Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18.8.2010 - 21.7.2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
During the study some deviations from microclimate limits in animal room were recorded. These microclimatic deviations did not affect the welfare of animals and had no impact on the results of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Reaction product of Distillates (petroleum), acid-treated heavy naphthenic and calcium oxide
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Smiles notation: not known - UVCB substance
- InChl: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 26.2.2010
- Expiration date of the lot/batch: 26.2.2012
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in PE bottle at room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Lysolaje 15, 165 00 Praha 6, závod Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 6 - 7 weeks
- Fasting period before study: none
- Housing: Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood.
- Diet (ad libitum): Complete peleted diet for rats and mice in SPF breeding - ST 1 BERGMAN. Diet was sterilised before using.
- Water (ad libitum): Drinking water. Water was sterilised before using.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-3°C
- Humidity: 30-70%
- Air changes: approximately 15 air changes per hour
- Photoperiod: 12-hour light/12 hour dark cycle

STUDY TIME SCHEDULE
The treated groups were administered daily for 90 days.
Experimental Data Collection:
Body weight: weekly
Food consumption: weekly
Water consumption: twice a week
Mortality control: twice daily
Health condition control: daily
Clinical observation: daily
Behavioural assessments: weekly
Functional observation: at the last week of administration and recovery period
Ophthalmological examination: at the 1st week of study and at the last week of administration and recovery period
Urinalysis: 90th (main group); 118th (satellite group) day of study
Haematology examination: 91st (main group); 119th (satellite group) day of study
Clinical biochemistry: 91st (main group); 119th (satellite group) day of study
Biometry of organs: 91st (main group); 119th (satellite group) day of study
Pathological examination: 91st (main group); 119th (satellite group) day of study



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The application form (test substance suspension in olive oil) was prepared daily just before administration. The mixture was mixed (cca 400 rpm) for 10 minutes. The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.

VEHICLE
- Olive oil (pharmaceutical quality)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days per week at the same time
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
160 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
400 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 females and 10 males per group, 5 females and 5 males in satellite group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study – 160, 400 and 1000 mg/kg/day were chosen on the basis of the results of the study: Toxicity to reproduction study ( Plodikova P.: Reproduction/Developmental Toxicity Screening Test, study No. 49/10/18, 2010)
- Post-exposure recovery period in satellite groups: 28 days.

Positive control:
No positive control

Examinations

Observations and examinations performed and frequency:
HEALTH CONDITION CONTROL: Yes
The health condition was controlled daily during the check-in, acclimatisation period, during the administration and during the recovery period in groups.

CLINICAL OBSERVATION: Yes
All rats were observed daily during the administration period.

DETAILED CLINICAL OBSERVATIONS: Yes
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, position of eyelids, breathing, tonic or clonic movements, stereotypes or bizarre behaviour.
The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

FUNCTIONAL OBSERVATION: Yes
This observation was done at the end of administration period and recovery period.
During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using dynamometer. Measurements were made on: 1) pectoral legs, 2) pelvis legs.

BODY WEIGHT: Yes
The body weight of animals was recorded weekly.

FOOD CONSUMPTION: Yes
Food consumption for animal/day was calculated of average values of each group.
Time schedule: weekly

FOOD EFFICIENCY: Yes
Calculation of food conversion in %: weight increment/food consumption x 100
Time schedule: weekly

WATER CONSUMPTION: Yes
Water consumption for animal/day was calculated of average values of each group.
Time schedule for examination: twice a week

OPHTHALMOSCOPIC EXAMINATION: Yes
Dose groups that were examined: Control and the highest dose level.
The following examinations were made: visual acuity, eyelids, palpebral fissure, eyelash, bulb, lacrimal apparatus, conjunction, sclera, cornea, iris, pupil, lens, eye ground.
Time schedule for examinations: During the first week of application and at the end of administration period and recovery period.

HAEMATOLOGY: Yes
Time schedule for collection of blood: 91st (main group); 119th (satellite group) day of study
Anaesthetic used for blood collection: Yes - light ether narcosis
Animals fasted: Yes
How many animals: all animals
Examined parameters:
Total erythrocyte count
Mean corpuscular volume
Haematocrit
Haemoglobin concentration
Total leucocyte count
Total platelets count
Partial thromboplastin time
Prothrombin time
Fibrinogen
Granulocytes
Lymphocytes
Monocytes

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: 91st (main group); 119th (satellite group) day of study
Animals fasted: Yes
How many animals: All
Examined parameters:
Glucose
Cholesterol, total
Urea
Bilirubin, total
Aspartate aminotransferase
Alanine amonitransferase
Alkaline phosphatase
Calcium
Phosphorus
Protein, total
Protein, albumin
Creatinine
Sodium
Potassium
Chloride

URINALYSIS: Yes
Time schedule for collection of urine: 90th (main group); 118th (satellite group) day of study
Metabolism cages used for collection of urine: Yes
Animals fasted: No
Examined parameters:
Volume
Colour
Cloud
Odour
Glucose
Protein
Bilirubin
Urobilinogen
pH
Specific gravity
Blood
Ketones
Nitrite
Leucocytes

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Biometry of Organs - The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart were recorded.
Pathological examination - the revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out.

HISTOPATHOLOGY: Yes
Organs for histopathological examination - see table "Organs for histopathological examination"


Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
see section Details on results
Mortality:
no mortality observed
Description (incidence):
see section Details on results
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see section Details on results
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Slight intergroup differences in food consumption of treated animals.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Slight intergroup differences in water consumption of treated animals.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see section Details on results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see section Details on results
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see section Details on results
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see section Details on results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see section Details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see section Details on results
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
No mortality, no clinical signs.

BODY WEIGHT AND WEIGHT GAIN:
Slight intergroup differences in body weight of treated animals.

FOOD CONSUMPTION:
Slight intergroup differences in food consumption of treated animals.

WATER CONSUMPTION:
Slight intergroup differences in water consumption of treated animals.

OPHTHALMOSCOPIC EXAMINATION:
No changes were observed.

HAEMATOLOGY:
Haematological examination revealed in treated males and females dose-dependent decreased total erythrocyte count, concentration of haemoglobin
and haematocrit. Increased value of MCV (mean corpuscular volume) was recorded in females after the recovery period. Increase concentration of fibrinogene was recorded in males after recovery period

CLINICAL CHEMISTRY:
Value of urea was reversibly decreased (probably decreased ureogenesis) at all treated groups of animals with statistical significance in males.
The concentration of cholesterol was reversibly increased in all treated groups.
Activity of all measured liver enzymes was decreased in both sexes of all dose levels.
Increased value of creatinine in blood (significantly persisted after the recovery period was recorded in both sexes.
Toxicologically important disbalance in ions (potassium, chloride) and minerals (phosphorus and calcium) were measured in both sexes but markedly in males of the middle and highest dose levels and females at the middle dose level. In females these changes were irreversible.

URINALYSIS:
Urinalysis revealed only sporadic changes without toxicological importance.

ORGAN WEIGHTS:
Dose-dependent increased weight of liver was recorded in males and females with statistical significance at the middle and highest dose levels.
The weight of kidneys was insignificantly increased in males at the highest dose level and females at the middle and highest dose levels.
Dose-dependent increased weight of spleen was recorded also in females (statistically significant in the highest dose level).

PATHOLOGY:
Macroscopic findings.
At the highest dose level the presence of focal macroscopic changes on stomach mucous membrane was recorded in one male and two females.

HISTOPATHOLOGY:
Pathological examination revealed negative influence of the test substance treatment on liver - affections of microscopic structure of liver were recorded. Microscopical examination of liver revealed two types of focal hepatocellular alteration: vacuolation of hepatocytes in treated males and inflammatory changes in females at the highest dose level.
Treatment related histopathological finding in kidneys was recorded only in females at the highest dose level – increased incidence of hydronephrosis.

HISTORICAL CONTROL DATA:
Source of data: haematological and biochemical values of control animals from seven 90-day oral toxicity studies performed during the last 2 years (Wistar rat, SPF breeding, VELAZ s.r.o., Koleč u Kladna)
Vehicle: rats were administered by olive oil, per os
Calculation of interval: mean of group ± 2×standard deviation (10th - 90th percentile)


Effect levels

Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The value was established mainly on the basis of changes in red blood cell component, biochemical parameters related to kidney function and histopathology of the liver.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The value of NOAEL (No Observed Adverse Effect Level) is 160 mg/kg/day.
The value was established mainly on the basis of changes in red blood cell component, biochemical parameters related to kidney function and histopathology of the liver.

Executive summary:

The test substance, Reaction product of Distillates (petroleum), acid-treated heavy naphtenic and calcium oxide, was tested for subchronic toxicity using the Method B.26 Sub-Chronic Oral Toxicity Test: Repeated Dose 90-day Oral Toxicity Study in Rodents, Council Regulation (EC) No. 440/2008, Published in O.J. L142, 2008. This sub-chronic oral toxicity test method is a replicate of the OECD Test Guideline No. 408: Repeated Dose 90-day Oral Toxicity Study in Rodents, Adopted 21st September 1998.

Wistar rats of SPF quality were used for testing. The test substance was administered in olive oil by stomach tube; oral application of rats was made daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 10 males and 10 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 160, 400, 1000 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day). The administration period lasted 90 days. After that animals of main groups were sacrificed and satellite animals were observed for the next 28 days without treatment.

The dose levels for the main study - 160, 400, 1000 mg/kg/day were chosen on the basis of results of the Study No.: Reaction product of Distillates (petroleum), acid-treated heavy naphtenic and calcium oxide – Reproduction/Developmental Toxicity Screening Test; VUOS-CETA Report No.10-257, 2010.

During the 90-day study clinical observation and health status control were performed daily. The body weight, food consumption were measured weekly and the detailed clinical observation was carried out in the same time interval. Water consumption was measured twice a week. Ophthalmologic examination was done at the beginning and at the end of the study. Before the end of study the functional observation was accomplished. The study was finished by urinalysis, haematological and biochemical analysis, and gross necropsy of animals. The selected organs for weighing and histopathological examination were removed.

 

Results

There were no treatment revealed changes for mortality, clinical observation, functional observations and ophthalmoscopic examinations.

Slight intergroup differences in body weight, food consumption and water consumption of treated animals were probably no toxicological relevance.

Haematological examination revealed marked symptoms of anaemia in treated males and females (dose-dependent decreased total erythrocyte count, concentration of haemoglobin and haematocrit). Increased value of MCV was recorded in females after the recovery period. Irreversible decreased concentration of bilirubin in blood serum in males with significance at the highest dose level was probably related to affection of red blood cells. Anaemia was connected with increased presence of rubiginous pigment (probably haemosiderin) in red pulp of spleen of females at the highest dose level. Dose-dependent increased weight of spleen was recorded also in females (statistically significantly in the highest dose level).

Biochemical and pathological examination revealed negative influence of the test substance treatment on liver. Value of urea was reversibly decreased (probably decreased ureogenesis) at all treated groups of animals with statistical significance in males. The concentration of cholesterol was reversibly increased in all treated groups. Activity of all measured liver enzymes was decreased in both sexes of all dose levels. Dose-dependent increased weight of liver was recorded in males and females with statistical significance at the middle and highest dose levels. Microscopic examination of liver revealed two types of focal hepatocellular alteration: vacuolation of hepatocytes in treated males and inflammatory changes in females at the highest dose level. Increase concentration of fibrinogene was recorded in males after recovery period and probably also related with changes in liver.

Biochemical examination revealed negative influence of the test substance treatment on kidneys. Increased value of creatinine in blood (significantly persisted after the recovery period was recorded in both sexes. Toxicologically important disbalance in ions (potassium, chloride) and minerals (phosphorus and calcium) were measured in both sexes but markedly in males of the middle and highest dose levels and females at the middle dose level. In females these changes were irreversible. The weight of kidneys was insignificantly increased in males at the highest dose level and females at the middle and highest dose levels. Urinalysis revealed only sporadic changes without toxicological importance. Treatment related increased incidence of hydronephrosis in kidneys was recorded only in females at the highest dose level.

Other findings or changes in organ weights were only occasional without treatment relation effect or without presence of related changes. In absence of a treatment-related distribution they were considered as changes of no toxicological significance.

 

Conclusion

The value of NOAEL (No Observed Adverse Effect Level) is 160 mg/kg/day. The value was established mainly on the basis of changes in red blood cell component, biochemical parameters related to kidney function and histopathology of the liver.