Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames bacterial mutation test: not mutagenic with and without metabolic activation

- Mouse lymphoma mammalian cell mutation test: not mutagenic with and without metabolic activation

- Micronucleus in mammalian cells: not clastogenic and not aneugenic

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:
22-April-2010 to 10-May-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
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
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Experiment 2:
TA1535
Without S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
TA1537
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate
TA98
Without S9-mix: 10, 33, 100, 333 and 1000 µg/plate

Experiment 3
TA1535
Without S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
With S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
TA1537
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate
TA98
Without S9-mix: 3, 10, 33, 100, 333 and 1000 µg/plate
With S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
TA100 and WP2uvrA
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate




Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The test substance was dissolved in Milli-Q water.
- Justification for choice of solvent/vehicle: Test compound was soluble in water and water has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Three independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat 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 independently repeated experiment.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 3330 μg/plate and above in the absence of S9-mix in tester strain TA100. Toxicity was observed at the dose level of 5000 μg/plate in the presence of S9-mix in tester strain TA100 and in the absence and presence of S9-mix in tester strain WP2uvrA. of

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without and with S9: 3330 µg/plate and above
TA1537: without and with S9: 1000 µg/plate and above
TA98: without S9: 333 µg/plate and above and with S9: 3330 µg/plate and above
TA100: without and with S9: 3330 µg/plate and above
WP2uvrA: without and with S9: 5000 µg/plate

Since in the tester strains TA1535 and TA98 in the absence of S9-mix and in TA1537 in the absence and presence of 5% (v/v) S9-mix in the first experiment too many dose levels with toxicity were tested, this part of the experiment was repeated (experiment 2).

Conclusions:
Under the test conditions, 202240/A is not mutagenic in Salmonella typhimurium TA100, TA1535, TA1537 and TA98 and Escherichia coli WP2uvrA in the absence or in the presence of metabolic activation.
Executive summary:

A bacterial reverse mutation assay (Ames Test) was performed according to the OECD guideline No. 471 and in compliance with GLP. The test material 202240/A was tested in Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in Escherichia coli (WP2uvrA). The test was performed in three independent experiments in the presence and absence of S9-mix (phenobarbital and ß-naphthoflavone-induced rat liver S9). The dose range finding study was reported as part of Experiment 1.

- Dose range finding:

TA100 and WP2uvrA : up to 5000 µg/plate with and without S9 mix (5%)

- Experiment 1:

TA1535, TA1537, TA98: up to 5000 µg/plate with and without S9 mix (5%)

- Experiment 2:

TA1535: up to 3330 µg/plate without S9 mix

TA1537: up to 1000 µg/plate with and without S9 mix (3.6%)

TA98: up to 1000 µg/plate without

- Experiment 3:

TA1535: up to 3330 µg/plate without S9 mix ; up to 5000 µg/plate with S9 mix (10%)

TA1537: up to 1000 µg/plate with and without S9 mix (10%)

TA98: up to 1000 µg/plate without S9 mix ; up to 5000 µg/plate with S9 mix (10%)

TA100 and WP2uvrA: up to 5000 µg/plate with and without S9 mix (10%).

202240/A did not precipitate on the plates at the highest dose levels.

Toxicity was observed in all tester strains in all experiments. Since in the strains TA1535 and TA98 in the absence of S9-mix and in tester strain TA1537 in the absence and presence of S9-mix too many dose levels with toxicity were tested, this part of the experiment was repeated in mutation experiment 2.

202240/A 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 tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in two independently repeated experiments.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the test conditions, 202240/A is not mutagenic in Salmonella typhimurium TA100, TA1535, TA1537 and TA98 and Escherichia coli WP2uvrA in the absence or in the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
21 October 1998 - 16 December 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test was conducted according to OECD Test Guideline No. 471, 1997, under GLP Standards, and QA
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
92/69/EEC
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
bacterial reverse mutation assay
Target gene:
His-gene: Amino acid histidine, and Trp-gene: amino acid tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: S. typhimurium TA 98: hisD3052, rfa, uvrB +R; TA 100: hisG46, rfa, uvrB +R; TA 1535: hisG46, rfa, uvrB; TA 1537: hisC3076, rfa, uvrB.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: E. coli WP2, uvrA: pKM101
Metabolic activation:
with and without
Metabolic activation system:
Male Sprague Dawley rat liver S9 induced by Aroclor 1254
Test concentrations with justification for top dose:
50, 160, 500, 1600 and 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test compound was dissolved in deionized water and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 ug/plate.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Strain TA100 and TA1535: sodium-azide; Strain TA1537: 9-aminoacridine; Strain TA98: 2-nitrofluorene; Strain WP2uvrA: 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). With metabolic activation: All strains: 2-aminoanthracene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: approx. 20 minutes
- Selection time (if incubation with a selection agent): approx. 48 hours

SELECTION AGENT (mutation assays): agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
Evaluation criteria:
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn.
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.
Statistics:
Mean and Standard Deviation
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Control plates (background control and positive controls) gave the expected number of colonies,
i.e. values were within the laboratory's historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the plate incorporation test toxicity was observed to all bacterial strains at a concentration of 5000 ug/plate with and without metabolic activation.
In the preincubation test toxicity was observed with and without metabolic activation. The test substance was toxic to the bacterial strains TA100 and TA1535 with metabolic activation at dose levels of 1600 ug/plate and above, and without metabolic activation at concentrations of 500 ug/plate and above. With the bacterial strains TA1537, TA98 and Escherichia coli toxicity was observed with metabolic activation at a concentration of 5000 ug/plate, and without metabolic activation at dose levels of 1600 ug/plate and above.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the test conditions, Locron P is not mutagenic in Salmonella typhimurium TA100, TA1535, TA1537 and TA98 and Escherichia coli WP2uvrA in the absence or in the presence of metabolic activation.
Executive summary:

Locron P was tested for mutagenicity with the strains TA100, TA1535, TA1537 and TA98 of Salmonella typhimurium and Escherichia coli WP2uvrA according to the OECD Guideline No. 471 and in compliance with GLP.

Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. Concentrations for both studies were 50, 160, 500, 1600 and 5000 ug/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range and similar to that described in the literature. All the positive control compounds showed the expected increase in the number of revertant colonies.

In the plate incorporation test toxicity was observed to all bacterial strains at a concentration of 5000 ug/plate with and without metabolic activation.

In the preincubation test toxicity was observed with and without metabolic activation. The test substance was toxic to the bacterial strains TA100 and TA1535 with metabolic activation at dose levels of 1600 ug/plate and above and without metabolic activation at 500 ug/plate and above. With the bacterial strains TA1537, TA98 and Escherichia coli toxicity was observed with metabolic activation at a concentration of 5000 ug/plate and without metabolic activation at dose level of 1600 ug/plate and above.

Mutagenicity: In the absence and in the presence of the metabolic activation system Locron P did not result in relevant increases in the number of revertants in any of the bacterial strains.

Under the test conditions, Locron P is not mutagenic in Salmonella typhimurium TA100, TA1535, TA1537 and TA98 and Escherichia coli WP2uvrA in the absence or in the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-Jul-2010 to 07-Sep-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 11, 36, 109, 363 and 725 µg/mL
Without S9-mix, 24 hours treatment: 11, 36, 109, 363 and 725 µg/mL
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 1, 3, 10, 33, 100, 250, 333 and 500 µg/mL
With S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium: RPMI 1640 (Hepes buffered medium (Dutch modification)
- Justification for choice of solvent/vehicle: Test compound was soluble in culture medium and culture medium has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 : 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 : 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
L5178Y/TK+/-3.7.2C
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
tested beyond the limit of the solubility
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
L5178Y/TK+/-3.7.2C
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 250 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- In the absence of S9, toxicity was only observed at dose levels of 109 µg/mL and above, 24 hours treatment.
- In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level (725 µg/mL)

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was only reduced by 72% compared to the total growth of the solvent controls after the 24 hours treatment period.

In the absence of S9-mix (3 hours treatment) and in the presence of S9-mix (both experiments), no toxicity was observed up to and including the highest tested dose level of 333 µg/ml .
Conclusions:
Under the test conditions, 202029/A was shown to be non-mutagenic to L5178Y cells at the TK locus in the presence or absence of a metabolic activation.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline No. 476 and in compliance with GLP, L5178Y/TK+/- -3.7.2C mouse lymphoma cells were exposed to test material at the following concentrations:

- Dose range finding test:
3 h treatment, with and without S9-mix: 11, 36, 109, 363 and 725 µg/mL
24 h treatment, without S9-mix: 11, 36, 109, 363 and 725 µg/mL


- First mutagenicity test:
3 h treatment, without and with 8 % (v/v) S9-mix: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL


- Second mutagenicity test:
3 h treatment, with 12 % (v/v) S9-mix: 0.1, 0.3, 1, 3, 10, 33, 100 and 333
μg/mL
24 h treatment, without S9-mix: 1, 3, 10, 33, 100, 250, 333 and 500 µg/mL

Vehicle and positive control groups were also included in each mutation test.

In the dose range finding test, after 3 h of treatment without S9-mix, the relative suspension growth was 77% at the highest test substance concentration of 725μg/mL compared to the suspension growth of the solvent control. With S9-mix, the relative suspension growth was 76 % at the highest test substance concentration of 725μg/mL compared to the relative suspension growth of the solvent control. After 24 h of treatment without S9-mix, the relative suspension growth was 14 % at the test substance concentration of 725μg/mL compared to the relative suspension growth of the solvent control. Test material precipitated in the exposure medium at concentrations of 363μg/mL and above in all experimental conditions.

In the first mutagenicity test, the relative total growth was 84% and 103% at 333μg/mL compared to the total growth of the solvent controls at 3 h treatment without and with 8% S9-mix, respectively. Precipitation was observed at the highest dose tested in both experimental conditions, i.e. 333 µg/mL.In the second mutagenicity test, the relative total growth was 28% at 500 µg/mL and 103% at 333μg/mL compared to the total growth of the solvent controls for the 24 h treatment without S9 -mix and for the 3 h treatment with 12% S9-mix, respectively. Precipitation was recorded from 250 µg/mL and at 333 µg/mLfor the 24 h treatment without S9-mix and for the 3 h treatment with S9-mix, respectively.

Test material did not induce a significant increase in the mutation frequency at any dose level either with or without metabolic activation in two independently repeated experiments. In all tests the concurrent vehicle and positive control were within acceptable ranges.

Under the test conditions, test material is not mutagenic at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells, in the absence and presence of S9-mix.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-mar-2010 to 03-may-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
OECD Guideline for the Testing of Chemicals, Draft proposal for a new Guideline No. 487: In Vitro Mammalian Cell Micronucleus Test (November 2, 2009).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin  was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hr exposure; 27 hr fixation: 10, 33, 100, 333 and 1000 µg/mL
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 33, 100, 333, 1000 and 1105 µg/mL
First experiment
Without S9-mix, 3 h exposure time, 27 h fixation time: 100, 300 and 600 µg/mL
With S9-mix, 3 h exposure time, 27 h fixation time: 300, 500 and 600 µg/mL
Second experiment
Without S9-mix, 24hr exposure; 24 hr fixation: 100, 300 and 600 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Exposure medium (RPMI 1640 medium)
- Justification for choice of solvent/vehicle: Test compound was soluble in the exposure mediu, this solvent has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 : 0.25 µg/mL for a 3 h exposure period, 0.15 µg/mL for a 24 h exposure period
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: colchicine: 0.1 µg/ml for a 3 hours exposure period and 0.05 µg/ml for a 24 hours exposure period
Remarks:
without S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 : 0.1 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9: 3 hr treatment, 24 hours recovery/harvest time
Continuous treatment
Without S9: 24 hours treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CBPI index).


OTHER:
- Interphase cells (mono and bi-nucleated cells) were analysed microscopically for the presence of micronuclei
Evaluation criteria:
A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.
Statistics:
The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics.
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 600 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was only observed at the dose level of 1000 µg/ml in the presence of S9, 3 hr treatment/27 hr fixation

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of mono and binucleated cells with micronuclei observed in 2000 cells found in the solvent and positive control cultures was within the laboratory historical control data range.


The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range, except in the presence of S9-mix. Although the number of binucleated cells with miconuclei was above the historical control data range in the presence of S9-mix, the number of binucleated cells with micronuclei was less than 10. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Conclusions:
Under the test conditions, NOTOX Substance 202029/A did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments. Therefore, test item is not clastogenic or aneugenic in human lymphocytes.
Executive summary:

An in vitro micronucleus assay was performed according to the most recent OECD draft proposal for a new guideline 487 and in compliance with GLP.

This report describes the effect of 202029/A on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity of 202029/A was tested in two independent experiments.

In the first cytogenetic assay, 202029/A was tested up to 600 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. 202029/A precipitated in the culture medium at this dose level.

In the second cytogenetic assay, 202029/A was also tested up to 600 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. 202029/A precipitated in the culture medium at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range, except in the presence of S9-mix. Although the number of binucleated cells with miconuclei was above the historical control data range in the presence of S9-mix, the number of binucleated cells with micronuclei was less than 10. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

202029/A did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Under the test conditions, test item is not clastogenic or aneugenic in human lymphocytes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Micronucleus in the mouse, bone marrow cells: not clastogenic and not aneugenic

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 September 1998 - 11 November 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test was conducted according to OECD Test Guideline No. 474, 1997, under GLP Standards, and QA
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
92/69/EEC
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Age at study initiation: male/female animals approx. 7 weeks
- Weight at study initiation: Males: 35 – 43 g; Females: 29 – 32 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: in fully air-conditioned rooms in makrolon cages type 3 (five animals) on soft wood granulate
- Diet: rat/mice diet ssniff R/M-H (V 1534), ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: 5 days under study conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: soluble in deionized water
- Concentration of test material in vehicle: (volume) 20% (w/v)
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the days of administration the test substance was dissolved in deionized water at the appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

DIET PREPARATION: no data
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Twice at an interval of 24 hours
Post exposure period:
Animals were killed by carbon dioxide asphyxiation 24 hours after dosing.
Remarks:
Doses / Concentrations:
2000 mg kg bw
Basis:
analytical conc.
No. of animals per sex per dose:
Five male and five female mice were used per sampling time in each treatment group (negative and positive control, and test substance)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (Endoxan)
- Route of administration: oral gavage
- Doses / concentrations: 50 mg kg bw / 0.5 % (w/v)
Tissues and cell types examined:
bone-marrow smears; micronucleated polychromatic erythrocytes, and ratio polychromatic erythrocytes (PCE)/total erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a preliminary dose range finding study, oral administration of 2000 mg Locron P per kg body weight did cause only slight toxic effects in male and female mice over an observation period of 7 days. This dose level was therefore the regularly limit dose, selected as the highest dose for the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): two oral administrations at an interval of 24 hours, and one sampling time (24 hours).

DETAILS OF SLIDE PREPARATION: One drop of the thoroughly mixed sediment of bone marrow was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours. Staining was performed as follows: 5 min. in methanol, 5 min. in May-Grunwald's solution; brief rinsing twice in distilled water, 10 min. staining in Giemsa solution, rinsing in distilled water, drying, and coating with Entellan.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
Evaluation criteria:
Both biological and statistical significances were considered together for evaluation purposes. A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
Averages and standard deviations were calculated. One-sided Wilcoxon tests were performed.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg kg bw
- Clinical signs of toxicity in test animals: Spontaneous activity decreased up to 6 hours after application, but no deaths were recorded (total 3 males and 3 females tested)
- Evidence of cytotoxicity in tissue analyzed: No macroscopic findings were observed.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Locron P did not statistically significantly raise the frequency of micronucleated polychromatic erythrocytes.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups which were treated with Locron P showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis.
The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained essentialy unaffected by the treatment with Locron P and was not less than 20% of the control value.
- Appropriateness of dose levels and route: In the preliminary dose range finding study, oral administration of 2000 mg kg bw did cause only slight toxic effects in male and female mice over an observation period of 7 days. This dose level was therefore the regularly limit dose, selected as the highest dose for the main study.

- Statistical evaluation: 2000 mg Locron P/kg bw did not show a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.
Conclusions:
Interpretation of results (migrated information): negative
The results lead to the conclusion that Locron P did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described in this report.
Executive summary:

The micronucleus test was carried out with Locron P. The test compound was dissolved in deionized water and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay. According to the test procedure the animals were killed 24 hours after administration. Endoxan (Cyclophosphamide) was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight. The number of polychromatic erythrocytes containing micronuclei was not increased following treatment with Locron P. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Locron P and was not less than 20% of the control value. The positive control, Endoxan, induced a marked and statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent. Under the conditions of the study the results indicate that Locron P is not mutagenic and not aneugenic in the micronucleus test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames Bacterial Mutation Test:

Two key studies were identified, both performed on the registered substance.

1) A bacterial reverse mutation assay (Ames Test) was performed according to the OECD guideline No. 471 and in compliance with GLP (NOTOX, 2010). The test material 202240/A was tested in Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in Escherichia coli (WP2uvrA). The test was performed in three independent experiments in the presence and absence of S9-mix (phenobarbital and ß-naphthoflavone-induced rat liver S9). Test material was tested up to 1000 to 5000 µg/plate depending on the strain and on the presence or absence of metabolic activation (S9 mix from 3.6 to 10%). 202240/A did not precipitate on the plates at the highest dose levels. Toxicity was observed in all tester strains in all experiments. Since in the strains TA1535 and TA98 in the absence of S9-mix and in tester strain TA1537 in the absence and presence of S9-mix too many dose levels with toxicity were tested, this part of the experiment was repeated in mutation experiment 2. 202240/A 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 tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in two independently repeated experiments. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the test conditions, 202240/A is not mutagenic in Salmonella typhimurium TA100, TA1535, TA1537 and TA98 and Escherichia coli WP2uvrA in the absence or in the presence of metabolic activation.

2) Locron P was tested for mutagenicity with the strains TA100, TA1535, TA1537 and TA98 of Salmonella typhimurium and Escherichia coli WP2uvrA according to the OECD Guideline No. 471 and in compliance with GLP (Stammberger, 1999). Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. Concentrations for both studies were 50, 160, 500, 1600 and 5000 ug/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range and similar to that described in the literature. All the positive control compounds showed the expected increase in the number of revertant colonies. In the plate incorporation test toxicity was observed to all bacterial strains at a concentration of 5000 ug/plate with and without metabolic activation. In the preincubation test toxicity was observed with and without metabolic activation. The test substance was toxic to the bacterial strains TA100 and TA1535 with metabolic activation at dose levels of 1600 ug/plate and above and without metabolic activation at 500 ug/plate and above. With the bacterial strains TA1537, TA98 and Escherichia coli toxicity was observed with metabolic activation at a concentration of 5000 ug/plate and without metabolic activation at dose level of 1600 ug/plate and above. In the absence and in the presence of the metabolic activation system Locron P did not result in relevant increases in the number of revertants in any of the bacterial strains. Under the test conditions, Locron P is not mutagenic in Salmonella typhimurium TA100, TA1535, TA1537 and TA98 and Escherichia coli WP2uvrA in the absence or in the presence of metabolic activation.

In vitro studies (micronucleus and mammalian cell gene mutation) performed on the analogue susbstance aluminium chloride:

An in vitro micronucleus assay was performed according to the most recent OECD draft proposal for a new guideline 487 and in compliance with GLP. This report describes the effect of 202029/A on the frequency of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity of 202029/A was tested in two independent experiments. In the first assay, 202029/A was tested up to 600 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. 202029/A precipitated in the culture medium at this dose level. In the second assay, 202029/A was also tested up to 600 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. 202029/A precipitated in the culture medium at this dose level. The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range, except in the presence of S9-mix. Although the number of binucleated cells with miconuclei was above the historical control data range in the presence of S9-mix, the number of binucleated cells with micronuclei was less than 10. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. 202029/A did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independent experiments. Under the test conditions, test item is not clastogenic and not aneugenic in human lymphocytes.

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline No. 476 and in compliance with GLP, L5178Y/TK+/- -3.7.2C mouse lymphoma cells were exposed to test material at the following concentrations:

- Dose range finding test: 3 h treatment, with and without S9-mix: 11, 36, 109, 363 and 725 µg/mL 24 h treatment, without S9-mix: 11, 36, 109, 363 and 725 µg/mL

- First mutagenicity test: 3 h treatment, without and with 8 % (v/v) S9-mix: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL

- Second mutagenicity test: 3 h treatment, with 12 % (v/v) S9-mix: 0.1, 0.3, 1, 3, 10, 33, 100 and 333μg/mL 24 h treatment, without S9-mix: 1, 3, 10, 33, 100, 250, 333 and 500 µg/mL

Vehicle and positive control groups were also included in each mutation test. In the dose range finding test, after 3 h of treatment without S9-mix, the relative suspension growth was 77% at the highest test substance concentration of 725μg/mL compared to the suspension growth of the solvent control. With S9-mix, the relative suspension growth was 76 % at the highest test substance concentration of 725μg/mL compared to the relative suspension growth of the solvent control. After 24 h of treatment without S9-mix, the relative suspension growth was 14 % at the test substance concentration of 725μg/mL compared to the relative suspension growth of the solvent control. Test material precipitated in the exposure medium at concentrations of 363μg/mL and above in all experimental conditions. In the first mutagenicity test, the relative total growth was 84% and 103% at 333μg/mL compared to the total growth of the solvent controls at 3 h treatment without and with 8% S9-mix, respectively. Precipitation was observed at the highest dose tested in both experimental conditions, i.e. 333 µg/mL. In the second mutagenicity test, the relative total growth was 28% at 500 µg/mL and 103% at 333μg/mL compared to the total growth of the solvent controls for the 24 h treatment without S9 -mix and for the 3 h treatment with 12% S9-mix, respectively. Precipitation was recorded from 250 µg/mL and at 333 µg/mLfor the 24 h treatment without S9-mix and for the 3 h treatment with S9-mix, respectively. The test material did not induce a significant increase in the mutation frequency at any dose level either with or without metabolic activation in two independent experiments. In all tests the concurrent vehicle and positive control were within acceptable ranges. Under the test conditions, test material was not mutagenic at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells, in the absence and presence of S9-mix.

In vivo Micronucelus study performed on the registered substance

The micronucleus test was carried out with Locron P. The test compound wasdissolved in deionized water and was given twice at an interval of 24 hours as an orallydose of 2000 mg per kg body weight to male and female mice, based on the results of aprevious dose range finding assay. According to the testprocedure the animals were killed 24 hours after administration.Endoxan (Cyclophosphamide)was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.The number of polychromatic erythrocytes containing micronuclei was not increased following treatment with Locron P. The ratio of polychromatic erythrocytes to total erythrocytes in both male and femaleanimals remained unaffected by the treatment with Locron P and was not less than20% of the control value. The positive control,Endoxan, induced a marked and statistically significant increase in the number ofpolychromatic cells with micronuclei, indicating the sensitivity of the test system. Theratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.Under the conditions of the study the results indicate that Locron P is notmutagenic and not aneugenic in the micronucleus test.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

The Key studies show no positive results for mutagenicity or genotoxicity. Based on the available data, the substance is not classified for mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.