Fatty acids, lanolin, esters with cholesterol-low lanolin alcs.

Endpoint summary

Description of key information

According to OECD 408 and under GLP conditions, Lanolin alcohols was administered by gavage to three groups, each of ten male and ten female rats, for 90 consecutive days, at dose levels of 100, 300 and 1000 mg/kg bw/day (Duster 2014). A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Observations and examinations of the animals included clinical signs, neurobehaviour, body weight, food consumption, hematology, blood chemistry, ophthalmoscopy, gross necropsy and histopathology.

There were no unscheduled deaths. Animals treated with 1000 mg/kg bw/day showed episodes of increased salivation (males/females) and episodes of red/brown staining around the snout (males). However, these observations are commonly observed following the oral administration of an unpalatable test item formulation and in isolation are not indicative of systemic toxicity.

Males treated with 1000 mg/kg bw/day and 100 mg/kg bw/day showed a statistically significant effects in functional performance (reduced mean forelimb grip strength, increased mean hindlimb grip strength. As 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. Thus, there were no treatment-related changes in behavioral parameters measured, sensory reactivity and no toxicologically relevant changes in functional performance.

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. Males treated with 100 mg/kg bw/day also showed a statistically significant reduction in body weight gain during Week 4. 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 slight reduction was considered not to be of toxicological significance. No adverse effects in overall food consumption, water consumption or food efficiency were detected in treated animals when compared to controls. There were no treatment-related ocular effects detected.

There were no toxicologically significant effects detected in the hematological and in the blood chemical parameters examined. Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in mean corpuscular hemoglobin concentration and a statistically significant increase in prothrombin time. Males from all treatment groups showed a statistically significant reduction in eosinophils. Males treated with 100 mg/kg bw/day also showed a statistically significant increase in erythrocyte count. 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.

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.

At necropsy, 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 hemorrhage in these animals which accounts for the macroscopic observations detected. These changes were not considered to represent a true treatment related effect. There were no treatment-related effects detected in the organ weights examined.

An increase in incidence of minimal or mild alveolar macrophages were 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.

In conclusion, the oral administration of Lanolin Alcohols to rats by gavage did not result in any toxicologically significant adverse effects. The NOAEL was therefore considered to be >= 1000 mg/kg bw/day for systemic toxicity.

A 90-day repeated dose toxicity study was performed in rats according to OECD 408, using Lanolin Fatty Acids (CAS 68424-43-1) (Braun, 2013). Ten Han-Wistar rats per sex and dose were administered 100, 300 and 1000 mg/kg bw/day of the test substance in corn oil by gavage, for 91 (females) or 92 (males) consecutive days. Control animals (10 per sex and dose) received the concurrent vehicle, corn oil. Observations and examinations of the animals included clinical signs, body weight, food consumption, haematology, clinical chemistry, urinalysis, organ weights, neurobehaviour, gross necropsy and histopathology. The daily oral administration of the test substance was tolerated without any adverse effects up to the high dose of 1000 mg/kg bw/day. One male of the 300 mg/kg bw/day dose group was killed in extremis subsequent to a dosing error. No further mortality was observed. There were no toxicologically significant effects on body weight, food consumption and clinical condition up to and including the highest dose level. There were no test item-related ophthalmoscopic changes at any dose level. There were no test item-related differences in the mean haematology, clinical chemistry and urine parameters at any dose level. Incidental changes of haematology parameters as reduced methaemoglobin levels (1000 mg/kg bw/day; males), reduced mean medium-fluorescene reticulocytes (1000 mg/kg bw/day; males), elevated mean relative eosinophils (300 mg/kg bw/day; females), and elevated mean and absolute eosinophils and reduced platelet count (100 mg/kg bw/day; females) were observed, which were statistically significant but remained within the range of the historical control values. The relative thrombin time was elevated in females of all dose groups and was outside the range of the historical controls. As the control values were also outside the historical control data, the effect was not considered to be treatment-related. 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. 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 ratio of these organs was not statistically significant. 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 oestrus cycling between control and treated animals. The male genital organs were also normal. In conclusion, as no adverse effects were observed up to the highest dose of 1000 mg/kg bw/day, a NOAEL of ≥1000 mg/kg bw/day was derived.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

(adopted 21 September 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Limit test:

no

Species:

rat

Strain:

other: Wistar Han(TM):RccHan(TM):WIST

Sex:

male/female

Details on test animals or test system 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

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

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

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)

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.

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

Critical effects observed:

not specified

 

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).

 

 

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: RccHanTM:WIST(SPF)

Sex:

male/female

Details on test animals or test system 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

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

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.

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.

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.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to the highest dose tested

Critical effects observed:

not specified

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

No study on the ester of lanolin alcohols and fatty acids is available. As the substance is expected to be hydrolysed by esterases and is hydrolysed abiotically, the toxicity of the hydrolysis products lanolin alcohols and lanolin fatty esters is representative for the toxicity of the substance

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

Mode of Action Analysis / Human Relevance Framework

The ester of lanolin alcohols and fatty acids can be hydrolysed abiotically and is expected to be hydrolysed by esterases. Therefore the toxicity of the hydrolysis products lanolin alcohols and lanolin fatty esters is considered representative for the toxicity of the substance

Justification for classification or non-classification

Based on the information available no classification for specific organ toxicity is considered necessary in absence of any treatment related adverse effects in the available studies on the hydrolysis products.