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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report Date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no
Remarks:
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): TK 10504 (DDM)
- Physical state: solid
- Analytical purity: > 98%
- Purity test date: July 24, 1980
- Lot/batch No.: 607212
- Stability under test conditions: stable

Test animals

Species:
rat
Strain:
other: RAIf (F3-hybrid of RII 1/Tif x RII 2/Tif) (Specified pathogen free)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Production, Ciba-Geigy LTD., 4332 Stein, Switzerland
- Age at study initiation: 4 weeks
- Weight at study initiation: 88-91 g (males); 90-91g (females)
- Fasting period before study: N/A
- Housing: Animals housed in groups of 5 in Macrolon cages type 4 with granulated wood bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2°C
- Humidity (%): 55 ±10 %
- Air changes (per hr): 15-17/hr
- Photoperiod (hrs dark / hrs light): 10 hrs light

IN-LIFE DATES: From: August 11, 1980 To: December 15, 1980

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

- Rate of preparation (frequency): daily
- Mixing appropriate amounts with: water
- Storage temperature of food: ambient
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Pretest samples were analysed for concentration and stability of the test material. Samples taken at random from the prepared dosing solutions were analysed for concentration in the Central Analytical Laboratories of Ciba-Geigy LTD., Basle, Switzerland
Duration of treatment / exposure:
90 days
Frequency of treatment:
continuous
Doses / concentrations
Remarks:
Doses / Concentrations:
Doses: 80, 400, 800 mg/kg bw/day Due to the small variation between nominal and actual measured concentrations, nominal values were used in calculation of test material intake.
Basis:
nominal in water
No. of animals per sex per dose:
80 males and 80 females per group
Details on study design:
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

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

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 15 (after 30 days exposure) and after 1 month recovery
- Anaesthetic used for blood collection: No
- Animals fasted: Yes (20 hours fasting)
- How many animals: 10 rats/sex/group
- Parameters checked: Erythrocytes, Leukocytes, Haemoglobin, Haematocrit, Reticulocytes, Thrombocytes, MCV, MCH, Differential, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 15 (after 30 days exposure) and after 1 month recovery
- Animals fasted: Yes (20 hours fasting)
- How many animals: 10 rats/sex/group
- Parameters checked: Glucose, urea nitrogen, sodium, potassium, calcium, ASP aminotransferase (GOT), ALT aminotransferase (GPT), alkaline phosphatase, creatinine, bilirubin, cholesterol, total proteins, protein electrophoresis

URINALYSIS: Yes
- Time schedule for collection of urine: week 15 (after 30 days exposure) and after 1 month recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Specific gravity, pH, chemical findings test st

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Hearing tests
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
For each time point and parameter, a uni-variate statistical analysis was conducted Each treated group was compared to control group in respect of dispersion and displacement. In addition, a trend test as applied considering all groups.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No clinical symptoms and no signs of local and/or systemic
toxicity were observed, except that the animals of groups 3 and 4
(400 and 800 ppm) failed to gain weight.
BODY WEIGHT AND WEIGHT GAIN
The mean body weight of male and female groups 3 and 4 (400 and
800 ppm) was markedly depressed, whereas the mean body weight of
male and female group 2 (80 ppm) was similar to that of the
respective controls. After replacement of the medicated water
with ordinary drinking water male and female groups 3 and 4 (400
and 800 ppm) did not recover essentially from this weight depression
during a 4 weeks recovery period.




WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The mean water consumption of male and female groups 3 and 4 (400
and 800 ppm) was markedly depressed, whereas the mean water consumption
of male and female group 2 (80 ppm) was similar to that
of the respective control groups. After replacement of the medicated
drinking water the water consumption of male and female
groups 3 and 4 (400 and 800 ppm) increased slowly to the amount
consumed by the respective control groups towards the end of the
4 weeks recovery period.

HAEMATOLOGY
The males and females of group 3 and 4 (400 and 800 ppm) showed reduced numbers of erythrocytes, a reduced haemoglobin concentration and a lower haematocrit still present at the end of the recovery period.
The mean cell haemoglobin was increased in males of group 3 and 4 (400 and 800 ppm) at the end of the recovery period.
The mean cell volume was increased in the males of group 3 and 4 (400 and 800 ppm) at the end of the recovery period.
The number of reticulocytes in females of group 3 and 4 (400 and 800 ppm) was increased. The number of reticulocytes was increased in the males of group 3 and 4 (400 and 800 ppm) and in females of group 4 (800 ppm) at the end of the recovery period.
The number of leucocytes was increased in the males and females
of group 4 (800 ppm). A slight increase of the number of the segmented neutrophils
(relative) was observed in the males and females of group 4 (800 ppm) and in the males of group 3 and 4 (400 and 800 ppm) at the end of the recovery period. A decrease in the number of lymphocytes (relative) was observed in the females of group 4 (800 ppm). The prothrombin time was prolonged in the males and females of group 4 (800 ppm).

CLINICAL CHEMISTRY
The activity of the alkaline phosphatase was increased in the males and females of group 3 and 4 (400 and 800 ppm) at the end of the treatment period as well as at the end of the recovery period.
The alanine aminotransferase (GPT) activity was slightly increased in males and females of group 3 and 4 (400 and 800 ppm). A similar increase of the alanine aminotransferase (GPT) activity was observed in the males of group 4 (800 ppm) at the end of the recovery period.
The aspartate aminotransferase (GOT) activity was increase in the males and females of group 3 and 4 (400 and 800 ppm) and in the males of group 3 and 4 (400 and 800 ppm) at the end of the recovery period.
The urea-nitrogen-concentration was increased in the males and females of group 3 and 4 (400 and 800 ppm) and in males of group 4 (800 ppm) at the end of the recovery period.
The bilirubin and cholesterin concentrations were increased in the males and females of group 3 and 4 (400 and 800 ppm).
The total protein concentration was slightly increased in the males of group 3 and 4 (400 and 800 ppm). The albumin fraction (relative) was increased in the females of group 2 and 4 (80 and 800 ppm). The A1 globulin fraction (relative) was decreased in the males of group 3 and 4 (400 and 800 ppm). The beta-globulin fraction (relative) was increased in the males of group 3 and 4 (400 and 800 ppm). The beta-globulin fraction (relative) was decreased in the females of group 2 and 4 (80 and 800 ppm). The gamma-globulin fraction (relative) was decreased in the females of group 4 (800 ppm).

URINALYSIS
The findings in the urine were generally unremarkable and
comparable to those of the control animals.

ORGAN WEIGHTS
Corresponding to the lower body weight in male and female groups 3 and 4 (400 and 800 ppm) the absolute organ weights of the animals in these groups were lower than in the respective control groups at the end of the treatment period. This influences also the organ weight ratios in that in male and female groups 3 and 4 (400 and 800 ppm) the organ to brain weight ratio was lower and the organ to body weight ratio was higher than in the respective control groups. No marked improvement in absolute and relative
Organ weights of males and females of group 3 and 4 (400 and 800 ppm) was observed at the end of the recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examination revealed the following findings in animals of group 4 (800 ppm):
In all animals marked biliary liver lesions and goitrogeneous effects on the thyroid were apparent. All histologically examined treated male and female rats showed moderate or marked hyperplasia of small biliary ducts with initial fibrosis in the peripheral parts of the liver lobules. These liver changes persisted also after the recovery period. Clear fluid in the abdominal cavity was found in one male and in 4 females. Slight or moderate oedema in the interstitium of the pancreas was seen microscopically in 5 males and 3 females. In nearly all males (18/20) and in all females slight, moderate or marked hypertrophy
of the thyroid follicular epithelial cells and diffuse hyperplasia of the glandular structures with marked colloid depletion was noted. In one male rat also focal nodular hyperplasia with reaccumulation of the colloid in some distended follicles developed.
After the recovery period the thyroid stimulation was much less pronounced. Only in 3/10 males and 2/10 females slight stimulation of the follicular epithelium was still evident.

Histopathological examination revealed the following findings in animals of group 3 (400 ppm):
All the males and the majority of females (16/20) showed slight or moderate hyperplasia of small biliary ducts. "The severity of these microscopical liver lesions was similar in rats sacrificed after the recovery period. In 4/20 males and in 17/20 females slight or moderate stimulation of the follicular epithelium in the thyroids was noted. In one male and one female also focal nodular hyperplasia of the thyroid parenchyma with colloid
reaccumulation in some follicles developed.
No compound related thyroid changes were noted after the recovery period, however.

Histopathological examination revealed the following findings in animals of group 2 (80 ppm):
No compound related liver lesions were noted. However in 2/20 males and in 2/20 females slight stimulation of the follicular epithelium in the thyroids was observed upon histopathological examination.
OTHER FINDINGS
Whereas nephrocalcinosis was evident in all females of treatment and control groups, mineralisation was seen in all males of the mid dose group and in 21 of 30 males of the high dose groups. One male of the 80 ppm group and none of the control males showed kidney mineralisation.

Simple hearing tests performed at the beginning of the treatment period and towards the end of the recovery period revealed no treatment related effects on the auditory perception.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
7.5 mg/kg bw/day (nominal)
Sex:
male
Dose descriptor:
LOAEL
Effect level:
8 mg/kg bw/day (nominal)
Sex:
female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
It can be inferred from the observations made during this study that a marginal "lowest observable effect level" for TK 10504 (MDA) when administered to rats in drinking water over a period of 3 months is 80 ppm (80 mg/kg water) corresponding to 7.5 mg/kg of bodyweight for males and 8.0 mg/kg of bodyweight for females respectively.