produce statistically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.
T001141 was considered to be genotoxic under the condition of the test.

 

 

Justification for classification or non-classification

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

Based on the available data, the criteria laid down in the CLP Regulation and the precautionary principle, the test item T001141 should be classified as Muta 2 - H341 Suspected of causing genetic defects.