Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was considered to be non-mutagenic in both in vitro assays - the reverse mutation (Ames) test using Salmonella Typhimurium and E. Coli, and the chromosome aberration test in human lymphocytes.

Read-accross from a supporting structural analogue did not induce mutation at the TK locus in the in vitro mouse lymphoma assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27.01.2009-21.10.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern GLP study conducted in accordance with OECD test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 from Liver of phenobarbitone & B-naphthoflavone Han Wister Rats
Test concentrations with justification for top dose:
Group 4(20)-hour without S9 - 0, 39.06, 78.13, 156.25, 312.5, 625, 1250, MMC 0.4 (pg/ml)
4(20)-hour with S9 - 0, 39.06, 78.13, 156.25, 312.5, 625, 1250, CP 5 (pg/ml)
24-hour without S9 - 0, 39.06, 78.13, 156.25, 312.5, 625, 1250, MMC 0.2 (pg/ml)
Vehicle / solvent:
Acetone- test material
Minimal Essential Medium (MEM)-positive control ( mitomycin C) in the absence of S9
Dimethyl sulphoxide (DMSO)- positive control ( Cyclophosphamide) in the presence of S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
MEM
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Absence of exogenous metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Presence of exogenous metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48h
- Exposure duration: i) 4-hour exposure to the test material without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest., ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest., iii) 24-hour continuous exposure to the test material without S9-mix prior to cell harvest.
- Fixation time (start of exposure up to fixation or harvest of cells): not stated

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays):5% Gurrs Giemsa


NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: The first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Microscopically determined after fixation.
- Determination of endoreplication: Not stated


Statistics:
The frequency of cells with aberrations (excluding gaps) and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: 4 hour exposure
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
32% growth inhibition at 625 ug/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: 4 hour exposure
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: 24- hour continous exposure
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
14% growth inhibition at 156.2ug/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test (Cell Growth Inhibition Test)

The dose range for the Preliminary Toxicity Test was 19.53 to 5000 pg/ml. The maximum dose was based on the maximum recommended dose level. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 156.25 pg/ml, in the 4(20)-hour pulse exposure groups and at and above 156.25 pg/ml in the continuous exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 pg/ml in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9) and also in the 24-hour continuous exposure group. The mitotic index data were recorded. The test material induced some mild evidence of toxicity in all of the exposure groups. The test material was observed to form a greasy oily precipitate at and above 2500 pg/ml and maximum exposure to the cells was considered to be achieved at 1250 pg/ml. The dose selection for the main experiment was based on the precipitating dose level before the onset of the greasy/oily precipitate where it was considered that the cells were not fully exposed to the test material. Therefore, the maximum dose selected was 1250 pg/ml for all of the exposure groups in the main experiment.

Chromosome Aberration Test

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Cell Growth Inhibition Test and that there were metaphases suitable for scoring present at the maximum test material dose level of 1250 pg/ml in the presence of metabolic activation (S9). In the absence of metabolic activation (S9) the maximum test material dose level with metaphases suitable for scoring was 1250 lag/ml. However due to a greasy/oily precipitate being observed at this dose level it was considered that the cells were not fully exposed to the test material. Therefore metaphase analysis was limited by this and the maximum dose level suitable for scoring in the absence and presence of S9 was 625 lag/ml. In the 24 hour continuous exposure in the absence of S9 again metaphase analysis was limited due to a greasy/oily precipitate observed at 625 lag/ml and, therefore, the maximum dose level scored was 312.5 lag/ml.
The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 1, See Appendix 2. These data show a 32% growth inhibition was achieved at 625 lag/ml in the absence of S9. However no toxicity was seen in the presence of S9. In the continuous exposure no toxicity was seen at the top dose however, 14% toxicity was seen at 156.25 lag/ml.
See Appendix 2, Form 1 for the chromosome aberration 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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test material induced small but statistically significant increases in the frequency of cells with aberrations in the presence of metabolic activation. However the significantincreases observed were within or close to the upper limit of the historical vehicle control range and well below the positive control response in the exposure group. They were also predominantly due to chromatid break type aberrations and were compared to a very low vehicle control value. It was therefore considered that these small increases had no biological relevance and were of no toxicological significance. No significant increases in the frequency of chromosome aberrations were observed in either of the exposure groups without metabolic activation.
The polyploid cell frequency data were also given in Appendix 2, Form 1. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce any toxicologically significant increases in the frequency of cell with aberrations. The test material was therefore considered to be nonclastogenic to human lymphocytes in vitro.
Executive summary:

The study was performed to evaluate the potential of the test substance to induce structural chromosomal aberrations in cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, ie. 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24 hours continuous exposure in the absence of metabolic activation. The test material induced small but statistically significant increases in the frequency of cells with aberrations in the presence of metabolic activation. However the significant increases observed were within or close to the upper limit of the historical vehicle control range and well below the positive control response in the exposure group. They were also predominantly due to chromatid break type aberrations and were compared to a very low vehicle control value. It was therefore considered that these small increases had no biological relevance and were of no toxicological significance The maximum dose level selected for metaphase analysis was limited by the formation of a greasy oily precipitate which was observed at and above 1250 pg/mI and, therefore, maximum exposure was considered to occur at 625 pg/ml. In concluison, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
January 2008 - May 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Modern GLP study conducted in accordance with OECD test guideline. Restriction due to the fact that the study was conducted on read-across substance.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
The tk (thymidine kinase) locus in mouse lymphoma L5178Y cells.
Test concentrations with justification for top dose:
Concentration of Final
Experiment treatment solution concentration
(mg/mL) (ug/mL)
Range finder: 0.4688 4.688
0.9375 9.375
1.875 18.75
3.75 37.5
7.5 75
15 150
1 0.25 2.5
0.5 5
1 10
2 20
3 30
4 40
5 50
7.5 75
15 150
30 300

2 1 10
2 20
4 40
6 60
7.5 75
10 100
15 150
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test substance was miscible with acetone at concentrations up to at least 500.2 mg/mL.
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: Final concentration in medium: 0.15 and 0.20 ug/mL
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: Final concentration in medium: 2.00 and 3.00 ug/mL
Details on test system and experimental conditions:
DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT (mutation assays): TFT (5-trifluorothymidine)

NUMBER OF REPLICATIONS: Two (except for positive control treatments, which only had single cultures).

NUMBER OF CELLS EVALUATED: 384 wells at 2,000 cells per well were plated for TFT resistance
Evaluation criteria:
PARAMETERS ASSESSED:
The following parameters were calculated:
- Suspension Growth (SG)
- Relative Suspension Growth (RSG)
- Relative Total Growth (RTG)
- Mutant Frequency (MF)

ACCEPTANCE CRITERIA
The assay was considered valid if all the following criteria were met:
1. The mean mutant frequencies in the negative (vehicle) control cultures fell within the normal range (50 to 170 mutants per 106 viable cells);
2. At least one positive control should show either an absolute increase in mean total MF of at least 300x10-6 (at least 40% of this should be in the
small colony MF), or an increase in small colony mutant frequency of at least 150x10-6 above the concurrent vehicle control;
3. The mean RTG for the positive controls should be greater than 10%;
4. The mean cloning efficiencies of the negative controls from the Mutation Experiments were between the range 65% to 120% on Day 2;
5. The mean suspension growth of the negative controls from the Mutation Experiments was between the range 8 to 32;
6. There should be no excessive heterogeneity between replicate cultures.

EVALUATION CRITERIA
For valid data, the test article was considered to be mutagenic in this assay if the MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF (Global Evaluation Factor) and the linear trend test was positive. This indicated a positive, biologically relevant response.

The test article was considered as negative in this assay if the MF of all test concentrations were less than the sum of the mean control mutant frequency plus GEF.

Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
All calculations were performed either manually or by cmoputer using validated software.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
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
- Precipitation:
In Experiment 1 (2.5-300 ug/mL), upon addition of the test article to the cultures, precipitation was observed at the six highest concentrations (30-300 ug/mL) in the absence and presence of S9. Following the 3-hour treatment incubation period, precipitate was observed at the highest two concentrations (150 and 300 ug/mL) in the absence and presence of S9. The lower concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S9 was retained and the higher concentration was discarded.
In Experiment 2 (10-150 ug/mL), upon addition of the test article to the cultures, precipitation was observed at the five highest concentrations (40-150 ug/mL) in the absence and presence of S9. Following the 3-hour treatment incubation period, precipitate was observed at the highest two concentrations in the absence of S9 (100-150 ug/mL) and at the highest concentration in the presence of S9 (150 ug/mL). The lower concentration at which precipitate was observed at the end of the treatment incubation period in the absence of S9 was retained and the higher concentration was discarded.

RANGE-FINDING/SCREENING STUDIES:
In the rangefinding study, precipitation was observed at the three highest concentrations (37.5-150 ug/mL). Following the incubation period, precipitate was observed in the supernatant at the highest two concentrations (75 and 150 ug/mL) in the absence and presence of S9.
The highest concentration tested (150 ug/mL) gave 110% Relative Total Growth in the absence and presence of S9.
No significant changes in osmolality or pH were observed at the highest concentration tested (150 ug/mL).

The acceptance criteria were met and the study was therefore accepted as valid.

In Experiments 1 and 2, the MF of the concentrations plated were all less than the sum of the mean control MF plus the GEF, indicating a negative result. In addition, for the negative and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony MF could be estimated and the proportion of small mutant colonies could be calculated. For the negative controls, the proportion of small colony mutants in the absence and presence of S-9 ranged from 25% to 28% in Experiment 1 and from 33% to 43% in Experiment 2. Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals NQO and B[a]P.

Conclusions:
Interpretation of results (migrated information):
negative

The test substance did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to the solubility limit in culture medium in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9).
Executive summary:

EC# 425-620-2 was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells in a GLP study conducted in accordance with OECD Test Guideline 476 using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S9). A 3-hour treatment incubation period was used for all experiments performed in the absence and presence of S9.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S9, ranging from 4.688 to 150 µg/mL (limited by solubility in culture medium). The highest concentration tested (150 µg/mL) gave 110% relative total growth (RTG) in the absence and presence of S9.

Accordingly, for Experiment 1 ten concentrations, ranging from 2.5 to 300 µg/mL, were tested in the absence and presence of S9. Two days after treatment, the highest concentration selected to determine viability and TFT resistance was 150 µg/mL, which gave 114% and 82% RTG in the absence and presence of S9, respectively. In Experiment 2 seven concentrations, ranging from 10 to 150 µg/mL, were tested in the absence and presence of S9. Two days after treatment, the highest concentrations selected to determine viability and TFT resistance were 100 µg/mL in the absence of S9 and 150 µg/mL in the presence of S9, which gave 76% and 88% RTG, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S9. Mutant frequencies in negative control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S9) and benzo[a]pyrene (with S9). Therefore the study was accepted as valid.

In Experiments 1 and 2, the mutant frequencies of the concentrations plated were all less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF, 126 mutants per 106 viable cells), indicating a negative result.

It is concluded that EC 425-620-2 did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to the solubility limit in culture medium in two

independent experiments in the absence and presence of a rat liver metabolic activation system (S9).

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:
May 2009- June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern GLP study conducted in accordance with OECD test guideline.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate
Vehicle / solvent:
Solvent: Acetone
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Migrated to IUCLID6: 2 pg/plate for WP2uvrA-, 3 pg/plate for TA100 and 5 pg/plate for TA1535
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: 80 pg/plate for TA1537
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: 0.2 pg/plate for TA98
Details on test system and experimental conditions:
The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 pg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations except a 20 minutes pre-incubation modification was performed.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
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:
no cytotoxicity
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

This study was designed to assess the mutagenic potential of the test material using a bacteria/ microsome test system. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coil strain WP2uvrA were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 pg/plate. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 pg/plate. A globular precipitate was observed at 5000 pg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or with or without pre-incubation. Therefore, the test material was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose:
read-across source
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

EXP0700332 did not induce mirconuclei formation in bone marrow cells when tested at the limit dose (2000 mg/kg/day) during the in vivo mammalian erythrocyte micronucleus test in rat bone marrow.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 August 2013 to 16 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: E00031-633
- Retest date of the lot/batch: 30 January 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in the dark
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rodent bone marrow micronucleus test is currently the most commonly performed assay for detecting the genotoxic potential of test items in vivo. The original protocol for bone marrow micronucleus tests using small laboratory rodents was developed independently in the laboratories of Schmid and Heddle over 30 years ago. Since then, the methods and principles of the assay have remained largely unchanged. Most discussions on study design have therefore focused on the effects that different dosing and sampling schedules have on the micronucleus yields (Mavournin et al, 1990; Hayashi et al, 1994; Gatehouse, 1994; Trzos, 1978). The need for using both sexes for testing has also been a point of debate (Parton et al, 1990; Kliesch and Adler, 1992).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Kent, England, UK.
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: Male: 201.7 - 239.4 g; Female: 174.3 - 201.2 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: All rats were housed in cages according to Charles River SOPs. Sterilised white wood shavings provided the bedding in the cages. Cages were changed on a weekly basis.
- Diet (e.g. ad libitum): Special Diet Services, Rat and Mouse (Modified) No. 1 SQC Expanded, was feely available.
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 - 21.4 °C
- Humidity (%): 50 - 73 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: Micronucleus test: From: 22 October 2013 To: 24 October 2013
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: peanut oil
- Justification for choice of solvent/vehicle: Vehicle control
- Amount of vehicle (if gavage or dermal): 10 mL/kg/day
Details on exposure:
The oral route was selected as this is the a possible route of administration in humans. Based on dose range finder, the maximum recommended dose level of 2000 mg/kg/day was selected for the micronucleus test. As there were no clinical signs in the dose range finding test this was 2000 mg/kg/day only dose level tested.
All treated animals were exposed to test or control materials via the oral dose route. The rats were weighed immediately before each exposure event and the dose volume adjusted using a graded syringe fitted with a gavage needle. Treated animals were exposed to dosing formulations at a dose volume of 10 mL/kg/day.
Duration of treatment / exposure:
24 hours - the rats were treated at 0 h and 24 hours via the oral route at a dose level of 2000 mg/kg/day. Bone marrow samples were taken 24 hours after the final dose.
Frequency of treatment:
0 and 24 hours
Post exposure period:
24 hours
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Vehicle control: 5 males and 5 females
EXP0700332: 7 males and 7 females
Positive control: 3 males
Untreated control: 3 males and 3 females
Control animals:
yes
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): An adequate positive control response for at least 2 animals and the dose group as a whole. This criterion was met in this micronucleus test.
- Route of administration: oral
- Doses / concentrations: 10 mL/kg/bw (dose 50 mg/kg; 5 mg/mL)
Tissues and cell types examined:
The frequenct of reticulocytes and micronucleated cells were determined. The number of micronucleated normochomatic erythrocytes in mature red blood corpulscles were also recorded as a control.
Scored micronuclei were also assigned based on size which provided a non-specific measure of compound induced spindle dsfunction
Details of tissue and slide preparation:
Rats were killed by increasing levels of carbon dioxide. The marrow was flushed out using a 1:1 mixture of foetal calf serum and trisodium citrate (0.8 %, w/v) in Sorenson's buffer (pH 6.8). Routine tissue culture antibiotics were included to prevent microbial growth. This mildly hypotonic treatment served to make the micronuclei clearly visible and to distinguish them from surrounding artefacts. Following completion of the sampling procedure the contents of the tubes were briefly agitated on a vortex mixer to allow separation of the cells.
The tubes were centrifuged to pellet the cells. The supernatant fluid was discarded and the cells were then resuspended on a vortex mixer in the residual liquid.
Clean slides were assigned numbers corresponding to the tube numbers. A drop of the suspension was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube (animal). The smear was left to air dry, fixed in methanol (for at least 5 min) and then stained with Giemsa stain (15%, v/v, in water) to give optimum erythrocyte discrimination. Permanent slide preparations were made by sealing cover slips onto the glass slides using mounting medium.
Evaluation criteria:
The average micronucleus incidence in vehicle control dosed and untreated CD rats, has in this laboratory been determined as 0.04% , a range of 0.01 – 0.13% per group of 5-7 animals and 0.02 - 0.11% per group of 10-12 animals.
These historical data have been used in the evaluation of response in this test. This frequency is also in agreement with published data for micronucleus tests with rats (Tamura et al, 1990; Salamone and Mavournin, 1994).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No micronucleus induction was detected in bone marrow erythrocytes of rats dosed with EXP0700332.
Conclusions:
In conclusion, EXP0700332 did not induce micronuclei in bone marrow cells when tested to the maximum recommended dose of 2000 mg/kg/day in male and female Sprague Dawley (CD) rats using a 0 h and 24 h oral dosing and 48 h sampling regimen.
Executive summary:

The in vivo genotoxic potential of EXP0700332 was evaluated in a micronucleus test in bone marrow erythrocytes of young, male and female rats following a 0 h and 24 h oral dosing and 48 h sampling regimen.

A Dose Range Finding study was undertaken to establish a suitable dose range for the main micronucleus experiment. Based on the findings of the Dose Range Finder, the maximum recommended dose of 2000 mg/kg/day was judged to be a suitable dose level for the micronucleus test.

In the micronucleus test, groups of rats were dosed at 0 h and 24 h via the oral route with EXP0700332 at the maximum recommended dose level, 2000 mg/kg/day. Bone marrow samples were taken 24 h after the final dose. Two control groups of rats were also dosed orally with either the vehicle, peanut oil (10 mL/kg/day), or the positive control agent, cyclophosphamide (50 mg/kg/day). An untreated control group was also included. The experimental schedule for the control groups followed that of the test item treated rats.

No micronucleus induction was detected in bone marrow erythrocytes of rats dosed with EXP0700332.

Animals treated with the vehicle alone showed normal background levels of micronuclei, while animals dosed with cyclophosphamide responded with substantial increases in the numbers of bone marrow micronuclei.

In conclusion, EXP0700332 did not induce micronuclei in bone marrow cells when tested to the maximum recommended dose of 2000 mg/kg/day in male and female Sprague Dawley (CD) rats using a 0 h and 24 h oral dosing and 48 h sampling regimen.

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

Additional information

The Registration material and the analogue substance both demonstrated no genetic toxicity potential in any of the tests conducted. Test substance was not found to be mutagenic in bacteria, and did not induce chromosome damage in human lymphocytes.. The analogue test substance was not found to induce chromosome damage in erythrocytes as sampled in bone marrow of rats. The results of genetic toxicity data on the analogue material were considered to be appropriate for the Registration material.


Short description of key information:
Negative for mutagenicity in bacterial cultures in GLP compliant short term in vitro tests (OECD 471)
Negative for induction of structural and numerical chromosome aberrations (OECD 473)
Negative for gene mutation in mouse lymphoma cells (OECD 476)
Negative for induction of chromosome damage in a GLP compliant in vivo micronucleus assay (OECD 474)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The Registration material and the analogue substance both demonstrated no genetic toxicity potential in any of the tests conducted. The results of genetic toxicity data on the analogue material were considered to be appropriate for the Registration material.

All results for genotoxic potential, in prokaryotic and eukaryotic (including mammalian) species were negative. Therefore, in accordance with the EC Regulation 1272/2008, no classification is required.