Bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative (OECD 471);

Chromosome aberration test: negative (OECD 473);

In vitro gene mutation test: negative (OECD 490).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-06-08 to 2009-08-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: 20090226
Purity: 99.38%

Target gene:

S. typhimurium: Histidine
E. coli: Trp operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary test: 0, 4, 20, 100, 500, 1500, 2500 µg/plate.
Main test: 0 (vehicle), 15.63, 31.25, 62.5, 125, 250, 500 µg/plate.

Vehicle / solvent:

- Vehicle/solvent used: Ethanol
- Justification for choice of solvent/vehicle: Sponsor indicated that test substance was insoluble in water. Solubility tests were conducted in DMSO, distilled water, minimum essential medium (MEM) and ethanol. The test material was soluble in ethanol, up to the highest tested concentration of 25 mg/mL, but not in any of the other solvents. Therefore, ethanol was chosen as the solvent/vehicle.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535, without metabolic activation only

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without metabolic activation only

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without metabolic activation only

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

E. coli WP2uvrA without metabolic activation only

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA1535, E. coli WP2uvrA, with metabolic activation only

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA100, TA98, TA1537 without metabolic activation only

Details on test system and experimental conditions:

TESTER STRAINS
- All strains were supplied by Molecular Toxicology, Inc. Boone, North Carolina, United States

MEDIA
- Each 25 mL of oxoid broth was innoculated with tester strain from master plates and then incubated in a gyrotary incubator (200 rpm) at 37 ± 1 °C for approx. 10 hrs
- Minimal glucose agar plates were prepared using 1.5 % Bacto agar (Difco), Vogel-Bonner medium E and 2 % glucose per L, and then 25 mL of agar placed in 100 × 15 mm petri dishes.
- The agar plates for the E. coli strain were supplemented with 0.25 mL of 0.1 % tryphtophan solution per L of agar.
- Top agar was prepared using 0.6 % agar and 0.5 % NaCl. Top agar for the Salmonella TA strains was supplemented with 0.05 mM L-histidine.HCl and biotin.

STORAGE OF TESTER STRAINS
- To each mL of suspension, 0.09 mL of DMSO was added. The mixture was stored at approx. -80 °C.
- After thawing, master plates of each strain were made and kept in a refrigerator to be used in the experiments.

CONFIRMATION OF GENOTYPES
- The genotypes of the tester strains were confirmed according to the methods of Maron and Ames (1983), prior to the start of experiments:
-- Auxotrophy: The S. typhimurium tester strains were histidine auxotrophs while E. coli WP2uvrA tester strain was a tryptophan auxotroph.
-- UV sensitivity (uvrB and uvrA): Tester strains TA98, TA100, TA1535, TA1537 and WP2uvrA exhibited sensitivity to UV light.
-- rfa mutation: All S. typhimurium strains exhibited resistance to crystal violet.
-- R-factor: Tester strains TA98 and TA100 exhibited resistance to ampicillin.
-- Each tester strain exhibited a characteristic number of spontaneous revertant colonies when plated.

METABOLIC ACTIVATION SYSTEM (S-9 MIX)
- S-9 was supplied by Molecular Toxicology, Inc. Boone, North Carolina, United States.
- Type: S-9 Aroclor 1254-induced Sprague-Dawley male rat liver
- Protein content: 42.5 mg/L
- Date of receipt: 2008-10-16
- Expiry date: 2010-07-03
- Storage conditions: Frozen (approx -20 °C)

- Cofactor-1 was supplied by Wako Pure Chem. Ind., Ltd, Japan
- Manufactured by Oriental Yeast Co., Ltd
- Date of receipt: 2009-04-30
- Expiry date: 2011-04-29

- Content of 1 mL S9-mix: 8 µmol MgCl2.6H2O, 33 µmol KCl, 5 µmol G-6-P, 4 µmol NADPH, 4 µmol NADH, 100 µmol sodium phosphate buffer (pH 7.4) and 50 µL S-9.
- Prepared S-9 mix was kept in an ice bath until use and used at 0.5 mL/plate.

PREPARATION OF TEST AND REFERENCE SUBSTANCES
- DEHCH was dissolved in ethanol to yield a 20-fold stock solution of the target concentrations. The highest test item solution was diluted with the same vehicle control.
- To determine test item concentration, 10 mL of each concentration was analysed. As a consequence, the first formulation of test material was rejected and a second batch prepared. This second batch was within ± 10 % of the nominal concentrations and were therefore considered valid and used.
- Positive control items were dissolved either in distilled water (sodium azide), or in DMSO (2-nitrofluorene, 9-aminoacridine, 4-nitroquinoline-N-oxide, 2-aminoanthracene, benzo(a)pyrene).
- Positive control substances were stored at -20 °C for up to 10 months and were thawed prior to use.
- Positive control substance concentrations were as follows:
-- TA 100 -S9: Sodium azide 0.5 µg/plate
-- TA 100 +S9: Benzo(a)pyrene 2 µg/plate
-- TA 1535 -S9: Sodium azide 0.5 µg/plate, 2-aminoanthracene 2 µg/plate
-- TA 1535 +S9: 2-aminoanthracene 2 µg/plate
-- TA 98 -S9: 2-nitrofluorene 2 µg/plate, benzo(a)pyrene, 2 µg/plate
-- TA98 +S9: Benzo(a)pyrene 2 µg/plate
-- TA1537 -S9: 9-aminoacridine 50 µg/plate
-- TA1537 +S9: Benzo(a)pyrene 2 µg/plate
-- WP2uvrA -S9: 4-nitroquinoline-N-oxide 0.5 µg/plate
-- WP2uvrA +S9: 4-nitroquinoline-N-oxide 4µg/plate

PRELIMINARY STUDY
- A preliminary dose range-finding study was performed to determine the concentration range for the confirmatory mutagenicity study.
- The highest concentration in the preliminary study was 2500 µg/plate.
- 500 µg/plate was chosen as the highest concentration for the main study.

MAIN STUDY
- 2 experiments were performed under identical conditions, each including vehicle and positive controls.
- Triplicate treatements were conducted for each concentration in the presence and absence of S-9 mix.

- Autoclaved top agar (2 mL) was dispensed to 12×75 mm tubes at 45 °C in a heating block. 0.05 mL of test item, 0.5 mL of S-9 mix or sodium phosphate buffer as appropriate, and 0.1 mL of bacterial culture were added to each tube. The mixture was stirred gently for 2-3 s using a Vortex mixer and was then poured onto minimal glucose agar plate.
- Vehicle control groups were treated with 0.05 mL of vehicle control and each of the positive control groups was treated with strain-specific controls (0.1 mL) instead of test item.
- Samples of the highest concentration tested (0.05 mL) and S-9 mix (0.5 mL) were also plated withouth bacterial culture to verify sterility.
- After the top agar was solidified, the plates were inverted, and incubated at 37 ± 1 °C for approx. 48 hrs. Revertants were then counted with a hand counter. Each concentration of test item was assayed in triplicate.

CHECKING
Each plate was evaluated for contamination by observation for any formation of a background lawn or any other abnormality.

EXPRESSION OF RESULTS
- Results were expressed as the mean number of revertant colonies from each triplicate of plates per dose with standard deviations.

Evaluation criteria:

A result was considered positive if there was a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation. In addition, any increases in revertant colonies were required to have occurred in the absence of toxicity (antibacterial effect) as defined as a clearing or diminution of background lawn, the appearance of microcolonies and/or a decrease of more than 50 % in the number of colonies compared to that of vehicle control.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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
- Precipitation of test substance was observed at 250 µg/plate when the test item was added to top agar.
- Precipitation of test substance was observed at 500 µg/plate in all studies after 48 hr incubation.

RANGE-FINDING/SCREENING STUDIES
- No antibacterial effects (cytogenicity) was observed at any concentration in the presence and absence of S-9 mix.
- Precipitation of test item was observed at concentrations of ≥ 500 µg/plate when test item solutions were mixed with top agar and incubated for 48 hrs.

COMPARISON WITH HISTORICAL CONTROL DATA
- The number of revertant colonies in the vehicle controls was within the range of KIT historical control data.

Sterility results:

No contaminant colonies were observed on the sterility plates for the highest concentration of the test substance or the S9 mix.

Confirmatory mutagenicity study:

There was no increase in the number of revertant colonies compared to the vehicle control at any concentration of the test substance either in the presence or absence of metabolic activation system in any strain tested. The precipitation of test item was observed at the concentration more than 250µg/plate in all strains used, when test item solutions were mixed with top agar. Precipitation was observed at the 500µg/plate concentration after 48 h incubation. The number of viable cells in the overnight cultures as determined by optical density was within acceptable limits: 1.2 to 2.3E09 cells/mL (S. typhimurium strains) and 2.3E09 cells/mL (E. coli strain); 1.1 to 2.2E09 cells/mL (S. typhimurium strains) and 2.1E09 cells/mL (E. coli strain) in the 1st and 2nd experiments respectively. The positive controls induced a significant increase in the number of revertant colonies indicating the assay was valid.

Dose Analysis:

Dosing solutions were analysed. The concentration analyses indicate that the actual concentration of the dosing solutions used were 101.81 % to 103.82 % and 94.47 % to 103.81 % of target concentration in the 1st and 2nd experiments, respectively.

Conclusions:

The test substance was assessed for genotoxicity according to OECD Guideline 471. The test substance was concluded to be negative in four test strains of Salmonella typhimurium and one test strain of Escherichia coli.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-06-22 to 2009-06-26

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)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch No.: 20090226
Purity: 99.38%

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Reconstituted minimum essential medium (MEM) supplemented with 2.2 g of NaHCO3, 2 mM L-glutamine, streptomycin sulfate (100 µg/L), penicillin G.Na (100 units) and 10 % foetal bovine serum (FBS) per litre.
- Periodically checked for Mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

With metabolic activation: 0 (control), 39.1, 78.1, 156.3, 312.5 µg/mL.
Without metabolic activation: 0 (control), 9.8, 19.5, 39.1, 78.1 µg/mL.

Vehicle / solvent:

- Vehicle/solvent used: Ethanol
- Justification for choice of solvent/vehicle: Sponsor indicated that test substance was insoluble in water. Solubility tests were conducted in DMSO, distilled water, minimum essential medium (MEM), ethanol. The test material was soluble in ethanol, up to the highest tested concentration of 25 mg/mL, but not in any of the other solvents. Therefore, ethanol was chosen as the solvent/vehicle.

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Without metabolic activation only

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

With metabolic activation only

Details on test system and experimental conditions:

CELL LINE
- Chinese hamster lung cells, CHL, Freeze No. 2184656 were obtained from American Type Culture Collection (Manassa, Virginia, United States) in 2004.
- The modal chromosome number of this cell line was 25 with a doubling time of approx. 15-17 hrs.
- The cells were thawed in culture medium and then grown as a monolayer.
- Confirmation of sterility was performed by using the inverted microscope and cells were screened for mycoplasma and cell seeding. The results showed that cells were negative for mycoplasma.

CULTURE CONDITIONS
- Cells were cryopreserved as a 0.9 mL cell suspension containing 0.1 mL of DMSO. ≥ 7 d prior to use, a vial of frozen cells was thawed, cultured and the absence of mycoplasma contamination verified.
- Cells were intubated at 37 °C ± 1 °C in a 5 % CO2 atmosphere as a monolayer in T-75 culture flasks and then subcultured twice-weekly. Rapidly growing cell cultures were trypsinised, suspended in culture medium and counted.
- 3 series of 25 cm² culture flask were seeded with 4 × 10⁴ cells each in 5 mL of medium and incubated for 3 d prior to treatment.

METABOLIC ACTIVATION SYSTEM (S-9 MIX)
- Type: Aroclor-induced Sprague-Dawley rat liver S-9
- Supplier: Molecular Toxicology, Inc., Boone, North Carolina, United States.
- Protein content: 42.5 mg/L
- Storage conditions: Approx. -20 °C

- Cofactor
- Supplier: Wako Pure Chem. Ind., Ltd, Japan
- Manufacturer: Oriental Yeast Co., Ltd
- Storage conditions: Refrigerated (2-8 °C)

- S-9 mixture content: 30 % v/v S-9
- Each mL of S-9 mixture contains 8 µmol MgCl2.6H2O, 33 µmol KCl, 5 µmol G-6-P, 4 µmol NADPH, 100 µmol sodium phosphate buffer (pH 7.4) and 0.3 mL S-9
- To each 0.5 mL ice cold S-9 mix was added 4.5 mL medium per T-25 flask.

TEST AND REFERENCE ITEM PREPARATION
- The test material, DEHCH, was dissolved in ethanol to yield a 200-fold stock solution of target concentrations.
- To determine the concentration in the formulations, analysis was performed. The analysis confirmed that the prepared formulations were within ± 10 % of labelled concentrations in the experiment.
- The formulations were considered stable at room temperature for 8 d, and were formulated 4 d prior to treatment.
- The positive control substances, CPA and EMS, were dissolved in sterile distilled water to prepare 100× stock solutions. The solutions were stored at approx. -20 °C for a maximum of 6 months. Positive controls were thawed immeditely before use.
- Positive control concentrations were 6 µg/mL for CPA +S9, 800 µg/mL for EMS 6 hrs -S9 and 600 µg/mL 22 hrs +S9.

PRELIMINARY DOSE RANGE-FINDING STUDY
- To determine the concentration that might inhibit cell growth by > 50 %, a dose range-finding study was performed with the highest concentration of 2500 µg/mL considering the solubility of the test item. The Relative Cell Count (RCC) was determined by comparing cell counts in the vehicle control treated structures to those treated with test material at concentrations of 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 µg/mL.

MAIN STUDY
- The treatment was 6 hrs for metabolic activation, and 6 and 22 hrs without metabolic activation.
- The presence of aberrant metaphases was evaluated in each treatment group at 3 concentrations that met the acceptance criteria for viability and analysable metaphases.
- Treatment at each concentration was in duplicate

- Treatment was initiated by the replacement of the original media in each flask in 3 series of 3 d old cultures (2 flasks/concentration) with 4.5 mL (+S9 mix) or 5.0 mL (-S9 mix) of fresh medium. After a minimum of 1 hr, vehicle, test substance, or positive control was added to each flask as follows:
-- Series I (6 hrs +S9): 4.5 mL culture medium, 0.025 mL test item, 0.5 mL S9 mix
-- Series II (22 hrs -S9): 5.0 mL culture medium, 0.025 mL test item
-- Series III (22 hrs -S9): 5.0 mL culture medium, 0.025 mL test item
- Series I and II were treated at 37 °C ± 1 °C for 6 hrs and Series III for 22 hrs
- At the end of treatment period, the mixture was removed, and the cultures washed once with Ca²⁺ and Mg²⁺ free Dulbecco's phosphate-buffered saline (CMF D-PBS) and replaced with 5 mL of fresh harvest.
- Approx. 22 hrs after the initiation of treatment, colchichine solution was added to each cell culture (final concentration 1 µM) and incubation cotinued for another 2 hrs.
- Cells were then harvested, approx. 24 hrs after initiation of treatment, for slide preparation and scoring.

SLIDE PREPARATION
- Cells were typsinised, counted and Relative Cell Count (RCC) calculated. Cells were then centrifuged at approx. 1000 rpm for 5 mins, and suspended in 5 mL of 75 mM KCl solution. After 10 mins at room temperature, 5 mL of fixative (methanol:glacial acetic acid, 3:1 v/v) was added to the cell suspension and then refrigerated for approx. 20 mins. The fixative was changed twice by centrifugation (approx. 1500 rpm for 5 mins). 2 slides were prepared from each fixed cell suspension. The slides were air dried, stained with 3 % giemsa solution, washed in tap water and distilled water, dried and mounted in DPX for chromosomal aberration scoring.

SLIDE SCORING
- 1/2 slides prepared was selected for evaluation based upon suitable microscopic appearance and coded so as to blind the evaluator to its treatment.
- Chromosomal aberrations were morphologically identified according to the principles described in "Atlas of Chromosome Aberration by Chemicals". Any structural aberrations were recorded; chromatid and chromosome gaps, chromatid type breaks and exchanges, and chromosome type breaks and exchanges.
- Cells with ≥ 4 aberrations of the same type were scored as multiple aberrations.
- Any metaphase with ≥ 1 aberrations, regardless of its type, was classified as a metaphase aberration.
- Slides were scanned systematically and each set of metaphases examined using 1000× magnification.

Structual aberrations
- Chromosomal aberrations were evaluated in 100 well-spread metaphases, each containin 23 to 27 chromosomes. The microscopic stage co-ordinates were recorded for each aberrant metaphase. Each type of aberration was recorded, and the number of aberrant metaphases (showing ≥ 1 aberrations, including and excluding gaps) calculated. The results were expressed as the number of findings per 100 metaphases.

Numerical aberrations
- Regardless of the presence of aberrations, an additional 100 metaphases were examined to determine the frequency of diplody (DP), polyploidy (PP, ≥ 37 chromosomes) and endoreduplication (ER).

Evaluation criteria:

The study would have been considered positive if there was a concentration-related increase or a clear, reproducible and statistically-significant increase in the number of cells with chromosome aberrations.

Statistics:

Analyses included the number of aberrant metaphases (excluding gaps), and frequency of polyploidy and endoreduplication (PP+ER). Threshold of significance: p < 0.05. The specific statistical tests used for various comparisons include:

Vehicle control v. test item-related groups: Pearson's χ² test and if p < 0.05, Fisher's exact test.
Dose-response: Cochran-Armitage trend test
Vehicle control v. positive control groups: Fisher's exact test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

RANGE-FINDING/SCREENING STUDIES:
- No cytotoxicity was observed at concentrations ≤ 2500 µg/mL, with or without metabolic activation, at 6 hrs and 22 hrs of treatment. However, at the beginning and end of the treatment period, the turbidity of the test item was observed at the concentration of > 312.5 µg/mL and 7.8 µg/mL in the presence (6 h + S9) and absence (6 h -S9; 22 h -S9) of S9 mix respectively. Thus, the highest concentration selected for use in confirmatory analysis of 312.5, 78.1 and 78.1 µg/mL for each treatment group (6 h+S9, 6 h -S9 and 22 h -S9).

CONFIRMATORY ASSAY (MAIN STUDY):
In the confirmatory assay, cytotoxicity was not observed at doses up to the highest concentration for each treatment group. However, at the beginning and end of the treatment period, the turbidity of the test item was observed at 312.5 µg/mL and 7.8 µg/mL in the presence (6 h + S9) and absence (6 h -S9; 22 h -S9) of S9 mix respectively.

RESULTS IN THE PRESENCE OF S-9 MIXTURE:
In the first experiment, the frequency of metaphases with structural aberrations was 0.5, 0.0, 1.0 and 0.5 in the vehicle control, 78.1, 156.3 and 312.5 µg/mL concentrations respectively. There was no statistically significant increase in the number of metaphases with structural aberrations at any dose level of test item tested. The frequency of polyploidy and endoreduplication (PP+EP) in the vehicle control was 1.0+0.0 and there was no significant increase in the number of aberrant metaphase cells at any concentration tested. As expected there was a clear increase in the number of aberrant metaphase cells in the positive control group (15.5/100 metaphases; P < 0.01) indicating that the assay was valid.

RESULTS IN THE ABSENCE OF S-9 MIXTURE:
After 6 h of treatment, the frequency of metaphases with structural aberrations was 0.5, 0.0, 0.5 and 11.0 in the vehicle control, 19.5, 39.1 and 78.1 µg/mL concentrations, respectively. There was no statistically significant increase in the number of metaphases with structural aberrations at any dose level of test item tested, The frequency of polyploidy and endoreduplication (PP+RR) in the vehicle control was 0.5+0.0 and there was no statistically significant increase in the number of aberrant metaphase cells at any concentration of test item.
After 22 h of treatment, the frequency of metaphases with structural aberrations was 0.5, 0.0, 1.5 and 0.0 in the vehicle control, 19.5, 39.1 and 78.1 µg/mL concentrations respectively. There was no statistically significant increase in the number of aberrant metaphase cells at any concentration of test item.
There was a clear increase in the number of aberrant metaphases in the positive control groups, after 6 h (19.5/100 metaphases, P<0.01) and 22 h (30.5/100 metaphases, P<0.01) of treatment indicating that the assay was valid.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The numbers of metaphases with structural aberrations in the vehicle and positive control were in the ranges established in historical data.

Conclusions:

The test substance was assessed for induction of chromosome aberrations according to OECD Guideline 473. Under the conditions of the study the test substance did not induce chromosome aberrations in cultured CHL cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-10 to 2017-02-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro gene mutation study in mammalian cells

Specific details on test material used for the study:

Batch: Not indicated
Purity: > 99%

Target gene:

Thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I (4 hours): 3.9, 7.8, 15.6, 31.3, 62.5, 125 μg/mL
Experiment II (24 hours): 31.3, 62.5, 125, 250, 500, 1000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1E+07 cells/flask

DURATION
- Exposure duration: 4 hours, 24 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 4E+03 cells/plate

EXAMINATION: Size Distribution of the Colonies
The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 25% of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 1E+06 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 1E+06 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic is considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics (p < 0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test medium was checked for precipitation and phase separation visible to the naked eye at the end of the 4 hours and 24 hours treatment just before the test item was removed. Phase separation was noted at 62.5 μg/mL and above in the presence and absence of metabolic activation after 4 hours treatment, and at 500.0 μg/mL and above in the absence of metabolic activation after 24 hours treatment.
No relevant cytotoxic effect indicated by a relative total growth of less than 50% of survival in both cultures was observed in both experiments. Two relative total growth levels just below 50% occurred at 250.0 and 1000.0 μg/mL in culture II of the second experiment. However, these isolated effects were judged as irrelevant fluctuation as they were not reproduced in parallel culture.
No substantial and reproducible increase of the mutation frequency was noted in the main experiments with and without metabolic activation. The mutation frequencies did not exceed the threshold of 126 above the corresponding solvent control at any experimental point.
MMS (19.5 μg/mL in experiment I and 13.0 μg/mL in experiment II) and CPA (3.0 μg/mL and 4.5 μg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity at at least one of the concentrations of the controls. The positive controls remained within the range of the historical positive control data throughout the study.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse micronucleus test: negative (OECD 474).

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:

2009-07-07 to 2009-07-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

No effect on study; slight problem with analysis of test concentrations

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Batch No.: 20090226
Purity: 99.38%

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio Inc., 143-1 Sangdeawon-Dong, Jungwon-Gu, Seongnam-Si, Gyeonggi-Do, Korea)
- Age at study initiation: Approx. 7 wks
- Weight at study initiation: Males 29.7-34.0 g; females 23.5-27.5 g
- Assigned to test groups under following basis: Using algorithm of Path/tox system v 4.2.2, so that body weights of each group will have similar weight distribution
- Fasting period before study:
- Housing: ≤ 10 (during acclimation period) and ≤ 6 (during dosing) animals per polycarbonate cage (240 mm width × 390 mm length × 180 mm height), including bedding.
- Diet: Ad libitum, pelleted diet
- Water: ad libitum, filtered, UV-radiated tap water
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature 22 ± 3 °C
- Humidity: 50-60 %
- Air changes: 10-20 times per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: Corn oil
- Justification for choice of solvent/vehicle: Sponsor indicated that test substance was insoluble in water. Solubility tests were conducted in DMSO, distilled water, corn oil. The test material was soluble in corn oil, up to the highest tested concentration of 200 mg/mL, but not in either of the other solvents. Therefore, corn oil was chosen as the solvent/vehicle.
- Volume of administered vehicle and test material: 10 mL/kg bw

Details on exposure:

RANGE-FINDING STUDY
- 4 males and 4 females were dosed with test material at 250, 500, 1000, 2000 mg/kg bw for 2 consecutive days.
- Based on the results of the range-finding study, three dose levels were selected as 500, 1000, 2000 mg/kg bw.

PREPARATION OF DOSING SOLUTIONS:

Duration of treatment / exposure:

2 d

Frequency of treatment:

Daily

Post exposure period:

Approx. 24 hrs

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 male, 6 female per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control substance: Cyclophosphamide
- Justification for choice of positive control: Widely used in this type of assay
- Route of administration: Intraperitoneally
- Dose: 70 mg/kg bw
- Volume: 10 mL/kg bw
- Frequency of administration: Single administration

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- Based on range-finding study

DETAILS OF SLIDE PREPARATION:
- Animals were sacrificed by CO₂ gas inhalation at 24 hrs after final administration. Bone marrow preparations were made according to Schmid (1975), and 2 slides of the cell suspensions per animal were made.
- Bone marrow cells were collected into 3 mL of foetal bovine serum using a syringe with a 23G needle, centrifuged at approx. 1000 rpm for 5 mins.
- After removing the supernatant, a small drop of viscous suspension was smeared onto clean microscopic slides.
- Preparations were air dried and fixed by submerging in absolute methanol for 5 mins.
- Fixed slides were stained as follows:
-- May Grünwald stain 3 mins
-- May-Grünwald stain (1:1 dilution) 2 mins
-- Giemsa stain (1:6 dilution) 10 mins
- Stained slides were rinsed with distilled water, dried and mounted with a mountant.

METHOD OF ANALYSIS:
- Slides with good staining were coded and examined by microscopy at 1000× magnification
- Small round or oval-shaped bodies with erythrocytes with a size of approx. ¹⁄₅ to ¹⁄₂₀ of the diameter of a PCE were regarded as micronuclei.
- Attention was given to the discriminate micronuclei from artefacts.
- Results were expressed as number of MNPCEs in 2000 PCEs.
- The mean number of MNPCEs ± standard deviation was calculated for each treatment group.
- The PCE/(PCE+NCE) ratio, indicating cytotoxicity to haematopoietic system was calculated by counting 500 cells.

OTHER:
- Mortality and external appearance of animals was checked and recorded daily during the study period.
- During the 24 hr post exposure period there were 3 sets of observations
- Bodyweights were measured on the day of reception, grouping, dosing and autopsy.

Evaluation criteria:

- The results would have been considered positive if there was a statistically significant and dose-related increase or a reproducible increase in the frequency of MNPCEs at at least one dose level.
- The study would have been accepted if PCE/(PCE+NCE) ratios were all >0.1, and the number of MNPCEs in the vehicle control group was within range of the laboratory's historical background data.

Statistics:

Performed according to Lovell et al. 1989 with minor modification, with a significance threshold of p < 0.05
- Test for differences in numbers between PCEs between treated group and vehicle control group: Kruskal-Wallace H-test and Dunn's rank sum test.
- Test for differences in numbers of PCEs between positive control group and vehicle control group: Mann-Whitney U-test
- Tests for differences of PCE/(PCE+NCE) ratio between treated group and vehicle control group: arcsine value of each datum was subjected to Fisher's ANOVA test and Dunnett's test.
- Test for differences of PCE/(PCE+NCE) ratio between positive control group and vehicle control group: arcsine value of each datum was subjected to Student's t-test
- Comparison of bodyweight of animals: Bartlett's test, Fisher's ANOVA if p > 0.05
- Dose-response: Cochran-Armitage trend test
- Vehicle control and test item groups: Pearson's χ² test, and Fisher's exact test if p < 0.05
- Test for dose-response: Cochran-Armitage trend test if p < 0.05 in the above analyses.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: None

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei per 2000 PCEs:
-- Vehicle control: Male 1.00; female 0.83
-- 500 mg/kg bw: Male 1.17; female 0.50
-- 1000 mg/kg bw: Male 1.00; female 1.17
-- 2000 mg/kg bw: Male 1.33; female 1.33
-- CPA: Male 50.17; female 43.17
- Ratio of PCE/NCE:
-- Vehicle control: Male 0.57; female 0.55
-- 500 mg/kg bw: Male 0.60; female 0.56
-- 1000 mg/kg bw: Male 0.57; female 0.57
-- 2000 mg/kg bw: Male 0.56; female 0.55
-- CPA: Male 0.44; female 0.44

- Statistical evaluation:
-- There were no statistically-significant increases in the number of MNPCEs at any dose of the test article compared to the vehicle control group
-- There was a significantly significant increase in the number of MNPCEs in the positive control groups (p < 0.01 in both males and females)
-- There was no statistically significant differences in the PCE/(PCE+NCE) ratio between vehicle control and article-treated groups
-- A statisticaly significant difference was observed in the PCE/(PCE+NCE) ratio between positive control and vehicle control groups (p < 0.01, in both males and females)
- Clinical signs: No clinical signs or behavioural alterations were noted during the observation period.
- Bodyweights: There were no statistically-significant differences in the mean body weights of test-item treated groups and positive control groups compared to those of the vehicle control.

VALIDITY OF TEST
- The test was considered valid as the PCE/(PCE+NCE) ratios were all > 0.1, and the number of MNPCEs in the vehicle control group was in the range of historical control data.

DOSING ANALYSIS
- The dosing concentrations were determined to be 105.50 % to 108.28 % of the target concentrations and the concentrations of test material were stable.

Conclusions:

The test susbtance was assessed for genotoxicity according to OECD Guideline 474. Under the conditions of the study the oral adminsitration of the test substance at doses up to 2000 mg/kg did not induce an increase in the incidence of micronucleated polychromatic erthrocytes in bone marrow. Therefore, the test substance was concluded to be negative in the micronucelus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Three in vitro genetic toxicity studies and an in vivo micronucleus assay have been conducted on the test material, as follows:

Ames Test (in vitro):

The test item was evaluated for its potential to induce reverse mutation using Salmonella typhimurium TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2uvrA in the presence or absence of metabolic activation with S9 mix using the plate incorporation method. Under the conditions of the study DEHCH was concluded to be negative. .

Chromosome aberration test (in vitro):

The potential of DEHCH to induce chromosomal aberrations in Chinese Hamster Lung (CHL) cells in the presence or absence of metabolic activation (S9 mix) was evaluated in the in vitro chromosome aberration assay. Under the conditions of the study DEHCH did not induce chromosomal aberrations in CHL cells.

In vitro gene mutation test:

This in vitro assay was performed to assess the potential of the test item to induce mutations at the thymidine kinase locus of the mouse lymphoma cell line L5178Y. In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Mouse micronucleus assay ( in vivo)

A micronucelus test of DEHCH was performed using bone marrow cells of specific pathogen free (SPF) 7 week old ICR mice The test item was orally administered twice at 24 h intervals at doses of vehicle control, 500, 1000 and 2000 mg/kg in both male and female mice (6/sex/group). The number of MNPCE (micronucleated polychromatic erythrocytes) was counted in 2000 PCEs (polychromatic erythrocytes) per animal. There was no statistically significant increase in the frequencies of MNCPEs at any dose of DEHCH compared to the vehicle control. In addition no significant difference was observed in the mean value for the ratio of PCE to total erythrocytes (PCE/(PCE+NCE)) in the DEHCH group. DEHCH was therefore concluded to be negative in the study.

Justification for classification or non-classification

Three in vitro genetic toxicity studies have been conducted on the test material, an Ames study (OECD 471), a chromosome aberration study (OECD 473), and in vitro gene mutation study (OECD 490) which were both negative. An in vivo mouse micronuclues assay (OECD 474) is also available which was also negative for mutagenicity.

All the in vitro and in vivo genetic toxicity studies showed the test material to have no significant effects for gentoxicity. As such, the test material can be considered to be non-classified.