Registration Dossier

Administrative data

Description of key information

One 7-day oral feeding study in rats and one 31-day dermal toxicity study in rabbits were available. In the 7-day oral toxicity study, rats treated at the highest dose showed a decrease in food intake, body weight loss, and decreased absolute and relative liver weight and decreased relative kidney weight. The NOAELs, therefore, were, 2800 and 3140 mg/kg bw for males and females, respectively. However, this study was considered of too limited duration and studies with TETA (read across) were used instead (see discussion). In the 31-day study rabbits were treated on their skin under occlusive conditions. The only lesions observed were local skin lesions; the degree of irritation was dose-related; effects in the 200 mg/kg group were generally more severe than in the 100 mg/kg group. There were no indications of systemic toxicity. Because no changes were seen in the 50 mg/kg group, the NOEL was 50 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed according to methods similar to OECD408. The study was not performed according to GLP. In the chronic sudy, 12 animals per sex/dose were used instead of 20. No information on test substance purity/composition.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
- At least 20 animals (ten female and ten male) should be used at each dose level. In this study 4 animals/sex were used in 4 and 8 week studies and 12 animals/sex weer used in 26 week studies.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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: for 5 days in a 4- or week study or for 14 days in a 26-week study

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, 8 or 26 weeks.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
100, 350, 1200 mg/kg bw/day 4 and 8 week study
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50, 175, 600 mg/kg bw/day 26 week study
Basis:
actual ingested
No. of animals per sex per dose:
5 in the 4 and 8 week study and 12 in the 26 week study
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: In the 26- week study, rats for reversibility study were treated similarly to those for the main study followed by a period of 13 weeks without treatment.
- 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. 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).
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
4-8 week studies
CLINICAL SIGNS AND MORTALITY
Signs considered to be related to treatment with hientine-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-2HC1.

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 w eek 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.

26 week studies
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 changein 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 periid

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.
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
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
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
43 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is not GLP compliant but comparable to guideline, with Klimisch score 2.

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

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April-May 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Purity of a.i. has been indicated; complete composition of the test substance is not available (available in departmental study file).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ray Nichols Rabbitry, Lumberton, TX, USA
- Age at study initiation: ca. 5 months
- Weight at study initiation: ca. 3.1 - 4.0 kg
- Fasting period before study: not applicable
- Housing: individually in stainless steel cages
- Diet (e.g. ad libitum): restricted to 4 oz per animal per day
- Water (e.g. ad libitum): ad lib
- Acclimation period: at least 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 5 degr F (22 ± 1.5 degr C)
- Humidity (%): 50
- Air changes (per hr): no info
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 9 April 1986 To: no info (most probably 31 days later)
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: 10 x 15 cm
- % coverage: 100
- Type of wrap if used: an occlusive bandage of absorbent gauze and non-absorbent cotton; the bandage was held in place using a lycra/spandex jacket
- Time intervals for shavings or clipplings: periodically as required

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no info
- Time after start of exposure: ca. 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): a 10% solution was applied with the volume adjusted to provide the required dose
- Concentration (if solution): 10%
- Constant volume or concentration used: yes, constant concentration

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Amount(s) applied (volume or weight with unit): 1 ml/kg BW

USE OF RESTRAINERS FOR PREVENTING INGESTION: no (but animals wore jackets)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test material concentration (100 mg/g) was measured 3 times during the study in triplo. % of target concentrations were 87.0, 95.3 and 90.9%, respectively. Stability was measured 2, 4 and 7 days after preparation. % of Day 0 was, 92.1, 59.4 and 59.9%, respectively.
Duration of treatment / exposure:
31 days
Frequency of treatment:
20 exposures, 5 days/week
Remarks:
Doses / Concentrations:
0, 50, 100 and 200 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a probe study using 2 female rabbits per group, at levels of 0, 50, 100 or 200 mg/kg bw/day for 4 days
- Rationale for animal assignment (if not random): computer-generated tables of random numbers
- Rationale for selecting satellite groups: not used
- Post-exposure recovery period in satellite groups: not used
- Section schedule rationale (if not random): no info
Positive control:
not used
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily on working days (one additional routine monitoring on these days and on weekend and holidays for dead animals and check for water and food availability)

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily upon removal when bandage and jacket were removed.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Since the animals were maintained on a restricted diet and normally ate all their ration, measurement of food consumption was not doen. Qualitative changes in consumption were monitored by a periodic visual check.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: not applicable

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to necropsy
- Anaesthetic used for blood collection: Yes (no info which was used)
- Animals fasted: No
- How many animals: all
- Parameters examined: RBC, WPC, platelet count, haemoglobin, packed cell volume

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to necropsy
- Animals fasted: No
- How many animals: all
- Parameters examined: blood urea nitrogen, ALP, ALAT, glucose, total protein, albumin, Cl, Na, K, globulin (calculated)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (including eyes)
HISTOPATHOLOGY: Yes (complete set) of control and high dose animals; skin (application site and a non-treated section), liver kidneys and gross pathologic lesions were aslo examined from low and mid dose animals
Organ weights: brain, liver, kidneys, adrenals, ovaries, testes
Other examinations:
None
Statistics:
Yes, for body weights, organ weights, clinical chemistry, haematology.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: one control female was removed from the study on day 14 because of a broken back.
There were no signs other than local dermal effects. These were dose-related and consisted of red dermal test sites on days 1-2. By study Day 6, animals tretaed with 200 mg/kg bw showed very red and slightly swollen test sites; on Day 16 the skin of some of these females was severely irritated with some crusting and bleeding. Other animals of the 200 mg/kg bw group and those of the 100 mg/kg group had irritated skin that did not progress further.

BODY WEIGHT AND WEIGHT GAIN: no effects

FOOD CONSUMPTION: no effects

FOOD EFFICIENCY: not measured

WATER CONSUMPTION: not measured

OPHTHALMOSCOPIC EXAMINATION: not applicable

HAEMATOLOGY: no effects

CLINICAL CHEMISTRY: no effects

URINALYSIS: not measured

NEUROBEHAVIOUR: not measured

ORGAN WEIGHTS: no effects

GROSS PATHOLOGY: confined to the skin of the 100 and 200 mg/kg groups, characterised by multifocal areas of epidermal and dermal necrosis often covered with crusts comprised of dry serum and necrotic debris.

HISTOPATHOLOGY: NON-NEOPLASTIC
Only dose-related changes in the skin consisting of: multifocal necrosis of the epidermis with extension into the dermis, acanthotic and hyperkeratotic areas, underlying dermis had a subacute inflammatory response consisting of lymphocytes, macrophages and heterophils. Dermal arterioles contained marginated leukocytes, hair folicles showed keratinization. Grading of the skin lesions reflected the extent if the lesion and the degree of functional damage.

HISTOPATHOLOGY: NEOPLASTIC (not applicable)

HISTORICAL CONTROL DATA (not applicable)

OTHER FINDINGS: none
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: skin effects; no systemic toxicity
Critical effects observed:
not specified
Conclusions:
At 100 and 200 mg/kg bw/day, the only lesions noted were skin lesions; the NOEL was 50 mg/kg bw/day.
Executive summary:

Groups of 5 male and 5 female rabbits were treated on their skin with 50, 100 or 200 mg TEPA per kg/bw ca. 6 h/day day, 5 days/week for a period of 31 days (under occlusive conditions). These levels were based on a 4 -day RF study using the same levels using 2 females per group. Controls were treated with the vehicle, distilled water. No changes were observed in body weights, food intake, haematology, clinical chemistry, and organ weights. The only lesions observed were local skin lesions; the degree of irritation was dose-related; effects in the 200 mg/kg group were generally more severe than in the 100 mg/kg group. There were no indications of systemic toxicity. Because no changes were seen in the 50 mg/kg group, the NOEL was 50 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The key study is GLP compliant and comparable to guideline, with Klimisch score 2.

Additional information

The repeated oral toxicity was of limited duration (7 days only). Effects on food intake, body weight gain and liver and kidneys weights were only seen at the high dose (5000 mg/kg bw). The 31 -day repeated dermal study in rabbits showed local effects only; no systemic effects were observed up to a level of 200 mg/kg bw. For read across purposes and to establish DNELS, oral studies with TETA are also included.

As indicated above, as there was only a limited oral study available for TEPA, read across was done with TETA.2HCl due to structural similarity.

In a 26 -week study (Yanagisawa, 1998) using dose levels of 50, 175 and 600 mg/kg bw/day, one male receiving 175 mg/kg bw/day and three males receiving 600 mg/kg/day died, showing lung changes. With regard to the lungs, histopathology revealed a dose-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. Apart from the histological changes found in the lung, all other changes were reversible. It was concluded that the NOAEL of TETA.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; the latter, therefore, was considered a LOAEL.

It was suggested that TETA.2HCl, a kind of polyamine, could be taken into bronchiolar epithelium and/or alveolar epithelium by an endogenous polyamine uptake process and be accumulated in the specific tissue site of the lung. Polyamines are generally irritating agents for mucous membranes, upper respiratory tract and skin (Lenga 1988; Maisey et al., 1988). In 4 - or 8 -week study, the histopathological changes observed in the stomach (submucosal inflammation within the glandular region) indicated that TETA.2HCl was slightly irritant. Therefore, it is likely that chronic interstitial pneumonitis is caused by the cytotoxic effect of TETA.2HCl which was accumulated into the bronchiolar epithelial cells and the alveolar pneumocytes.

In a 13 week study (Greenman, 1996), mice and rats received TETA.2HCl in the drinking water at concentrations of 0, 120, 600, or 3000 ppm for up to 92 days; they were fed diets containing nutritionally adequate levels of copper. An additional control group of rats and mice received a Cu-deficient diet. This low copper diet resulted in Cu-deficiency symptoms, such as anemia, liver periportal cytomegaly, pancreatic atrophy and multifocal necrosis, spleen hematopoietic cell proliferation, and increased heart weight, together with undetectable levels of plasma copper in rats but not in mice. TETA.2HCl lowered plasma copper levels somewhat (at 600 and 3000 ppm) in rats, but did not induce the usual signs of copper deficiency. TETA.2HCl caused an increased frequency of uterine dilatation at 3000 ppm in rats fed the adequate copper diet but was not noted in females fed the Cu-deficient diet. TETA.2HCl toxicity occurred only in mice in the highest dose group. Increased frequencies of inflammation of the lung interstitium and liver periportal fatty infiltration were seen in both sexes, and hematopoietic cell proliferation was seen in the spleen of males. Kidney and body weights were reduced in males as was the incidence of renal cytoplasmic vacuolization. There were no signs of copper deficiency in mice exposed to TETA.2HCl. Based on the effects in mice the NOAEL was 600 ppm or 92 mg/kg bw/day for males and 99 mg/kg bw/day for females.

In a 26 -week toxicity study (Maemura, 1998) Beagle dogs received TETA.2HCl orally at dosages of 50, 100 or 200 mg/kg bw/day for 26 weeks followed by a 13 -week reversibility phase. However, in view of the severe signs which resulted in the sacrifice for humane reasons of two males and one female receiving 200 mg/kg bw/day during week 9 of treatment, surviving dogs of this group were only treated for 10 weeks. Signs before killing included marked underactivity, body tremors, abnormal gait, limited use of limbs and prone posture. The ante mortem neurological

examination generally indicated depressed postural and flexor withdrawal reactions. The signs were rapidly reversible except in one female which was killed humanely on day 2 of the reversibility period. Abnormal "stiff legged" gait and underactivity were evident, from week 23 of treatment, in two males and one female receiving 100 mg/kg bw/day. In the absence of any macroscopic or histopathologic findings, even after the examination of additional samples of muscle and nerve, the exact nature of this condition could not be elucidated. In all treated groups low copper and zinc concentrations in the livers and high urinary copper and zinc concentrations were found. The NOAEL was considered to be 50 mg/kg bw/day.

Overall, the results from the 26 -week key study in rats (Yanagisawa, 1998) and another 13 -week study in mice (Greenman, 1996) showed lung toxicity. The 26 -week study in dogs (Maemura, 1998) did not show lung toxicity but showed neurological effects instead. The NOAELs in these studies were in the same order of magnitude, i.e. between 50 and 99 mg/kg bw/day, although for male rats the level of 50 mg/kg bw/day should be considered a LOAEL. The level of 50 mg TETA.2HCl per kg bw per day corresponds to (146.23/219.16) x 50 = 33 mg TETA per kg bw per day.

This corresponds to 189/146 x 33 = 43 mg TEPA per kg bw per day for long term oral exposure.

One 28 -day dermal toxicity study in rabbits with TEPA as testsubstance was available (Szabo JR, et al (1986))

Groups of 5 male and 5 female rabbits were treated on their skin with 50, 100 or 200 mg TEPA per kg/bw ca. 6 h/day day, 5 days/week for a period of 31 days (under occlusive conditions). These levels were based on a 4 -day RF study using the same levels using 2 females per group. Controls were treated with the vehicle, distilled water. No changes were observed in body weights, food intake, haematology, clinical chemistry, and organ weights. The only lesions observed were local skin lesions; the degree of irritation was dose-related; effects in the 200 mg/kg group were generally more severe than in the 100 mg/kg group. There were no indications of systemic toxicity. Because no changes were seen in the 50 mg/kg group, the NOEL was 50 mg/kg bw/day.

At 100 and 200 mg/kg bw/day, the only lesions noted were skin lesions; the NOEL was 50 mg/kg bw/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
This is the key study

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
This is the only study available.

Repeated dose toxicity: via oral route - systemic effects (target organ) respiratory: lung

Repeated dose toxicity: dermal - systemic effects (target organ) other: skin

Justification for classification or non-classification

Based on the results observed, the oral toxicity study with TEPA was of too limited duration to conclude about classification. With regard to the dermal study, the NOAEL for systemic effects was at least 200 mg/kg bw, but its duration was only 31 days instead of 90 days, so also insufficient information for classification. With regard to TETA, the changes observed were not considered to be sufficient for labeling with R48/20 (old classification), or STOT repeated Cat. 2, we therefore suggest to do the same for TEPA, thus no classification for STOT repeated exposure needed.