(1S,4R,4aS,9aR)-4,4a,9,9a-tetrahydro-1H-1,4-methanofluorene

Endpoint summary

Description of key information

The no observed effect level (NOEL) of MTF was judged to be 4 mg/kg for males and 20 mg/kg for females.  Treatment-related effects in the liver and kidneys of male rats receiving 20 mg/kg/day and in the liver of female rats receiving 100 mg/kg/day and above were observed.

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:

14 November 1996 to 26 December 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to Guidelines for chemical substance control law in Japan and in compliance with GLP

Qualifier:

according to guideline

Guideline:

other: Guidelines for the Chemical Substances Control Law, Japan (1986)

Deviations:

no

GLP compliance:

yes

Remarks:

Compliance to "GLP for Chemical Substance Control Law Guidelines, Japan (1984, 1988) claimed.

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 weeks
- Weight at study initiation: Males = 162-193 g; females = 128-160 g
- Housing: 2 per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 55%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

IN-LIFE DATES: From: 14 November 1996 To: 12 December 1996

Route of administration:

oral: gavage

Vehicle:

other: 0.1% Tween 80 solution

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance in the dosing suspensions at 0.4 and 100 mg/ml were confirmed to be stable for 8 days. Homogeneity of the test substance in
dosing suspensions was confirmed on the day of preparation.
The dosing suspensions obtained on the first preparation were analysed and the concentration of the test substance confirmed.

Duration of treatment / exposure:

Administration was carried out by oral gavage.

Frequency of treatment:

The test substance was administered once daily for 28 days in the morning with a disposable syringe connected to a gastric tube.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

6 males and 6 females per dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Preliminary 2-week oral dose-finding study at 0, 100, 500 and 1000 mg/kg in 3 males and 3 females. One female recieving 1000 mg/kg was sacrificed on Day 2 due to significant deterioaration in clinical signs, 2 further females died on Day 9. Scheduled necropsy on Day 15 indicated enlarged liver and increased liver weight in males and females of the 100 mg/kg group and above. Therefore dose levels set at 4, 20, 100 and 500 mg/kg
- Post-exposure recovery period in satellite groups: 14-day recovery period for satellite groups in the control, 100 and 500 mg/kg groups.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily during dosing and once daily during other periods

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
Gross weight of each feeder was measured once weekly and the mean daily food consumption for each animal calculated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Thiopental sodium anaesthesia
- Animals fasted: No
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- Animals fasted: No
- How many animals:all animals

URINALYSIS: Yes
- Time schedule for collection of urine: Day 27
- Metabolism cages used for collection of urine: No
- Animals fasted: No

Sacrifice and pathology:

GROSS PATHOLOGY: Organs were weighed and the following relative organ weights were calculated: Brain, liver, kidneys, adrenals, thymus, spleen, testes or ovaries.
HISTOPATHOLOGY:Heart, liver, spleen, kidneys, adrenals and other organs and tissues judged to be necessary for the histopathological examination according to the results of the examinations.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation noted in males and females from the 100 and 500 mg/kg groups during the administration period. However this was a transient change often occuring immediately after administration and not at termination of dosing. Not considered to have any toxicological significance and excluded from evaluation of NOEL.

Mortality:

mortality observed, treatment-related

Description (incidence):

Salivation noted in males and females from the 100 and 500 mg/kg groups during the administration period. However this was a transient change often occuring immediately after administration and not at termination of dosing. Not considered to have any toxicological significance and excluded from evaluation of NOEL.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

body weight of each treatment group was comparable to that of control groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of each treatment group was comparable to that of the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

at the end of the adminstraton period, shortened prothrombion time was noted in males receiving 100 and 500 mg/kg/day, and decreased haemoglobin was noted in females receiving 500 mg/kg/day. At the end of the recovery period, these changes were not noted

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

increased total choloesterol, increased total protein, increased gamma GT, increased albumin, decreased ALP and increased calcium. At end of recovery period also increased cholesterol and total protein in high dose groups, increased albumin.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased liver weights in 100 and 500 mg/kg/day groups. Increased relative liver weights

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Changes noted in liver and kidney

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Key result

Dose descriptor:

LOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Key result

Dose descriptor:

LOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: High total cholesterol in the blood, increased relative liver weight, and centrilobular hypertrophy of hepatocytes and increased mitotic figures.

Key result

Critical effects observed:

no

Conclusions:

MTF was administered repeatedly by oral gavage at doses of 0, 4, 20, I 00, and 500 mg/kg for 28 days to male and female SD rats to examine the potential toxic effects of MTF and their reversibility.
In the clinical observations, salivation was noted in males and females of the I 00 and 500 mg/kg groups at postdose during the administration period. However, this was a transient change that often occurs right after administration, and doesn't occur by termination of dosing. Therefore, this change was judged to have resulted from stimulation such as taste of the test substance and was not considered to have any toxicological significance. The data were excluded from the evaluation of the no observed effect level (NOEL).

In the hematological examination, shortened prothrombin time was noted in males of the I 00 and 500 mg/kg groups, and low hemoglobin concentration was noted in females of the 500 mg/kg group at the end of the dosing period. However, since these were slight changes within the physiological variations, and no abnormalities were observed in hematological parameters associated with these changes, it was considered that they had little toxicological significance. At the end of the recovery period, the following changes were noted: decreased reticulocyte count in males of the 500 mg/kg group, increased red blood cell count, hemoglobin concentration, and hematocrit value in females of the I 00 mg/kg group, and increased platelet count and prolongation of prothrombin time in females of the 500 mg/kg group. However, these were slight changes within the physiological variations, and no such changes were noted at the end of the administration period. Moreover, since any changes noted in erytlu·oid parameters in females of the I 00 mg/kg group were not observed in the 500 mg/kg group, it was judged that the changes were incidental and unrelated to the test substance.

In the blood chemistry examination, the following changes were noted: high total cholesterol level in females of the I 00 mg/kg group and males and females of the 500 mg/kg group; high total protein, albumin, and/or yGT levels in males of the I 00 mg/kg group and males and females of the 500 mg/kg group. Since hypertrophic changes in the liver were observed in males and females of both groups, the above-mentioned changes in lipid and protein parameters were considered to be the results of increased lipid or protein synthesis. At the end of the recovery period, high total cholesterol, total protein, and/or albumin levels were also observed in males of the 100 mg/kg group and males and females of the 500 mg/kg group. These changes in females of the 500 mg/kg group were accompanied by low A/G ratio.

A high calcium level was noted in males of the 100 mg/kg group and males and females of the 500 mg/kg group. Since histopathological changes in the kidneys were noted in males of both groups, it was suggested that they may have been associated with a variation in calcium. Moreover, low ALP level was noted in females of the 500 mg/kg group; however,.no changes in calcium or ALP were noted at the end of the recovery period.

In the histopathological examination, enlargement of the liver accompanied by increased absolute and relative liver weights was observed in males and females of the I 00 and 500 mg/kg groups. In the histopathological examination, centrilobular hepatocyte hypertrophy in the liver was noted in males of the 20 mg/kg group and males and females of the I 00 mg/kg group and above. Centrilobular hepatocyte hypertrophy in the liver is considered to be adaptive phenomenon in viva which can be seen in general when a drug-metabolizing enzyme is induced. Since the hypertrophy of hepatocytes observed in this study was centrilobular, and cytoplasm was eosinophilic, it was considered that these changes may have been the result of drug-metabolizing enzyme. Moreover, cytoplasmic vacuolation and intracytoplasmic eosinophilic inclusion bodies in hepatocytes observed at the same time were considered to be changes as a result of increased agranular endoplasmic reticulum associated with induction of drug-metabolizing enzyme. Furthermore, the single cell necrosis in hepatocytes was considered to be due to an overload of the induction of drug-metabolizing enzyme. Since centrilobular hepatocyte hypertrophy in the liver were reduced by termination of dosing, and the other changes described above disappeared, the changes observed in the liver were considered to be reversible.

Enlargement of the kidneys accompanied by increased absolute and relative liver weights was observed in males of the I 00 and 500 mg/kg groups. In the histopathological examination, enhancement of hyaline droplets in the proximal tubular epithelium was observed in males of the 20 mg/kg group and above. Hyaline droplets in the proximal tubular epithelium is the result of reabsorption of proteins including a20 globulin, which is observed spontaneously in adult male rats.3 It is known that this change is enhanced by administration of various drugs and chemicals. It is considered that this change may be caused by increased reabsorption in the proximal renal tubule as a result of increased a2u globulin synthesis in the liver and by decreased catabolism as a result of binding of a20 globulin to drugs or their metabolites phagocytized in lysosome in epithelial cells of the uriniferous tubule. Moreover, it is known that chronic progressive nephropathy is enhanced by degeneration and regeneration of epithelial cells of the uriniferous tubule as a result of excessive accumulation of hyaline droplets. Furthermore, associated with chronic progressive nephropathy, eosinophilic bodies were observed. Enhancement of basophilic renal tubule and eosinophilic bodies in the tubular epithelium observed in males of the 20 mg/kg group and above in this study was considered to be changes having the meanings described in the above paragraph. At the end of the recovery period, enhancement of basophilic change in the tubular epithelium was observed in males of the I 00 and 500 mg/kg groups; however, hyaline droplets were comparable to the control group in degree and showed a tendency to recover.

At the end of the dosing period, high absolute and relative spleen weights were observed in males of the 500 mg/kg group, and low absolute spleen weight was observed in females of the 500 mg/kg group. At the end of the recovery period, high relative testis weight was observed in the 500 mg/kg group, and high absolute and relative kidney weights were observed in females of the 500 mg/kg group. However, no abnormalities associated with these changes were observed in the histopathological examination.

No changes considered attributed to the test substance were noted in body weight, food consumption, or urinalysis data.
As described above, centrilobular hepatocyte hypertrophy in the liver, increased mitotic figures, cytoplasmic vacuolation in hepatocytes, enhancement of hyaline droplets and eosinophilic bodies in the proximal tubular epithelium in the kidneys were observed in males of the 20 mg/kg group. As for females, high total cholesterol in blood, high relative liver weight, centrilobular hepatocyte hypertrophy in the liver, and increased mitotic figures were observed in the I 00 mg/kg group. Therefore, the no observed effect level (NOEL) of MTF was judged to be 4 mg/kg for males and 20 mg/kg for females under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A 28 -Day Repeated Dose Oral Toxicity study of MTF in Rats was performed by the Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd. (6L351, 2007).

The study was conducted according to 'Guidelines for the Chemical Substances Control Law (1986)' and in compliance with 'GLP for the Chemical Substances Control Law Guidelines (1984, 1988)'.

The aim of this study was to observe changes in biological functions and morphological forms, and to examine the potential toxic effects of MTF and their riversibility by repeated oral administration to rats daily for 28 days.

The no observed effect level (NOEL) of MTF was judged to be 4 mg/kg for males and 20 mg/kg for females. Treatment-related effects in the liver and kidneys of male rats receiving 20 mg/kg/day and in the liver of female rats receiving 100 mg/kg/day and above were observed.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Treatment-related effects were observed in the livers of male rats receiving a daily oral dose of 20 mg/kg/day and above for 28 days. With reference to Annex I, Section 3.9 of the CLP Regulation because significant toxic effects in orgns were seen below 30 mg/kg bodyweight/day in a subacute study it is considered that MTF meets the criteria for classification for Specific Target Organ Toxicity following Repeated Exposure, Category 1. The corresponding hazard phrase is H372: Causes damage to organs through prolonged or repeated exposure.

It is noted that in addition to effects seen in the liver, the male rats treated with MTF also showed signs of effects in the kidneys. Due to the nature of the findings observed and on the basis that corresponding effects were not seen in the kidneys of the female animals treated concurrently, the effects in kidneys were due to alpha-2u globulin nephropathy which is not relevant in human toxicity. For the purposes of classification it is considered that the available data does not indicate that kidneys should be considered a target organ.