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Toxicological information

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
Study period:
90 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Actual guideline not stated in the report
Deviations:
not specified
Remarks:
No deviations were recorded
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wi-AF7 Han (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Not stated
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 5 weeks
- Weight at study initiation: Part I - males 101-155g, females 111-139g; Part II - males 120-143g, females 112-132g
- Fasting period before study: Not stated
- Housing: Individually housed in wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not stated

DETAILS OF FOOD AND WATER QUALITY:
- Details of analyses were not given

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Part I - 22-30 oC; Part II - 21-26 oC
- Humidity (%): Part I - 35-55%; Part II - 33-50%
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): Not stated

IN-LIFE DATES: Part I - From: August 1982 To: December 1982; Part II - From: January 1983 To: May 1983

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The test material was administered to the animals via incorporation into the diet. Concentrations of the test material in the diet (ppm) were varied on a weekly basis in accordance with the growth and feed consumption of the animals such that the dose levels in terms of mg/kg bw/day were maintained at a constant level throughout the study.
Vehicle:
not specified
Remarks:
The test material was incorporated and mixed directly into the standard feed (Altromin Standard-diat TPF N 1321 fortified)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: NA

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Altromin Standard-diat TPF N 1321 fortified (ex Altromin, Lage/Lippe)
- Storage temperature of food: Not stated

VEHICLE
- Justification for use and choice of vehicle (if other than water): NA
- Concentration in vehicle: NA
- Amount of vehicle (if gavage): NA
- Lot/batch no. (if required): NA
- Purity: NA
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In the 3rd, 6th, 9th and 10th weeks of the study, random samples of the feed mixes were analysed. It was determined that the feed contained the requisite quantities of the test material. The concentration determinations yielded levels of 90-105% of the nominal value.
Duration of treatment / exposure:
3 months or 90 days
Frequency of treatment:
Treated feed was available ad libitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Part I - Control (Group1)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Part I - Low dose level (Group 2)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Part I - Intermediate dose level (Group 3)
Dose / conc.:
312 mg/kg bw/day (actual dose received)
Remarks:
Part I - High dose level (Group 4)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Part II - Control (Group 1A)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
Part II - Low dose level (Group 2A)
No. of animals per sex per dose:
20 males and 20 females per group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Not stated
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Additional 10 males and 10 females per group were included to remain after the treatment period was completed to be assessed for signs of recovery from any treatement-related effects during a 4 week off-treatment period
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): Random
- Since a NOAEL was not determined in Part 1, a separate study was commenced in male and female rats with a new control group and a new low dietary inclusion concentration equivalent to an achieved dose level of 25 mg/kg/day.
Positive control:
No positive control was required

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Twice weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes were examined for all individuals at the autopsy at the end of the treatment period.
- Dose groups that were examined: All treated and control animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 6 and 12/13
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Yes
- How many animals: 10 males and 10 females per group
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6 and 12/13
- Animals fasted: Yes
- How many animals: 10 males and 10 females per group
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6 and 12/13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: NA
- Dose groups that were examined: NA
- Battery of functions tested: sensory activity / grip strength / motor activity / other: NA

IMMUNOLOGY: No
- Time schedule for examinations: NA
- How many animals: NA
- Dose groups that were examined: NA

OTHER:
THYROID HORMONES: In Week 7 blood was withdrawn under halothane anaesthesia from 10 males and 10 females per group for the measurement of thyroid hormones (total T3, total T4 and TSH). The same animals were sampled at termination after 3 months treatment and after the recovery period, for the same parameters.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)
- Full autopsy on any animals dying during the study and all animals killed at scheduled sacrifices after 3 months, and at the end of the 1 month recovery period

HISTOPATHOLOGY: Yes (see table 5)
- The list of tissues specified in Table 5 were processed and examined for each animal killed at the scheduled sacrifices
Statistics:
Body weight, food and water consumption, organ weights and clinicochemical data - analysed using Dunnett's multiple t-test. Differences in group sizes were taken into account by correcting the critical t-values and also the degrees of freedom.
Haematological data - analysed either by using Dunnett's multiple 2-sided t-test with differences in group sizes or variance in homogeneity taken into account by correcting the critical t-values and the degrees of freedom OR, a 2-sided alternative based on a linear rank test.
Statistical significances were quoted at the 5% or 1% level of probability.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Non treatment-related minor incidences of hair loss, spontaneous injury and one case of skin lesions were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Five animals from the control or 125 mg/kg/day groups died following the diagnostic blood aspiration. Three animals receiving 125 mg/kg/day group died during halothane anaesthesia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Transient reduced body weight gain in females treated at 125 mg/kg/day from the 4th week (77.5%), and in females receiving 312 mg/kg/day from the start of the study (94%) when compared with the control females. Recovery in the body weight of females from these treatment groups was evident during the follow-up 4-week off-treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food intake by females treated at 312 mg/kg/day (89.8%) when compared with the controls.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Increased water intake by males treated at 312 mg/kg/day when compared with the controls.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly reduced haematocrit, haemoglobin and erythrocyte values were recorded for males and females treated with 312 or 125 mg/kg/day and for females only treated with 50 mg/kg/day. No dependence on dose level was reported for these slight deviations from control values.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Elevated cholesterol levels in males and females treated with 312 mg/kg/day at both week 6 and week 12/13 investigations. Normal values were recored at the end of the recovery period.
Elevated GPT activity in males and females treated with 312 mg/kg/day and females treated with 125 mg/kg/day at both week 6 and week 12/13 invesigations. Normal values were recorded at the end of the recovery period.
Reduced serum albumin levels in females treated with 312 mg/kg/day at both investigations. Normal values were recorded at the end of the recovery period.
Treatment with higher doses resulted in elevation of serum TSH and T3 levels, with the females being more sensitive to this effect (elevated concentrations measured >/= 50 mg/kg/day). Elevated concentrations were seen in males only at 312 mg/kg/day. Apart from a few isolated cases, T4 levels were normal.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased absolute and relative thyroid weights were observed in males and females treated at 312 or 125 mg/kg/day, with a slight increase also in males treated at 50 mg/kg/day. The increased thryoid weights were still apparent at the end of the recovery period in rats previously treated at 312 mg/kg/day.
Relative liver weights were increased in females treated at 125 mg/kg/day and in males treated at 312 mg/kg/day. In males and females treated at 312 mg/kg/day adrenal weights were reduced and spleen weights increased. Reduced prostate weights and increased thymus weights (females) were also observed. All these changes proved to be reversible at the end of the follow-up observation period.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In male and female rats treated at 312, 125 or 50 mg/kg/day, hypertrophy and hyperplasia of the follicular epithelium of the thyroid was observed, with the change rated as slight to moderate in the 50 and 125 mg/kg/day groups and strongly pronounced in the 312 mg/kg/day group. These changes were considered consistent with hormonal stimulation. This change in the thyroid was not observed in those animals treated at 25 mg/kg/day and, whilst the effect did show signs of regression at the end of the 1-month follow-up period, the pathological change was still apparent in animals from these three treatment groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Elevated serum TSH and total T3 levels were recorded for males and females treated with 312 mg/kg/day and for females only treated with 125 or 50 mg/kg/day at week 7. At the week 12/13 investigation elevated TSH and T3 levels were only recorded in males treated at 312 mg/kg/day.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
> 25 - < 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 50 - < 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: The effect on the thyroid is considered an adaptive response to signs of liver enzyme induction at dose levels of >125 mg/kg/day. Reduced body weight gain and food intake was also recorded for females treated at 125 mg/kg/day and above.

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
The principal effect related to treatment with the test material under the conditions of this study was evidence of slight liver enzyme induction at 125 and 312 mg/kg/day and indications of a significant stimulatory effect on the thyroid gland in both sexes indicated by increases in circulatory levels of T3 and TSH, increased thyroid weights and follicular hypertrophy and hyperplasia observed microscopically. None of these findings relating to the liver or thyroid gland were recorded for animals treated at 25 mg/kg/day, therefore, this dose level was designated the no-observed-effect-level.
Executive summary:

Groups of male and female rats were fed the test material in the diet for a 3-month period at varying concentrations in order to maintain a daily intake of 0, 50, 125 or 312 mg/kg/day. At the end of the 3-month treatment period, some animals from each treatment group were maintained off-treatment for a period of 1 month to assess the progression or regression of any treatment-related changes. Since no no-effect-level was established in this initial study, a separate study was commenced in male and female rats with a new control group and a new low dietary inclusion concentration equivalent to an achieved dose level of 25 mg/kg/day.

At the dose level of 50 mg/kg/day and above there were strong indications of a stimulatory effect on the thyroid gland in males and females comprising increases in circulating T3 and TSH levels, increased thyroid weights and follicular hyperplasia and hypertrophy noted histopathologically. The changes showed some dose dependency with the pathological lesions graded as slight to moderate in the groups treated at 50 and 125 mg/kg/day and as strongly pronounced at 312 mg/kg/day. The changes in the thyroid were still apparent in these groups at the end of the follow-up recovery period albeit less pronounced. The increases in circulating GPT levels and increases in liver weight noted in rats treated with 125 or 312 mg/kg/day were considered indicative of a weak hepatic enzyme induction at these dose levels which could, at least in part, be responsible for the thyroid response at these dose levels although there was no significant reduction in circulating T4 levels reported. All changes were shown to be reversible at the end of the follow-up period although examination of the thyroid did not demonstrate complete resolution of the treatment-related changes.

Under the given experimental conditions of this study, the rats showed no treatment-related effects at the dose level of 25 mg/kg/day and this dose is, therefore, designated the no-observed-effect-level. Since the effect on the thyroid, including pathological changes, was dose dependent in degree and was still apparent, to a lesser degree, in the 50 mg/kg/day dose level group at the end of the follow-up off-treatment period, it is postulated that the no-observed-adverse-effect-level (NOAEL) in this study would be >50 but <125 mg/kg/day. This study was assigned a reliability rating of 1: reliable without restrictions.