Registration Dossier

Administrative data

Description of key information

In a reliable study, in which rats were treated with hexadecanol via the diet for 13 weeks, a NOAEL of >4400 mg/kg bw/day (highest dose tested) was determined. Reduced weight gain, food consumption and organ weight changes were deemed to be secondary to the high dose administered but not specific to the test substance (Sci Assoc 1966).

A 28 -day study with hexadecanol in rats showed no effects up to the highest dose tested, 1000 mg/kg bw (Henkel 1985).

In a test according to OECD 408 and under GLP conditions, Lanolin alcohols was administered by gavage at dose levels of 100, 300 and 1000 mg/kg bw/day (Duster 2014). No toxicologically relevant adverse effects were reported at any of these doses. The NOAEL was therefore considered to be >= 1000 mg/kg bw/day for systemic toxicity.

NOAELs following repeated exposure to acetic acid and its salts range from 210 mg/kg bw/day (2-4 month acetic acid drinking water study; systemic toxicity) to 3600 mg/kg bw/day (acetic acid, sodium salt, 4 week dietary study; no effects reported). Signs of irritation/corrosion at the site of contact as well as systemic toxicity have been reported (HSDB acetic acid)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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:
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
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
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).

 

 

Conclusions:
The NOAEL is 1000 mg/kg bw
Executive summary:

In a test 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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not stated
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions. Report in German language, English summary page.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
(no neurobehavioural testing; limited range of endpoints assessed in other examinations)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: M 84-98 g; F 81-93g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- no data

VEHICLE
- Justification for use and choice of vehicle (if other than water): not stated
- Concentration in vehicle: 0, 2, 10 or 20%
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, 5 days/week
Remarks:
Doses / Concentrations:
100, 500, and 1000 mg/kg bw
Basis:
other: nominal conc.
No. of animals per sex per dose:
10 (main study) + 5 (satellite groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: reversibility
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random): no data
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: daily

FOOD EFFICIENCY: No data

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at end of study
- Dose groups that were examined: no data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 21/22 daily doses
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters examined.: Haematocrit, MCV, Hb, RBC, WBC, Thrombocytes, differential white count.

CLINICAL CHEMISTRY: Yes 
- Time schedule for collection of blood: After 21/22 daily doses
- Animals fasted: No data
- How many animals: No data
- Parameters examined: Serum urea, creatinine, Na, K, calcium, alkaline phosphatase, ALAT, ASAT, GT, bilirubin, chloride, albumin, total protein, cholesterol

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: thyroid, adrenals, thymus, kidney, spleen, heart, brain, testes, liver

HISTOPATHOLOGY: Yes, all organs from the control and top dose animals were examined plus the animals from the reversibility study.
Statistics:
T-test. U-test for organ weights
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No effects on mortality
Unremarkable other than top dose females appearing rather defensive when handled

BODY WEIGHT AND WEIGHT GAIN
No effects

FOOD CONSUMPTION
No effects

FOOD EFFICIENCY
No data

WATER CONSUMPTION
No effects

OPHTHALMOSCOPIC EXAMINATION
No effects

HAEMATOLOGY
No differences between treated and control animals other than an increase in neutrophils containing rodlike bodies observed in top dose females (confidence level 95%*). Values obtained (% rod like cells) were controls 2.5, low dose 3.3, mid dose 2.9, high dose 5.3*

CLINICAL CHEMISTRY
Statistically significant changes (*95% ** 99% confidence) in some clinical chemistry parameters were noted as follows:
- 500 mg/kg bw/day males increased potassium*,
- 500 mg/kg/day females increased GGT*, cholesterol** and chloride*
- glucose was elevated in top dose males (mmol/l): Control 6.03, Low  6.20, Mid 6.25, High 7.28**
These changes were not dose and/or sex related and not correlated with any histopathological findings and are therefore not considered of toxicological significance.

URINALYSIS
No effects

NEUROBEHAVIOUR
No data

ORGAN WEIGHTS
Both absolute and relative organ weights were essentially comparable in treated and control animals.
Sporadic changes were observed as follows (*95% ** 99% confidence):
- increase in absolute male kidney weight at 500 mg/kg/day*
- increase in absolute testis weight at 1000 mg/kg/day*; mean relative (absolute) testis weight: Control 0.856 (3.207), Low 0.839 (3.186), Mid 0.908 (3.455), High 0.893 (3.474*)
- the only change in relative organ weight was an increase in male adrenal weight at 1000 mg/kg/day*; mean relative (absolute) adrenal weight: Control 0.013 (0.050), Low  0.014 (0.054), Mid 0.014 (0.055), High 0.015* (0.058)

GROSS PATHOLOGY
No effects

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment related histopathological changes in test, control or reversibility groups.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not applicable

HISTORICAL CONTROL DATA (if applicable)
No data
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified
Conclusions:
In a reliable study, performed according to a protocol similar to OECD guideline 407, a 28-day oral NOAEL of 1000 mg/kg bw/day was determined in the rat. The study was performed in compliance with GLP.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not stated
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Valid with restrictions including lack of biochemical investigations and limited reporting of statistical findings. Study reasonably well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats treated via the diet for 90 days with limited evaluation
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc.
- Age at study initiation: no data but "young"
- Weight at study initiation: males 104.1 g, females 90.5 g
- Fasting period before study: no data
- Housing: individually in suspended wire-mesh cages
- Diet (e.g. ad libitum): Purina Laboratory Chow, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data but "controlled within narrow limits"
- Humidity (%): no data but "controlled within narrow limits"
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: no data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): basal laboratory diet (Purina Laboratory Chow)
- Storage temperature of food: no data
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuous in diet
Remarks:
Doses / Concentrations:
1%, 2.5%, 5-10%
Basis:
nominal in diet
No. of animals per sex per dose:
10 (treated), 20 (controls)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: no satellite groups
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 5 days/week
- Cage side observations included: general physical appearance, gross signs of systemic toxicity and/or pharmacological effect, behaviour, mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as mg/kg bw/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 30 and 90
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5/sex per group
- Parameters examined: microhaematocrit, haemoglobin, total and differential leukocyte count

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: days 30 and 90
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- How many animals: 5/sex per group (samples pooled)
- Parameters examined: albumin, acetone, bilirubin, colour, occult blood, sugar, pH, appearance, microscopic examination of sediment

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: brain, thyroid, heart, liver, spleen, kidneys, adrenals, gonads (testes or ovaries)

HISTOPATHOLOGY: Yes
- brain, thyroid, parathyroid, heart, lung, liver, spleen, stomach, small intestine, large intestine, pancreas, kidney, urinary bladder, adrenal, gonad, lymph node, bone, bone marrow
- all listed tissues from 5/sex from high dose and controls examined
Other examinations:
none
Statistics:
Chi-squared test for comparing relative organ weights. Original organ weight analyses using the Chi square test were supplemented by Tukey tests carried out by the Weinberg group (see 'Any other information on materials and methods')
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- all animals survived the 13 week treatment  period.
- all surviving animals appeared normal

BODY WEIGHT AND WEIGHT GAIN
- significantly reduced (84.7 - 89.8% of controls) in top dose males for most study weeks, in mid dose females at weeks 4-13 and high dose females (81.7-89.7%) throughout the study
- changes were attributed at least in part to reduced food consumption and the high content of test material in the diet.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- significantly reduced (76.4 - 89.2% of controls) in  top dose males at weeks 1 and 12, in mid dose males at week 13, in mid dose females at week 1 and high dose females weeks 1 and 12 (79.1 - 89.9%  of controls).

FOOD EFFICIENCY
- no data

WATER CONSUMPTION
- not examined

OPHTHALMOSCOPIC EXAMINATION
- not examined

HAEMATOLOGY
- no effects

CLINICAL CHEMISTRY
- not examined

URINALYSIS
- no effects

NEUROBEHAVIOUR
- not examined

ORGAN WEIGHTS
- the original report indicated that there were significant differences in some relative organ weights from treated groups compared to controls. These were reanalysed by the Weinberg Group using the Tukey test (see 'Remarks on results' section)

GROSS PATHOLOGY
- unremarkable

HISTOPATHOLOGY: NON-NEOPLASTIC
- there were no treatment related histopathological changes in the control and top dose animals examined (including testes & ovaries). 

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
- not applicable

HISTORICAL CONTROL DATA (if applicable)
- no data
Dose descriptor:
NOAEL
Effect level:
> 4 257 other: mg/kg (bw) based on highest dose tested.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
> 4 567 other: mg/kg (bw) based on highest dose tested.
Sex:
female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
In a reliable study, in which rats were treated with Alfol 16 via the diet for 13 weeks, an NOAEL of >4400 mg/kg bw/day (highest dose tested) was determined. Reduced weight gain, food consumption and organ weight changes were deemed to be secondary to the high dose administered but not specific to the test substance.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
210 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The substance under consideration is Reaction mass of Acetic acid, esters with lanolin alcs. and hexadecyl acetate and octadecyl acetate and oleyl acetate. It is expected that this ester will be hydrolysed to the alcohols (hexadecanol, octadecanol and lanolin alcohol) and acetic acid (by esterases). No adverse effects were observed in repeated dose studies with hexadecanol and lanolin alcohol.

It cannot be excluded that the hydrolysis product acetic acid will contribute to the overall toxicity of Reaction mass of Acetic acid, esters with lanolin alcs. and hexadecyl acetate and octadecyl acetate and oleyl acetate after oral administration. Therefore in the DNEL calculation the starting point will be the lowest NOAEL found for acetic acid in a worst case approach (210 mg/kg bw in rats 2 -4 months exposure). The contribution of acetic acid is expected to be much lower when exposure to the target substance is via the dermal route (as formation of acetic acid is expected only after absorption via the skin). Route-to-route extrapolation needs to be applied to account for this difference in absorption (and thus systemic exposure to the hydrolysis product acetic acid).

Additional information

Justification for classification or non-classification

Based on the information available the substance does not need to be classified for toxicity safter repeated dose according to EC Regulation 1272/2008 (CLP).