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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: pre-GLP study but well documented.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1953
Report Date:
1953

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not specified
Principles of method if other than guideline:
The study was conducted before GLP but the results are documented in a GLP-like manner. Many specifications of modern guidelines for subchronic toxicity testing were followed.
GLP compliance:
no
Remarks:
pre-GLP study
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
other: CR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: immature
- Weight at study initiation: approximately 100 g
- Housing: 6 animals per cage
- Acclimation period: 5 days

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Stock solutions of the test item were prepared weekly in 8.0 % concentrations. It was acidified with hydrochloric acid so that when added to the drinking water, the latter posessed a pH not less than 5.0 nor greater than 6.0. Because of the apparent "Buffering capacity" of the test item, at least 24 h was allowed after addition of the HCl before pH determination was made. The stock solution was kept constantly refrigerated. Dilutions were prepared from the stock solution in such concentrations as to result in the consumption of the intended dosage levels; the dilutions served as the sole drinking-water supply of the experimental animals throughout the investigation. These dilutions were freshly prepared every day and the remainder of the previous day's dilutions was discarded.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The substance was administered via drinking water ad libitum for 13 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
200, 50 and 20 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
Six animals per sex and per dose
Control animals:
yes, concurrent no treatment
Details on study design:
The test substance was investigated simultaneously in both male and female rats of the CR strain; experiments were initiated when the animals were still immature (approximately 100 grams) in order to observe more readily any effects on the normal growth pattern. The compound was offered to the experimental animals for voluntary consumption following solution at appropriate concentrations in the drinking-water. Such solutions served as the sole water supply of the animals. This procedure was adopted in place of the usual dietary method because of two fundamental virtues: (1) daily measurements could be made of the volumes consumed, thus permitting accurate determinations of the quantities of drug ingested, and (2) the thirst drive is stronger than the hunger drive, thus tending to eliminate the variations in growth pattern which result from impalatability or poor acceptance of products when incorporated in the diet. Six male and six female animals were used at each of three dosage levels with each of the compounds: two hundred mg/kg, fifty mg/kg, and twenty mg/kg, and a similar group of animals received no drug for control purposes.

The drug concentrations in the drinking-water were at such levels as to approximate the ingestion of the desired doses; as the animals gained in body weight it was necessary to alter these concentrations accordingly in order to maintain the mg/kg basis of drug consumption. Every precaution was applied to assure stability of the solutions prepared. Body weights were recorded twice weekly during the initial two-week period, and then weekly until investigations were terminated. Hemotological studies were conducted immediately preceding start of the investigation, again at the mid-point of the study, and finally at termination. After ninety experimental days, all surviving animals were sacrificed and individual weights of the vital organs were recorded. Gross examinations of the viscera were made to note any deviations from normal appearance or structure and representative sections of various organs and tissues were removed for histological examination.

Examinations

Observations and examinations performed and frequency:
1 - Body weights were recorded weekly
2 - Hemograms: RBC, WDG, Ha and differential, 3 male and 3 female rats from each group
3 - Organ weights: Liver, Kidneys, Spleen and Adrenals
4 - Tissue sections for Histopathology: Liver, Kidney, Spleen, Adrenal, Heart, Stumach, Intestine, Gonad, Thyroid, Femoral Bone Marrow and Brain. In addition, Sections of the Lung and mesenteric Lymph Node were taken from the female animals.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
The dosage levels actually consumed by the experimental animals closely approximated the intended dosage levels (Table 1). The foregoing values represent averages of the entire group of animals in each instance. The growth pattern of all experimental groups did not differ significantly from their respective control groups. Mortalities were observed only among the males during the course of the experiment; these were one male at the high dose, two males at the median dose, and one male at the low dose. Among the controls 4/l0 male rats and 1/14 female rats died before the test was terminated. Therefore, it was considered that the mortalities in treated groups were not test item related.No significant alterations in the hemogram were observed. With the possible exception of a mild eosinophila which was present among the females at each of the three dosage levels and among the males at the high dosage level only. This mild eosinophilia was not considered significant. Of the organs weighed at the termination of the experiment, the only marked deviations observed were in reference to the livers of the treated animals as the average weight of this organ was less than the controls among the male rats at the high dosage level and also at the low dosage level. The difference does not appear to be of sufficient magnitude to be significant. Gross examination of the various organs at autopsy did not reveal any deviations from normal appearance or structure. Consideration of all the data recorded and presented herein does not permit any clear-cut conclusions in reference to untoward effects resulting from the voluntary consumption by rats of the compounds in doses as high as approximately 200 mg/kg per day for ninety days. In essence, the results obtained were negative.

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed during testing

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1

   Intended dose  Actual dose  Difference  Total drug consumed
   mg/kg bw/day  mg/kg bw/day  %  mg/kg bw/day
Males         200 187  -7  4480 
50  49  -2  1300 
20  18  -10  450 
Females 200  183  -8  2780 
50  51  +2  840 
 20 21  +5  340 
         

Applicant's summary and conclusion

Conclusions:
Based on the results obtained from oral subchronic toxicity testing the NOAEL was considered to be 200 mg/kg bw/day.
Executive summary:

The test item was administered to male and female Charles River rats in drinking water for 13 weeks at nominal doses of 0 (vehicle control), 20, 50 and 200 mg/kg body weight/day (Pharmacology Research Inc., 1953). The test material was administered at nominal concentrations in slightly acidified drinking water. Six male and six female rats per dose group received the test solution as the only drinking supply ad libitum; the control group received drinking water only.

Data and dose calculation:

The fluid volumes consumed by each group of animals were averaged on a weekly basis and divided by the number of animals composing the group and were stated as cc/rat. These values were converted to cc/kg and further to mg/kg bw/day uptake of the test item. The intended doses had been approximated in every instance with a maximum deviation not greater than 12 %.

Body weights were recorded twice weekly during the initial two-week period and then weekly until termination of the study. Hematological studies were conducted immediately preceding start of the study, again at the mid-point of the study, and finally at termination. After ninety experimental days, all surviving animals were sacrified and individual weights of the vital organs were recorded. Gross examinations of the viscera were made at this time to note any deviations from normal appearance or structure and representative sections of various organs and tissues were removed for histological examination.

The following substance-related findings were obtained:

The growth pattern of all experimental groups did not differ significantly. Mortalities were observed only among the males during the course of the experiment; these were one male at the high dose, two males at the median dose, and one male at the low dose. This was not considered to be significant and was not appeared to be related to drug consumption in any instance.

No significant alterations in the hemogram were observed with the possible exception of a mild eosinophilia which was present among the females at each of the three dosage levels and among the males at the high dosage level only. However, this mild eosinophilia was not considered significant. Of the organs weighed at termimation of the experiment, the only marked deviations observed were in reference to the livers of the treated animals: The average weight of this organ was less than the controls among the rats at the high dosage level and also at the low dosage level. The difference does not appear to be of sufficient magnitude to be significant.

Gross examination of the various organs at autopsy did not reveal any deviations from normal appearance or structure.

Conclusions:  

The subacute toxicity following voluntary consumption of the test item by male and female rats in drinking water in the concentrations of 200, 50 and 20 mg/kg bw/day for a 90 day period showed essentially negative results. The test item was concluded to be relatively innocuous under the conditions of the experiment. Signs of a mild eosinophilia appeared to be present among the experimental animals, but this has been seriously questioned. The no observed adverse effect level (NOAEL) under the conditions of this study was considered to be 200 mg/kg bw/day.