(1R)-1-[(4R,4aR,8aS)-2,6-bis(3,4-dimethylphenyl)tetrahydro[1,3]dioxino[5,4-d][1,3]dioxin-4-yl]ethane-1,2-diol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January 1995 to 18 July 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 28-day Repeated Dose Toxicity Study in Mammalian Species of The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances (Notification No. 700 of Kanpogyo, No. 1039 of Yakuhatsu, and No. 1014 of of 61 Kikyoku, Dec. 5, 1986)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

413-110-2

EC Name:

-

Cas Number:

135861-56-2

Molecular formula:

C24H30O6

IUPAC Name:

1-[2,6-bis(3,4-dimethylphenyl)-hexahydro-[1,3]dioxino[5,4-d][1,3]dioxin-4-yl]ethane-1,2-diol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 1,3:2,4-bis-O-(3,4-dimethlbenzlidene)-D-solbitol; (3,4-DMBS)
- Substance type: white powder
- Physical state: solid
- Analytical purity: ≥99.0 w/w %
- Lot/batch No.: 4073003
- Storage condition of test material: a cold and dark place

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 5 week
- Weight at study initiation: males: 144.4- 177.4 g; females: 116.4- 137.7 g
- Housing: animals were housed individually in hanging stainless steel cages with wire-mesh floor
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: animals were quaratined and acclimatised

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 10- 15
- Photoperiod (hrs dark / hrs light): 12/ 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was weighted accurately and mixed with olive oil to make a concentration of 10.0 w/v %. This was prepared once a week. Other three concentrations of 2.0, 0.4 and 0.08 w/v % were prepared every time from 10.0 w/v %.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and uniformity of the test substance in these preparations was confirmed in the laboroatory.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily for 28 day, and the subsequent 14 days were used as a recovery period

Doses / concentrations

Remarks:

Doses / Concentrations:
8, 40, 200, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

6 males and 6 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 14-day repeated-dose preliminary toxicity study was carried out at 3 doses of 50, 250 and 1000 mg/kg. There were abnormalities in organ weights in the 250 mg/kg and higher groups, and blood chemical examinations in the 50 mg/kg and higher groups.
In the main study, the maximum dose was chosen at 1000 mg/kg and 3 lower doses at 200, 40 and 8 mg/kg. Recovery groups were set at 1000 mg/kg and the vehicle control groups.

Positive control:

None.

Examinations

Observations and examinations performed and frequency:

The day of the start of dosing was defined as day 1, and the day before as day -1. The week of the start of dosing period was defined as week 1. Also, the next day of final dosing was defined as recovery day 1, and the week of the start of recovery period as recovery week 1.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once per day

BODY WEIGHT: Yes
- Time schedule for examinations
- Before dosing: day -2 (at the time of grouping)
- During the dosing period: days 1 (at the start of dosing), 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26 and 28
- During the recovery period: days 1 (at the start of the recovery period), 3, 5, 8, 10, 12 and 14
- Immediately before necropsy for calculation of relative organ weights

FOOD CONSUMPTION:
- Time schedule for measurement of food consumption
- Before dosing: Once
- During the dosing and recovery periods: Twice a week

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, overnight (16- 20 h) at the end of the dosing and recovery periods
- How many animals: All
- Blood samples were taken via abdominal aorta from rats under ether anesthesia. Sodium citrate was used as an anticoagulant for examinations of prothrombin time and activated partial thromboplastin time, and EDTA-2K was used for another parameters.
- The following parameters were examined: red blood cell count (RBC), white blood cell count (WBC), haemoglobin conc. (Hb), haematocrit value (Ht), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), platelet count, reticulocytes count, prothrombin time (PT), activated partial thromboplastin time (APTT), differentiation of leukocytes (band form neutrophils (N-Band), segmental neutrophils (N-Seg), eosinophils (Eosino), basophils (Baso), lymphocytes (Lymph), monocytes (Mono)).

CLINICAL CHEMISTRY: Yes
Sera were separated from the blood samples used in hematological examinations and examined as follows.
- Parameters checked: GOT, GPT, alkaline phosphatase (ALP), cholinesterase (ChE), γ-GTP, total cholesterol (T-Cho), triglyceride (TG), glucose, total protein (T-Ptotein), albumin, A/G ratio, blood urea nitrogen (BUN), creatinine, total bilirubin (T-Bil), Ca, IP, Na, K, Cl

URINALYSIS: Yes
- Time schedule for collection of urine: 16-h urine samples were collected from all animals at day 28 and recovery day 14
- Metabolism cages used for collection of urine: Yes
- Urine was examined for: volume, colour, additional items of pH, protein, ketone bodies, bilirubin, occult blood, glucose and urobilinogen

ORGAN WEIGHTS
- The following organs were weighed wet in all animals: brain, liver, spleen, kidneys, adrenal glands, testes (or ovaries)

HISTOPATHOLOGICAL EXAMINATIONS
- The following organs and tissues from all animals were preserved in 10 % formalin: brain (cerebrum, cerebellum), hypophysis, eyeball, thyroid glands (with parathyroid glands), heart, lung, liver, kidneys, spleen, adrenal glands, stomach, intestine (duodenum to rectum), testes (or ovaries), urinary bladder, bone marrow (femur), gross lesions
- Light microscopic examinations were performed on the following organs and tissues after paraffin embedding and sectioning followed by hematoxylin and eosin staining:
- Dosing period
- Vehicle control and 1000 mg/kg groups: liver, spleen, kidneys, heart, stomach, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), adrenal glands
- Gross lesions
- Dosing period, male 8 mg/kg group: glandular stomach; male 40 mg/kg group: cerebrum; male 200 mg/kg group: skin; female 8 mg/kg group: glandular stomach
- Recovery period, male 1000 mg/kg group: glandular stomach

Sacrifice and pathology:

NECOROSPY:
All animals were necropsied in detail to record.

ORGAN WEIGHTS
- The following organs were weighed wet in all animals: brain, liver, spleen, kidneys, adrenal glands, testes (or ovaries)

HISTOPATHOLOGICAL EXAMINATIONS
- The following organs and tissues from all animals were preserved in 10 % formalin:
brain (cerebrum, cerebellum), hypophysis, eyeball, thyroid glands (with parathyroid glands), heart, lung, liver, kidneys, spleen, adrenal glands, stomach, intestine (duodenum to rectum), testes (or ovaries), urinary bladder, bone marrow (femur), gross lesions

- Light microscopic examinations were performed on the following organs and tissues after paraffin embedding and sectioning followed by hematoxylin and eosin staining:
Dosing period:
- Vehicle control and 1000 mg/kg groups: liver, spleen, kidneys, heart, stomach, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), adrenal glands
Gross lesions:
- Dosing period, male 8 mg/kg group: glandular stomach; male 40 mg/kg group: cerebrum; male 200 mg/kg group: skin; female 8 mg/kg group: glandular stomach
- Recovery period, male 1000 mg/kg group: glandular stomach

Statistics:

Data regarding body weights, food consumption, hematological examination, blood chemical examination, urine volume and organ weights were analyzed using Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5 %, one way analysis of variance was performed. When there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment group was analyzed by Dunnett's test (equal number of data) or Scheffe's test (unequal number of data).

If the variances were not homogeneous in the Bartlett's test, Kruskal-Wallis's test was used. When there was a significant difference in this test, the difference between the vehicle control group and each of the treatment group was analyzed by nonparametric Dunnett's test (equal number of data) or nonparametric Scheffe's test (unequal number of data).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically significant effects

Mortality:

no mortality observed

Description (incidence):

No toxicologically significant effects

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically significant effects

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects

Urinalysis findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased relative liver weight in males in the 1000 mg/kg group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No toxicologically significant effects

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths.

During the dosing period:
- Male: Salivation immediately after dosing was noted in the vehicle control group (3/12, days 12, 24 and 25) and the 1000 mg/kg group (2/12, days 25 and 28). Scab formation (2/6), loss of hair (2/6) and exudate (1/6) were also noted in the 200 mg/kg group.
- Female: Salivation was noted in the vehicle control (5/12, between day 13 and 27) , the 40 mg/kg group (3/6, between day 12 and 26) and the 1000 mg/kg group (1/12, days 27 and 28). These conditions appeared immediately after dosing.

During the recovery period;
- Male: Dark reddish change of right eyeball (1/6) was noted in the vehicle control group.
- Female: Loss of hair (1/6) was noted in the 1000 mg/kg group.

BODY WEIGHT AND WEIGHT GAIN
During the dosing period
- No abnormalities were noted in either sex of any dosing group.

During the recovery period
- Male: No abnormalities were noted in any dosing group.
- Female: A significant increase of body weight gain on day 3 was noted in the 1000 mg/kg group.

FOOD CONSUMPTION
During the dosing period
- No abnormalities were noted in either sex of any dosing group.

During the recovery period
- Male: No abnormalities were noted in any dosing group.
- Female: A significant increase of food consumption gain on day 4 was noted in the 1000 mg/kg group.

HAEMATOLOGY
- The blood sample of 1 female was not used in a statistical analysis because of a part of coagulation.

At the end of the dosing period
- No abnormalities were noted in either sex of any dosing group.

At the end of the recovery period
- Male: No abnormalities were noted in any dosing group.
- Female: A decrease in mean corpuscular haemoglobin concentration was noted in the 1000 mg/kg group

CLINICAL CHEMISTRY
At the end of the dosing period
- Male: No abnormalities were noted in any dosing group.
- Female: An increase in K was noted in the 40 and 200 mg/kg groups

At the end of the recovery period
- Male: No abnormalities were noted in any dosing group.
- Female: Decreases in Cholinesterase, albumin and A/G ratio and increases in γ-GTP and creatinine were noted in the 1000 mg/kg group.

URINALYSIS
- There were no change considered to be due to the test substance during the dosing period and at the end of the recovery period.

ORGAN WEIGHTS
At the end of the dosing period
- Male: An increase of absolute liver weight was noted in the 40 mg/kg or higher groups and an increase of relative liver weight was noted in the 1000 mg/kg group.
- Female: No abnormalities were noted in any dosing group.

At the end of the recovery period
- Male: No abnormalities were noted in any dosing group.
- Female: An increase of absolute liver weight and decreases of relative kidney and brain weights were noted in the 1000 mg/kg group.

GROSS PATHOLOGY (NECROSPY)
At the end of the dosing period
- Male: Blackish spots of mucosa in the glandular stomach (1/6) in the 8 mg/kg group, dilatation of ventricules in the cerebrum (1/6) in the 40 mg/kg group and scab formation (2/6) in the 200 mg/kg group were noted.
- Female: Blackish spot of mucosa in the glandular stomach (1/6) was noted in the 8 mg/kg group.

At the end of the recovery period
- Male: Blackish spot of mucosa in the glandular stomach (1/6) was noted in the 1000 mg/kg group.
- Female: No abnormalities were noted in any dosing group.

HISTOPATHOLOGY
At the end of the dosing period
- Male: Cyst formation in the kidney in the vehicle control group (2/6) and the 1000 mg/kg group (1/6), necrosis of mucosa in the glandular stomach in the 8 mg/kg group, dilatation of ventricles in the cerebrum (1/1) in the 40 mg/kg group and necrosis and scab formation in the skin (2/2) in the 200 mg/kg group.
- Female: Cyst formation in the kidney in the vehicle control group (2/6) and the 1000 mg/kg group (1/6) and necrosis of mucosa in the glandular stomach in the 8 mg/kg group were noted.

At the end of the recovery period
- Necrosis of mucosa in the glandular stomach was noted in males of the 1000 mg/kg group. Females were not examined.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increased relative liver weight in males of the 1000 mg/kg group

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increase in relative liver weight in males at 1000 mg/kg is not considered to be a serious adverse effect as there was no associated organ dysfunction and no elevated liver weight in 1000 mg/kg males at the end of the recovery period.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The No Observed Effect Level (NOEL) of the test material for rats was determined to be 200 mg/kg/day under the conditions tested.

Executive summary:

A 28-day repeated-dose oral toxicity study of the test material followed by a 14-day recovery test was conducted in male and female Crj: CD (SD) rats (6/sex/ group), 5 weeks of age at the start of dosing. The highest dose was set at 1000 mg/kg, and 3 lower doses at 200, 40 and 8 mg/kg. Recovery groups were separately provided for vehicle control and 1000 mg/kg groups.

There were no deaths on account of administration of the test material.

No abnormalities were noted in general conditions, body weights, food consumption, haematological examinations, blood chemical examinations, urinalysis, necropsy and histopathological examinations.

In organ weights, increased absolute and relative liver weights were noted in males of the 1000 mg/kg group.

In the recovery test, no abnormalities were noted.

In conclusion, No Observed Effect Level of the test material for rats was considered to be 200 mg/kg/day under the conditions tested.

The No Observed Adverse Effect Level was considered to be 1000 mg/kg/day.