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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1975
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was equivalent to internationally accepted test guidelines and is considered relevant, adequate and reliable. The study was not conducted under GLP and therefore did not contain all information (preceded implementation of GLP); some parameters were limited compared to current test guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1975
Report Date:
1975

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Principles of method if other than guideline:
Limited parameters measured for haematology, serum analysis and urinalysis.
Only gross lesions were examined histopathologically.
GLP compliance:
no
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
other: CD Spraque Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age at study initiation: Not provided
- Weight at study initiation: Not provided
- Fasting period before study: Not provided
- Housing: Individual wire-mesh suspended cages
- Diet (e.g. ad libitum): Charles River Rat, Mouse and Hamster Meal
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not provided
- Humidity (%):Not provided
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): Not provided
The animals were housed in an environmentally controlled room with fresh air make-up

IN-LIFE DATES: From: To: Not provided

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: For purposes of this study, [Trade name] was furnished to Pharmacopathics Research Laboratories, Inc., in its commercial form, namely, as a 50% solids solution in water. The solution was added to the animals’ diet in sufficient concentrations to achieve dietary levels of 0.25, 1.0 and 4.0% on a solids basis.

DIET PREPARATION
- Rate of preparation of diet (frequency): fresh diets were prepared weekly
- Mixing appropriate amounts with (Type of food): Charles River Rat, Mouse and Hamster Meal
- Storage temperature of food: Not provided
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0.25%, 1.00, 4.00 %
Basis:
nominal in diet
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
The percentages in the diet were based on a ‘solids’ basis. Dose levels in % were recalculated to test arcticle intake based upon mean body weight and mean food consumption. E.g. rat of (mean =) 250 g eats (mean =) 20 g feed/day (Based on Derelanko M.J. The toxicologist’s pocket Handbook).
- 0.25 % in feed = 0,25 g/100g feed = 2,5 mg/g feed = 50 mg/rat/day = 200 mg/kg bw/day ; 188 mg/kg bw is taken as conservative value.
- 1 % in feed = 1 g/100g feed = 10 mg/g feed = 200 mg/rat/day = 800 mg/kg bw/day; 750 mg/kg bw is taken as conservative value.
- 4 % in feed = 4 g/100g feed = 40 mg/g feed = 400 mg/rat/day = 3200 mg/kg bw/day; 3000 mg/kg bw is taken as conservative value.

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: No (but mean test article intake)

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 AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 6 weeks and 13 weeks
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 40 (5 male and 5 female from each group)
- Parameters examined: hematocrit, hemoglobin, WBC count and differential WBC count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: when sacrificed at 13 weeks
- Animals fasted: No data
- How many animals: 40 (5 male and 5 female from each group), same animals as hematology test
- Parameters examined: glucose, blood ure nitrogen, alkaline phosphatase and sGOT (AST)

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the sixth and thirteenth week on test
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: color, appearance, pH, specific gravity, sugar, protein and microscopic examination of the sediment

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
ORGAN WEIGHTS: Yes (see table 14-17)
GROSS PATHOLOGY: Yes (see table 20-21)
HISTOPATHOLOGY: Only gross lesions (see table 20-21)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All rats except one survived the duration of the experiment. A female rat from the high dose group, died at the end of the fifth week of the experiment. Histopathological examination of this rat revealed the presence of mild, chronic renal disease.
Mortality:
mortality observed, treatment-related
Description (incidence):
All rats except one survived the duration of the experiment. A female rat from the high dose group, died at the end of the fifth week of the experiment. Histopathological examination of this rat revealed the presence of mild, chronic renal disease.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
It is obvious that the intermediate and high level groups, both male and female, did not gain as much as the control and low dose groups did.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The feed consumption for the high dose (4.00%) female group was higher than the feed consumption of the other female groups.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The feed efficiency for the high dose (4.00%) group of rats both male and female, was less than the feed efficiency of the other groups.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Although slight decrease in haematocrit, hemoglobin and white blood cells in females dosed at 4%.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Although indication of increased alkaline phosphatase and SGOT in males and females dosed at 1 and 4%.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
0.25%: incr. relative kidney weight (F) - 1%: decr. relative heart weight (M&F) & adrenal weight (F); incr. relative kidney (F) & liver weight (M&F) - 4%: incr. relative testes (M), liver (M&F) and kidney weight (F); decr. relative ovary weight (F)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Except for gross lesions (e.g. respiratory disease observed in control and high dose groups; renal disease observed in high dose groups).
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All rats except one survived the duration of the experiment. A female rat from the high dose (4.00%) group, rat #466, died at the end of the fifth week of the experiment. Histopathological examination of this rat revealed the presence of mild, chronic renal disease.

BODY WEIGHT AND WEIGHT GAIN
The mean body weight, weight range and weight gain of the rats during the course of the experiment are summarized in Table 1.
The male control group gained an average of 393 g during the course of the experiment, the 0.25% group gained an average of 394 g, the 1.00% group gained an average of 363 g and the 4.00% group gained an average of 213 g during the experiment.
The female control group gained an average of 218 g during the course of the experiment, the 0.25% group gained an average of 205 g, the 1.00% group gained an average of 193 g and the 4.00% group gained an average of 115 g during the experiment.
It is obvious that the intermediate and high level groups, both male and female, did not gain as much as the control and low dose groups did.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The feed consumption data are summarized in Table 2. The mean feed consumption in g/rat/week for the male control rats was 171, for the 0.25% group 171, for the 1.00% group 166 and for the 4.00% group 172g. The corresponding feed consumption for the female control group was 137, for the 0.25% group 139, for the 1.00% group 136 and for the 4.00% group 156g.
The feed consumption for the high dose (4.00%) female group was higher than the feed consumption of the other female groups.

FOOD EFFICIENCY
The feed efficiency data are summarized in Table 3. The mean feed efficiency in g of feed/week/ g of body weight for the male control group was 0.52, for the 0.25% group 0.51, for the 1.00% group 0.54 and for the 4.00% group 0.69. The corresponding feed efficiency for the female control group was 0.62, for the 0.25% group 0.62, for the 1.00% group 0.63 and for the 4.00% group 0.93.
The feed efficiency for the high dose (4.00%) group of rats both male and female, was less than the feed efficiency of the other groups.

HAEMATOLOGY
A. Hematocrit
The normal hematocrit concentration in the albino rat in our experience has been 33% to 55%. Several rats from all groups at six weeks had hematocrit values lower than normal. By 13 weeks, however, all rats except three exhibited normal hematocrit values; rat #425 (1.00%group), as well as rats #468 and #480 (4.00% group). In addition rat #387 (0.25% group) as well as rats #445 and #474 (4.00% group) also had low hematocrit values, with high dose female mean remaining below normal (28.7%). Histologically the bone marrow of these rats exhibited normal cellularity and composition.
B. Hemoglobin
The normal hemoglobin concentration in the albino rat in our experience has been 12 to 18g%. Female rats #438 (1.00% group) and #476 (4.00% group) had low hemoglobin values at six weeks, however, by 13 weeks these were normal. Two female rats (4.00% group), rat #468 and #474, had low hemoglobin values at six as well as 13 weeks. In addition, the following rats had low hemoglobin values at 13 weeks: #387 (0.25% group), #409 and #425 (1.00% group) and #445 and #480 (4.00% group), with the high dose female mean remaining below normal (10.3 g%). Histologically, however, the bone marrow of these rats exhibited normal cellularity and composition.
C. White Blood Cell Count
The normal white blood cell count in the albino rat in our experience has been 5000 to 25000 cells/mL. All rats except one had normal WBC counts at both time intervals. Rat #459 (4.00% group) had a WBC count of 25800 cells/mL at 13 weeks. Its bone marrow, however, was histologically normal.
D. Differential White Blood Cell Count
The albino rat in our experience displays lymphocytosis normally throughout its lifetime. All rats examined at six and thirteen weeks except control rat #346 at six weeks as well as control rat #357 and 0.25% rat #387 at 13 weeks displayed lymphocytosis. The bone marrow of these rats, however, was not remarkable histologically.

CLINICAL CHEMISTRY
A. Blood Glucose
The normal serum glucose for the adult albino rat in our experience has been 50-250 mg%. All rats except four (4.00% group) had normal glucose values; rats #445, #453, #468 and #479 had glucose values of 40 mg%, 22 mg%, 39 mg% and 40 mg%, respectively. Histologically the liver and pancreas of these rats were normal.
B. Blood Urea Nitrogen
The albino rat in our experience has a normal range of blood urea nitrogen of 5-35 mg%. All rats examined had normal BUN values.
C. Alkaline Phosphatase
In our experience the adult albino rat has an alkaline phosphatase range of 10-80 units per liter. Most rats, including most of the controls, had abnormally high serum alkaline phosphatase values.
D. Serum Glutamic Oxaloacetic Transaminase
The normal adult albino rat has an SGOT range of 10-100 units per liter according to our experience. Almost all rats, including the control, had abnormally high SGOT values. Because of the high levels of serum alkaline phosphatase and SGOT, a diligent histopathological search was made of the H&E sections of the high dose rats to find out any underlying pathology. Particular emphasis was placed on the parathyroid-skeletal renal system and on the hepato-biliary system. Since some histopathological lesions were found –see below under Histology- which could account for three values, the following additional study was performed to clarify this matter.

Five one year old male control rats as well as five one year old male rats which had been receiving 4.00% of [Trade name] for one year (terminal sacrifice from a reproduction study) had the same assays performed with the results tabulated in Table 9A. As can be seen from this table, all rats had normal glucose, BUN and alkaline phosphatase values. The SGOT values, however, continue to be abnormally high, both in the control as well as in the high dose rats. Since our control sera on the day of the assays were in phase, we consider this SGOT “elevation” to be spurious findings not related to the administration of [Trade name].

URINALYSIS
The six-week urinalysis results were basically negative for both male and female rats. At 13 weeks, however, several rats in all groups exhibited proteinuria to a certain degree. Certain of these rats at autopsy exhibited mild chronic renal disease, an entity not uncommon in this strain of rats ( see below under Histology).

ORGAN WEIGHTS
The mean body weight for the male control rats was 502 g, for the 0.25% group 495 g, for the 1.00% group 464g and for the 4.00% group 346g. The corresponding mean body weight for the female control rats was 301 g, for the 0.25% group 296 g, for the 1.00% group 293 g and for the 4.00% group 215g.
It is, again, obvious that the intermediate and high dose group gained less than the other two groups.
The mean thyroid weight for the male control group was 0.04 g, for the 0.25% group 0.03 g, for the 1.00% group 0.03 g and for the 4.00% group 0.02 g.
The mean heart weight for the male control group was 1.39 g, for the 0.25% group 1.28g, for the 1.00% group 1.15g and for the 4.00% group 0.90 g. The corresponding mean heart weight for the female control group was 0.96 g, for the 0.025% group 0.94 g, for the 1.00% group 0.83 g and for the 4.00% group 0.69 g.
The mean adrenal weight for the male control group was 0.08 g, for the 0.25% group 0.08 g, for the 1.00% group 0.07 g and for the 4.00% group 0.07 g. The corresponding mean adrenal weight for the female control group was 0.10 g, for the 0.25% group 0.10 g, for the 1.00% group 0.08 g and for the 4.00% group 0.06 g.
The mean kidney weight for the male control group was 3.14 g, for the 0.25% group 3.06 g, for the 1.00% group 3.29 g and for the 4.00% group 2.39 g. The corresponding mean kidney weight for the female control group was 1.87 g, for the 0.25% group 2.05 g, for the 1.00% group 2.09 g and for the 4.00% group 1.67 g.
The mean liver weight for the male control group was 17.56 g, for the 0.25% group 17.76 g, for the 1.00% group 19.18 g and for the 4.00% group 15.88 g. The corresponding mean liver weight for the female control group was 9.74 g, for the 0.25% group 10.91g, for the 1.00% group 12.98 g and for the 4.00% group 12.21 g.
The mean testicular weight for the male control group was 3.46 g, for the 0.25% group 3.38 g, for the 1.00% group 3.31 g and for the 4.00% group 3.15 g. The corresponding mean ovarian weight for the female control group was 0.21 g, for the 0.25% group 0.23 g, for the 1.00% group 0.18 g and for the 4.00% group 0.13 g.
The organ weights of the high dose groups were in general, smaller than those of the control group. Histologically, however, these organs were not remarkable.

GROSS PATHOLOGY
The high dose animal which died on the 35th day of dosing (rat #466) had mild chronic renal disease. The rest of the findings consisted of six cases of respiratory disease and one case of fatty infiltration of the liver in the control group as well as five cases of respiratory disease, seven cases of renal disease and one case of cloudy swelling of the liver in the high dose group. None of these findings was compound related, since these conditions are endemic in this strain of rat.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
188 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: 0.25% in the diet
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: 1% in the diet; increased relative liver weights in both sexes.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The findings in the 90-day rat study with CAS No. 37294-19-8 up to 1% in the diet are not really adverse. The organ weight changes at 1% were mainly seen in females, showing marginal weight gain loss but no gross pathological changes. Findings were more pronounced at 4%, and the chronic renal disease in males and females may be considered – possibly partly – test article related.

Applicant's summary and conclusion

Conclusions:
When the test item (purity ca. 50%) is fed to rats at concentrations as high as 4.00%, it causes a significant difference in body weight gain only at the high dose (4.00% ) level. This finding is probably related to an effect by the compound during absorption from the gastro-intestinal tract.
Executive summary:

The test item containing 50% active ingredient, a colorless, clear viscous liquid with a nut-like odor, was fed to three groups of 40 young random bred albino rats (20 male and 20 female) at 0.25%, 1.00% and 4.00% with a control group also consisting of 40 rats (corresponding to mean test article intake of 188, 750 and 3000 mg act.ingr./kg bw/day). The rats were housed individually in an environmentally controlled room. The experiment lasted for 90 days during which time feed consumption and body weights were recorded weekly. At six and 13 weeks five hematological parameters and complete urinalysis (five parameters) were performed while at 13 weeks an additional four biochemical parameters were examined. The rats were subsequently autopsied and 20 rats (10 of each sex) from the control and the high dose groups had careful histological examination performed on 30 separate tissues from each. Organ weights were taken from 20 rats (10 of each sex) per level. The following parameters were calculated: feed consumption, feed efficiency and organ weight to body ratios.
From these results we conclude that the substance caused toxicity by means of a significant difference in body weight gain only at the high dose (4.00% ) level. This finding is probably related to an effect by the compound during absorption from the gastro-intestinal tract. There was one mortality in the high dosed females. Haematology and serum analysis also indicated toxicity at the high dose, whereas at the medium dose, only some organ-to-body weight changes were observed (e.g. liver weight increases in both sexes). Other organ-to-body weight changes were also observed at the high dose group (e.g. increased gonad weights in males; decreased gonad weight in females); increased relative kidney weights were observed in all female dose groups. Chronic renal disease (mild) was observed in 1/20 males and 6/20 females of the high dose groups.

The high dose was considered to be toxic, whereas the changes of the medium dose may be considered adaptive. Therefore 0.25% (188 mg/kg) is considered NOEL, whereas 1% (750 mg/kg) may be considered as NOAEL.