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Toxicological information

Repeated dose toxicity: other routes

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Administrative data

Endpoint:
short-term repeated dose toxicity: other route
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study that meets basic scientific principles
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1965
Report Date:
1965

Materials and methods

Principles of method if other than guideline:
Rabbits were administered the test substance intravenously 5 days/week over 4 weeks. The animals were observed for 24 hours or 20 days after the exposure ended. The body weight and clinical signs were reported. Specified hematological and urinary parameters were examined and gross pathology and histopathology of target organs was performed.
GLP compliance:
no
Remarks:
performed prior to GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tris(hydroxymethyl)aminomethane, THAM
- Physical state: White, crystalline solid from Abbott Laboratories

Test animals

Species:
rabbit
Strain:
other: New Zealand
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 3.0-4.0 kg
- Acclimation period: 14 days

Administration / exposure

Route of administration:
intravenous
Vehicle:
water
Details on exposure:
The animals were administered 500 mg/kg bw/day of the test substance (0.3M) or saline to which 5 m Eq of KCl/L was added (control group). The dosage and rate of administration were equal in volume and time on a body weight basis; 500 mL/min. Intravenous infusions were given 5 days per week for 4 weeks. The test solution was 'Sterile Anti-Acidosis Lyophilised' containing 36.3 g tris(hydroxymethyl)aminomethane, 1.75 g NaCl and 0.37 g KCl dissolved in 1000 mL water for injection.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily, 5 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
Two males and 2 females from each group were sacrificed and necropsied 24 hours after the completion of the last treatment. The remaining animals in each group were observed for a total of 20 days after the last infusion, prior to necropsy.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes. Rectal temperature was recorded twice daily during the acclimatisation and observation periods, and immediately before and hourly until temperatures returned to normal after all infusions given during the 28-day treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION: Recorded daily

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Heart blood samples were collected for BSP retention tests once a week during the acclimatisation and observation periods, and during the second week of the exposure period. Blood samples were collected from an ear vein once during the acclimatisation and observation periods and twice a week during the exposure period.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters examined: BSP retention (in heart blood), total serum proteins, A/G ratios, serum bilirubin, cephalin flocculation, serum transaminase (SGOT), hemoglobin, hematocrit, white blood cells, red blood cells, differential and platelet counts.

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: Daily. Urine output was recorded daily during the whole acclimatisation and study period. During the acclimatisation and observation periods, urinalysis was performed once per week on the first urine voided in the morning. During the exposure period, the urinalysis was performed on the first urine voided after infusion, on the first day of infusion and twice a week thereafter for a total of 3 weeks. On two days a week during the exposure period, a postinfusion urinary excretion of the test substance was measured on 4 (2 males and 2 females) rabbits in each group. The postinfusion time at which the specimen was collected varied between rabbits.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters examined: pH, albumin, glucose, benzidine test for hemoglobin on centrifuged sediment and supernate, and microscopic examination for red and white blood cells and casts. Method by Linn and Roberts (In vitro and in vivo effects of amino buffers. Annals N.Y. Acad. Sci. 1940; 92: 333-812 ) was used.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Organs and tissued were examined for gross lesions.

HISTOPATHOLOGY: Yes. Specimens of all organs and tissues were fixed in neutral buffered 10% formalin. Tissues from the liver and kidneys were fixed in formalin-calcium solution. From these specimens, paraffin embedded tissue sections were prepared and stained by Harris' hematoxylin and eosin technique. Histochemical methods to demonstrate the sites of activity of enzymes (alkaline phosphatase, acid phosphatase, esterase, peptidase, DPN diaphorase, TPN diaphorase) were perfomed using the frozen tissue sections of the rabbit liver and kidney speciments fixed in formalin-calcium.

All histopathologic methods used were described by Thompson (Selected histochemical and histopathological methods, Springfield, Charles C. Thomas, 1965).

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No animals died during the study period. 6/8 rabbits had temperatures in excess of 40.6 ºC (105 ºF) at some point during the exposure period, but the effect was not treatment-related.

BODY WEIGHT AND WEIGHT GAIN
The body weight of the animals fluctuated between 200 g and 400 g during the study in both groups, with no specific trend (no individual animal data reported). The effect is not considered to be treatment-related.

FOOD CONSUMPTION
The food consumption was similar between the treatment and control groups (no individual animal data reported).

WATER CONSUMPTION
The food consumption was similar between the treatment and control groups (no individual animal data reported).

HAEMATOLOGY
The results of the weekly analyses on blood samples were within the normal ranges (those reported during the acclimatisation period) for the following parameters: total serum proteins, A/G ratio, serum bilirubin, cephalin flocculation, serum transaminase, RBC, differential counts, hemoglobin, hematocrit, and platelet counts. White blood cell counts in excess of 13,000 were seen in 5/8 rabbits in the treatment group. In all the cases, elevated WBC levels were noted in the animals with dry gangrene in the external ear.

CLINICAL CHEMISTRY

URINALYSIS
No diuretic effect of the test substance was observed and no significant changes between the control and treatment groups were noted for any of the parameters tested during urinalysis. (No individual animal data reported).

GROSS PATHOLOGY
Seven of 8 rabbits receiving the test substance had inflammatory lesions of the external ear, around the infusion site. The lesions varied from swelling and redness to dry gangrene and erosion (no individual animal data reported). This is considered to be treatment-related local effect. The test substance is alkaline and is known to cause irritation at the site of exposure in an acute oral and a subcutaneous toxicity study. 2/4 treatment animals necropsied 20 days after the last treatment had visible infarcts in the kidneys. No gross lesions were noted in any other organ or tissue in any other treated animal and no gross lesions were noted in the control animals.

HISTOPATHOLOGY: NON-NEOPLASTIC

In the 7 test substance treated animals that had gross lesions of the ear, there were microscopic lesions of chronic cellulites and necrosis in the subcutaneous tissues of the ear at the injection sites.
The 2 animals with gross kidney lesions had histopathologic kidney lesions visible at the microscopic examination of the tissue samples. These animals also had chronic interstitial nephritis. Infiltrations of lymphocytes were observed in the tissue sections of the liver and kidney of 3 additional rabbits in the treatment group. The infiltrations were seen in animals that were sacrificed 20 days after the exposure ended, as well as in those sacrificed immediately following the final treatment. Peracute toxic nephrosis was observed in 1 rabbit sacrificed 20 days after exposure ended. Urolithiasis was also observed in this animal. Cases of peracute toxic nephrosis were also observed in rats treated with the test substance (Thompson, 1965). The kidney effects are probably related to the alkalinity of the substance, which is excreted primarily via the urine.

The control animals had incidental lesions that were not related to the injections: periportal lymphocytic infiltration (1/8), bilateral chronic interstitial nephritis (1/8) and Encephalitozoonosis- compatible lesions in the brain (1/8).

No difference in staining reaction was observed for the enzyme activity measured in liver and kidney tissue.

Effect levels

Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: histopathology (kidney lesions, chronic interstitial nephritis). Only one dose was used.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion