Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Lanolin Fatty Acids (LANFA 2)
- Physical state: brown waxy solid
- Analytical purity: > 90%
- Purity test date: 18 Jul 2012
- Lot/batch No.: 0000678698
- Expiration date of the lot/batch: 18 Jul 2013
- Storage condition of test material: Room temperature in the dark, in the original container (tightly closed, dry and well-ventilated)

Test animals

Species:
rat
Strain:
other: RccHanTM:WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B. V., Horst, The Netherlands
- Age at study initiation: 8 weeks
- Weight at study initiation: 207 - 239 g (males), 130 - 149 g (females)
- Housing: group housing (up to 4 animals) housing in Makrolon cages, type-4
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 20/12) rat/mouse maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The solutions were prepared weekly.

VEHICLE
- Concentration in vehicle: 20, 60, and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis was performed by Harlan Laboratories Ltd. using a GC method. After experimental start and during week 13, samples of the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Samples of about 1 g of each concentration were taken after experimental start to confirm stability (4 hours and 8 days).
The samples were delivered to the analytical department (Harlan Laboratories Ltd., Analytics, Itingen / Switzerland) and stored at -20 ± 5 °C until analysis. The test item was used as analytical standard.
Duration of treatment / exposure:
91 days (females, 92 days (males)
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a 14-day range-finding toxicity study no test-item related efects were observed at doses of 100, 300, and 1000 mg/kg bw/day.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- General clinical observations were made at least once a day. Twice daily all animals were observed for morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter.
- Weekly behavioral observations included motor activity (circling, spasm), behavior (increased exploration, reduced grooming, vocalisation, reflexes (blink, pinna, iridic light reflex, push-off, pain response, startle/hearing, righting reflex), and further parameters (lacrimation, limbs cyanotic, mydriasis, miosis, exophthalmos, reduced muscle tone).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional Observational Battery (screen) was made during week 13 in all animals (analoguous to the weekly behavioral observations). Forelimb and hindlimb grip strength measurements were performed during the last treatment week using a push-pull strain gauge (Mecmesin, AFG 25N). Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the last treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study.

FOOD CONSUMPTION:
Food consumption was measured weekly.

WATER CONSUMPTION AND COMPOUND INTAKE : No data

HAEMATOLOGY: Haematological parameters, prothrombin time and activated partial thromboplastin time were examined in all animals after 13 weeks after a 18 h fasting period. The following haematological parameters were checked: haematocrit, haemoglobin concentration, erythrocyte count, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular hemoglobin concentration, haemoglobin concentration distribution width, reticulocyte count, reticulocyte maturity index (low, medium, high fluorescence), leukocyte count (total and differential), methaemoglobin.

CLINICAL CHEMISTRY: Clinical chemistry parameters were examined in all animals after 13 weeks after qa 18 h fasting period. The following parameters were checked: glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, bile acids, gamma-glutamyl-transferase, creatine kinase, sodium, potassium, chloride, calcium, phosphorus, total protein, albumin, globulin, albumin/globulin ratio.

URINALYSIS: A urinalysis was performed with samples collected from all animals. Urine volume (18 h), color, appearance, and specific gravity (relative density) were checked. In addition, parameters as pH value, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, erythrocytes, and leukocytes were determined.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatisation (all animals), during week 13 (control and high dose animals)
Sacrifice and pathology:
GROSS NECROPSY
All animals were subjected to a detailed gross necropsy after sacrifice after week 13.

HISTOPATHOLOGY / ORGAN WEIGHTS
Slides of all organs and tissues collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the principal investigator for histopathology. Organ and tissue samples taken from animals which died spontaneously were evaluated similarly to those organs taken from animals of the high-dose group. The stage of estrus was also evaluated during examination of the vagina and reported in the pathology report.
The following tissues were prepared for microscopic examination and the weight was determined (w): adrenal glands (w), aorta, bone (femur including joint), bone and bone marrow (sternum), brain (including section of medulla/pons, cerebral and cerebellar cortex; w), cecum, colon, duodeum, epididymides (fixed in Bouin´s solution; w), esophagus, eyes with optuc nerve (fixed in Davidson´s solution), harderian gland (fixed in Davidson´s solution), heart including auricles (w), Ileum with Peyer´s patches, jejunum, kidneys (w), larynx, lacrimal gland (exorbital), liver (w), lungs (filled with formalin at necropsy), lymph nodes (mesenteric and mandibular), mammary gland area, nasal cavity, ovaries (w), pancreas, pharynx, pituitary gland, prostate gland and seminal vesicles incl. coagulating glands, rectum, salivary glands (mandibular, sublingual), sciatic nerve, skeletal muscle, skin, spinal cord, (cervical, midthoracic, lumbar), spleen (w), stomach, testes (fixed in Bouin´s solution; w), thymus (w), thyroid (incl. parathyroid gland, if possible), tongue, trachea, ureters, urinary bladder (filled with formalin at necropsy), uterus (incl. oviducts, cervix and vagina; w), all gross lesions.
Statistics:
The following statistical methods were used as appropriate to analyze body weight, food consumption, grip strength, locomotor activity, clinical laboratory data, ophthalmoscopic findings, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
300 mg/kg bw/day: mortality and clinical signs were observed in one male due to a dosing error.
Mortality:
no mortality observed
Description (incidence):
300 mg/kg bw/day: mortality and clinical signs were observed in one male due to a dosing error.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
sporadic visible weight loss was observed in single animals due to misdosing or by chance
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:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
incidental effects on methaemoglobin levels, reticulocytes, eosinophils and platelets count (all values within historical control value), effect on relative thrombin time (all groups outside historical control data)
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
incidental effects on mean sodium, chloride, phosphorus levels, and AST/LDH levels (all within historical control values)
Urinalysis findings:
no effects observed
Description (incidence and severity):
incidental effects on volume, pH, and relative density (all within historical control values)
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
incidental effects only on absolute liver, thymus, brain weights
Gross pathological findings:
no effects observed
Description (incidence and severity):
incidental findings within the normal range of background findings
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
incidental findings within the normal range of background findings
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One male treated with 300 mg/kg bw/day was killed in extremis on day 71 of treatment. The cause of death was considered to be dosing error and was unrelated to any systemic toxicity. All other animals survived.
At 100 mg/kg/day, a nodule in the left thoracic region were noted in one male in week 13 of treatment. At 300 mg/kg bw/day, decreased activity, ruffled fur, breathing noises and localized swelling in the axillary region were noted in one male in week 10/11 of treatment and breathing noises were noted in one male in week 13 of treatment. These findings were considered to be related to misdosing and unrelated to the treatment. All other males and all females were without findings.

BODY WEIGHT AND WEIGHT GAIN
At 100 mg/kg bw/day visible weight loss were noted in one male in week 13 of treatment. At 300 mg/kg bw/day visible weight loss was noted in one male in week 10/11 of treatment. These findings were considered to be related to misdosing and unrelated to the treatment. At 1000 mg/kg bw/day, visible weight loss was noted in one male in week 14 of treatment. All other males and all females were without findings.

FOOD CONSUMPTION
There were no test item-related differences in the mean daily food consumption or mean relative food consumption of the males and females at any dose level.

FOOD EFFICIENCY
Not examined.

WATER CONSUMPTION
Not examined.

OPHTHALMOSCOPIC EXAMINATION
There were no test item-related ophthalmoscopic changes at any dose level. Typical background findings were noted (uni- or bilateral corneal opacity, persistent hyaloid vessel or persistent pupillary membrane) in males and females of all groups. The severity and incidence of these findings at the end of the treatment period were similar.

HAEMATOLOGY
There were no test item-related differences in the mean hematology parameters at any dose level.

1000 mg/kg bw/day:
Significantly elevated mean relative prothrombin time (p<0.01), was noted in females. Similar findings were not seen in males at this dose level. Significantly reduced mean methemoglobin levels (p<0.05) were noted in females. Significantly reduced mean medium-fluorescence reticulocytes (p<0.05) were noted in males. Females were not affected.
300 mg/kg bw/day:
Significantly elevated mean relative eosinophils of the relative differential leukocyte count (p<0.01) and significantly elevated relative prothrombin time (p<0.05) were noted in females.
100 mg/kg bw/day:
Significantly elevated mean relative and absolute eosinophils (p<0.01) and reduced mean basophils (p<0.05) of the relative differential leukocyte count, as well as significantly elevated relative prothrombin time (p<0.01) and reduced mean platelet count (p<0.05) were noted in females.

With the exception of the difference in relative prothrombin time, all values in any dose group remained within the range of the historical control values. The relative thrombin time in the control group was also slightly elevated when compared with the historical control values.

CLINICAL CHEMISTRY
There were no test item-related differences in the mean clinical biochemistry parameters at any dose level.
1000 mg/kg bw/day: Significantly elevated mean sodium, chloride and phosphorus levels (p<0.05) were noted in males. In females significantly elevated mean phosphorus level (p<0.05) was noted.
300 mg/kg bw/day: Significantly reduced mean sodium level (p<0.01) was noted for females. Males were unaffected.
100 mg/kg bw/day: Significantly elevated mean aspartate amino transferase (p<0.05), lactate dehydrogenase (p<0.01) and phosphorus (p<0.05) and significantly reduced mean sodium level (p<0.05) were noted in females, whereas males were unaffected.
All values in any dose group remained within the range of the historical control values.

URINALYSIS
There were no test item-related differences in the mean urinalysis parameters at any dose level.
In males at 100 and 1000 mg/kg bw/day, significantly reduced volume and pH as well as increased relative density at 1000 mg/kg bw/day (p<0.05) were noted when compared with the mean values of the control rats. The values remained within the range of the historical control values and the differences were considered to be unrelated to the test item. The females were unaffected.

NEUROBEHAVIOUR
There were no test item-related clinical observations evident during the functional observational battery (week 13) at any dose level. The mean hind limb grip strength values of female rats treated with 1000 mg/kg bw/day was statistically reduced (p<0.05) when compared with the mean values of the control rats. The mean fore- and hind limb grip strength values of the remaining test item-treated rats compared favorably with those of the respective control rats. There were no test item-related differences in the mean locomotor activity at any dose level.

ORGAN WEIGHTS
There were no test item-related changes in the mean absolute or relative organ weights at any dose level.
In females treated with 300 mg/kg bw/day, significantly elevated mean absolute liver and thymus weights (p<0.05) were noted. The mean liver and thymus brain weight ratios were significantly elevated (p<0.05 or p<0.01) when compared with the controls. The organ to body weight ratios of these organs were not statistically significant.

GROSS PATHOLOGY
There were no treatment-related changes.
The findings in male no. 26 of group 3 which was killed in extremis appeared to be related to dosing error and were not considered to be test item related. The remaining findings distributed among control and treated animals were considered to be within the range of normal background alterations.

HISTOPATHOLOGY: NON-NEOPLASTIC
The microscopic findings recorded in this study were considered to be within the normal range of background alterations that is seen in untreated animals of this age and strain. In particular, a small increase in incidence of brown pigment in hepatocytes and macrophages in the liver of high dose group males and females was considered unrelated to treatment as this is a common incidental finding in control rats of this strain and age.
The examination of the ovaries and the female genital tract did not reveal any difference in estrus cycling between control and treated animals. The male genital organs were also normal.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Results on haematology (coagulation parameters) of male and female animals.

Results after 13 weeks [mean]

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Historical controls

[mean±SD]

 

Males

 

 

 

 

 

 

PT

0.95

0.98

1.02

0.99

0.81±0.07

 

PTT [sec]

21.6

21.2

22.6

21.3

21.2±4.0

 

Females

 

 

 

 

 

 

PT

1.02

1.18**

1.18*

1.22**

0.83±0.07

 

PTT [sec]

34.6

31.8

31.4

30.7

24.1±7.0

 

 

 

 

 

 

 

 

*/**: significant at 5% (*) or 10% (**)

PT: prothrombin time

PTT: partial thromboplastin time

Applicant's summary and conclusion