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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

2-({2-[bis({2-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-({2-[bis({4-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-{[2-({2-[(oxiran-2-yl)methoxy]phenyl}({4-[(oxiran-2-yl)methoxy]phenyl})methyl)phenoxy]methyl}oxirane

EC Number:

942-639-6

IUPAC Name:

2-({2-[bis({2-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-({2-[bis({4-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-{[2-({2-[(oxiran-2-yl)methoxy]phenyl}({4-[(oxiran-2-yl)methoxy]phenyl})methyl)phenoxy]methyl}oxirane

Test material form:

solid

Details on test material:

Name: EPICLON EXA-7250
Batch/Lot number: B006
Appearance: Brown solid (in smaller amount colour is yellow)
Purity: >99%
Manufacture date: 02 October 2014
Expiry date: 02 March 2017
Storage condition: Room temperature 15-25°C, below 70 RH%
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face
mask) for unknown materials were applied to assure personnel
health and safety.

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.