Amines, polyethylenepoly-, triethylenetetramine fraction

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The study was performed similar to OECD408. The study was not performed according to GLP.No information on test substance purity/composition.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The study was performed similar to OECD407. The study was not performed according to GLP. No information on test substance purity/composition.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

5 animals per sex used

Principles of method if other than guideline:

Also 8 weeks of treatment were observed.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Triethylenetetramine dihydrochloride, TJA-250, N,N'-bis(2-aminoethyl)- 1,2-ethanediamine dihydrochloride (trientine-2HCl) is a white to pale yellow crystalline powder easily soluble in water.

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Kent, England
- Age at study initiation: 33-42 days
- Weight at study initiation: no data
- Fasting period before study: not applicable
- Housing: The animals were housed four or five of one sex per cage in stainless steel cages (54 x 38 x 20 cm)
- Diet (e.g. ad libitum): ad libitum, expanded rodent diet, RM1(E) SQC
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5-25.5
- Humidity (%): 42-66
- Air changes (per hr): 14
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Trientine-2HCl was dissolved in reverse osmosis water, all formulations were prepared freshly each day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Trientine-2HCl was shown to be stable in reverse osmosis water for at least 96 hr at both ambient temperature and at 4°C. No further details.

Duration of treatment / exposure:

4 and 8 weeks

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: range finding study
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: no
- Section schedule rationale (if not random): no data

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected at least twice daily for evidence of treatment-related signs or ill-health during the first week of treatment, twice weekly during weeks 2 to 4 of treatment and once weekly thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a more detailed weekly examination, which included palpation, was performed on each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was measured on the day that treatment commenced and at twice weekly intervals throughout the treatment period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each cage, that remaining, and an estimate of the amount spilled were recorded for each week throughout the treatment period
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was recorded (over a 3-day period on each occasion), during the first 4 weeks of treatment in the 4- or 8-week study. In addition, during week 8 of treatment in the 4- or 8-week study water consumption measurements were performed for all surviving animals over an approximate 24-hour period, as two separate intervals (after dosing until 5 p.m. [Interval 1] and 5 p.m. until 9 a.m. the following day [Interval2]. The measurements were performed in conjunction with urine collection.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before commencement and after 24 days (4- or 8- week study) of treatment, both eyes were examined by means of an indirect ophthalmoscope.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained from all surviving animals, after overnight starvation, during weeks 4 and 8 of treatment in the 4- or &week study and during week 24 (males) or week 25 (females) of treatment and during week 12 of the reversibility period in the 26-week study.
- Anaesthetic used for blood collection: Yes, halothane/nitrous oxide anesthesia
- Animals fasted: Yes
- How many animals: all survinving animals
- Parameters examined:
packed cell volume (PCV), hemoglobin (Hb), erythrocyte count (RBC), total leucocyte count (WBC), differential leucocyte count (neutrophils (N), lymphocytes (L), eosinophils, basophils, monocytes and a small proportion of large unstained cells) and platelet count, Prothrombin time (PT), activated partial thromboplastin time (APTT). Mean cell hemoglobin concentration, mean cell hemoglobin and mean cell volume were calculated.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters examined: alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate amino-transferase (AST), creatinine phosphokinase (CPK), lactate dehydrogenase (LDH), urea (Urea), creatinine (Crea), glucose (Gluc), total bilirubin (TBil), triglyceride (Trig), total cholesterol (Chol), total protein (TP), sodium (Na), potassium (K), chloride (Cl), calcium (Ca) and inorganic phosphorus (P)

URINALYSIS: Yes
- Time schedule for collection of urine:
> In the 4- or 8 week study overnight during week 4 of treatment (approximately 5 p.m. until 9 a.m. the following morning) urine samples were collected from all animals in an individual metabolism cage without food or water.
> During week 8 of treatment in the 4- or 8 week study urine was collected from all surviving animals over approximately 24 hr, as two separate intervals (after dosing until 5 p.m. [interval 1] and 5 p.m. until 9 am. the following day [interval 2]).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
> The individual samples were examined in respect to appearance and volume, pH by a pH meter, specific gravity (SG) using an Atago refractometer, protein by precipitation with sulphosalicylic acid, total reduction substances by Clinitest, glucose, ketones, bilirubin, urobilinogen, nitrite and blood by Multistix, the sediment from centrifugation with microscopy, sodium (Na), potassium (K) and chloride (Cl) using the Technicon Axon System, Toshiba Ltd., Japan.
> The urine collections were performed in conjunction with water consumption measurements. The Interval 1 sample was analyzed in respect to volume, sodium, potassium and chloride concentrations in the 4- or 8 week study. The Interval 2 sample was examined in respect to all of those parameters described above.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
> In the 4- or 8-week study, during weeks 3 and 4 of treatment over 12 consecutive days, the duration and regularity of the estrus cycle of all females was assessed by vaginal smear.

Sacrifice and pathology:

GROSS PATHOLOGY:
On completion of the treatment/reversibility period, all surviving animals were killed for detailed necropsy by exsanguination under halothanelnitrous oxide anesthesia in the 4- or 8- week study. Animals killed or found dead during the treatment period were also subjected to necropsy.

The following organs were dissected and weighed for each animal: adrenals, brain, epididymides, heart, kidneys, liver, lungs with mainstem bronchi, ovaries, pituitary, prostate, submandibular gland, seminal vesicles, spleen, testes, thymus, thyroid with parathyroids (after fixation) and uterus with cervix.

HISTOPATHOLOGY:
Samples of the following tissues and any grossly abnormal tissues were preserved in 4% neutral buffered formaldehyde, except eyes, optic nerve and Harderian glands, which were placed in Davidson's fluid and sub sequently retained in 70% methylated spirit. In the 26- week study, testes and epididymides were initially preserved in Bouin's fluid; adrenals, brain, caecum, colon, duodenum, epididymides, eye and optic nerve, femoral bone and marrow, Harderian glands, heart, ileum, jejunum, kidneys, lachrymal gland, liver, lungs with mainstem bronchi, lymph nodes (mandibular, mesenteric), mammary gland, esophagus, ovaries, pancreas, pituitary, prostate, rectum, submandibular gland, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, sternum, st~mach, testes, thymus, thyroid with parathyroids, tongue, trachea, urinary bladder, uterus with cervix and vagina. Histopathological examination was performed on the above-mentioned tissues from all animals in the control group and the high dosage group. Examination of the lungs and stomach was extended to animals in the remaining dosage groups. Samples of any abnormal tissues were also retained for histopathological examination.

Statistics:

The significance of inter-group differences in hematology, blood chemistry and urinalysis was assessed by Student's t-test using a pooled error variance. For organ weights and body weight changes, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant, a Behrens-Fisher test was used to perform pairwise comparisons; otherwise a Dunnett's test (Snedecor et al., 1967) was used. Metal analyses were assessed using Dunnett's test. Inter-group differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test(Fisher, 1950).

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

4-8 week studies
CLINICAL SIGNS AND MORTALITY
Signs considered to be related to treatment with trientine-2HCl were confined to a slightly higher incidence of yellow perigenital staining in females receiving 350 or 1200 mg/kg bw/day which was usually apparent from the first week of treatment. During the latter half of the treatment period, a hunched posture and thin build was evident in four and three males receiving 1200 mg/kg/day, respectively. During week 8 of treatment.
In animals receiving 1200 mg/kg bw/day, one male was killed in extremis and another male was found dead. Previous signs in these animals consisted of thin build, underactive behavior, reduced body temperature, piloerection, pallor, pale eyes, a hunched posture and brown staining around the muzzle. Shallow and irregular respiration was also observed prior to death in the animal that was killed. Increased lung weights and dark lung were recorded at necropsy, and bronchiolar epithelium hypertrophy and bronchio-alveolar pneumonia and increased mucus-secreting neck cells in the stomach were shown in both animals dying before the end of the treatment period.

BODY WEIGHT AND WEIGHT GAIN and FOOD/WATER CONSUMPTION
During the first four weeks of the treatment period the body weight gain and the food consumption of treated animals was similar to those of the control animals. During weeks 5 to 8 of treatment the body weight gain and the food consumption of males receiving 1200 mg/kg/day was decreased. Water consumption was slightly increased for males receiving 1200 mg/kg/day during weeks 1 to 4 of treatment.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopy examination during week 4 of treatment did not reveal any findings considered to be related to treatment with trientine-2HCl.

HAEMATOLOGY
In hematological examinations, small, statistically significant increases were seen on both occasions of examination in respect to packed cell volume, hemoglobin concentration and erythrocyte count in males treated at 1200 mg/kg bw/day when compared with contemporary controls. A number of other statistically significant intergroup differences were seen, such as increased activated partial thromboplastin times and increased lymphocyte and platelet numbers. These probably represent chance variations but could reflect minor effects of treatment.

CLINICAL CHEMISTRY
Blood chemistry investigations revealed some differences statistically significant from the control values which were consistently seen and therefore attributed to treatment; these consisted of low alkaline phosphatase activity in animals treated at 350 or 1200 mg/kg bw/day and high alanine amino-transferase activity in rats treated at 1200 mglkglday. Other changes that were clearly attributable to treatment were confined to animals receiving the high dosage. These changes consisted of high triglyceride and urea concentrations and low glucose concentrations, high cholesterol and low
total protein concentrations confined to males and females, respectively.

URINALYSIS
Urinalysis during week 4 of treatment revealed low urinary pH and a slightly high specific gravity in animals receiving 350 or 1200 mg/kg bw/day; and increased sodium and chloride and decreased potassium output in animals receiving 1200 mg/kg bw/day. Results of urinalysis performed during week 8 of treatment were similar to those during week 4 of treatment except that during Interval 1 there was a markedly decreased urinary output in animals receiving 1200 mg/kg bw/day and an increased potassium output in treated animals receiving 1200 mg/kg bw/day. There were no other changes in clinical examinations which could be associated with treatment.

ORGAN WEIGHTS
Slightly increased relative kidney weights in animals receiving 1200 mg/kg bw/day were noted after 4 and/or 8 weeks of treatment. Slightly increased relative andor absolute lung weights were noted in animals receiving 1200 mg/kg bw/day after 8 weeks of treatment. Other changes of organ weight were also confined to animals receiving the high dosage. These changes consisted of slightly increased relative liver weights for females after 4 weeks of treatment, and slightly decreased absolute and relative thymus weight and slightly increased relative adrenal weights for males after 8 weeks of treatment. There were no other changes in organ weights which could be associated with treatment.

GROSS PATHOLOGY
Macropathological findings that were attributable to treatment were confined to animals receiving 1200 mg/kg bw/day. These findings consisted of areas of change in the lung of one female killed after 8 weeks of treatment, dark lung in one male that died during the treatment period and a thin appearance in one male killed after 8 weeks of treatment and in one male that died during the treatment period.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological changes considered to be related to treatment were found mainly in the lungs and stomach. A high incidence of bronchiolar epithelium hypertrophy and broncho-alveolar pneumonia were found in the lungs of males and females receiving 1200 mg/kg bw/day, with a higher incidence in the males. In addition, there was a slightly higher than expected incidence of accumulation of alveolar macrophages in males receiving 1200 mg/kg kg/day. There were no similar changes observed in the lungs from animals receiving 100 or 350 mg/kg bw/day. In the stomach, submucosal acute inflammation within the glandular region was recorded for males that received 350 and 1200 mg/kg bw/day and in all treated female groups. For animals receiving 100 and 350 mg/kg bw/day, this finding was found only in animals killed after 4 weeks of treatment, with the exception of one male killed after 8 weeks that received 350 mg/kg bw/day. There was also an increased incidence of mucus-secreting neck cells of animals receiving 1200 mg/kg bw/day killed after 4 or 8 weeks of treatment. It is also considered to be treatment-related that cortical atrophy of the thymus was found in one male killed after 8 weeks of treatment and in the two males that died during the treatment period, and that lymphocytolysis of the mandibular lymph nodes was found in the two males that died during the treatment period.

OTHER FINDINGS
There was no evidence of any dosage- or treatment-related effects on estrous cycle.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: irreversible toxic changes in the lung

Critical effects observed:

yes

Lowest effective dose / conc.:

1 200 mg/kg bw/day (actual dose received)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Oral administration of trientine-2HCl to rats was associated with death and irreversible toxic changes in the lung in the highest dose group. A dosage of 350 mg/kg bw/day was considered to be the NOAEL for females and for males.

Executive summary:

Triethylenetetramine dihydrochloride (trientine-2HCl, TJA-250), a copper chelating agent used to treat Wilson's disease, was administered orally to male and female F-344 rats for 4 or 8 weeks at dosages of 0, 100, 350 or 1200 mg/kg/day or for 26 weeks at dosages of 50, 175 or 600 mg/kg/day. 4 or 8-week study. Two males receiving 1200 mg/kg/day died during week 8 of treatment. In males receiving 1200 mg/kg/day during weeks 5 to 8 of treatment, body weight gain and food consumption were decreased and hunched posture and thin build were observed. During week 4 or 8 of treatment urinalysis revealed, for males receiving 100 mg/kg/day or animals receiving 350 mg/kg/day or more, increased electrolyte outputs possibly due to the hydrochloride nature of trientine-2HCl, with low plasma alkaline phosphatase activities evident in animals receiving 350 or 1200 mg/kg/day. After 4 and 8 weeks, and during 8 weeks of treatment, high lung weights and bronchiolar epithelium hypertrophy and broncho-alveolar pneumonia were recorded for animals receiving 1200 mg/kg/day, and submucosal acute inflammation within the glandular region of the stomach was recorded for males receiving 350 or 1200 mg/kg/day and in all treated female groups. 26 -week study. One male receiving 175 mg/kg/day and three males receiving 600 mg/kg/day died, showing lung changes. The body weight gain of animals receiving 600 mg/kg/day was slightly decreased. Blood chemistry and urinalysis examinations showed changes similar to those indicated in the 4- or 8-week study. The low plasma copper concentrations seen in males receiving 600 mg/kg/day, the slightly low liver copper concentrations found in animals receiving 600 or 175 mg/kg/day and the high urinary copper concentrations found in all treated groups, are attributed to the pharmacological action of trientine-2HCl. Histopathology revealed a dosage-related incidence and severity of focal chronic interstitial pneumonitis accompanied by fibrosis of the alveolar walls in females receiving 175 mg/kg/day or more and all treated male groups, but no significant pathological changes in the stomach. Apart from the histological changes found in the lung, all the above changes were reversible. In conclusion, the NOAEL of trientine-2HCl in this 26-week study was considered to be 50 mg/kg/day for females and less than 50 mg/kg/day for males.

Data source

Reference

Reference Type:

publication

Title:

Subacute and chronic toxicity studies of triethylenetetramine dihydrochloride (TJA-250) by oral administration to F-344 rats

Author:

Yanagisawa, T., Maemura, S., Sasaki, H., Endo, T., Okada, M., East, P.W., Virgo, D.M., Creasy, D.M.

Year:

1998

Bibliographic source:

The Journal of Toxicological Sciences, 619 Vol. 23, Supplement IV, 619-642

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

12 animals/sex; 26-weeks tested

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(2-aminoethyl)ethylenediamine dihydrochloride

EC Number:

253-854-3

EC Name:

N,N'-bis(2-aminoethyl)ethylenediamine dihydrochloride

Cas Number:

38260-01-4

Molecular formula:

C6H18N4.2ClH

IUPAC Name:

N,N'-bis(2-aminoethyl)ethane-1,2-diamine dihydrochloride

Test material form:

solid

Specific details on test material used for the study:

Triethylenetetramine dihydrochloride, TJA-250, N,N'-bis(2-aminoethyl)- 1,2-ethanediamine dihydrochloride (trientine-2HCl) is a white to pale yellow crystalline powder easily soluble in water.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Kent, England
- Age at study initiation: 33-42 days
- Weight at study initiation: no data
- Fasting period before study: not applicable
- Housing: The animals were housed four or five of one sex per cage in stainless steel cages (54 X 38 X 20 cm)
- Diet (e.g. ad libitum): ad libitum, expanded rodent diet, RM1(E) SQC
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5-25.5
- Humidity (%): 42-66
- Air changes (per hr): 14
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Trientine-2HCl was dissolved in reverse osmosis water, all formulations were prepared freshly each day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Trientine-2HCl was shown to be stable in reverse osmosis water for at least 96 hr at both ambient temperature and at 4°C. No further details.

Duration of treatment / exposure:

26 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: range finding study
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: yes for control group and high dose group (13 weeks without treatment).
- Section schedule rationale (if not random): no data

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected at least twice daily for evidence of treatment-related signs or ill-health during the first week of treatment, twice weekly during weeks 2 to 4 of treatment and once weekly thereafter. During weeks 14 to 26 and the reversibility period in the 26- week study, animals were inspected once every two weeks.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a more detailed weekly examination, which included palpation, was performed on each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was measured on the day that treatment commenced and at twice weekly intervals throughout the treatment period in the 4- or 8 week study or at weekly intervals throughout the treatment and reversibility periods in the 26-week study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each cage, that remaining, and an estimate of the amount spilled were recorded for each week throughout the treatment period in both studies and the reversibility period in the 26-week study.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was recorded (over a 3-day period on each occasion), during the first 4 weeks of treatment in the 4- or 8-week study, during weeks 1,6, 13 and 25 of treatment and week 13 of the reversibility period in the 26-week study. In addition, during week 8 of treatment in the 4- or 8-week study and during weeks 12 (males), 13 (females) and 25 of treatment and week 13 of the reversibility period in the 26-week study, water consumption measurements were performed for all surviving animals over an approximate 24-hour period, as two separate intervals (after dosing until 5 p.m. [Interval 1] and 5 p.m. until 9 a.m. the following day [Interval2]. The measurements were performed in conjunction with urine collection.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before commencement and after 24 days (4- or 8- week study) or after 25 weeks (26-week study) of treatment, both eyes were examined by means of an indirect ophthalmoscope.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained from all surviving animals, after overnight starvation, during weeks 4 and 8 of treatment in the 4- or &week study and during week 24 (males) or week 25 (females) of treatment and during week 12 of the reversibility period in the 26-week study.
- Anaesthetic used for blood collection: Yes, halothane/nitrous oxide anesthesia
- Animals fasted: Yes
- How many animals: all survinving animals
- Parameters examined:
packed cell volume (PCV), hemoglobin (Hb), erythrocyte count (RBC), total leucocyte count (WBC), differential leucocyte count (neutrophils (N), lymphocytes (L), eosinophils, basophils, monocytes and a small proportion of large unstained cells) and platelet count, Prothrombin time (PT), activated partial thromboplastin time (APTT). Mean cell hemoglobin concentration, mean cell hemoglobin and mean cell volume were calculated.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters examined: alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate amino-transferase (AST), creatinine phosphokinase (CPK), lactate dehydrogenase (LDH), urea (Urea), creatinine (Crea), glucose (Gluc), total bilirubin (TBil), triglyceride (Trig), total cholesterol (Chol), total protein (TP), sodium (Na), potassium (K), chloride (Cl), calcium (Ca) and inorganic phosphorus (P)

URINALYSIS: Yes
- Time schedule for collection of urine:
> In the 4- or 8 week study overnight during week 4 of treatment (approximately 5 p.m. until 9 a.m. the following morning) urine samples were collected from all animals in an individual metabolism cage without food or water.
> During week 8 of treatment in the 4- or 8 week study and during weeks 12 and 25 of treatment and during week 13 of the reversibility period in the 26- week study, urine was collected from all surviving animals over approximately 24 hr, as two separate intervals (after dosing until 5 p.m. [interval 1] and 5 p.m. until 9 am. the following day [interval 2]).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
> The individual samples were examined in respect to appearance and volume, pH by a pH meter, specific gravity (SG) using an Atago refractometer, protein by precipitation with sulphosalicylic acid, total reduction substances by Clinitest, glucose, ketones, bilirubin, urobilinogen, nitrite and blood by Multistix, the sediment from centrifugation with microscopy, sodium (Na), potassium (K) and chloride (Cl) using the Technicon Axon System, Toshiba Ltd., Japan.
> The urine collections were performed in conjunction with water consumption measurements. The Interval 1 sample was analyzed in respect to volume, sodium, potassium and chloride concentrations in the 4- or 8 week study and, additionally, appearance and pH were measured in the 26-week study. The Interval 2 sample was examined in respect to all of those parameters described above.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
> In the 4- or 8-week study, during weeks 3 and 4 of treatment over 12 consecutive days, the duration and regularity of the estrus cycle of all females was assessed by vaginal smear.
> Metal analysis In the 26-week study, concentrations of copper, zinc and iron in plasma, urine, liver and kidney were analyzed. During week 26 of treatment and week 13 of the reversibility period, overnight urine samples were collected from all surviving animals in an individual metabolism cage without food and water (approximately 5 p.m. until 9 a.m. the following morning). Immediately before necropsy, blood samples were obtained from all surviving animals via the retro-orbital sinus and collected into sodium heparin as an anticoagulant. At necropsy, sections of the liver (left and median
lobes) and right kidney (cranial pole) were taken. The plasma, urine, liver and kidney samples were stored deep-frozen (-20°C) until analysis. Five animals in each group were examined at the end of each period. Samples of plasma or urine were oxidized and diluted with nitric acid. Concentrations of copper, zinc and iron in their samples were determined by an atomic absorption spectrophotometer (Perkin Elmer Model 280). Samples of liver or kidney tissues were destroyed and oxidized by heating in nitric acid and perchloric acid and diluted with hydrochloric acid. Measurements of the elements of these diluted samples were made using an inductively coupled plasma emission optical spectrometer (Thermo Electron Plasma 300).

Sacrifice and pathology:

GROSS PATHOLOGY:
On completion of the treatment/reversibility period, all surviving animals were killed for detailed necropsy by exsanguination under halothanelnitrous oxide anesthesia in the 4- or 8- week study and by carbon dioxide inhalation in the 26-week study. Animals killed or found dead during the treatment period were also subjected to necropsy.

The following organs were dissected and weighed for each animal: adrenals, brain, epididymides, heart, kidneys, liver, lungs with mainstem bronchi, ovaries, pituitary, prostate, submandibular gland, seminal vesicles, spleen, testes, thymus, thyroid with parathyroids (after fixation) and uterus with cervix.

HISTOPATHOLOGY:
Samples of the following tissues and any grossly abnormal tissues were preserved in 4% neutral buffered formaldehyde, except eyes, optic nerve and Harderian glands, which were placed in Davidson's fluid and sub sequently retained in 70% methylated spirit. In the 26- week study, testes and epididymides were initially preserved in Bouin's fluid; adrenals, brain, caecum, colon, duodenum, epididymides, eye and optic nerve, femoral bone and marrow, Harderian glands, heart, ileum, jejunum, kidneys, lachrymal gland, liver, lungs with mainstem bronchi, lymph nodes (mandibular, mesenteric), mammary gland, esophagus, ovaries, pancreas, pituitary, prostate, rectum, submandibular gland, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, sternum, st~mach, testes, thymus, thyroid with parathyroids, tongue, trachea, urinary bladder, uterus with cervix and vagina. Histopathological examination was performed on the above-mentioned tissues from all animals in the control group and the high dosage group. Examination of the lungs and stomach was extended to animals in the remaining dosage groups. Samples of any abnormal tissues were also retained for histopathological examination.

Statistics:

The significance of inter-group differences in hematology, blood chemistry and urinalysis was assessed by Student's t-test using a pooled error variance. For organ weights and body weight changes, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant, a Behrens-Fisher test was used to perform pairwise comparisons; otherwise a Dunnett's test (Snedecor et al., 1967) was used. Metal analyses were assessed using Dunnett's test. Inter-group differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test(Fisher, 1950).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no signs that could be unequivocally associated with treatment except in the following animals which were found dead or killed in extremis. Two males receiving 600 mg/kg bw/day were found dead during weeks 13 or 23 of treatment. Two males, one each receiving 175 or 600 mg/kg bw/day, were killed in extremis during weeks 14 or 19 of treatment, respectively. One control male was killed in extremis during week 2 of the reversibility period. Of the animals killed in extremis, signs prior to dispatch included reduced body temperature, shallow or fast respiration, pallor and underactivity. At necropsy, dark lungs were recorded for the two high dosage males that died. All of the treated males that died, or were killed, showed perivascular edema, necrosis and regenerative hyperplasia of the terminal bronchioles, acute interstitial pneumonitis, bronchiolar hyperplasia and bronchiolar epithelium hypertrophy of the lungs. These deaths were considered to be treatment- related. The histopathological findings for the control male that was killed during the reversibility period included monocytic leukaemia, and this was considered to be the cause of death.

BODY WEIGHT AND WEIGHT GAIN and FOOD/WATER CONSUMPTION
The body weight gain of animals receiving 600 mg/kg bw/day was slightly decreased; this was most marked in the males. Body weight gain during the reversibility period was similar between the control group and animals previously receiving 600 mg/kg bw/day.
The overall food intake, during both the treatment and reversibility periods, was similar between the control and treated groups. Water consumption measurements, performed over three days, showed that the water intake of animals receiving 600 mg/kg bw/day was slightly increased during week 1 of treatment (male; 18.2 ±1.07 vs 16.1 ± 0.96 mL/rat/day, female;15.2±0.66 vs 13.5±0.84 mL/rat/day) with males at this dosage similarly affected during week 6 of treatment (18.2±0.78 vs 16.5±0.89 mL/rat/day). Water consumption, when measured during urine collection, was unaffected by treatment.

OPHTHALMOSCOPIC EXAMINATION
There were no ocular changes considered to be related to treatment.

HAEMATOLOGY
Hematological examinations at the end of both the treatment and reversibility periods showed several small, statistically significant changes at the high dosage; however, these changes were minor and the values were within the normal range expected.

CLINICAL CHEMISTRY
Blood chemistry investigations at the end of the treatment period revealed low alkaline phosphatase activities in animals receiving 600 mg/kg bw/day and males receiving 175 mg/kg bw/day. Animals receiving 600 mg/kg bw/day also showed low concentrations of cholesterol, total protein, creatinine (male only) and urea (female only). Low cholesterol concentrations were also noted in males receiving 175 mg/kg bw/day. There was evidence of electrolyte disturbance in all treated groups, although these changes often lacked dosage relationship or were confined to one sex.

URINALYSIS
Results of urinalysis during Interval 1 at the end of the treatment period were similar to those found during the 4- or 8-week study. On both sampling occasions, decreased urinary output and low pH, and increased electrolyte outputs (sodium, potassium and chloride) were evident in animals receiving 600 mg/kg/day. Low pH and increased chloride output were also evident, at both sampling occasions, in animals receiving 175 mg/kg bw/day. During Interval 2 on both sampling occasions, slightly low pH, decreased potassium output and increased chloride output were noted in animals receiving 600 mg/kg bw/day. All these changes seen in the blood chemistry examination and urinalysis at the end of the treatment period were not evident or clearly reduced after the reversibility period. There were no other changes in clinical examinations which could be associated with treatment.

ORGAN WEIGHTS
Relative kidney weights were slightly increased in males receiving 600 mg/kg bw/day. This was still evident at the end of the reversibility period. Slightly decreased absolute and relative spleen weights were noted in males receiving 600 mg/kg bw/day, although in the absence of any change in the females or any histopathological correlate, this was considered to be of no toxicological significance.
All other organ weight changes were either associated with the difference in body weight at necropsy, confined to one sex or lacked dosage relationship and were therefore considered to be fortuitous.

GROSS PATHOLOGY
There was an increased incidence of pale areas of the lungs in animals receiving 600 mg/kg bw/day. This was still evident at the end of the reversibility period.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related histopathological findings were seen in the lungs, with males more affected than females. There was a dosage-related incidence and severity of focal chronic interstitial pneumonitis which was frequently accompanied by fibrosis of the alveolar walls. The lesion was present in the lungs of all animals receiving 600 mg/kg bw/day and in the majority of animals receiving 175 mg/kg bw/day. There were no lung changes in females receiving 50 mg/kg bw/day, but an increased number of the males from this group exhibited minimal pneumonitis, four of them showing evidence of alveolar fibrosis. One control male also showed minimal focal interstitial pneumonitis, but this was not associated with any fibrosis. The interstitial pneumonitis in the treated animals was characterized by focal areas of alveolar collapse with varying degrees of fibrosis. The focal interstitial lesions were generally localized around bronchioles which were frequently increased in number (bronchiolar hyperplasia) and lined by hypertrophic and hyperplastic epithelium.
Other changes included a benign tumor of the adrenal medulla which occurred in a male in the high dosage group. This is a common age-related spontaneous tumor in this strain of rat, and the single occurrence in this study is not considered to be related to treatment.
In addition, a reduced incidence of focal inflammation associated with hepatocyte degeneration in the liver was seen in males from the high dosage group. This lesion is a common background finding in male rats of this age and strain, the reduced incidence in the treated males is not considered to be of toxicological significance.
There were no pathological changes in the stomach considered to be related to treatment.
Pathological changes for animals killed after 13 weeks of reversibility included pulmonary lesions characterized by bronchiolar hyperplasia, focal chronic interstitial peumonitis and alveolar fibrosis observed in all the males and the majority of females previously treated with trientine-2HCl at 600 mg/kg/day. There was no evidence of recovery.

OTHER FINDINGS
Metal analysis
Low plasma copper concentrations were found in males receiving 600 mg/kg bw/day. A high urinary copper concentration was found in all treated groups, with dosage relationship being evident. Slightly low copper concentrations were found in the liver of treated animals receiving 600 or 175 mg/kg bw/day. Females receiving 600 or 175 mg/kg bw/day showed low plasma iron and slightly low plasma zinc concentrations. Low urinary iron and high urinary zinc concentrations were noted in all treated male groups with a dosage relationship being evident. High urinary zinc concentrations were seen in females receiving 600 mg/kg bw/day and high urinary iron concentrations in females receiving 600 or 175 mg/kg bw/day. None of these changes were evident at the end of the reversibility period. There were no changes in metal concentrations in the kidneys considered to be related to treatment.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: irreversible toxic changes in the lung

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: irreversible toxic changes in the lung

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of trientine-2HCl to rats was associated with death and irreversible toxic changes in the lung. A dosage of 50 mg/kg bw/day was considered to be the NOAEL for females and for males the NOAEL was considered to be less than 50 mg/kg/day.

Executive summary:

Triethylenetetramine dihydrochloride (trientine-2HCl, TJA-250), a copper chelating agent used to treat Wilson's disease, was administered orally to male and female F-344 rats for 4 or 8 weeks at dosages of 0, 100, 350 or 1200 mg/kg/day or for 26 weeks at dosages of 50, 175 or 600 mg/kg/day. 4 or 8-week study. Two males receiving 1200 mg/kg/day died during week 8 of treatment. In males receiving 1200 mg/kg/day during weeks 5 to 8 of treatment, body weight gain and food consumption were decreased and hunched posture and thin build were observed. During week 4 or 8 of treatment urinalysis revealed, for males receiving 100 mg/kg/day or animals receiving 350 mg/kg/day or more, increased electrolyte outputs possibly due to the hydrochloride nature of trientine-2HCl, with low plasma alkaline phosphatase activities evident in animals receiving 350 or 1200 mg/kg/day. After 4 and 8 weeks, and during 8 weeks of treatment, high lung weights and bronchiolar epithelium hypertrophy and broncho-alveolar pneumonia were recorded for animals receiving 1200 mg/kg/day, and submucosal acute inflammation within the glandular region of the stomach was recorded for males receiving 350 or 1200 mg/kg/day and in all treated female groups. 26 -week study. One male receiving 175 mg/kg/day and three males receiving 600 mg/kg/day died, showing lung changes. The body weight gain of animals receiving 600 mg/kg/day was slightly decreased. Blood chemistry and urinalysis examinations showed changes similar to those indicated in the 4- or 8-week study. The low plasma copper concentrations seen in males receiving 600 mg/kg/day, the slightly low liver copper concentrations found in animals receiving 600 or 175 mg/kg/day and the high urinary copper concentrations found in all treated groups, are attributed to the pharmacological action of trientine-2HCl. Histopathology revealed a dosage-related incidence and severity of focal chronic interstitial pneumonitis accompanied by fibrosis of the alveolar walls in females receiving 175 mg/kg/day or more and all treated male groups, but no significant pathological changes in the stomach. Apart from the histological changes found in the lung, all the above changes were reversible. In conclusion, the NOAEL of trientine-2HCl in this 26-week study was considered to be 50 mg/kg/day for females and less than 50 mg/kg/day for males.