REAKTIV OLIV F00-149

Endpoint summary

Description of key information

GLP guideline study for subacute oral toxicity in Sprague Dawley rats (28 d), resulting in a NOAEL for systemic effects of 250 mg/kg/d and a NOAEL of 62.5 mg/kg bw/d for local effects in the stomach.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-Jan-8 to 2002-Jun-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH Gartenstrasse 27 D-33178 Borchen
- Age at study initiation: approximately 6 weeks
- Housing: In transparent macrolon cages (type IV) on soft wood granulate in an air-conditioned rooms, 5 animals per cage, separated according to sex
- Diet (e.g. ad libitum): ssniff R/M-H (V 1534), exept fot the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): Tap water in plastic bottles ad libitum, exept fot the period in which the animals were kept in diuresis cages
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12 hours light / dark cycle

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 62.5, 250, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Male: 10 animals at 0 mg/kg bw/day
Male: 5 animals at 62.5 mg/kg bw/day
Male: 5 animals at 250 mg/kg bw/day
Male: 10 animals at 1000 mg/kg bw/day
Female: 10 animals at 0 mg/kg bw/day
Female: 5 animals at 62.5 mg/kg bw/day
Female: 5 animals at 250 mg/kg bw/day
Female: 10 animals at 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Acute oral toxicity testing of Reaktiv Oliv F00-0149 at a dose of 2000 mg/kg in the rat (only this dose was tested) showed that the median lethal dose (LD50) is above 2000 mg/kg body weight in both male and female animals. All the animals tolerated the dose of 2000 mg/kg without any signs of intoxication.Based on these results, dose levels of 0, 62.5, 250 and 1000 mg/kg body weight per day were selected for the present study.
- Rationale for selecting satellite groups: control and high dose group
- Post-exposure recovery period: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Survival control of the animals was examined twice daily (on weekends and public holidays once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The behavior and general health condition of the animals were observed once daily.
- Once before the first treatment and once a week thereafter, detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa, and impairment of dental growth.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of all animals were determinated before the start of the study and then twice weekly throughout the study.

FOOD CONSUMPTION:
-Food consumption was determined continuously (two times per week).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period, hematological examinations were performed on all animals
- Anaesthetic used for blood collection: Yes - Blood samples were taken from the retrobulbar venous plexus in narcosis (intraperitoneal injection of 67 + 6.7 mg/kg body weight Ketamine-Hydrochloride + Xylazine)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Erythrocyte counts (RBC), Heinz Body Counts, Hematocrit (packed cell volume), Hemoglobin, Mean corpuscular hemoglobin
(MCH), Mean corpuscular hemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Reticulocyte counts, Differential leukocyte counts, Leukocyte counts (WBC), Coagulation time (clotting time), Thrombocyte counts (platelets)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After blood sampling for hematological testing, the animals were killed by section of the vena cava cranialis in deep narcosis and exsanguinated.
- Parameters checked: γ-Glutamyltranspeptidase, Alanine Aminotransferase (ALAT or GPT), Albumin, Albumin / Globulin ratio (calculated), Alkaline phosphatase, Aspartate Aminotransferase (ASAT or GOT), Bilirubin direct, Bilirubin total, Calcium, Chloride (Cl-), Cholesterol, Creatinine, Globulin (calculated), Glucose, Inorganic Phosphorous, Potassium (K+), Sodium (Na+), Total Protein, Triglycerides, Urea, Uric Acid

URINALYSIS: Yes
- Time schedule for collection of urine: Urine analysis was performed in all animals a few days before termination of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Appearence, Bilirubin, Blood, Color, Glucose, Ketone bodies, Microscopic Examination (Sediment), pH, Protein, Urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first treatment and thereafter once a week detailed clinical observations were performed in all animals outside the home cage in a standard arena
- Battery of functions tested: Each animal was assessed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, salivation, nasal discharge, piloerection, pupil size, and unusual respiratory pattern. Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, tremor, and any other abnormal motor movements (such as excessive grooming, repetitive circling or other stereotypes) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded. In addition, defecation and urination were evaluated. At the termination of the study sensory reactivity to stimuli of different types (auditory, visual, and proprioceptive) was evaluated including startle reflex (click response), response to approach with the finger to the nose of the animal, and righting reflex. The presence and absence of pupillary constriction was assessed using a pen flashlight directed into the eye. Assessments of motor function were performed including measurement of motor activity, and forelimb and hindlimb grip strength. The animals were evaluated for motor activity during a 60-minute period in an 16-station automated motor activity monitoring device. Activity counts were recorded by the interruption of photocells in 3-minute-intervals to give a total of 20 intervals. A strain gauge device measured fore- and hindlimb grip strength.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- After exsanguination, all animals were necropsied and checked for macroscopically visible abnormalities. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.
- Endotracheal fixation of the lungs
- Organ weights of Adrenals, Brain, Epidymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOPATHOLOGY: Yes
- From all animals, tissues and organs were preserved in a suitable fixative (buffered formalin) and/or further processed for histopathological investigations.
- Adrenals, Bone marrow / sternum, Brain with medulla oblongata, Epididymides, Heart, Small Intestine 2 jejunum, Large Intestine 2 colon, Kidneys, Liver, Lungs, Lymph nodes 1 mandibular, Lymph nodes 2 iliac, Nerve sciatic nerve, Ovaries with oviducts, Prostate, Seminal vesicle, Spinal cord 1 cervical, Spleen, Stomach,
Testes, Thymus, Thyroid gland with parathyroids, Trachea, Urinary bladder, Uterus, All other gross lesions

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

Clinical observations:
No mortality occurred during the study period.

There were no effects reported in behavior and state of health. At 1000 mg/kg bw/d, black discolored feces were observed in males from study day 15 onwards up to approx. one week of the recovery period. In addition males and females exhibited broken-off incisors during the 2nd week of
recovery.

Body weights were statistically significantly decreased in male rats receiving 1000 mg/kg bw/d. This finding persisted during the recovery period. In male rats receiving 250 mg/kg bw/d body weights were slightly decreased, attaining statistically significance only on days 5 and 8. The food consumption was distinctly decreased in the males receiving 1000 mg/kg bw/d and slightly decreased in females at the same dose level during the recovery period.

Neurotoxicological measurements including "open field" observations, assessment of sensory function, and forelimb and hindlimb grip strength were not influenced by the administration of the test substance in all groups.

Laboratory findings:
Hematology revealed statistically significant increased red blood cell counts for the 1000 mg/kg bw/d males and hemoglobin for the males and females as well as decreased reticulocytes for the males, respectively at the end of the recovery period.

Clinical biochemistry revealed the following statistically significant findings in males and females receiving 1000 mg/kg bw/d that were considered to be toxicologically significant: increased of total bilirubin (males, final and recovery), increased of direct bilirubin (males, recovery), increased of urea nitrogen (males and females, recovery), decreased glucose (females, final and recovery), decreased triglycerides (males, final and recovery).

No treatment-related findings of toxicologically significance were noted by urinalysis.

Effects in organs:
In male rats receiving 1000 mg/kg bw/d decreased liver, spleen, and thymus weights were observed at the end of the study and also after the recovery period. Increased testes and epididymides weights were detected only at the end of the recovery period. These findings were considered to be
primarily due to the decrease in terminal body weights in this group.

At necropsy grey to dark brown discolored kidneys, testes and ovaries were observed in all males and females receiving 1000 mg/kg bw/d. This discoloration was still present after the recovery period of two weeks.

Histopathology revealed marked dose dependent mixed-cellular infiltration in the submucosa of the glandular stomach was observed in animals of both sexes given 250 and 1000 mg/kg bw/d. These findings has been attributed to (local) irritating properties of the test substance.

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: cellular infiltrations in the stomach which can be attributed to irritating properties of the test substance

Dose descriptor:

LOAEL

Remarks:

(local)

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on mixed-cellular infiltration in the submucosa of the glandular stomach.

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Dose descriptor:

LOAEL

Remarks:

(systemic)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs, body weight changes, food consumption, hematologic parameters, clinical chemistry, urinalysis, organ weights, gross pathology

Critical effects observed:

not specified

Conclusions:

Under conditions of this study the systemic NOAEL for subacute oral administration of Reactive Olive F00-0149 is 250 mg/kg bw/d. The LOAEL is 1000 mg/kg/d, based on clinical signs, body weight changes, hematologic parameters, clinical chemistry, urinalysis, organ weights, and gross pathology.

Executive summary:

In a subacute toxicity study Reactive Olive F00-0149 was administered to 5 Sprague Dawley rats/sex/dose by gavage in deionized water at doses of 0, 62.5, 250, 1000 mg/kg bw/day for 28 days. 5 animals/sex in the control and high dose group was used as recovery groups. 

Adverse effects were seen in clinical signs, body weight changes, food consumption, hematologic parameters, clinical chemistry, urinalysis, organ weights, gross pathology, and non-neoplastic histopathology. Repeated oral administration of the test item at dose levels of 62.5 or 250 mg/kg body weight to rats was generally well tolerated troughout the treatment and recovery period. Doses of 1000 mg/kg bw caused impairment of body weight development in particular for the males as well as cellular infiltrations in the stomach which can be attributed to irritating properties of the test substance. A threshold dose for these findings was identified at 250 mg/kg body weight. With regard to the present study the NOAEL is 62.5 mg/kg bw/d for local (irritating) effects. The NOAEL for systemic effects is 250 mg/kg/d.

This subacute toxicity study in rats is acceptable and satisfies the guideline requirement for a subacute oral study (OPPTS 870.3050; OECD 407) in rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a subacute toxicity study Reactive Olive F00-0149 was administered to 5 Sprague Dawley rats/sex/dose by gavage in deionized water at doses of 0, 62.5, 250, 1000 mg/kg bw/day for 28 days. 5 animals/sex in the control and high dose group was used as recovery groups. 

Adverse effects were seen in clinical signs, body weight changes, food consumption, hematologic parameters, clinical chemistry, urinalysis, organ weights, gross pathology, and non-neoplastic histopathology. Repeated oral administration of the test item at dose levels of 62.5 or 250 mg/kg body weight to rats was generally well tolerated troughout the treatment and recovery period. Doses of 1000 mg/kg bw caused impairment of body weight development in particular for the males as well as cellular infiltrations in the stomach which can be attributed to irritating properties of the test substance. A threshold dose for these findings was identified at 250 mg/kg body weight. With regard to the present study the NOAEL is 62.5 mg/kg bw/d for local (irritating) effects in the stomach. The NOAEL for systemic effects is 250 mg/kg/d.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP guideline study examining subacute repeated dose toxicity in male and female rats.

Justification for classification or non-classification

No specific target organ toxicity has been reported in an OECD 407 GLP guideline study.