oligomeric reaction product of 2-hydroxybenzaldehyde with 1-chloro-2,3-epoxypropane and phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

12 June 2015- 29 June 2016

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

The S9 was prepared from the livers of 7-week old male Sprague-Dawley rats treated with phenobarbital (PB, 4 times at 24-hour intervals) and 5, 6-benzoflavone (once at a single i.p. dose of 80 mg/kg on the same day of the third PB injection)

Test concentrations with justification for top dose:

A preliminary test was performed at 5000 µg/plate as the maximum dose using 7 doses with a common ratio of 4. As the results, the number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at the following dose levels.

Without S9 mix; 5000 µg/plate (TAlOO)
1250-5000 µg/plate (TA1535)

With S9 mix; 1250-5000 µg/plate (TAlOO)
313-5000 µg/plate (TA1535)
1250-5000 µg/plate (WP2uvrA)

Based on the results of the preliminary test, the main test I was performed at 5000 µg/plate as the maximum dose using 5-9 doses with a common ratio of 2. As the results, the number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at thefollowing dose levels.

Without S9 mix; 5000 µg/plate (TAlOO)
1250-5000 µg/plate (TA1535)

With S9 mix; 625-5000 µg/plate (TAlOO)
156-5000 µg/plate (TA1535)
1250-5000 µg/plate (WP2uvrA)

The main test II was performed at using the same doses as the main test I.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2-AA) and 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylarnide (AF-2)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

Details on test system and experimental conditions:

METHOD OF APPLICATION: This study was performed using the pre-incubation method with and without S9 mix.

After pre-culture, bacterial density of each strain was measured by a digital photometer (TAITEC Co., Ltd. Mini photo 518R), and then the bacterial suspension for counting the number of cells was prepared by the stepwise dilution method. The suspension was overlaid to a minimal glucose agar plate and incubated at 37°C for 48 hours. The number of cells was calculated by counting the number of colonies on the plate. The bacterial suspension for the reverse mutation test was stored at room temperature.

Preparation of liquid growth medium
Oxoid Nutrient Broth No.2 (Oxoid Ltd., Lot No. 1239615) 2.5 g was dissolved in 100 mL of purified water. The medium was autoclaved for 15 minutes at 121°C and stored in a cold storage.

Preparation of soft agar
Bacto-agar (Becton, Dickinson and Company, Lot No.3220297) 0.6 g and sodium chloride (Wako Pure Chemical Industries, Ltd.) 0.5 g were added to 100 mL of purified water. This mixture was autoclaved for 15 minutes at 121°C and then was stored at 45°C until use.

Preparation of top agar
Each of the following each amino acid solution was added to the soft agar at a volume ratio of 1 to 10 just before use. The top agar was kept at 45°C until use.

Salmonella typhimurium: 0.5 mmol/L D-biotin and 0.5 mmol/L L-histidine solution mixture
Escherichia coli: 0.5 mmol/L L-tryptophan solution

The S9 was prepared from the livers of 7-week old male Sprague-Dawley rats (body weight: 204 to 244 g) treated with phenobarbital (PB, 4 times at 24-hour intervals at i.p. doses of 30, 60, 60 and 60 mg/kg, respectively) and 5, 6-benzoflavone (once at a single i.p. dose of 80 mg/kg on the same day of the third PB injection).

1mL of S9 was added to 9 mL of the cofactor mix to give the proper ratio for the S9 mix. The S9 mix was freshly prepared just prior to use in each test and was kept in an ice bath until use. The ingredients and their contents per mL of the S9 mix preparation were as follows:

S9
Magnesium chloride
Potassium chloride
D -Glucose 6 -phosphate
13-NADPH
13-NADH
Sodium phosphate buffer (pH 7.4)

(1) The test substance was weighed and dissolved in DMSO with vortex mixing and ultrasonic wave treatment to make a 50 mg/mL solution.
(2) A portion of this solution was diluted stepwise with DMSO to make the test substance solutions of each concentration.
(3) All procedures, including those for weighing the test substance, dilution, dispensation and treatment of the solutions, were performed under a yellow light at room temperature.

The storage period and temperature after preparation of the test substance solutions to the time of use were as follows;

Preliminary test: 50 minutes at room temperature (23°C)
Main test I: 1 hour and 50 minutes at room temperature (23°C)
Main test II:- 1 hour and 20 minutes at room temperature (23°C)

As the results of the preliminary test, the number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at the following dose levels.

Without S9 mix; 5000 µg/plate (TAlOO)
1250-5000 µg/plate (TA1535)

With S9 mix; 1250-5000 µg/plate (TAlOO)
313-5000 µg/plate (TA1535)
1250-5000 µg/plate (WP2uvrA)

Based on these results, the main test I and main test Il were performed 5000 µg/plate as the maximum dose using 5-9 doses with a common ratio of 2

Pre-incubation method
(1) For each treatment, 0.1 mL of the test substance solution, negative (solvent) control or positive control solution was added into a sterilized test tube with aluminum cap.
(2) For assays without S9 mix, 0.5 mL of 0.1 mol/L sodium phosphate buffer (pH 7.4) was mixed and 0.1 mL of the bacterial suspension was added to this mixture.
(3) For assays with S9 mix, 0.5 mL of S9 mix was mixed and 0.1 mL of the bacterial suspension was added to this mixture.
(4) The mixture was incubated with gentle shaking (Shaking frequency: 120 times/minute) for 20 minutes at 37°C (pre-incubation).
(5) After pre-incubation, 2 mL of the molten top agar was added to this mixture and then poured onto a minimal glucose agar plate.
(6) After the overlaid agar had solidified, the plates were incubated for 48 hours at 37°C.

Preliminary test: 1 plate/dose (2 plates/dose for the negative (solvent) and positive controls)
Main test I: 3 plates/dose
Main test II: 3 plates/dose

Evaluation criteria:

Sterility test
In the preliminary and two main tests, bacterial contamination was examined using one plate for each of the highest dose of the test substance solution, S9 mix, and the 0.1 mol/L sodium phosphate buffer.
(1) 0.1 mL of the highest dose of the test substance solution or 0.5 mL of the S9 mix orthe 0.1 moVL sodium phosphate buffer was mixed with 2 mL of the molten top agar.
(2) The mixture was poured onto a minimal glucose agar plate.
(3) After the overlaid agar had solidified, the plates were incubated for 48 hours at 37°C and then checked for bacterial contamination macroscopically.

Observation
Precipitation: Precipitation was judged by observation of the plate surface macroscopically.
Microbial growth inhibition: Background lawn of the bacterial cells that have amino-acid requirement was observed by a stereoscopic microscope (Nikon, SMZ-10), and microbial growth inhibition was judged by the relationship between the test substance treated plates and the negative (solvent) control plate.

Measurement of colony number
Revertant colonies were measured using an automatic colony counter (System Science Co., Ltd., CA-1lD) or manual counting. Revertant colonies in positive control treatment groups, in TAlOO of test substance treatment groups without precipitation, and in the test substance treatment groups in which many revertant colonies were observed were measured using an automatic colony counter and the others measured by manual counting. Correction of the measuring area was employed using instrumental analysis.

Statistics:

No statistical analysis was performed with the test results in this study.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Maintest I

Number of revertant colonies: The number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at the following dose levels.

Without S9 mix; 5000 µg/plate (TAIOO)

1250-5000µg/plate(TA1535)

With S9 mix;      625-5000 µg/plate(TAlOO)

156-5000 µg/plate (TA1535)

1250-5000 µg/plate (WP2uvrA)

 Mutation activities: The mutation activities were calculated in the following dose, the tester strain. (The mutation activities were adopted the maximum in the main test I)

Without S9 mix; 5000 µg/plate (TAlOO): 31.0 number ofrevertant colonies/mg

WithS9mix;      625 µg/plate (TAlOO): 309 number ofrevertant colonies/mg

 

Microbial growth inhibition: Microbial growth inhibition was not observed in all tester strains with or without S9 mix.

Precipitation: Precipitation of the test substance was observed at the following dose levels.

Without S9 mix; 313 µg/plate or more (all tester strains)

With S9 mix;     1250µg/plateormore(alltesterstrains)

Maintest II

Number of revertant colonies: The number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at the following dose levels.

Without S9mix;5000 µg/plate (TAIOO)

1250-5000µg/plate(TA1535)

WithS9mix;      625-5000 µg/plate(TA!OO)

156-5000 µg/plate (TA1535) 1250-5000 µg/plate (WP2uvrA)

 Mutation activities: The mutation activities were calculatedinthe following dose, the tester strain. (The mutation activities were adopted the maximum in the main testII)

WithoutS9mix;5000µg/plate(TAI00):34.6numberofrevertantcolonies/mg

WithS9mix;      625µg/plate(TAIOO):312numberofrevertantcolonies/mg

 Microbial growth inhibition: Microbial growth inhibition was not observedinall tester strains with or without S9 mix.

Precipitation: Precipitation of the test substance was observed at the following dose levels.

Without S9mix;313 µg/plate or more (all tester strains) WithS9mix;      1250µg/plateormore(alltesterstrains)

 

Conclusions:

In the main test I, the number of revertant colonies induced by the test substance was great r than 2-fold of that of the corresponding negative (solvent) control value in TAlOO, TA1535 without S9 mix, in TAlOO, TA1535 and WP2uvrA with S9 mix. The same result was obtained also in the main test II, and the reproducibility of the test results was confmned. The maximum mutation.activities was 312 at 625 µg/plate doses in TAlOO with S9 mix of main test II. Furthermore, both tests were performed accurately because the acceptable criteria were satisfied.

From the results described above, it was concluded that EPICLON EXA-7250 was mutagenic under the conditions employed in this study.

Executive summary:

Mutagenicity of EPICLON EXA-7250 was assessed in the bacterial reverse mutationtest using five strains,Salmonella typhimuriumTAlOO, TA1535, TA98 and TA1537, and Escherichia coli WP2uvrA.The test was conducted by the pre-incubation method with and without S9mix.

 

A preliminary test was performed at 5000 µg/plate as the maximum dose using 7 doses with aco=onratio of 4. As the results, the number of revertant colonies treated withthe test substance was greater than 2-fold that treated with the negative (solvent) control at the following doselevels.

Without S9 mix; 5000 µg/plate (TAlOO)

1250-5000 µg/plate (TA1535)

WithS9mix;      1250-5000µg/plate(TAlOO)

313-5000 µg/plate (TA1535)

1250-5000 µg/plate(WP2uvrA)

Microbial growth inhibition of the test substance was not observed in all tester strains with or without S9 mix.

Precipitation of the test substance was observed at 313 µg/plate or more in all tester strains without S9 mix, at 1250 µg/plate or more in all tester strains with S9 mix.

 

Based on the results of the preliminary test,the main test I was performed at 5000µg/plate as the maximum dose using 5-9 doses with a common ratio of 2.

As the results,the number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at the following dose levels.

Without S9 mix; 5000 µg/plate (TAlOO)

1250-5000 µg/plate (TA1535) WithS9mix;     625-5000µg/plate(TAlOO)

156-5000 µg/plate (TA1535)

1250-5000 µg/plate(WP2uvrA)

Microbial growth inhibition of the test substance was not observed in all tester strains with or without S9 mix.

Precipitation of the test substance was observed at 313 µg/plate or more in all tester strains without S9 mix, at 1250 µg/plate or more in all tester strains with S9 mix.

 

The main test II was performed at using the same doses as the main test I. The results of the main test II were same as the main test I.

 

From the results described above, it was concluded that EPICLON EXA-7250 was mutagenic under the conditions employed in this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 June 2015 - 9 October 2015

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: An in vitro chromosomal aberration study of EPICLON EXA-7250 was conducted using human lymphocytes from peripheral blood as indicator cells

Species / strain / cell type:

mammalian cell line, other: Human lymphocytes from peripheral blood

Details on mammalian cell type (if applicable):

CELLS USED

Donor

(1) Adult female (age: 39 to 41 [cell growth inhibition test], 39 to 41 [chromosomal aberration test])
(2) Non-smoker
(3) Registered donor according to the SOP of the testing facility
(4) Did not take any pharmaceutical drugs from one day before the day of blood sampling
(5) Healthy on the day of blood sampling

Blood sampling
The blood sampling was conducted at "Iryou Houjin Zaidan Sanyu-kai, Tobu-chiku Kyodo Shinryo-jyo" (Medical Corporation "Toujin-kai", East Area Clinic) by nurses on June 22, 2015 (cell growth inhibition test) and on September 8, 2015 (chromosomal aberration test). The procedures were not conducted under GLP conditions. The blood samples were collected as 20 mL per donor in blood sampling tubes treated with heparin. Blood samples were collected from four donors in each test. The blood samples from donors were mixed and used.



MEDIA USED
Culture medium
RPMI1640 and RPMI1640 medium were prepared during the study. These media were used in common for the chromosomal aberration test with human lymphocyte.

RPMl1640
For 990 mL of RPMI1640 liquid medium (Life Technologies Corp., Lot No. 1621337, 1666713, 1694256), 10 mL of penicillin/streptomycin solution (penicillin: 10000 unit/mL, streptomycin: 10000 µg/mL, Life Technologies Corp., Lot No. 1647113, 1655311, 1688260) was added.

RPMl1640 medium
(1) A 100 mL portion of heat-inactivated (56°C, 30 minutes) fetal bovine serum (Life Technologies Corp., Lot No. 1034554) was added to 900 mL ofRPMI1640.
(2) On the day of use, phytohemagglutinin-P (PHA-P, Wako Pure Chemical Industries, Ltd., Lot No. AWFI493, KPK2879) was added to give a concentration of 0.05 vol%.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

-S9 mix assay: 15.6, 31.3, 62.5, 125,250,500, 1000 and 2000 µg/mL
+S9 mix assay: 15.6, 31.3, 62.5, 125,250, 500, 1000 and 2000 µg/mL
24-hour assay: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL

According to the applied guideline, 2000 µg/mL as the highest concentration and the concentrations described above were selected.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Cell growth Inhibition test.
Preparation of the test substance solutions
(1) The test substance was melted in warmed water.
(2) The melted test substance was weighed and dissolved in DMSO by vortex mixing and ultrasonication.
(3) The final volume of the solution was adjusted with DMSO to prepare the highest test substance solution of 100-fold concentration of the final concentration.
(4) A portion of the highest test substance solution was serially diluted with DMSO to prepare each test substance solutions of 100-fold concentration of the final concentration.

The concentrations of prepared solutions were as follows;
Cell growth inhibition test: 200,100, 50, 25, 12.5, 6.25, 3.13 and 1.56 mg/mL
Chromosomal aberration test: 20, 18, 16, 14, 12, 10, 8, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2,1.5, 1 and 0.5 mg/mL

(5) All the procedures, including those for weighing the test substance and dilution of the solutions, were performed under UV-cut light at room temperature. The test substance solutions were used immediately after preparation. The storage period after preparation to the time of use was as follows;

Cell growth inhibition test: 30 to 45 minutes
Chromosomal aberration test: 25 to 55 minutes

Cell growth inhibition test
-S9 mix assay: Short-term treatment assay (3-hour treatment and 21-hour recovery) without S9 mix
+S9 mix assay: Short-term treatment assay (3-hour treatment and 21-hour recovery) with S9mix
24-hour assay: Continuous treatment assay (24-hour treatment) without S9 mix

Cell treatment
(1) Blood samples were collected.
(2) All of the collected blood samples were mixed and added to RPMil 640 medium at 10 v/v%. The resulting cell suspension was mixed well.
(3) The cell suspension was separated into plastic centrifuge tubes (volume: 50 mL) at 10 mL. They were cultured in a C0 2 incubator for 2 days.
(4) After the completion of the culture in Step (3), the cell suspension was mixed by shaking with a pipette.
(5) The test substance solution and S9 mix were added to the cell suspension as shown in the table below. All assays were performed in duplicate. Each tube was distinguished by the numbering to the tubes.
(6) The cells were incubated for 24 hours in the continuous treatment assay and for 3 hours in the short-term treatment assay. During the cell treatment, each centrifuge tube was closed with a cap, and was mixed gently by a mechanical shaker (tilt angle: about 8 degrees; tube rotation rate: about 30 rpm).
(7) In the short-term treatment assay, after the 3-hour incubation, the treatment mixture was transferred to a plastic centrifuge tube (volume: 15 mL). The cells were washed [the cell suspensions were centrifuged (1000 rpm for 5 minutes; this was applied to the following centrifugation steps) and the precipitated cells were suspended in RPMI1640] twice and further incubated in 5 mL of fresh RPMI1640 medium for an additional 21 hours without shaking.
(8) At the beginning and end of the treatment, all plastic centrifuge tubes were examined macroscopically for possible precipitation of the test substance.

Preparation of specimens
(1) Two hours before the end of culture, colcemid was added to the medium in each tube at a final concentration of 0.1 µg/mL to accumulate the metaphase cells.
(2) At the end of the culture, the cell suspension was centrifuged.
(3) After the supernatant was removed, the cells were subjected to hypotonic treatment with 4 mL of a 0.075 mol/L solution of potassium chloride at 37°C for 15 minutes.
(4) The cells were then fixed with 0.5 mL of an ice-cold fixative mixture (ethanol - acetic acid [3:1, v/v]), centrifuged and the supernatant was removed.
(5) The cells were treated with 4 mL of the fresh fixative mixture, centrifuged and the resulting supernatant was removed.
(6) The procedure in (5) was conducted again.
(7) The cells and 4 mL of the fixative mixture were mixed. The resulting mixtures were allowed to stand for 21 hours and 15 minutes in a refrigerator.
(8) The mixtures were centrifuged and the supernatants were removed. The cells were suspended in an appropriate amount of the fixative mixture.
(9) The cell suspension was dropped onto two sites on a slide glass placed on wet cloth, and then was dried.One slide was prepared from each tube.
(10) The cells were stained for 20 minutes with 3% Giemsa's solution. The slides were washed with water and dried.
(11) The slides were mounted with a cover glass and the mounting medium.

Measurement of mitotic index and relative mitotic index

The number of metaphase cells was counted by examining 500 cells per tube (i.e., 1OOO cells per concentration). The mitotic index (%) was calculated for each concentration according to the following equation.
Mitotic index = (Number of metaphase cells)/(Number of cells examined) x 100
From the mitotic index, the relative mitotic index (%) at the concentration was calculated according to the following equation.
Relative mitotic index = (Mitotic index in cells treated with the test substance)
/(Mitotic index in cells treated with the negative control) x 100
Because of cytotoxicity, mitotic index could not be measured at 500 µg/mL or more in the -S9 mix assay and 24-hour assay and 2000 µg/mL in the +S9 mix assay.

Calculation of 50% cell growth inhibition concentration
The IC50 were calculated in all treatment conditions. The IC5o value was calculated from a liner equation derived from the two data points showing relative mitotic indices higher and lower than 50%, but closest to 50%.

Chromosomal aberration test
Treatment conditions
-S9 mix assay, +S9 mix assay, 24-hour assay

Test substance concentrations
In the cell growth inhibition test, the IC50 values were 40.8 µg/mL, 151.3 µg/mL and 26.2
µg/mL in the -S9 mix assay, the +S9 mix assay and the 24-hour assay, respectively. Based on these results, the concentrations described below were set for the chromosomal aberration test.
-S9 mix assay: 20, 25, 30, 35, 40, 45, 50, 55 and 60 µg/mL
+S9 mix assay: 60, 80,100, 120, 140, 160, 180 and 200 µg/mL
24-hour assay: 5, 10, 15, 20, 25, 30, 35 and 40 µg/mL

Cell treatment

Cells were treated as described above for the cell growth inhibition test.
Specimens were prepared as described above for the cell growth inhibition test.
Measurement of mitotic index and relative mitotic index was conducted as described above for the cell growth inhibition test.

Observation
Selection of concentrations for metaphase analysis
The lowest concentrations at which the relative mitotic indices were less than 50% were 45 µg/mL, 120 µg/mL and 25 µg/mL in the -S9 mix, +S9 mix and 24-hour assay, respectively. Based on the results of the measurement of relative mitotic index, the specimens of the following concentrations were used for the metaphase analysis in the test substance treatment group.

-S9 mix assay: 35, 40 and 45 µg/mL
+S9 mix assay: 80, 100 and 120 µg/mL
24-hour assay: 15, 20 and 25 µg/mL

Evaluation criteria:

Criteria for selection of observable metaphase cells
(1) Chromosomes should be well spread.
(2) Structural aberrant cell: Number of centromeres should be 46±2
Numerical aberrant cell: Number of centromeres should be 46±2 or 69 or more.

7.8.8.3.3 Number of cells analyzed
150 cells per tube (300 cells per concentration)

Structural aberration [ 1 1
(1) Chromatid break
(2) Chromatid exchange
(3) Chromosome break

Structural aberration [ 1 1
(1) Chromatid break
(2) Chromatid exchange
(3) Chromosome break
(4) Chromosome exchange (dicentric, ring etc.)
(5) Fragmentation
A "gap" was defined as an achromatic region in a single chromatid that was narrower than the width of the chromatid.
The number of gaps was listed in a separate column from the other aberrations. Gaps were not included in the structural aberrations.

Numerical aberration
(1) Polyploid cells with 69 or more centromeres
(2) Endoreduplicated cells

Statistics:

Statistical analysis was performed to detect the differences in the incidences of aberrant cells between the negative control and the test substance groups, and between the negative control and the positive control groups. As at least one of the observation frequency (number of normal cells or number of aberrant cells) was 4 or less in each analysis, statistical analysis was performed by Fisher's exact probability test using the computer program package "Package software EXSUS, ver. 7.7.1". Statistically significant differences were evaluated at a 2-tailed significance level of 5%. When no significant difference from the negative control value was detected, the response was judged as negative.
Since a significant difference from the negative control value was detected in the above statistical analysis in the test substance treatment group, the dose-response relationship was examined by Cochran-Armitage test in the -S9 mix assay and 24-hour assay.

Key result

Species / strain:

mammalian cell line, other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Cell growth inhibition test

Precipitation of the test substance was observed in the treatment mixture at the beginning and the end of the treatment at 1OOOµg/mL or more in all treatment conditions. The IC5o values were 40.8 µg/mL, 151.3 µg/mL and 26.2 µg/mL in the-S9 mix assay, the +S9 mix assay and the 24-hour assay, respectively.

Chromosomal aberration test

Precipitation of the test substance was not observed in the treatment mixture at the beginning and the end of the treatment at any concentration in any treatment conditions. The lowest concentrations at which the. relative mitotic indices were less than 50% were 45 µg/mL, 120 µg/mL and 25 µg/mL in the -S9 mix, +S9 mix and 24-hour assay,respectively.

Based on the results of the relative mitotic index, the concentrations for the metaphase analysis were selected at 35, 40 and 45 µg/mLinthe -S9mixassay, 80, 100 and 120

µg/mLinthe +S9mixassay, and 15, 20 and 25 µg/mL in the 24-hour assay.Inthe preliminary observation, all selected specimens were acceptable to perform metaphase analysis. In addition, the specimens of the positive control group in each treatment condition were confirmed to perform properly. The concentrations of the positive control groupforthemetaphaseanalysiswereselectedat1.2µg/mLinthe-S9mixassay,6µg/mLinthe+S9mixassayand0.5µg/mLinthe24-hourassay.

As a result, significant increase of the incidence of chromosomal structural aberrant cells compared to the negative control group was observed in all test substance treatment group in the -S9 mix assay and 24-hour assay, and dose-dependent increase was also detected by Cochran-Armitage test in the-S9 mix assay and 24-hour assay.

In the negative control group, the incidences of both structural aberrant cells and numerical aberrant cells were less than 5% in the respective treatment condition. In the positive control group, the incidence of chromosome aberrant cells was more than 5% and increased significantly compared to the negative control group in the respective treatment conditions.

Conclusions:

An in vitro chromosomal aberration study with human lymphocytes was conducted to assess the clastogenicity ofEPICLON EXA-7250.
As a result, significant increase of the incidence of chromosomal structural aberrant cells compared to the negative control group was observed in all test substance treatment group in the -S9 mix assay and 24-hour assay, and dose-dependent increase was also observed in the-S9 mix assay and 24-hour assay.
The incidences of cells with chromosomal aberration were as expected in the negative control and positive control groups. These results indicated that this study was conducted properly.
In conclusion, EPICLON EXA-7250 was considered to have the potential to induce chromosomal aberration in human lymphocytes under the conditions employed in this study.

Executive summary:

Anin vitrochromosomal aberration study of EPICLON EXA-7250 was conducted using human lymphocytes from peripheral blood as indicator cells.

 

The cell growth inhibition test was conducted at 15.6, 31.3, 62.5, 125,250, 500, 1000 and 2000 µg/mL in the absence ofS9mix(-S9mix assay) and in the presence ofS9mix(+S9mix assay) in the short-term treatment assay and in the continuous treatment assay for 24 hours (24-hour assay).

Asaresult ofthecellgrowthinhibitiontest,the50%cellgrowth inhibitionconcentration(IC5o)values were 40.8 µg/mL in the-S9mix assay, 151.3 µg/mL in the+S9mix assay,and26.2µg/mLinthe24-hourassay.

 

Based on the results of the cell growth inhibition test, chromosomal aberration test was conducted at 20, 25, 30, 35, 40, 45, 50, 55 and 60 µg/mL in the-S9    mix assay, 60, 80,100, 120, 140, 160, 180 and 200 µg/mL in the+S9mix assay, and 5, 10, 15, 20, 25, 30, 35 and 40 µg/mL in the 24-hour assay.

Based on the results of the relative mitotic index and the preliminary observation, the concentrations for the metaphase analysis were selected at 35, 40 and 45 µg/mL in the-S9mix assay, 80, 100 and 120 µg/mL in the+S9mix assay, and 15, 20 and 25 µg/mL in the 24-hour assay.

As a result, significant increase of the incidence of chromosomal structural aberrant cells compared to the negative control group was observed in all test substance treatment group in the-S9mix assay and 24-hour assay, and dose-dependent increase was also observed in the-S9mix assay and 24-hour assay.

 

In conclusion, EPICLON EXA-7250 was considered to have the potential to induce chromosomal aberration in human lymphocytes under the conditions employed in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 December 2015-6 February 2016

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:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 7 weeks old
- Weight at study initiation: 246.7 to 277.5 g
- Assigned to test groups randomly: Five animals were allocated to each group by the stratified-by-weight randomization method based on the body weights measured on the day of the assignment, so that the mean body weight of each group became almost homogeneous.
- Housing:Polycarbonate cages
- Diet (e.g. ad libitum): Pellet diet for experimental animals. The diet was given to animals ad libitum and replaced at the animal assignment.
- Water (e.g. ad libitum):The drinking water was supplied to animals in the watering bottles ad libitum and replaced together with the watering bottles at the animal assignment.
- Acclimation period:Quarantine period was set for 5 days after the animal receipt. The animals were also acclimated during the quarantine period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7°C to 22.6°C
- Humidity (%): 51.4% to 58.6%
- Air changes (per hr): 6 to 20 times/h
- Photoperiod (hrs dark / hrs light): 12 h/day, 7:00 to 19:00

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 20 v/v% Cremophor aqueous solution
- Lot/batch no. (if required): BCBM8568V

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test article was weighed at 36.0 g into a mixer cup.
The cup containing the weighed test article was warmed at approximately 70°C (65°C to 75°C) and an appropriate volume of Cremophor was added to the cup. Then, the test article and the Cremophor were mixed with a conditioning mixer (AR-250, Thinky) for 5 min.
An appropriate volume of Cremophor and water for injection were added to the cup and mixed with the mixer for 2 min.
The resultant mixture was transferred into a measuring glass washing with Cremophor and/or water for injection.
Cremophor and/or water for injection were further added to the mixture prepared in the (6) procedures to make the suspension at the final volume of 180 mL including at 20 v/v% of Cremophor (36 mL in total) and 200 mg/mL of the test article.
The 200 mg/mL-formulation was stirred with a magnetic stirrer to make it homogenous.
A part of the 200 mg/mL-formulation was diluted with the vehicle (20% Cremophor) to prepare a 100 mg/mL-formulation, and the 100 mg/mL-formulation was diluted with the vehicle to prepare a 50 mg/mL-formulation.
Samples at approximately 5-mL each were collected from the top, middle and bottom layers of each test article formulation and given to the analysis of concentration and homogeneity described in Section 7.3.4.
Each remaining formulation was divided into three PP containers for each day of dosing and used within the period for which stability of the test article formulations was certified, actually with 7 days after the preparation.
The test article formulations were stirred gently so as to avoid foaming. A magnetic stirrer was used for stirring when the formulations were sampled and divided into the containers.

DIET PREPARATION
A 3-mL disposable syringe attached with a gastric tube for rats was used for administration to all the treatment groups by oral gavage.
The test article formulations were collected into a syringe while being stirred with a magnetic stirrer.
The administration procedure is generally used for oral dosing to rats and enables to administer correctly to the animals.

Duration of treatment / exposure:

3 days

Frequency of treatment:

Three times; 48, 24 and 3 h before sampling of the target organs (liver, stomach and bone marrow)
Double dosing at 48 and 24 h before the specimen preparation adequately induces micronuclei one-time sampling of the erythrocytes in the bone marrow [4]. It was confirmed that a combination study of the comet-micronucleus assay using three-time dosing was validated in the International Validation Study of the comet assay with additional dosing at 3 h before the specimen preparation [1]. This dosing regimen is also accepted in the applied guideline.

Post exposure period:

The animals were observed for their clinical signs before each administration and at approximately 1 h after the dosing.

Dose / conc.:

0 mg/kg bw/day (nominal)

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:

5 animals per dose

Positive control(s):

ethylmethanesulphonate;
- Justification for choice of positive control(s): This chemical was used as the positive control in the International Validation Study of the comet assay, in which the combination study of the comet-micronucleus assay was validated its usefulness [1]. In addition, this chemical is recommended in the applied guidelines of the comet and micronucleus assays. There are sufficient laboratory historical data for the liver and stomach comet assay and bone marrow micronucleus assay.
- Route of administration: Oral gavage
- Doses / concentrations: The positive control, ethyl methanesulfonate, was treated at 200 mg/kg/day

Tissues and cell types examined:

Stomach is the first organ to be contacted by a test article when it is administered by oral route.Liver is the main metabolism organ.The stomach and liver were selected in the present study to evaluate the comet assay.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

In the toxicity study entitled "A14-Day Repeated Dose Oral Toxicity Study of EPICLON EXA-7250 in Rats (Preliminary Study)" (Study No.: B140906) conducted at the test facility, no mortality was observed and there were no notable changes in clinical signs or body weights at 1000mg/kg/day.Based on these results, the high dose level used in the present study was selected at 2000 mg/kg/day defined as the highest limit dose in the applied guidelines. The lower doses were set at 1000 and 500 mg/kg/day with a common ratio of 2. In addition to the test article, the vehicle alone was administered to the negative control group and EMS known to have potentials to induce DNA damages and micronuclei was treated to the positive control group at 200mg/kg/day.


DETAILS OF SLIDE PREPARATION:
(1) Each of the treated rats was deeply anesthetized with an intraperinoneal injection of sodium thiopental (Ravonal®, Lot No. X002, Mitsubishi Tanabe Pharma Corporation). The abdominal cavity was opened, and then the abdominal aorta was cut for exsanguination.
(2) The liver and stomach were removed from the animal, and the samples of the liver and glandular stomach for the comet assay were prepared.
(3) A sheet corresponding between the animal and coding numbers was created to code each sample with writing the coding number.
(4) A 40-µL of the sample was mixed with a 360-µL of 0.5 w/v% low melting agarose gel, and a 40-µL of the mixture was placed onto each well of comet slides (CometSlide™ HT, 20 Well Slide, Trevigen Inc.).
(5) Each sample was allocated to three slides.


METHOD OF ANALYSIS: All the treated animals were subjected to microscopic observation or image analysis.

Evaluation criteria:

Comet assay
If the comet parameter (mean and/or median% tail DNA) in the test article group(s) was statistically higher than that in the negative control group with dose-dependency, the study director would evaluate whether cytotoxic effects were related to the higher value of the comet parameter based on the incidences of hedgehogs and histopathological examination if it did. The comet assay would be judged positive if the test article induced a statistically and biologically significant increase in the comet parameter(s) caused by non-cytotoxic effects.

Micronucleus assay
If the significant increase of MNIMEs was observed with a dose-dependent response, the micronucleus assay would be judged positive.

If the statistical significance was detected in the test article group for both the comet and micronucleus assays, biological relevance would be assessed comprehensively on the basis of the laboratory historical negative control data and the distribution of individual values in the corresponding group and/or distribution of each data of the % tail DNA in each animal.

Statistics:

% tail DNA- The EXSUS software (Version 7.7.1, CAC EXICARE Corporation) was used for the statistical analysis for the% tail DNA. Dunnett's test and Student t-test were applied at two-tailed significance levels of 0.05 and 0.01.
The mean and median % tail DNA of each slide were calculated for each organ, and the individual means of the mean and median % tail DNA of the two slides were calculated. The group means of the mean and median% tail DNA were calculated from these individual means.
Dunnett's test was performed to compare the mean values in each of the test article groups with those of the negative control group. Liner regression analysis was not applied to confirm dose-dependent responses since there were no statistical significances in the mean or median% tail DNA by Dunnett's test.
To analyze the meari and median % tail DNA between the negative and positive control groups, Student's t-test was performed.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No deaths occurred in this study. Loose stool was observed in one animal 1 h after the third dosing at 2000 mg/kg/day of the test article. This animal also showed soiled perineal region at that time. Soiled perineal region was noted in other three animals in this group and one animal in the 1000 mg/kg/day group before and 1 h after the third dosing. In these groups, no abnormal clinical signs were found at other observation time points. In the other groups, there were no abnormal clinical signs throughout the experimental period.

Body weghts
The mean body weights in the 2000 mg/kg/day group and positive control group were significantly lower than that in the negative control group before the third dosing. There were no statistically significant differences in the mean body weights in other test article groups compared to the negative control group.

Gross necropsy
No abnormal findings were macroscopically observed in either the liver or stomach of any animal.

Liver comet assay
The group means of the mean % tail DNA were 1.56 ± 0.12%, 1.56 ± 0.27%, 1.55 ± 0.18% and 1.60 ± 0.25% at O (negative control), 500, 1000 and 2000 mg/kg/day of the test article, respectively. The group means of the median % tail DNA were 0.28 ± 0.07%, 0.32 ± 0.12%, 0.22 ± 0.06% and 0.30 ± 0.15% in the same order to the mean% tail DNA, respectively. There were no statistically significant differences in either of the mean or median% tail DNA between the test article groups and the negative control group. In contrast, the positive control group showed significantly higher values in the mean and median % tail DNA (20.01 ± 0.77% and 19.45 ± 0.98%, respectively) than those in the negative control group.
The frequencies (%) of hedgehogs were lower than 1% in all the animals.

Glandular stomach comet assay
The group means of the mean % tail DNA were 6.67 ± 0.78%, 6.35 ± 0.57%, 5.83 ± 0.35% and 6.52 ± 0.37% at O (negative control), 500, 1000 and 2000 mg/kg/day of the test article, respectively. The group means of the median % tail DNA were 2.99 ± 0.55%, 2.71 ± 0.22%, 2.70 ± 0.40% and 2.94 ± 0.35% in the same order to the mean% tail DNA, respectively. There were no statistically significant differences in either of the mean or median% tail DNA between the test article groups and the negative control group. In contrast, the positive control group showed significantly higher values in the mean and median % tail DNA (30.51 ± 0.86% and 29.69 ± 1.16%, respectively) than those in the negative control group.
The frequencies (%) of hedgehogs were 2.00% or less in all the rats except for two animals in the positive control group.

Bone marrow micronucleus assay
The group means of the MNIMEs-incidences were 0.150 ± 0.018%, 0.145 ± 0.041%,
0.135 ± 0.029% and 0.140 ± 0.029% at O (negative control), 500, 1000 and 2000 mg/kg/day of the test article, respectively. No statistically significant increases in the incidences of MNIMEs were detected in the test article groups compared to the negative control group. The mean ratios of IMEs were 64.2 ± 1.7%, 65.l ± 0.6%, 64.0 ± 2.0% and 63.9 ± 1.4% in the same order to the MNIMEs-incidences, respectively. No statistically significant differences were observed in the test article groups compared to the negative control group. In the positive control group, the incidence of MNIMEs was 1.390 ± 0.358% (total number: 278 MNIMEs) showing a statistically significant increase compared to the negative control value. The ratio of IMEs in the positive control group (48.6. ± 2.5%) was significantly lower than that in the negative control group.

Conclusions:

A combination study of the comet-micronucleus assay was conducted in male rats to assess the in vivo genotoxic potential of the test article, EPICLON EXA-7250. The comet assay was performed to investigate the ability to induce DNA damages in the liver and glandular stomach cells. Simultaneously, the micronucleus assay was done to examine the chromosomal aberration inducibility in the erythrocytes among the bone marrow cells.

The laboratory historical control data of the comet assay in the liver and glandular stomach are shown in Appendix 1 and Appendix 2, and that of the micronucleus assay are shown in Appendix 3. The individual comet parameters (mean and median% tail DNA) and MNIMEs-incidences of all animals in the concurrent negative control group ranged within the laboratory historical control data (mean ± 2SD). Those in the concurrent positive control group were higher than those of the minimum historical positive control data, and the mean values in the concurrent positive control group were statistically significantly higher than those in the concurrent negative control group in both the comet and micronucleus assays. These results obtained from the concurrent control groups indicated that the present study was conducted under the appropriate conditions and supported the validity of the study. Additionally, in both the liver and glandular stomach comet assays, the individual frequencies of the hedgehogs in the test article group showed low values and were comparable to those in the negative control group. Based on the applied guideline (OECD TG489), the frequency of hedgehogs is one of the parameters of tissue damages or cytotoxicity, although the etiology of the hedgehogs is uncertain. There were no evidences of tissue damage or cytotoxicity in either of the livers or glandular stomachs, or in the bone marrow as well in this study. Under the circumstances, the comet parameters and incidences of MNIMEs in the test article groups were comparable to those in the negative control group without statistical significances showing negative responses in the comet assay in the liver and glandular stomach and in the bone marrow micronucleus assay.

Based on the results of this study, EPICLON EXA-7250 was judged negative in both the comet and micronucleus assays, and the test article was concluded to have neither in vivo genotoxic potential to induce DNA damage nor in vivo genotoxic ability to produce chromosomal aberrations under the conditions employed in this study.

Executive summary:

A combination study of the comet-micronucleus assay was conducted in male Crl:CD(SD) rats (7 weeks old at dosing) to assess thein vivagenotoxic potential of the test article, EPICLON EXA-7250. The comet assay was performed to investigate the ability to induce DNA damages in the liver and glandular stomach cells. Simultaneously, the micronucleus assay was done to examine the chromosomal aberration inducibility in the erythrocytes among the bone marrowcells.

 

The test article was suspended in the vehicle (20 v/v% Cremophor aqueous solution) and administered orally by gavage to five animals per group at dose levels ofO(vehicle alone; negative control), 500, 1OOOand 2000 mg/kg/day for three consecutive days; 48,

24 and 3 h before the specimen preparation. The positive control, ethyl methanesulfonate, was treated at 200 mg/kg/day in the same manner with the testarticle.

 

Neither mortality nor severe toxic signs were observed in the treated animals, whereas the mean body weight at 2000 mg/kg/day was significantly lower than the negative control group before the  final  dosing.  Before  and  after  the  final  dosing  at 2000 mg/kg/day, soiled perineal region was found in three and four animals, respectively. This finding was also noted in one animal at 1000 mg/kg/day at those times.. In addition, loose stool was observed in one of four rats showing soiled perineal  region  at 2000 mg/kg/day. Consequently, all the treated animals were used for the specimen preparation to evaluate the comet parameters and micronucleus induction. In the comet assay, 150 nuclei (cells) of the liver and glandular stomach per animal were analyzed to calculate the comet parameters; mean and median % tail DNA. In the bone marrow micronucleus assay, 4000 immature erythrocytes (IMEs) were counted to calculate the incidence of micronucleated IMEs (MNIMEs) in eachrat.

 

As a result, there were no significant differences in the comet parameters or micronucleus induction between the negative control group and the test article groups. The control groups satisfied the acceptance criteria of both the comet and micronucleus assays.

 

Based on the results of this study, the test article was judged negative in both the comet and micronucleus assays, and EPICLON EXA-7250 was concluded to have neither genotoxic potential to induce DNA damagesin viva nor genotoxic ability to produce

·   chromosomal aberrationsin vivaunder the conditions employed in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)