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Description of key information

In a 90 day feeding study in rats similar to OECD guideline 408 (WARF Institute, 1971), a NOAEL of 162 mg/kg bw/day and a LOAEL of 292 mg/kg bw/day based on decrease in body weight was determined.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1971-06-06 to 1971-10-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1998-09-21
Deviations:
yes
Remarks:
preserved tissues devergent from guideline
GLP compliance:
no
Remarks:
Pre-GLP study
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Sprague-Dawley, Madison, Wisconsin, USA
- Females: non-pregnant: yes
- Age at study initiation: ~3 - 4 weeks
- Weight at study initiation: 45 - 55 g
- Housing: individually in metal screen bottom cages.
- Diet: ad libitum, Purina Lab Chow
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2


Route of administration:
oral: feed
Details on route of administration:
Feed, mixed and presented fresh weekly. It was offered ad libitum from specially capped clean glass jars that limit spillage and contamination of feed.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The TFM (HB) was ground by mortar and pestle with lab chow to obtain a 10 % premix, then admixed dry with the basal diet to obtain the desired level. The amount of TFM (HB) used for each level was determined using a purity for this TFM (HB) of 90 %.

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: Purina Lab Chow
Duration of treatment / exposure:
90 days
Frequency of treatment:
continuously
Dose / conc.:
500 ppm
Remarks:
= 50.0 mg/kg bw/day
Dose / conc.:
900 ppm
Remarks:
= 90.0 mg/kg bw/day
Dose / conc.:
1 620 ppm
Remarks:
= 162.0 mg/kg bw/day
Dose / conc.:
2 916 ppm
Remarks:
= 292.0 mg/kg bw/day
Dose / conc.:
5 248 ppm
Remarks:
= 525.0 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: day 21 and day 90

BODY WEIGHT: Yes
- Time schedule for examinations: initially and weekly through 90 days.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean weekly diet consumption.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 21 and day 90
- Animals fasted: No
- How many animals: 5/group
- Parameters examined: RBC, WBC electronically by Coulter Counter; Hgb by the cyanomethemoglobin method; Hematocrit by centrifugation; Differential cell count - 100 white cells identified

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 21 and day 90
- How many animals: 5/group
- Parameters examined: SGOT, SAP by micro method on Eskalab system

URINALYSIS: Yes
- Time schedule for collection of urine: day 21 and day 90
- How many animals: 5/group
- Parameters examined: blood, biliruben, ketone, glucose, protein, pH




Sacrifice and pathology:
GROSS PATHOLOGY: Yes, liver, heart, kidneys, adrenal glande, gonad, spleen,

HISTOPATHOLOGY: Yes, brain, pituitary, lymph nodes, salivary glands, thyroid & parathyroid, lungs, heart, spleen, liver, stomach, small intestine, colon, mesenteric lymph nodes, pancreas, adrenals, kidneys, gonads, urinary bladder, uterus or prostate, muscle (skeletal), bone marrow, bone
Statistics:
Organ weight data were punched onto data cards and submitted with the appropriate program to a Univac 1108 for computing of organ to body weight ratios, averages, and comparisons of test group to controls by t-test. T-tests of body weights and feed consumptions were calculated on a Hewlett-Packard 9100 A Calculator, program 70809-modified, with reference to: Statistical Theory and Methodology for Science and Engineering, Second Edition, K. A. Brownlee, John Wiley & Sons, Inc. 1965,
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormal behavior was observed in the animals nor signs of illness or other displays in any particular group which indicated a response to a specific stress.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight data for males in dose group 3 - 6 showed a dose related depression of body weights in test groups when compared to the control group. Body weight curves for females did not show the marked differentiation seen in the male curves.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Description (incidence and severity):
No remarkable differences exist here for males or females with the exception of the 2 higher levels which show slightly poorer efficiencies.
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Though a strong dose response in male was not evident, tendencies for increased liver weights in the test groups and increased kidney weight in the high level test group were seen. Data and analyses for females revealed almost no significant difference's regarding organ weights and organ weight ratios for the organs measured. The single instance of notable differences was the increased liver weight and liver weight ratio of the high level group when compared to the control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross observations are minimal and of low incidence. Alterations observed are of a non-specific type and not related to administration of test compound. No gross alterations were observed in any other animals of all groups.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Liver alterations of swelling, and cloudy swelling, were seen in both control and test animals and are believed to result from the cellular changes that occur from the time the animal was euthanized until the tissues were fixed. Vacuolation had a very low incidence and is believed to be due to the nutritional status of the animal at the time of death. Pigmentation was again seen in control and test animals and was usually the result of red blood cell destruction. The kidney alteration of congestion of the medulla was seen with a very high incidence in all groups of animals and was believed to be a result of the type of euthanasia. Other histopathological alterations observed had a very low incidence of occurrence. Since histologic observations noted were minimal, apparent in both test and control animals, and incidence was essentially the same in all groups, the alterations were considered spontaneous and not related to the administration of the test material.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
162 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
LOAEL
Effect level:
292 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Conclusions:
In this 90 day feeding study in rats similar to OECD guideline 408, a NOAEL of 162 mg/kg bw and a LOAEL of 292 mg/kg bw was determined, which is based on the observed decrease in body weight
Executive summary:

In a pre-GLP 90-day feeding study in rats similar to OECD guideline 408 (WARF Institue, 1971), groups of weanling SD rats (10/sex/group) were fed diets containing the test substance (90 %) at concentrations of 50.0, 90.0, 162.0, 292.0, or 525.0 mg/kg bw/day for 90 days. The control groups (20/sex) received the untreated diet. The results showed that body weights of the 292 and 525 mg/kg bw/day groups were consistently decreased (10 - 13 %) in males from week 3 to the end of the study. The decrease was statistically significant. Food consumption, and hematological parameters were similar to those of the controls. Clinical signs were not seen in the treated or control rats. There was a decrease in the aspartate aminotransferase (SGOT) activity in both males and females at 525.0 mg/kg bw/day on the 21 day examination period, but by 90 day examination period the SGOT values of 525.0 mg/kg bw/day animals were similar to those of the controls. The alkaline phosphatase level was slightly increased in both males and females of 525.0 mg/kg bw/day groups, but no statistical significance was found. At sacrifice, liver weights of the 292.0 and 525.0 mg/kg bw/day females were slightly increased. No gross pathology and histological changes were observed. The LOAEL of this study was 292 mg/kg bw/day which is based on decreased in body weights; the NOAEL was 162 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
162 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch code 2

Additional information

In a pre-GLP 90-day feeding study in rats similar to OECD guideline 408 (WARF Institute, 1971), groups of weanling SD rats (10/sex/group) were fed diets containing the test substance (90 %) at concentrations of 50.0, 90.0, 162.0, 291.6, or 524.8 mg/kg bw/day for 90 days. The control groups (20/sex) received the untreated diet. The results showed that body weights of the 291.6 and 524.8 mg/kg bw/day groups were consistently decreased (10 - 13 %) in males from week 3 to the end of the study. The decrease was statistically significant. Food consumption, and hematological parameters were similar to those of the controls. Clinical signs were not seen in the treated or control rats. There was a decrease in the aspartate aminotransferase (SGOT) activity in both males and females at 524.8 mg/kg bw/day on the 21 day examination period, but by 90 day examination period the SGOT values of 524.8 mg/kg bw/day animals were similar to those of the controls. The alkaline phosphatase level was slightly increased in both males and females of 524.8 mg/kg bw/day groups, but no statistical significance was found. At sacrifice, liver weights of the 291.6 and 524.8 mg/kg bw/day females were slightly increased. No gross pathology and histological changes were observed. The LOAEL of this study was 291.6 mg/kg bw/day (based on 1 ppm = 0.1 mg/kg bw/day) is based on decreased in body weights; the NOAEL was 162 mg/kg bw/day.

Regarding the mortality, the findings are supported by another subchonic repeated dose toxicity study in Sprague-Dawley rats (WARF Institute, 1971), since no mortality was observed up to the highest dose group (525.5 mg/kg bw/day).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The NOAEL was 162 mg/kg bw/day. As a result the test substance is considered not to be classified for repeated dose oral toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.