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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 June 2013- 07 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(adopted 21 September 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Lanolin Alcohol
- Physical state: yellow solid
- Analytical purity: >90%
- Batch No.: 0000664004
- Expiration date of the batch: 19 Feb 2014
- Storage condition of test material: room temperature in the dark
- Other: Label: SUPER HARTOLAN-PA-(RB) 0000664004 BATCH

Test animals

Species:
rat
Strain:
other: Wistar Han(TM):RccHan(TM):WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: six to eight weeks old
- Weight at study initiation: males: 189 - 219 g; females: 156 - 197 g
- Housing: The animals were housed in groups of three to four by sex in solid-floor polypropylene cages with stainless steel lids and softwood flake bedding (Datesand Ltd., Cheshire, UK)
- Diet: pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK); ad libitum
- Water: drinking water; ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 55±15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 July 2013 To: 02 Oct 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Arachis oil BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd., Project Number 41301173). Results showed the formulations to be stable for at least twenty days. Formulations were therefore prepared fortnightly and stored at approximately +4°C in the dark.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test item formulation and were analyzed for concentration of Lanolin Alcohols at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The test item concentration in the test samples was determined by HPLC with UV detection using an external standard technique. The results indicate that the prepared formulations were within ± 4% of the nominal concentration.
Duration of treatment / exposure:
90 days
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

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing throughout the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12).
- Dose groups that were examined: all control and high dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (Day 90).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all animals from each test and control group
- The following parameters were examined:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Methaemoglobin (Meth)
Reticulocyte count (Retic)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: only at the end of the study (Day 90)
- Animals fasted: No
- How many animals: all animals from each test and control group
- The following parameters were examined:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot. Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Battery of functions tested: sensory activity, grip strength, motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, uterus, liver)
HISTOPATHOLOGY: Yes (adrenals, ovaries, aorta (thoracic), pancreas, bone & bone marrow (femur including stifle joint), pituitary, bone & bone marrow (sternum), prostate, brain (including cerebrum, cerebellum and pons), rectum, caecum, salivary glands (submaxillary), colon, sciatic nerve, duodenum, seminal vesicles, epididymides, skin (hind limb), esophagus, eyes, spinal cord (cervical, mid-thoracic and lumbar), spleen, heart, stomach, ileum (including Peyer’s patches), testes, jejunum, thymus, kidneys, thyroid/parathyroid, liver, tongue, lungs (with bronchi), trachea, lymph nodes (mandibular and mesenteric), urinary bladder, mammmary glands (with cervix), muscle (skeletal), vagina)
Statistics:
Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: increased salivation (males + females); episodes of red/brown staining around the snout (males) (non-adverse effects)
Mortality:
mortality observed, treatment-related
Description (incidence):
1000 mg/kg bw/day: increased salivation (males + females); episodes of red/brown staining around the snout (males) (non-adverse effects)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
only males: 100, 300, 1000 mg/kg bw/d: significant reduction in body weight gain during week 8; 100 mg/kg bw/day: significant reduction in body weight gain during week 4 (non-adverse effects)
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
only males: 1000 mg/kg bw/d: significant reduction in MCHC and increase in prothrombin time; all dose groups: significant reduction in eosinophils; 100 mg/kg bw/d: significant increase in erythrocyte count (non-adverse effects)
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
1000 mg/kg bw/d: significant reductions in phosphorus and bilirubin (males); significant increase in glucose (females) (non-adverse effects)
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
only males: 1000 mg/kg bw/d: significant reduction in mean forelimb grip strength; 100 mg/kg bw/d: significant increase in mean hindlimb grip strength; 300 mg/kg bw/d: significant increase in overall activity (non-adverse effects)
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/d (2 males), 300 mg/kg bw/d (2 females): reddened lungs; microscopic examination revealed agonal congestion and haemorrhage in these animals (effect is considered not to be treatement-related)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
only females: 300, 1000 mg/kg bw/d: increase in incidence of minimal or mild alveolar macrophages in the lungs (non-adverse effects)
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no adverse clinical signs of toxicity detected.
Animals of either sex treated with 1000 mg/kg bw/day showed episodes of increased salivation from Day 9 (females) and Day 18 (males) onwards (tables 1 and 2). Seven males treated with 1000 mg/kg bw/day also showed episodes of red/brown staining around the snout between Day 16 and Day 56 (table 1). Observations of this nature are commonly observed following the oral administration of an unpalatable test item formulation and in isolation are not indicative of systemic toxicity.
There were no unscheduled deaths.

BODY WEIGHT AND WEIGHT GAIN
No adverse effects were detected in body weight gain in treated animals when compared to controls.
Males from all treatment groups showed a statistically significant reduction in body weight gain during week 8 (table 3). Males treated with 100 mg/kg bw/day also showed a statistically significant reduction in body weight gain during week 4 (table 3). The intergroup differences did not show a true dose related response and overall body weight gain for these males was comparable to control males. Therefore this reduction was considered not to be of toxicological significance. Females from all treatment groups showed no differences compared to control animals.

FOOD CONSUMPTION AND FOOD EFFICIENCY
No adverse effect in overall food consumption or food efficiency was detected in treated animals when compared to controls.

WATER CONSUMPTION
There were no treatment-related effects detected in water consumption.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ocular effects detected.

HAEMATOLOGY
There were no toxicologically significant effects detected in the hematological parameters examined.
Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in mean corpuscular heamoglobin concentration and a statistically significant increase in prothrombin time (table 4). The majority of individual values were within normal background ranges for these parameters and in the absence of any associated changes the intergroup differences were considered not to be of toxicological importance. Males from all treatment groups showed a statistically significant reduction in eosinophils (table 4). Males treated with 100 mg/kg bw/day also showed a statistically significant increase in erythrocyte count (table 4). The majority of individual values were within normal background ranges for these parameters and in the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance.

CLINICAL CHEMISTRY
There were no toxicologically significant effects detected in the blood chemical parameters examined.
Males treated with 1000 mg/kg bw/day showed statistically significant reductions in phosphorus and bilirubin. The majority of individual values were within normal background ranges for these parameters and in the absence of any associated histopathological changes the intergroup differences were considered not to be of toxicological importance. Females treated with 1000 mg/kg bw/day showed a statistically significant increase in glucose. Although the majority of individual values were outside of the normal range for this parameter, in the absence of any associated histopathological changes the intergroup difference was considered not to represent an adverse effect of treatment.

NEUROBEHAVIOUR
Behavioral Assessments: There were no treatment-related changes in behavioral parameters measured.
Functional Performance Tests: There were no toxicologically significant changes in functional performance. Males treated 1000 mg/kg bw/day showed a statistically significant reduction in mean forelimb grip strength. Males treated with 100 mg/kg bw/day showed a statistically significant increase in mean hindlimb grip strength. The intergroup differences were confined to one out of the three tests and in the absence of any associated clinical signs to suggest a neurotoxic effect, the intergroup differences were considered not to be of toxicological importance. Males treated with 300 mg/kg bw/day showed a statistically significant increase in overall activity. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological significance.
Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.

ORGAN WEIGHTS
There were no treatment-related effects detected in the organ weights examined.
Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY
No toxicologically significant macroscopic abnormalities were detected. Two males treated with 1000 mg/kg bw/day and two females treated with 300 mg/kg bw/day had reddened lungs at necropsy. Microscopic examination revealed agonal congestion and haemorrhage in these animals which accounts for the macroscopic observations detected. These changes were not considered to represent a true treatment related effect.

HISTOPATHOLOGY
The following treatment related microscopic abnormality was detected:
Lungs: An increase in incidence of minimal or mild alveolar macrophages was evident in females treated with 1000 and 300 mg/kg bw/day. No such effects were detected in males treated with 1000 or 300 mg/kg bw/day or animals of either sex treated with 100 mg/kg bw/day.
The affected macrophages had abundant foamy cytoplasm and may have arisen from the accidental inhalation of small amounts of the test item during dosing or possibly phospholipidosis. However, apart from occasional perivascular infiltration of mononuclear inflammatory cells adjacent to the foci there was no evidence of associated inflammation or damage to surrounding alveoli. It is, therefore, considered likely that the change was adaptive rather than an adverse effect of treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: corresponding to the highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

 

Table 1: Summary incidence of daily clinical observations for males

Dose level [mg/kg bw/day]

Number of animals

Number of animals which have died or have been killed due to animal welfare reasons

Clinical observations

Number of animals showing effect (day of observation)

0

10

0

no abnormalities detected

-

100

10

0

no abnormalities detected

-

300

10

0

no abnormalities detected

-

1000

10

0

Increased salivation

10 (18-88)

Staining around the snout

7 (16-56)

 

 

Table 2: Summary incidence of daily clinical observations for females

Dose level [mg/kg bw/day]

Number of animals

Number of animals which have died or have been killed due to animal welfare reasons

Clinical observations

Number of animals showing effect (day of observation)

0

10

0

no abnormalities detected

-

100

10

0

no abnormalities detected

-

300

10

0

no abnormalities detected

-

1000

10

0

Increased salivation

10 (9-87)

 

 

Table 3: Increase in body weight in [g] for males (10 animals per group)

Day numbers

0 (Control)

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

1-8

34.4 ± 4.8

29.2 ± 5.2

36.7 ± 5.6

30.9 ± 6.2

8-15

25.3 ± 3.8

22.0 ± 6.0

27.6 ± 9.1

25.8 ± 4.6

15-22

21.3 ± 5.4

20.3 ± 6.5

22.2 ± 9.3

23.7 ± 5.3

22-29

23.9 ± 4.6

17.3 ± 5.4*

22.1 ± 5.9

23.4 ± 4.5

29-36

17.1 ± 4.7

13.8 ± 5.9

16.5 ± 3.8

15.0 ± 6.3

36-43

16.6 ± 4.8

16.1 ± 5.5

18.2 ± 4.5

16.9 ± 3.2

43-50

13.9 ± 4.3

13.6 ± 3.1

13.1 ± 5.1

13.0 ± 2.7

50-57

14.0 ± 4.1

11.3 ± 3.1*

11.5 ± 2.1*

10.9 ± 3.4*

57-64

8.1 ± 4.5

9.3 ± 2.3

10.5 ± 5.8

7.4 ± 3.9

64-71

 7. 7 ± 3.3

4.7 ± 3.5

4.6 ± 4.8

5.5 ± 4.9

71-78

8.3 ± 3.4

6.2 ± 5.6

10.2 ± 4.0

8.0 ± 1.9

78-85

5.5 ± 3.2

7.7 ± 4.5

7.4 ± 5.1

101.0 ± 3.0*

85-91

4.5 ± 4.9

4.0 ± 4.5

5.1 ± 3.0

6.6 ± 4.5

Abs. Gain 1-91

200.6 ± 27.0

175.5 ± 31.5

205.7 ± 42.9

197.1 ± 27.8

% Gain

96.0 ± 11.4

85.7 ± 13.7

98.0 ± 19.8

96.1 ± 10.9

* Significantly different from control: p < 0.05

 

 

Table 4: Group mean hematological values for males (10 animals per group)

Parameters

0 (Control)

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Hb [g/dL]

16.43 ± 1.11

17.02 ± 0.55

16.76 ± 0.46

16.41 ± 0.69

RBC [10^12/L]

8.806 ± 0.652

9.429 ± 0.315*

9.003 ± 0.219

9.111 ± 0.519

Hct [%]

46.93 ± 3.23

48.97 ± 1.67

48.29 ± 1.69

48.25 ± 2.33

MCH [pg]

18.68 ± 0.87

18.06 ± 0.66

18.63 ± 0.5

18.04 ± 0.76

MCV [fL]

53.32 ± 1.4

51.97 ± 1.53

53.62 ± 1.5

53.00 ± 1.53

MCHC [g/dL]

35.02 ± 1.15

34.73 ± 0.49

34.74 ± 0.64

34.04 ± 0.64**

WBC [10^9/L]

7.69 ± 1.19

7.03 ± 1.61

7.18 ± 1.21

6.78 ± 0.88

Neut [10^9/L]

1.359 ± 0.448

1.123 ± 0.619

1.173 ± 0.384

1.192 ± 0.347

Lymph [10^9/L]

6.232 ± 1.097

5.871 ± 1.396

5.948 ± 1.076

5.568 ± 0.707

Mono [10^9/L]

0±0 n

0±0 n

0±0 n

0±0 n

Eos [10^9/L]

0.102 ± 0.072

0.0358 ± 0.055*

0.061 ± 0.037*

0.022 ± 0.036**

Bas [10^9/L]

0±0 n

0±0 n

0±0 n

0±0 n

CT [seconds]#

9.25 ± 0.41

9.56 ± 0.53

9.51 ± 0.67

9.79 ± 0.63*

PLT [10^9/L]

539.0 ± 120.8

555.3 ± 70.1

562.0 ± 55.5

590.6 ± 62.4

APTT [seconds]#

15.74 ± 1.86

15.53 ± 1.65

15.61 ± 1.54

16.65 ± 1.94

* Significantly different from control: p < 0.05

** Significantly different from control: p < 0.01

n: data not appropriate for statistical analysis

# group mean coagulation values

 

Erythrocyte count (RBC)

Hematocrit (Hct)

Erythrocyte indices - mean corpuscular hemoglobin (MCH)

- mean corpuscular volume (MCV)

- mean corpuscular hemoglobin concentration (MCHC)

Total leucocyte count (WBC)

Differential leucocyte count - neutrophils (Neut)

- lymphocytes (Lymph)

- monocytes (Mono)

- eosinophils (Eos)

- basophils (Bas)

Platelet count (PLT)

Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed.

Prothrombin time (CT) was assessed by ‘Innovin’ and activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

 

 

Applicant's summary and conclusion