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

Endpoint summary

Description of key information

In conclusion, no toxicologically significant changes were observed at 11,000 ppm; therefore, no-observed-adverse-effect-level (NOAEL) of EPICLON EXA-7250 was estimated to be 11,000 ppm, equivalent to 1000 mg/kg/day, under the conditions of this study.  

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 November 2015 - 18 March 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

This strain is widely used in toxicity studies using rodents, and there is plenty of historical data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Laboratories Japan, Inc. (Atsugi)
- Age at study initiation: 5 weeks old
- Weight at study initiation:
Males: 163.2 to 186.6 g
Females: 117.4 to 140.7 g
The body weights of all animals on the initial day of administration were confirmed to be within the mean ± 20% for each sex.
- Fasting period before study:
- Housing:Polycarbonate cages (maximum [outside] dimensions: 257W × 387D × 197H mm, minimum [inside] dimension: 220W × 380D × 183H mm, Tokiwa Kagaku Kikai Co., Ltd.) Sterilization before use: Autoclave
- Diet (e.g. ad libitum):Basal diet (powder diet for experimental animals, CR-LPF, radiation sterilized, Oriental Yeast Co., Ltd., Lot No. 150929). The diet was replaced once on the day of grouping and once a week after the initiation of administration.
- Water (e.g. ad libitum):Tap water passed through a 5 µm filter and disinfected by UV irradiation
- Acclimation period:8 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20.8°C to 22.6°C
- Humidity (%):47.6% to 61.3%
- Air changes (per hr):6 to 20 times per hour with all fresh air The ventilation frequency is measured periodically (twice/year) and the results were confirmed to meet the specification of the standard operation procedure (SOP) of the test facility.
- Photoperiod (hrs dark / hrs light):12 hours per day (7:00 to 19:00)

IN-LIFE DATES: From: 18 November 2015 To: 8 January 2016

Route of administration:

oral: feed

Details on route of administration:

The test substance was administered orally by feeding the test substance mixed diet.

Vehicle:

not specified

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
The diet preparation was prepared once 6 days before the initiation of administration in the following procedures.
(1) The container containing the test substance was put in a plastic bag and warmed up in warm water at about 40°C (permissible range: 35°C to 45°C) to melt and take out the test substance.
(2) The prescribed amounts of the test substance and the basal diet were weighed accurately.
(3) The test substance was put in a container and heated up in hot water at about 70°C (permissible range: 65°C to 75°C), and then the basal diet of a 10 times amount of the test substance was gradually added while mixing the test substance and diet.
(4) Appropriate amount of the basal diet was further added and the mixture was mixed and ground with a compact mill (Sample Mill, SK-M10R and SK-M10, Kyoritsu Riko Co., Ltd.).
(5) The basal diet was further added and mixed with a tabletop universal mixer (Kenmix: KM-230, Aicohsha Manufacturing. Co., Ltd.) for about 10 minutes.
(6) Finally, remaining basal diet was added and mixed for about 30 minutes with a V type mixer (FM-V-130, Powrex Co., Ltd.) to make the diet preparation containing 1,200, 3,600, or 11,000 ppm of the test substance.
(7) The diet preparation was divided into polyethylene bags and used for administration within 34 days after preparation (expiration date: 5 weeks [35 days] after preparation).
Storage conditons: Cold place (actual temperature: 4.3°C to 5.4°C, permissible range: 1°C to 10°C), well-closed bag

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

After the storage, analytical samples were collected from the top, middle, and bottom layers of each diet preparation and analyzed by the high performance liquid chromatograph (HPLC). The concentrations and homogeneity of the test substance in the diet preparations were confirmed at the test facility.

Duration of treatment / exposure:

28 Days

Frequency of treatment:

Dosing period: Days 1, 8, 15, 22, and 28

Dose / conc.:

1 200 ppm

Dose / conc.:

3 600 ppm

Dose / conc.:

11 000 ppm

No. of animals per sex per dose:

5 males and 5 females at each dose level and concentration.

Control animals:

yes

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): The animals were assigned to test groups by the stratified-by-weight randomization method based on body weights measured on the day of grouping so that they were evenly assigned with respect to the mean body weight. Upon assignment, 1 male and 1 female with the lowest body weight and 1 male and 1 female with the highest body weight were rejected. On the initial day of administration, a decrease in body weight was noted in 1 male (No. 10203) assigned to the low dose group; therefore, this animal was excluded from the study and one of males rejected at assignment, a male (No. 10206) closest to the body weight of the excluded animal, was included in the low dose group. Thirty males and 30 females were used in this study.

Animals not used for this study were excluded from the study on the initial day of administration, and were euthanized by severing the abdominal aorta and exsanguination under anesthesia with intraperitoneal injection of sodium thiopental (Ravonal®, Mitsubishi Tanabe Pharma Corporation) on the initial day of administration.

- Post-exposure recovery period in satellite groups: Five males and 5 females each in the control and high dose groups were subjected to the recovery study for 14 days after the end of the dosing period.

Observations and examinations performed and frequency:

All animals were observed every day for clinical signs from the initiation of administration to necropsy. Frequencies of the observation are listed below. Dosing period: Twice a day (in the morning and afternoon)
Other days: Once a day

BODY WEIGHT: All animals were weighed on the days shown below with an electronic balance (PB3002-S, Mettler-Toledo International Inc.).
- Time schedule for examinations:
Dosing period: Days 1, 8, 15, 22, and 28
Recovery period: Days 29, 36, and 42
In addition, the final body weights were measured on the day of necropsy (Days 29 and 43) to calculate the relative organ weight (organ weight to body weight ratios).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The gross weight of the diet (including feeder) was measured with an electronic balance (PB3002-S, Mettler-Toledo International Inc.), and the food consumption during the measurement periods shown below was calculated.
Dosing period: Days 1 to 8, 8 to 15, 15 to 22, and 22 to 25
Recovery period: Days 29 to 36 and 36 to 39
The mean daily food consumption was calculated based on the difference in the gross weight between the measurement days.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

HAEMATOLOGY:
(1) Day of examination At the scheduled necropsy (Days 29 and 43)
(2) Sample collection Blood was sampled and processed as follows to collect the examination samples. Fasting before blood sampling: Overnight (about 20 to 23 hours, from about 17:00 on the day before necropsy to necropsy)
Anesthesia at blood sampling: Sodium thiopental (Ravonal®, Mitsubishi Tanabe Pharma Corporation, intraperitoneal injection)
Blood sampling site: Posterior vena cava
Sampling volume: About 4 to 5 mL (including sample for blood chemistry test)
Anticoagulation with EDTA: Blood sample (about 0.8 mL) was anticoagulated with EDTA-2K. Plasma collection: Blood sample (0.6 mL) was anticoagulated with 3.2 w/v% trisodium citrate solution, and then plasma sample was obtained by centrifugation (12000×g for 3 min at 4°C).
(3) Examination items and methods Examination items and methods are listed in the following table. PT and APTT were examined using plasma samples, and the other items were examined using blood samples anticoagulated with EDTA-2K.
(4) Handling of remaining samples Remaining blood and plasma samples were discarded after examination.

CLINICAL CHEMISTRY:
Day of Examination: At the scheduled necropsy (Days 29 and 43) (2)
Sample collection Blood sample: A portion of the blood samples obtained at the hematology test Serum sampling: Blood samples (about 2 mL) were left to stand at room temperature for about 30 to 60 minutes and serum samples were obtained by centrifugation (1500×g for 10 min at 4°C). The serum samples were stored at about −80°C (actual temperature: −70.7°C to −78.3°C, permissible range: −60°C or below), when examination could not be performed on the day of sampling.

Storage of serum samples for hormone measurement:
A portion of serum samples for blood chemistry was stored in a freezer at −80°C (permissible range: −60°C or below) for the measurement of serum thyroid stimulating hormone (TSH), triiodothyronine (T3) and thyroxine (T4). However, the serum samples were discarded because no effects on the pituitary or thyroid were suspected based on the results of various examinations in this study.

URINALYSIS:
Urinalysis was conducted on 5 males and 5 females, in the ascending order of animal number, in each group. Since no treatment-related changes were observed, further examinations were not conducted.
Dosing period: Week 4 (Day 25)

NEUROBEHAVIOURAL EXAMINATION:
Detailed clinical observations, function tests, and motor activity measurement were conducted on all animals. Schedule of the examinations is shown below. Examinations were carried out in the afternoon.

Detailed clinical observations: Once before the initiation of administration, once a week during the dosing and recovery periods Function tests and motor activity measurement: Once in the final week (Week 4) of the dosing period The function tests and motor activity measurement in the recovery period were not conducted, because no treatment-related changes were noted in the results of these examinations in the dosing period. At the detailed clinical observations and function tests in the dosing and/or recovery period, the label listing original animal number and dose level was removed and a label listing temporary animal number during the examination was attached to the cages for blind test. A corresponding table of original and temporary animal numbers was prepared.

Detailed clinical observations
(1) Hand-held observations Reactivity on removal from the cage, reactivity to handling, aggression, skin (trauma, color of skin), fur (soiled fur), eyes (exophthalmos, palpebral closure), conjunctiva (color of conjunctiva), secretion, lacrimation, salivation, piloerection, pupil size Criteria for each examination item are shown in Section 12.3.

(2) Open field observations (observation period: 2 minutes per animal) Rearing, arousal, urination, defecation, posture/body position, breathing, coordination movement, gait, tremor, clonic convulsion, tonic convulsion, stereotypy, bizarre behavior.

(1) Sensory reactivity to stimuli The following items were examined under open field conditions: approach response, touch response, auditory response, tail pinch response, aerial righting reaction.

(2) Grip strength measurement The grip strength of the forelimb and hindlimb was measured twice per animal using a digital force gauge (Imada Co., Ltd.), and a higher value was adopted. This examination was performed after the completion of sensory reactivity to stimuli.

Motor activity measurement Animals transferred to clean polycarbonate cages shortly before the measurement. Motor activity was recorded for 1 hour with a motor activity-measuring device (SUPERMEX, Muromachi Kikai Co., Ltd.). Data were calculated every 10 minutes from the start of measurement, while a total of 1 hour was calculated. During the measurement, the diet and drinking water were not given to the animals.


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

(1) Organs/tissues collection and preservation The organs/tissues were removed. The organs/tissues were fixed and preserved in 10 vol% phosphate-buffered formalin; however, the testes were fixed in Bouin’s solution and the eyeballs/optic nerve/Harderian glands were fixed in Davidson’s solution and preserved in 10 vol% phosphate-buffered formalin.

(2) Preparation of specimens and microscopic examination The organs/tissues of all males and females in the control and 11,000 ppm groups collected at the end of the dosing period were processed by the usual method to prepare hematoxylin and eosin-stained sections and examined by microscopy. In addition, the cervical skin, at which crust was observed at necropsy, of 2 males (No. 10107 and 10108) in the control group was histopathologically examined in the same manner. Since histopathological change in the mesenteric lymph node, which was suspected to relate to treatment, was noted in the 11,000 ppm group, the mesenteric lymph node of all group collected at the end of the dosing and recovery periods was examined.

All animals were euthanized by exsanguination from the abdominal aorta after blood sampling for hematology and blood chemistry tests, and then they were subjected to the necropsy.

Other examinations:

The organs/tissues shown in Section 7.10.10.1 collected from animals subjected to the scheduled necropsy were weighed using an electronic balance (AW120, Shimadzu Corporation). Bilateral organs were weighed together. The thyroid/parathyroid was weighed after fixation in 10 vol% phosphate-buffered formalin. The relative organ weight (organ weight to body weight ratios) was calculated using the body weight on the day of necropsy.

Statistics:

Safety Study Support System (Provantis, Instem LSS Ltd.) was used for the statistical analyses.

Homogeneity of the variance among the groups was first tested by Bartlett’s test (significance level: 5%), and the mean values of the control group and the treatment groups were compared by Dunnett test (significance level: 1 and 5%, two-sided test) when the variance was homogeneous. When the variance was heterogeneous, Bartlett’s test was applied again using the log-transformed values, and Dunnett test was performed using the log-transformed values when the variance of the log-transformed values was homogeneous. When the variance of the log-transformed values was heterogeneous, Steel multiple comparison test (significance level: 1 and 5%, two-sided test) was performed using the rank-transformed values.

Numerical data of 2 groups during the recovery period was tested by the F test (significance level: 5%) for homogeneity of variance, and Student t-test (if the variance was homogeneous) or Aspin-Welch t-test (if the variance was heterogeneous) was performed (significance level: 1 and 5%, two-sided test).

Categorical data: Categorical data of urinalysis was analyzed by Steel multiple comparison test (significance level: 1 and 5%, two-sided test) was performed using the rank-transformed values. Categorical data of histopathology was analyzed by Fisher exact test (significance level: 1 and 5%, two-sided test).

The statistical analysis was conducted for data listed below.
Numerical data:
• Detailed clinical observations data (number of rearing)
• Function tests data (grip strengths of the forelimb and hindlimb)
• Motor activity
• Body weight
• Food consumption
• Hematology data
• Blood chemistry data
• Organ weight (absolute and relative weights)
Categorical data:
• Urinalysis data (paper test)
• Histopathology data

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormality in clinical sign was observed in males or females in the treatment groups during the dosing or recovery period.

Nonspecific change, erosion of the skin at the neck (cervical area), was observed in 2 males (No. 10107 and 10108) in the control group from the dosing period (Day 14), and it turned into crust by the end of the recovery period.

Mortality:

no mortality observed

Description (incidence):

No death occurred in males or females during the dosing or recovery period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No abnormality in body weight was noted in males or females during the dosing or recovery period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A lower food consumption was noted in females in the 11,000 ppm group from Day 1 to 8; however, this was not considered to be treatment related as it was a slight and transient change observed only shortly after the initiation of the administration. Nor was lower food consumption noted in females in the 3,600 and 11,000 ppm groups from Day 22 to 25 considered to be treatment related because the mean value was higher than that of the control group in the before (Day 15 to 22) and after (Day 29 to 36) periods and the statistical significance was ascribable to a higher value in the control group from Day 22 to 25. Higher food consumption noted in males in the 11,000 ppm group from Day 36 to 39 in the recovery period was not considered to be treatment related because a similar tendency was not observed in the dosing period and the mean value was within the range of the control group during the dosing period

Food efficiency:

not specified

Description (incidence and severity):

The mean test substance intakes during the dosing period at 1,200, 3,600, and 11,000 ppm were 106.07, 312.76, and 975.20 mg/kg/day for males and 113.67, 340.88, and 1026.13 mg/kg/day for females, respectively.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the dosing period, a higher MCHC was noted in males and females at 11,000 ppm; however, this was not considered to be treatment related as it was a change within the normal range of the historical control data in the test facility (see Section 12.4, (3)) and no abnormality was observed in other erythrocyte parameters at this dose level. Lower MCV and MCH in males at 1,200 ppm, longer APTT in females at 1,200 ppm, and higher neutrophil count in females at 3600 ppm were also not considered to be treatment related because they were changes within the normal range of the historical control data and no dose dependent-change was observed in these parameters. At the end of the recovery period, a higher neutrophil count was noted in females at 11,000 ppm; however, this was not considered to be treatment related as it was a change within the normal range of the historical control data and the statistical difference was ascribable to a lower value in the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the dosing period, a lower total bile acid concentration was noted in females at all dose levels; however, this was not considered to be treatment related as it was a change within the normal range of the historical control data in the test facility and not accompanied with other changes in blood chemistry parameters suggesting decreased intestinal absorption involved in reduction of bile acid reabsorption. At the end of the recovery period, a higher K and a lower Cl concentrations in males and a higher ASAT concentration in females were noted at 11,000 ppm; however, these were not considered to be treatment related because they were changes within the normal range of the historical control data and no similar tendencies were observed at the end of the dosing period.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no significant difference in urinalysis parameters in males or females between the control and any of the treatment groups.

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

no effects observed

Description (incidence and severity):

(1) Sensory reactivity to stimuli Sensory reactivity to stimuli was comparable among the groups in males and females, and no abnormality was observed in any animal.
(2) Grip strength There was no significant difference in grip strength of males or females between the control and any of the treatment groups in males or females.
There was no significant difference in motor activity of males or females between the control and any of the treatment group.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

At the end of the dosing period, minimal blood absorption in the mesenteric lymph node was observed in 2 males at 11,000 ppm.
At the end of the recovery period, no treatment-related change was observed in males or females.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic crust at the cervical area in 2 males (No. 10107 and 10108) in the control group, which observed at necropsy at the end of the recovery period, was evaluated as crust in the histopathological examination. Other various histopathological changes in the control and 11,000 mg/kg groups observed at the end of the dosing period were judged not to be treatment related because they are nonspecific changes sporadically observed in rats and there was no apparent difference in the incidence between the control and 1000 mg/kg groups.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the dosing period, distention of the cecum was observed in 3 males at 11,000 ppm. At the end of the recovery, no treatment-related change was observed in males or females. Besides, crust of the skin at the cervical area was observed in 2 males (No. 10107 and 10108) in the control group at the end of recovery period as it was observed in the clinical observation

Details on results:

At the end of the recovery period, no test substance-related abnormalities were observed in any examination at 11,000 ppm.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

urinalysis

Key result

Critical effects observed:

no

Conclusions:

EPICLON EXA-7250 was administered orally (dietary administration) at dose levels (test substance concentration in the diet) of 0 (control group), 1,200, 3,600, 11,000 ppm to male and female Crl:CD(SD) rats for 28 days to assess the toxicological effects of EPICLON EXA-7250 and its reversibility. In this study, the mean test substance intakes at 1,200, 3,600, 11,000 ppm were 106.07, 312.76, and 975.20 mg/kg/day for males and 113.67, 340.88, and 1026.13 mg/kg/day for females, respectively.

At the end of the dosing period, the following changes were observed at 11,000 ppm. At necropsy, distention of the cecum was observed in males; however, this was judged not to be toxicologically significant as there were no histopathological changes in the cecum or any associated change such as change in feces, and complete reversibility was seen after the 14-day recovery period. Cecal distention/enlargement in rodents caused by oral dose of poorly digestible/absorbable chemical substances due to change in osmotic activity is widely known [1, 2]; therefore, the cecal distention in this study that attributable to change in cecal osmotic changes was judged not to be toxic change. In the histopathological examination, blood absorption in the mesenteric lymph node was observed in males; however, this was also judged not to be toxicologically significant as it was a minimal change and there were no associated histopathological changes. The absorbed blood was considered to originate from tissues drained by the lymph node [3]; however, no histopathological change suggesting hemorrhage was observed in the abdominal organs or tissues at necropsy or in the histopathological examination.
At the end of the recovery period, no test substance-related abnormalities were observed in any examination at 11,000 ppm.

In conclusion, no toxicologically significant changes were observed at 11,000 ppm; therefore, no-observed-adverse-effect-level (NOAEL) of EPICLON EXA-7250 was estimated to be 11,000 ppm, equivalent to 1000 mg/kg/day, under the conditions of this study.

Executive summary:

EPICLON EXA-7250 was administered orally (dietary administration) to male and female Crl:CD(SD) rats for 28 days to assess the toxicological effects of EPICLON EXA-7250 and its reversibility by observing functional and morphological changes. The dose levels (test substance concentration in the diet) were set at 1,200, 3,600, and 11,000 ppm, and animals in the control group were given the basal diet. Each group consisted of 5 males and 5 females. A 14-day recovery period (5 animals/sex/group) was set for the control and 11,000 ppm groups. In this study, the mean test substance intakes at 1,200, 3,600, and 11,000 ppm were 106.07, 312.76, and 975.20 mg/kg/day for males and 113.67, 340.88, and 1026.13 mg/kg/day for females, respectively.  

No test substance-related abnormalities were observed in the clinical observation, detailed clinical observations, function tests, motor activity measurement, body weight measurement, food consumption measurement, urinalysis, hematology test, blood chemistry test, or organ weight measurement.

 

At the end of the dosing period, distention of the cecum was observed at necropsy in 3 males at 11,000 ppm; however, this was judged not to be toxicologically significant because of the absence of histopathological change in the cecum or any associated change, and its complete reversibility. In the histopathological examination, blood absorption in the mesenteric lymph nodes was observed in 2 males at 11,000 ppm; however, this was also judged not to be toxicologically significant as it was a minimal change and there was no a hemorrhagic change in the abdominal organs.  

At the end of the recovery period, no test substance-related abnormalities were observed in any examination item at 11,000 ppm.  

In conclusion, no toxicologically significant changes were observed at 11,000 ppm; therefore, no-observed-adverse-effect-level (NOAEL) of EPICLON EXA-7250 was estimated to be 11,000 ppm, equivalent to 1000 mg/kg/day, under the conditions of this study.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat