1,1'-[[3-(dimethylamino)propyl]imino]bispropan-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: The key study is performed according to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA (Dakoulas, 2012). According to the results of the study, the test substance is not mutagenic in the Ames test with and without metabolic activation. One supporting study performed according to OECD guideline 471 is available.

Chromosome aberration test: A study was performed as per the OECD guideline 473 in Chinese hamster lung fibroblasts (V79) were tested for chromosome aberrations in two chromosome aberration tests at different concentrations. As per the results of this study, the test item does not induce chromosome aberrations.

 

Mammalian cell gene mutation test: The study is performed according to a method similar to OECD Guideline 476 in Chinese hamster Ovary (CHO) cells (Clarke, 2012). The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, the test substance was concluded to be negative with and without metabolic activation. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005-03-30 to 2005-04-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA (TSCA) OPPTS

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK Department of Health

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: 050303
- Expiration date of the lot/batch: not indicated
- Purity test date: not indicated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: The test material was soluble in sterile distilled water at 50 mg/ml (the most concentrated stock solution) in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle of choice.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
The test material was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity contect (4%) of the test material.

Target gene:

Histidine locus (S. tymphimurium strains) and tryptophan locus (E. coli strain)

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:

Aroclor 1254-induced rat liver S9 (10% (v/v))

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate with and without S9-mix.
Initial Toxicity - Mutation Assay 1: 15, 50,150,500, 1500 and 5000 µg/plate with and without S9-mix (Salmonella strains); 50, 150, 500, 1500 and 5000 µg/plate with and without S9-mix (Escherichia strains);
Mutagenicity Assay 2 : 50,150,500, 1500 and 5000 µg/plate with and without S9-mix;
In the initial toxicity-mutation assay, no precipitate was observed. The test material was toxic only at 5000 µg/plate to TA100 without S9-mix only. TA100 (with S9-mix) and WP2uvrA exhibited no toxicity. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: The test material was soluble in sterile distilled water at 50 mg/mL (the most concentrated stock solution) in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle of choice.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix; 2 μg/plate (WP2uvrA-), 3 μg/plate (TA100) and 5 μg/plate (TA1535)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9; 80 μg/plate (TA1537)

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9-mix; 0.2 μg/plate (TA98)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

With S9-mix; 1 μg/plate (TA100), 2 μg/plate (TA1535 and TA1537) and 10 μg/plate (WP2uvrA)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9-mix; 5 μg/plate (TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Preliminary toxicity test & Mutation assay 2:
The assay was performed by mxing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 0.1 mL of test material formulation, 0.5 mL of S9-mix or phosphate buffer and 2 mL of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Monomal agar (30 mL/plate). Ten (preliminary toxicity test) or five (Mutation assay 2) concentrations of the test material and a vehicle control (sterile distilled water) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.
- Mutation assay 1:
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.


DURATION
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium) or Tryptophan (E. coli)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the growth of the bacterial background lawn

Rationale for test conditions:

see above in specific details on test material, test concentrations and details on test system and conditions

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 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.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

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
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: Soluble at 50 mg/mL
- Precipitation: no test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix
- Other: sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES: Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies
- Negative (solvent/vehicle) historical control data: Results for the negative controls (spontaneous mutation rates) were considered to be acceptable

Conclusions:

All criteria for a valid study were met. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test substance did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-01 to 2011-12-06

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): JEFFCAT DPA
- Substance type: Yellowish liquid
- Physical state: Liquid
- Analytical purity: 100%
- Composition of test material, percentage of components: 0.24 wt% water, 8,8 meq/g total amine
- Lot/batch No.: 0H519
- Storage condition of test material: Room temperature, stored protected from light without desiccant

Target gene:

Histidine dependence (S. typhimurium) -> frameshift mutations
Tryptophan locus (E. coli) -> basepair mutations

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:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate
Confirmatory mutagenicity assay: 50, 150, 500, 1500 and 5000 µg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Water was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in water at approximately 50 mg/mL, the maximum concentration tested in the solubility test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (1 µg/plate for TA98, TA1535 and TA1537), (2.0 µg/plate for TA100) and (15 µg/plate for WP2 uvr A)

Remarks:

with metabolic activation

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 Migrated to IUCLID6: 1.0 µg/plate for TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation Migrated to IUCLID6: 1.0 µg/plate for TA100 and TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation Migrated to IUCLID6: 75 µg/plate for TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation Migrated to IUCLID6: 1,000 µg/plate for WP2 uvrA

Details on test system and experimental conditions:

An initial toxicity-mutation assay and a confirmatory mutagenicity assay was performed

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours

SELECTION AGENT (mutation assays): selective minimal agar

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: A dose level is considered toxic if one or both of the following criteria are met: (1) A >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn.

OTHER: Automated data collection system were used for the collection of data or analysis.

Evaluation criteria:

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0 times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase mean revertants at the peak of the dose response was greater than or equal to 2.0 times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test article formed a clear solution in water at approximately 50 mg/mL, the maximum concentration tested in the solubility test
- Precipitation: No precipitation was observed.
- Sterility results: Contaminant colonies were observed on the pre-dosing sterility plates for the vehicle and Sham mix in the confirmatory mutagenicity assay. Since no contamination was observed on the assay plates, the Study Directer has concluded that this had no adverse impact on the integrity of the data or the validity of the study conclusions. No contaminant colonies were observed on the remaining sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES: In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg/plate; this dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Neither precipitate nor toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 µg per plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Excessive toxicity, as reductions in revertant counts beginning at 150 µg per plate was observed, therefore tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was spilled by the technician during dosing and there was not enough remaining to dose tester strains Wp2uvrA in the presence of S9 activation. Therefore, tester strain WP2uvrA in the presence of S9 activation was also retested in the retest of confirmatory mutagenicity assay.In the retest experiment, no positive mutagenic responses were observed with tester strain TA1537 in the absence of S9 activation and tester strain WP2 uvrA in the presence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 µg/plate. Neither precipitate nor toxicity was observed.

Conclusions:

Under the conditions of this study, the test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-14 to 2016-08-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted September 26, 2014

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Chemical name: Reaction product of 3-dimethylaminopropylamine and propylene oxide with 1,1'-[(3-dimethylaminopropyl)imino]-bis-2-propanol and 1-[(3-dimethylaminopropyl)(2-hydroxy-1-methylethyl)amino]-2-propanol as main component
- Other name: DMAPA-2PO
- CAS number: 63469-23-8
- Source and lot/batch No.of test material: 5Y1024
- Purity: 100%
- Supplier: Tosoh Corporation
- Storage condition of test material: into a light-resistant and airtight container and stored at room temperature
- Stability under test conditions: Stable, Stable at storage condition
- Molecular formula: C11H26N2O2 (main component)
- Molecular weight : 218.3 (main component; ratio below molecular weight 1000: >99.9%
- Vapor pressure: 13 Pa (20°C)
- Melting point: <-20°C
- Boiling point: 250-270 °C
- Specific weight: 0.940
- Density: 0.940
- Appearance: Yellow liquid
- Solublity in vehicle water: >=200 mg/mL
- Stability in vehicle water : Stable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Remarks:

CHL/IU cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Health Sciences Research Resources Bank, Japan Health Sciences Foundation
- Suitability of cells: CHL/IU cells have been recommended in section 5 of the guideline
- Cell cycle length, doubling time or proliferation index: about 15h (doubling time)
- Number of passages if applicable: 13 for the cell growth inhibition test, 6 for first chromosomal aberration test, 8 for second chromosomal aberration test
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: 25 per cell
- Normal (negative control) cell cycle time:
- Spontaneous frequencies of cells with structural aberrations and the numerically aberrant cells: <5%

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were suspended in medium [Eagle's minimum essential medium (Nissui Pharmaceutical) and 10 vol% heat-inactivated newborn calf serum (NBCS, HyClone Laboratories)] including 10vol% DMSO and were frozen in liquid nitrogen. L-Glutamine 0.292 g/L and sodium hydrogen carbonate 1.95 g/L were added to the Eagle's minimum essential medium (where cells were already suspended) with 10 vol% heat inactivated new born calf serum and BioWest
- Properly maintained: Yes
- culture condition (setting value): incubator: CO2 incubator; temperature: 37°C, humidity: under humid condition; CO2 concentration: 5%
- subculture: culture vessel: 90-mm diameter Petri dish; frequency of passage: two times a week; passage number of cells: 13 for the cell growth inhibition test, 6 for the first chromosomal aberration test, 8 for the second chromosomal aberration test
- Periodically checked for Mycoplasma contamination: Yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 for chromosomal aberration test

Test concentrations with justification for top dose:

- Cell Growth inhibition test: 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/ml.
Highest dose = 2000 µg/mL (maximum dose in case of no cytotoxicity on test methods)
- Chromosomal aberration test: dose levels of test substance were set based on results of cell growth inhibition test. Cytotoxicity defines as RPD was less than 50%. Cytotoxicity was observed in all treatment methods. Doses were set to obtain the dose which RPD was 40% or more and 50% or less
- First chromosomal aberration test:
-short-term without S9-mix: 110, 130, 150, 170, 190, 210, 230, 250, 260 and 270 µg/mL
-short-term with S9 mix: 110, 130, 150, 170, 190, 210, 230, 250 and 270 µg/mL
-24 hours continuous: 30, 40, 50, 60, 70, 80, 90, 100 and 110 µg/mL
-the dose which RPD was 50% or less was not obtained in any treatment method. Specimen preparation was not carried out in '-S9 mix' and 24 hours continuous treatment. In '+S9 mix', specimens were prepared because a cytotoxicity that RPD was near to 50% was obtained. In '+S9 mix', it was possibly thought that adequate evaluation results was not obtained because the dose that RPD was 50% or less was not obtained. Second chromosomal aberration test was carried out in all treatment methods.
- Second chromosomal aberration test:
-short-term without S9 mix: 170, 190, 210, 230, 250, 270, 290, 310 and 330 µg/mL
-short-term with S9 mix: 150, 170, 190, 210, 230, 250, 270, 280, 290, 310 and 330 µg/mL
-24 hours continuous: 70, 90, 100, 110, 120, 130, 140, 150, 170, 190 and 210 µg/mL
- observation : Specimens were observed in all treatment methods. All specimens of the negative and positive controls were observed. Cytotoxicity was obtained in all treatment methods. Highest dose was selected at lowest dose that RPD was 40% or more and 50% or less.
-short-term without S9 mix: 210, 230, 250 µg/mL
-short-term with S9 mix: 250, 270 and 280 µg/mL
-24 hours continuous: 100, 110 and 120 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water (Otsuka Pharmaceutical factory, Lot number: K5K72, for injection)
- Justification for choice of solvent/vehicle: The test substance was soluble in distilled water at 200 mg/mL. The test substance solution of 200 mg/mL prepared with distilled water was considered to be stable from the facts that there were no changes in color, exothermic reaction nor gas generation at room temperature within 2 hours after preparation. Therefore, distilled water was selected as a vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix, short term treatment, concentration 1 mg/mL, volume 12µL, dose 4 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

1) short term treatment without S9 mix, concentration 0.01 mg/mL, volume 15µL, dose 0.05 µg/mL 2) 24 hours continuous treatment, concentration 0.01 mg/mL, volume 25µL, dose 0.05 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable): Cell growth inhibition test: 5 X 10^3 cells/ml; First and second chromosomal aberration tests: 1.5 X 10^4 cells/ml

DURATION
- Preincubation period: 2 or 3 days
- Exposure duration: short term treatment= 6h; continuous treatment= 24h
- Expression time (cells in growth medium): short term treatment=18h, continuous treatment=24h
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):not specified

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine solution (50µL of a 10µg/mL solution), at 2 hours before the end of the culture

STAIN (for cytogenetic assays): 2 vol% Giemsa solution

NUMBER OF REPLICATIONS:was measured in terms of Relative Population Doubling

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Leftover cells were collected by centrifugation and were subsequently given a hypotonic treatment.
Fixation comprised two steps (pre-fixation and complete fixation) and the cells were dropped onto a glass slide in a suspension made with fixative solution.
One specimen per dose was prepared. The specimens were then dried and stained with 2 vol% Giemsa solution.

NUMBER OF CELLS EVALUATED:
-structural aberration: 300 metaphase cells per dose (75 cells per speciment) containing 25 +/- 2 chromosomes were observed using a microscope. The total number of cells with structural aberrations and the number of aberrant cells in each aberration category were recorded.
- numerical aberration: the number of polyploid cells with triploid or more (38 or more chromosomes) and endoreduplicated cells among 300 metaphase cells per dose (75 cells per specimen) was recorded.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 50 metaphase cells per dose

DETERMINATION OF CYTOTOXICITY
- Method: Cell growth rate (measurement of cells in the beginning and end of the culture) for cultures exposed to test item were compared with
the negative control and the population doubling (PD) and the relative population doubling (RPD) were calculated using the formulae:
PD = [log {(No. of cells at the preparation of the specimens) / (Number of cells at the start of the treatment)}]/log 2
RPD = (PD in test substance group)/(PD in negative control group) x 100

OTHER EXAMINATIONS:
- Determination of polyploidy:Yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No. of cells with structural aberrations (chromatid and chromosome breaks and exchange and fragmentation) was recorded.

Rationale for test conditions:

No data

Evaluation criteria:

Judgement criteria of test results
A test substance was judged to be clearly positive when:
1. At least one of the doses of the test substance exhibits a statistically significant increase compared with the concurrent negative control
2. The increase of the frequencies of cells with chromosomal aberrations is dose-related
3. Any of the results are outside the distribution of the negative control group of the historical data

However, a test substance was judged to be clearly negative when:
1. None of the doses of the test substance exhibits a statistically significant increase compared with the concurrent negative control
2. There is no dose-related increase
3. All results are inside the distribution of the negative control group of the historical data

Acceptable criteria of the test
1. The frequency of cells with chromosomal aberrations in the concurrent negative control are 'under control' in quality control method used for control chart in the historical data
2. The frequencies of cells with chromosomal aberrations in the concurrent positive control are 'under control' in quality control method used for control chart in the historical data. Concurrent positive controls produced a statistically significant increase compared with the concurrent negative control
3. Cell proliferation criteria in the negative control should be fulfilled
4. If the findings are not judged to be positive by the result of the short-term treatments, the continuous treatment was carried out

Statistics:

-Fisher's exact test was performed to compare the frequencies of the cells with structural aberrations and numerically aberrant cells.
-In cases where chromosomal aberrations in test substance groups were significantly high (in comparison to negative control groups), Cochran-Armitage trend test was used.
-Significance level: 1-5% both sides

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

when RPD <50%

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
Cell growth inhibition test: 7.31 (0 µg/ml) to 8.9 (2000 µg/ml)
Chromosomal aberration test: 7.24 (0 µg/ml) to 7.55 (330 µg/ml)
- Precipitation: none of the specimens showed precipitation
RANGE-FINDING/SCREENING STUDIES:
Results from the cell growth inhibition test were used to set the dose levels in the chromosome aberration tests.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: There were no statisitcally significant increases in the frequencies of cells with structural aberrations and numerically aberrant cells in groups treated with the test substance as compared to the negative controls. In addition, the frequencies of cells with structural aberrations and numerically aberrant cells were within the range of the negative control of the historical data in the testing facility. Therefore, strucutural aberration and numerical aberration were judged to be negative
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: table in the section “Any other information on results incl. tables”
- Negative (solvent/vehicle) historical control data: “Any other information on results incl. tables”

In each treatment method, the frequencies of cells with chromosomal aberrations in the negative and the positive control groups were within the range of the historical data. The frequency of cells with structural aberrations in the positive control was showed a statistically significant increase at both sides of 1% level. Cell proliferation criteria in the negative control met the criteria in the testing facility, indicating that the present study was appropriately performed.

-Negative control

Treatment method		Range of frequency of cells with chromosomal aberrations (%)
		Structural aberration		Numerical aberration
		Lower control limit	Upper control limit	Lower control limit	Upper control limit
Short term treatment	Without S9 mix	<0	4.2	<0	1.2
	With S9 mix	<0	3.0	<0	1.2
24h long term continuous treatment		<0	4.2	<0	1.6

The minimum range below 0 was shown <0

-Positive control

 

Treatment method		Substance	Dose (µg/ml)	Frequency of cells with chromosomal aberrations (%, mean)
				Structural aberration	Numerical aberration
Short term treatment	Without S9 mix	MMC	0.05	29.1	0.4
	With S9 mix	CPA	4.00	33.9	0.4
24h of continuous treatment		MMC	0.05	62.6	0.1

 

Treatment method		Range of frequency of cells with chromosomal aberrations(%)
		Structural aberration		Numerical aberration
		Lower control limit	Upper control limit	Lower control limit	Upper control limit
Short-term treatment	Without S9 mix	19.8	38.4	<0	1.5
	With S9 mix	23.8	43.9	<0	1.5
24h continuous treatment		48.9	76.3	<0	0.8

The minimum range below 0 was shown as <0

Conclusions:

There were no significant statistical increases in the frequencies of cells with structural aberrations and numerically aberrant cells in groups treated with the test substance as compared to the negative controls. The frequencies of cells with structural aberrations and numerical aberrant cells were within the range of the negative control of the historical data in the testing facility. Structural aberration and numerical aberration were judged to be negative. It was concluded that the test substance did not induce chromosomal aberrations under the present test conditions.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-24 - 2011-12-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well performed GLP study performed according to OECD Guideline 476. Dosing formulation analysis for concentrations and stability was not conducted (but, the solubility test showed that the substance is soluble in water at a conc of 50 mg/mL). The concentration of the 6-thioguanine cannot be confirmed (but mutant frequency in both negative and positive control fell within the validation criteria).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

CHO/HGRT Assay

Deviations:

no

Principles of method if other than guideline:

The mutagenesis assay was performed according to a protocol developed from published methodologies (Hsie et al., 1981; and O'Neill et al., 1977).
Hsie, A.W., D.A. Casciano, D.B. Couch, B.F. Krahn, J.P. O'Neill, and B.L.Whitfield (1981). The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals. A report of the Gene-Tox Program, Mutation Research 86:193-214.
O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch, and A.W. Hsie (1977) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system, Mutation Research 45:91-101.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Jeffcat DPA
- Physical state: light yellow liquid
- Analytical purity: 100 %
- Lot/batch No.: Batch # 0H519
- Expiration date of the lot/batch: not mentioned
- Storage condition of test material: room temperature, in the dark

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT)
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes. cells used were between 1 and 3 subpassages from cleansing in order to assure karyotypic stability
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

250-500-1000-1500-2100 µg/mL without metabolic activation
500-1000-1200-1400-1500-2100 mg/mL S9-activated cultures

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: A solubility test was conducted to select the vehicle. The selection is based on the solubility of the test article and the compatibility with the target cells. The test article was soluble in water at a concentration of 50 mg/mL, the maximum concentration tested.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation Migrated to IUCLID6: in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation Migrated to IUCLID6: in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5h
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 7-10 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7-10 days

SELECTION AGENT (mutation assays): 6-thioguanine (TG, 2-amino-6-mercaptopurine)

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: For the selection of the TG-resistent phenotype, the replicates from each treatment condition were trypsinized and replated, in quintuplicate, at a density of 2 x 1E05 cells/100 mm dish in F12FBS5-Hx containing 10 µM 6-thioguanine. For cloning efficiency determinations at the time of selection, 100 cells/60 mm dish were plated in triplicate in F12FBS5+Hx without TG.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency

Evaluation criteria:

Following criteria are presented as a guide to interpretation of the data:
• The test article was considered to induce a positive response if there was a concentrationrelated increase in mutant frequencies with at least two consecutive concentrations showing mutant frequencies of > 40 mutants per 1E06 clonable cells.
• If a single point above 40 mutants per 1E06 clonable cells was observed at the highest concentration, the test article was considered equivocal.
• If no culture exhibited a mutant frequency of > 40 mutants per 1E06 clonable cells, the test article was considered negative.

Statistics:

no data

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The cultures treated with 2100 µg/mL with S9 activation were too toxic to clone for mutant selection

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality:osmolality of the solvent control was 267 mmol/kg; osmolality of the top conc (2100 µg/mL) was 282 mmol/kg
- Evaporation from medium: no data
- Water solubility: the test article was soluble in water at a concentration of 50 mg/mL, the maximum concentration tested
- Precipitation: no test article precipitate was observed at any concentration in treatment medium

RANGE-FINDING/SCREENING STUDIES:
preliminary toxicity assay performed: cells were exposed to solvent and to nine concentrations of the test substance ranging from 0.5 to 2100 µg/mL in the absence and presence of S9 reaction mixture. Cloning efficiency was measured: 97% of solvent control without activation, 70% with S9 activation.The conc chosen for the mutagenesis assay ranged from 250 - 2100 µg/L for both with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control articles have been characterized as per Certificate of Analysis on file with the testing facility. The stability of the negative and positive control articles and their mixtures was demonstrated by acceptable results that met the criteria for a valid test.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Under the conditions of this study, the substance was concluded to be negative in the CHO/HGPRT mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In the micronucleus test according to OECD guideline 474 (Krsmanovic and Divi (2011)), performed on male or female ICR mice, a single oral administration of the test item up to a dosage of 2000 mg/kg. Hence the test item was concluded to be non-mutagenic.

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:

2011-10-11 to 2011-11-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented GLP study performed according to OECD Guideline 474 without deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Jeffcat DPA
- Substance type: Light yellow liquid
- Physical state: Liquid
- Analytical purity: 100%
- Composition of test material, percentage of components: 0.24 wt % water, total amine: 8.8 meq/g
- Lot/batch No.: 0H519
- Stability under test conditions: no data
- Storage condition of test material: The substance is stored at ambient (15 to 30°C) in the dark without desiccant.
- Other: synonym: 2-propanol, 1, 1'-((3-(dimethylamino)propyl)imino)bis- ; CAS# 63469-23-8

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 7 weeks old
- Weight at study initiation: range:
dose range finding study: males: 31.1 - 34.0 grams, females: 26.3 - 28.1 grams
definitive micronucleus study: males: 29.9 - 34.0 grams, females: 23.9 - 29.9 grams
- Assigned to test groups randomly: yes, in each study, mice were assigned to the appropriate number of treatment groups using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals did not exceed ± 20% from their group mean.
- Fasting period before study: no
- Housing: Animals were housed in an AAALAC-accredited facility with a controlled environment
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ± 22°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour.
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Purified water

Details on exposure:

All dose formulations were administered at a dose volume of 20 mL/kg by a single oral administration using appropriate size disposable polypropylene syringes with gastric intubation tubes (needles). The oral route of administration has been routinely used, and is widely-accepted for use in the mammalian bone marrow erythrocyte micronucleus assay.

Duration of treatment / exposure:

One single dose

Frequency of treatment:

Once

Post exposure period:

Dose range finding study: mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration on Study Day 0 and on study Days 1 and 3. Following the last observation on Study Day 3, animals were euthanized by exposure to CO2 verified by toe pinch reflex and discarded without further examination.
Definitive micronucleus study: mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity, which were recorded. Bone marrow was collected 24 hours post-dose (all groups) and 48 hours post-dose (vehicle control and high dose group).

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Dose range finding study: 5 (2000 mg/kg)
Definitive study: 10 (vehicle control), 5 (500 and 1000 mg/kg), 15 (2000 mg/kg), 5 (positive control)

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide monohydrate
- Route of administration: oral: gavage
- Doses / concentrations: 50 mg/kg

Tissues and cell types examined:

Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a labeled centrifuge tube containing aprpoximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were air dried and fixed in methanol. One set of slides was stained with a nucleid acid-specific stain, acridine orange, and was used in microscopic evaluation.

Details of tissue and slide preparation:

To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Using a fluorescent microscope and medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. using oil immersion (1000 X), the following cell populations and cellular components were evaluated and enumerated:
- polychromatic erythrocytes (PCEs; 2000 PCEs per animal were scored)
- normochromatic erythrocytes (NCEs; number of NCEs and micronucleated NCEs determined by scoring 1000 total erythrocytes per animal)
- micronuclei
The proportion of polychromatic erythrocytes to total erythrocytes were also determined per total of 1000 erythrocytes (PCEs/ECs ratio) for each animal.

Evaluation criteria:

The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10000 PCEs for each treatment group was determined.

Statistics:

Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time. In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each mouse and treatment group (PCEs/ECs ratio). The proportion of polychromatic erythrocytes to total erythrocytes in test substance animals should not be less than 20% of the control value. The test substance was judged negative if no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groupos relative to the concurrent negative (vehicle) groups is obersved. The test substance would have been considered to induce a positive response if the incidence of micronucleated polychromatic erythrocytes at one or more doses is statistically elevated relative to the vehicle control (p< = 0.05, binomial distribution, Kastenbaum-Bowman Tables).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

apart from piloerection in all animals.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
No mortality was observed. Piloerection was seen at a dose of 2000 mg/kg, in all males following dose administration and on Study Day 1, and in 3/5 females on Study Day 1. No appreciable changes in the group mean body weights were seen.

RESULTS OF DEFINITIVE STUDY
No mortality was observed in any of the treatment groups. All mice in the control substance (vehicle or positive) groups and all female mice in the 500 and 1000 mg/kg treatment groups appeared normal during the study period. Piloerection was seen in all male mice at 500, 1000 and 2000 mg/kg. This clinical sign persisted in the male mice at 2000 mg/kg at 24 hours post dose.
No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test substance groups relative to the respective vehicle control groups were observed, suggesting that the test substance did not inhibit erythropoiesis.
No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (p > 0.05, binomial distribution, Kastenbaum-Bowman Tables).
CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p≤ 0.05, binomial distribution, Kastenbaum-Bowman Tables) in both male and female mice. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

Conclusions:

Under the conditions of the study conduct, a single oral administration of the test substance at doses up to and including 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test substance was concluded to be negative in the micronucleus test using male or female ICR mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

Bacterial reverse mutation assay:

BioReliance (2012) performed an Ames test (plate incorporation test) with S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli strain WP2 uvr A with and without metabolic activation (key, K1, OECD guideline 471).

Following test concentrations were applied in triplicate: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate (initial toxicity-mutation assay) and 50, 150, 500, 1500 and 5000 µg/plate (confirmatory mutagenicity assay). Solvent and positive controls were also run in triplicate and were considered to be valid. According to the results of the study, the test substance is not mutagenic in the Ames test with and without metabolic activation.

A reliable, supporting study was performed according to OECD guideline 471 following GLP requirements (Bowles, 2005). In this Ames test, the mutagenic potential of the test substance was investigated in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli strain WP2 uvrA with and without metabolic activation. All criteria for a valid study were met. under the conditions of the study, the test substance did not cause a mutagenic response in either presence or absence of metabolic activation.

 

Chromosome aberration test:

Suzuki (2016) performed a K1 study in which a chromosome aberration test was performed as per the OECD guideline 473 using Chinese hamster lung fibroblasts (V79) following a cell growth inhibition test.

In this study scheme, two chromosome aberration tests were performed with the following concentrations:

- First chromosomal aberration test:

   -short-term without S9-mix: 110, 130, 150, 170, 190, 210, 230, 250, 260 and 270 µg/mL

   -short-term with S9 mix: 110, 130, 150, 170, 190, 210, 230, 250 and 270 µg/mL

   -24 hours continuous: 30, 40, 50, 60, 70, 80, 90, 100 and 110 µg/mL

- Second chromosomal aberration test:

    -short-term without S9 mix: 170, 190, 210, 230, 250, 270, 290, 310 and 330 µg/mL

    -short-term with S9 mix: 150, 170, 190, 210, 230, 250, 270, 280, 290, 310 and 330 µg/mL

     -24 hours continuous: 70, 90, 100, 110, 120, 130, 140, 150, 170, 190 and 210 µg/mL

Within this test, the test item did not show any statistically significant increase in the number of cells with structural and numerical aberrations, both with and without metabolic activation when compared to number of cells in a valid test with negative control. Hence, it was concluded that the test item did not induce chromosomal aberrations.

 

CHO/HGPRT Assay:

BioReliance (2012) performed an in vitro mammalian cell gene mutation assay in Chinese hamster Ovary (CHO) cells with and without metabolic activation. Following concentrations were tested in duplicate: 250, 500, 1500, 2100 µg/mL without metabolic activation and 500, 1000, 1200, 1400, 1500, 2100 µg/mL S9-activated cultures (5 hours of exposure). A vehicle control (sterile distilled water) and positive control (ethylmethanesulphonate without metabolic activation; benzo(a)pyrene with metabolic activation) were scored as well. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, the test substance is negative with and without metabolic activation. Positive and negative controls were considered valid. The cultures treated with 2100 µg/mL with S9 activation were too toxic to clone for mutant selection.

 

Genetic toxicity in vivo:

Micronucleus test according to OECD guideline 474 (Krsmanovic and Divi (2011)): Under the conditions of the study, a single oral administration of the test substance at doses up to and including 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test substance was concluded to be negative in the micronucleus test using male or female ICR mice. Following clinical signs were observed: piloerection was seen in all male mice at 500, 1000 and 2000 mg/kg. This clinical sign persisted in the male mice at 2000 mg/kg at 24 hours post dose.Vehicle and positive controls were valid.

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation (EC) 1272/2008, the test substance should not be classified for mutagenicity.