Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10.09.2004 - 14.01.2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Total number of animals: 50 males and 50 females (plus 2/2 reserve animals)
Age at delivery: 5 weeks
Body weight range at acclimatization: males: 95.9 -115.0 grams (mean 104.2 grams) / females: 81.3 - 102.2 grams (mean 93.0 grams)
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with target ranges for temperature between 19 to 25°C and for relative humidity between 30-70%. 12 hours fluorescent light/12 hours dark, music during the light period.
Accomodation: In groups of five (allocation A and B) in Makroion cages type 4 with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
Diet: Pelleted standard Provimi Kliba 3433, ad libitum
Water: Community tap water, ad libitum

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: bidistilled water
Details on oral exposure:
Dose volume: 10 ml/kg bw
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control group)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
The groups comprised 10 animals per sex, which were sacrificed after 90 days of treatment. Additional 5 rats per sex and group were used in the control and high dose groups. These animals were treated for 90 days and then allowed a 28-day treatment­free recovery period after which they were sacrificed.
Control animals:
yes

Results and discussion

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, a NOEL (no-observed-effect-level) could not be established. In the absence of morphological changes in target organs, the minimal changes to plasma electrolyte concentrations in low dose animals were considered of a non-adverse nature. Therefore, the NOAEL (no-observed-adverse-effect-level) is defined at 50 mg/kg body weight/day.
Executive summary:

In this subchronic toxicity study, the test item A 130 was administered orally by daily gavage to SPF-bred Wistar rats of both sexes at target doses of 50, 100, and 200 mg/kg body weight/day for a period of 90 days. A control group was treated similarly with the vehicle, bidistilled water, only. The groups comprised 10 animals per sex, which were sacrificed after 90 days of treatment. Additional 5 rats per sex and group were used in the control and high dose groups. These animals were treated for 90 days and then allowed a 28-day treatment­free recovery period after which they were sacrificed.

Clinical signs, food consumption and body weights were recorded periodically during acclimatization, treatment and recovery periods. Water consumption was recorded once weekly during weeks 1, 5, and 12. Ophthalmoscopic examinations were performed during acclimatization, at treatment end and at recovery end. Functional observational battery, locomotor activity and grip strength were performed at the end of the treatment period.

At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalysis. All animals were killed, necropsied and examined post martern. Histological examinations were performed on organs and tissues from all controi and high dose animals, target organs of the low and mid dose groups, recovery groups, and all gross lesions from all animals.

DOSE FORMULATION ANALYSIS

The overall mean concentrations determined by chemical analyses were close to the nominal values. The investigations on homogeneity and stability revealed values within the required rang es.

MORTALITY / VIABILITY

All animals survived to their scheduled necropsy.

CLINICAL SIGNS

Daily cageside observations and weekly detailed clinical observations revealed deeply yellow colored urine in all test item treated animals (except for the lowest dose males). lncidences of salivation were noted in test item-treated males (at any dose level) and females (at mid­and high dose) with a higher incidence in male rats at the high dose.

There were no other clinical signs of toxicological relevance.

FUNCTIONAL OBSERVATIONAL BATTERY

Detailed observations at week 13: A deeply yellow colored urine was noted in male and female rats of the high dose group (200 mg/kg/day).

Grip Strength: Slightly reduced mean hind limb grip strength measured for mid and high dose males and reduced forelimb grip strength in high dose males were considered associated with the lower body weights.

Locomotor Activity: In the absence of a dose-dependent effect, no findings were considered to be related to the treatment with the test item.

FOOD CONSUMPTION

Similar mean values were recorded for the absolute and relative mean food consumption for the control and treatment groups.

WATER CONSUMPTION

Although not statistically significant, the mean water intakes were considered increased in high dose males and females at the week 12 measurement. This resulted in slightly higher overall mean water intakes for high dose males and females.

In high dose animals, a normalization of the water consumption was observed during the recovery period.

BODY WEIGHTS

A depressed mean body weight gain was recorded for mid and high dose males during the whole treatment period. However, significantly lower absolute mean weights were noted for high dose males from week 7 onwards only. The final mean body weights (week 13) were minimally lower in mid and high dose males, when compared to the control mean.

During recovery, a trend towards normalization of the mean body weight was noted for high dose males.

OPHTHALMOSCOPY

No test item-related changes of toxicological relevance were noted in males and females during the ophthalmoscopic examinations.

CLINICAL LABORATORY INVESTIGATIONS

Hematology: Higher reticulocyte counts (Reti) were determined at treatment end for mid and high dose males and high dose females and persisted to recovery end in both sexes.

In males, the changes to the reticulocyte counts were associated with a shift in the maturity index in L Reti, M Reti, and H Reti in mid and high dose animals. At recovery end, the maturity index was not considered to be affected.

lncreased platelet counts (Plt) were recorded at treatment end for both sexes at the mid and high dose groups. In addition, the prothrombin time (PT) was increased in high dose males at treatment end. Both parameters returned to normal values during recovery.

Clinical biochemistry: Minimally higher concentrations were recorded for high dose males at treatment end for the total protein (Prot) concentration and the pertaining albumin fraction (Alb).

Changes to the lipid metabolism were substantiated by higher cholesterol levels (Chol) at treatment end in mid and high dose males and in high dose females, a higher triglyceride level (Trigly) in high dose males, and increased phospholipids concentrations (Phos-Lip) in mid and high dose males, and in high dose females. No relevant changes were noted at recovery end.

The plasma electrolyte balance was considered affected at all dose levels tested. Whereas the sodium levels (Na+) were elevated in all treated male groups, they were depressed in all female groups. Potassium levels (K+) were elevated in all treated male and female groups. In high dose females, an elevated concentration persisted to recovery end.

Effects on the calcium levels (Ca++) were restricted to all male groups, whereas chloride levels (Cl-) were affected in all females groups.

An elevated phosphate level (P04-in) was noted in high dose males only.

At treatment end, the glutamatdehydrogenase activity (GLDH) was depressed in low, mid, and high dose males, and in mid and high dose females. This finding, together with a minimally depressed aspartate-aminotransferase activity (ASAT) in high dose males and females remains of equivocal toxicological relevance.

Urinalysis: An increased urine volume (Vol) was recorded for high dose males and females, accompanied by a significantly lower relative density (Rel dens) in high dose females only.

Turbid and/or cloudy appearance (Appear) as weil as an intense discoloration (yellow/ brown, brown, red/brown) was noted in rats of both sexes and at any dose level. These changes reverted during the recovery period.

Minimally elevated pH-values were measured for high dose males and females. This change, although within the physiological range, was considered associated with the excretion of the test item or metabolites thereof.

The following changes were considered to be artifacts associated with the excretion of the test item (or metabolites thereof) and the resulting urine discoloration: lncreased bilirubin (BIL) concentrations in rats of both sexes at all dose levels and persisted to recovery end in high dose animals; increased nitrite (NIT) concentrations in both sexes at all dose levels; higher leukocyte counts (LEU) for males and females at all dose levels.

ORGAN WEIGHTS

At treatment end: Absolute and relative mean liver weights were minimally to slightly affected in both sexes at all dose levels. The changes to absolute and/or relative mean weights for brain, heart, thymus, and testes in high dose males were considered associated with the lower final mean body weight.

At recovery end: Minimally lower mean body weights in high dose males and females, minimally higher absolute and relative mean weights for liver and kidney, and an increased heart to body weight ratio in males persisted to recovery end.

MACROSCOPIC / MICROSCOPIC FINDINGS

At necropsy, a dark red discoloration of the thyroid gland was seen in males of groups 3 and 4 (100 and 200 mg/kg) and in females of group 4. After 4 weeks of recovery this change was still present in animals of group 4.

Microscopically, centrilobular hypertrophy of the hepatocytes was observed in all treated groups and still present in animals of group 4 after the 4 weeks recovery period. Higher incidence and severity of hemosiderin deposits was present in the red pulp of males and females of groups 3 and 4, and persisted in animals of group 4 after the 4 weeks recovery period. lncreased incidence and severity of tubular basophilia was seen in the kidneys of males of group 4. This change was still present in recovery males of group 4. Follicular hypertrophy of the thyroid gland and hypertrophy of the TSH-producing cells of the pituitary gland were considered secondary to the hypertrophy of the hepatocytes that most likely induced an indirect perturbation of the pituitary-thyroid axis.

Referring to pathology investigations only, the NOAEL corresponded to 50 mg/kg in male and female animals.