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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: modern GLP study with no significant protocol deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Batch number: E09K006
Date of receipt: 31 January 2012
Expiry date: January 2013

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Crl:CD (SD) rats (a total of 45 male and 45 female) were received from Charles River (UK)
Ltd. The rats were ordered at 29 to 35 days of age and within a weight range of 118 to 145 g
for males and 108 to 135 g for females. On receipt all animals appeared healthy and a sample
of 15 males and 15 females were weighed. All animals were found to be within the
acceptable weight range for this study.
On arrival, the animals were removed from the transit boxes and allocated to study cages.
Using the sequence of cages in the battery, one animal was placed in each cage. Each sex
was allocated separately.
Each animal was assigned a number and identified uniquely within the study by a tail tattoo.
Each cage label was colour-coded according to group and was numbered uniquely with cage
and study number, as well as the identity of the occupant.
Before the start of treatment, one male with non-resolving ophthalmic lesions and one female
with a bodyweight at the extreme of the weight range were replaced with spare animals of
suitable weight from the same batch.
The animals were allowed to acclimatise to the conditions described below for 12 days before
treatment commenced. For those animals selected for this study, their age at the start of
treatment was 41 to 47 days and their bodyweights were in the range of 212 to 261 g for
males and 164 to 213 g for females.
The spare animals were removed from the study room after treatment commenced.
2.3.2 Animal housing, diet and water supply
Animals were housed inside a barriered rodent facility (Building 8, Room 0816). The
facility was designed and operated to minimise the entry of external biological and chemical
agents and to minimise the transference of such agents between rooms. Before the study the
room was cleaned and disinfected.
Each animal room was kept at positive pressure with respect to the outside by its own supply
of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature
and relative humidity controls were maintained within the range of 19 to 230C and 40 to 70%
respectively and monitored continuously. There were no deviations from these ranges.
Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours
continuous dark per 24 hours.
Alarms were activated if there was any failure of the ventilation system, or temperature limits
were exceeded. A stand-by electricity supply was available to be automatically brought into
operation should the public supply fail.
The animals were housed individually. The cages were made of a polycarbonate body with a
stainless steel mesh lid. Softwood bark-free fibre was used as bedding provided at a depth of
2 cm (estimated by eye) and was sterilised by autoclaving and changed at appropriate
intervals each week. Cages, food hoppers and water bottles were changed at appropriate
intervals.
The cages constituting each group were blocked together by sex and the groups were
dispersed in batteries so that possible environmental influences arising from their spatial
distribution were equilibrated, as far as was practicable. Additionally, batteries of cages were
rotated around the room at weekly intervals to further minimise possible spatial variations.
The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1
Maintenance Diet) except overnight before routine blood sampling. This diet contained no
added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles
fitted with sipper tubes.
Each animal was provided with an Aspen chew block for environmental enrichment. The
soft white untreated wood blocks were provided throughout the study and were replaced
when necessary. A plastic shelter (tunnel) was also supplied for environmental enrichment;
and removed from the cage during periods of occlusion after administration and when an
Elizabethan collar was fitted, during the first five weeks of treatment. From the sixth week of
treatment, in an attempt to reduce the animal tampering with the semi-occlusive dressing,
nestlets (bedding material) were supplied to each cage, and were replaced when necessary,
and plastic shelters (tunnels) were provided at all times, with the exception of the 30 minute
period when an Elizabethan collar was worn. Following administration, two pellets of diet
were placed in the animal cage as forage food.
Each batch of diet was analysed routinely by the supplier for various nutritional components
and chemical and microbiological contaminants. Supplier's analytical certificates were
scrutinised and approved before any batch of diet was released for use. The quality of the
water supply is governed by regulations published by the Department for Environment, Food
and Rural Affairs. Certificates of analysis were received routinely from the water supplier
and the suppliers of the bedding, nestlets and Aspen chew blocks. Since the results of these
various analyses did not provide evidence of contamination that might have prejudiced the
study, they are not presented.
No other specific contaminants that were likely to have been present in the bedding, nestlets,
chew blocks, diet or water were analysed, as none that may have interfered with or prejudiced
the outcome of the study was known.

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
Treated animals received the test substance as a dermal administration followed by six hours
semi-occlusion of the dermal test site. Administration was performed on five days of each
week during Weeks 1 to 12 and daily thereafter, until the day of dispatch to necropsy in
Week 14. Individual dose volumes were measured using a micropipette (Days 1-3 only) or a
1 mL syringe (graduated to 0.01 mL). The test substance was initially applied to the middle
of the application site (6x6 cm; approximately 10% of the body surface area) to ensure the same area of skin was treated, and distributed evenly, as far as possible across the site, using
the tip of the micropipette (Days 1-3 only) or a metal spatula.
The control (sham-dosed) group was similarly treated, with the absence of the test substance.
All animals were dosed in sequence of animal/cage-number within each group. The volume
administered to each animal was calculated from the most recently recorded scheduled
bodyweight.
A record of the weight of each formulation dispensed and the amount remaining after dosing
was made. The balance of these two weights was compared with the predicted usage as a
check that the doses had been administered correctly. It is considered that any discrepancy in
the weight of material used was not significant.
Immediately after administration, unmedicated gauze dressing was held in position around
the trunk with a cotton wool pad, an open woven bandage and a tubigrip bandage. Sufficient
tension was used to ensure that the dose remained in contact with the skin. The dressing was
used to maximise the potential for absorption of the test material across the dermal barrier
and minimise the risk of oral ingestion.
After no less than six hours, the semi-occlusive dressing was carefully removed. To reduce
the risk of oral ingestion and to prevent residual test material being moved to the skin
adjacent, during grooming, the test site was cleansed with copious quantities of warm tap
water (approximately 30-35oC) and a mild, dilute soap (Simple™), using cotton wool. The
test site and surrounding hair were dabbed-dry with disposable paper towels. Fresh tubigrip
was used for each administration.
After the application site had been washed and dried an Elizabethan collar was fitted to each
animal for no less than 30 minutes. This procedure ensured that the application site was
completely dry before possible grooming could occur. The test site then remained
non-occluded until the next administration.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Substance applied neat. No need for analytical validation.
Duration of treatment / exposure:
6 hours / day, 5 days/week for 13 weeks
Frequency of treatment:
6 hours / day, 5 days/week for 13 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
125 mg/kg bw/day
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal per unit area
No. of animals per sex per dose:
15 animals per sex per dose.
Control animals:
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
Serial observations
Dated and signed records of all activities relating to the day by day running and maintenance
of the study within the animal unit as well as to the group observations and examinations
outlined in this experimental procedure were recorded in the Study Day Book. In addition,
observations relating to individual animals made throughout the day were recorded.
All observations described below were performed in animal/cage number sequence except
where otherwise indicated.
2.4.1 Clinical observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Any deviation from normal was recorded at the time in respect of nature and
severity, date and time of onset, duration and progress of the observed condition, as
appropriate.
Daily (on the five days the animals were treated/sham dosed) during the first week of
treatment and twice weekly (during the five day/week period when the animals were
treated/sham dosed) from Week 2 of treatment until termination, detailed observations were
recorded at the following times in relation to dose administration:
Immediately before dosing X
Immediately after dosing on return of the animal to its cage
On completion of dosing of each group
Between one and two hours after completion of dosing (bandage application)
As late as possible in the working day (following removal of all Elizabethan
collars) X
In addition, a more detailed weekly physical examination was performed on each animal to
monitor general health.
On each day of treatment all animals were assessed visually at approximately 3 to 4 hours
after dose administration in order to ensure that the dressings were still in place (see
Deviations from protocol).
X The dermal application site of each animal was examined for signs of irritancy before each
administration (see Deviations from protocol) and at removal of the semi-occlusive dressing,
according to the Draize, J.H. (1965) scoring system, as follows:
Erythema and eschar formation:
No erythema 0
Slight erythema 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4
Oedema formation
No oedema 0
Slight oedema 1
Well-defined oedema (area well-defined by definite raising) 2
Moderate oedema (edges raised approximately 1 mm) 3
Severe oedema (raised more than 1 mm and extending beyond the area of exposure) 4

2.4.2 Mortality
Debilitated animals were observed carefully and killed for reasons of animal welfare where
necessary. A complete necropsy was performed in all cases as described below.
2.4.3 Bodyweight
The weight of each rat was recorded one week before treatment commenced (Week -1), on
the day that treatment commenced (Week 0), weekly throughout the treatment period and
before necropsy. More frequent weighings were instituted, when appropriate, for animals
displaying ill-health, so that the progress of the observed condition could be monitored.
These data are not reported here.
2.4.4 Food consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for the week before treatment started (Week -1), and each week throughout the
treatment period until Week 13 (forage food (two pellets offered at dose administration) was
included in the calculations. Animals were fed ad-libitum during Week 14 until termination.
From these records the mean weekly consumption per animal (g/rat/week) was calculated for
each cage.
2.4.5 Ophthalmic examination
Before treatment commenced, the eyes of all animals allocated to the study (including spare
animals) were examined by means of a binocular indirect ophthalmoscope. One rejected
male was replaced with a male that had no adverse ocular abnormality, selected from the
spare animals for the study. During Week 13 of treatment the eyes of all animals of Groups 1
(Sham control) and 4 (1000 mg/kg/day) were similarly examined.
Prior to each examination, the pupils of each animal were dilated using tropicamide
ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber,
iris (pupil dilated), lens, vitreous and fundus were examined.
As no treatment-related changes were observed, the examination was not extended to animals
of Groups 2 or 3 (125 or 500 mg/kg/day).
Sacrifice and pathology:
Macroscopic pathology
All animals were subject to a detailed necropsy.
After a review of the history of each animal, a full macroscopic examination of the tissues
was performed. All external features and orifices were examined visually. The cranial roof
was removed to allow observation of the brain, pituitary gland and cranial nerves. After
ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and
pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position,
morphology or interaction was recorded.
The requisite organs were weighed and external and cut surfaces of the organs and tissues
were examined as appropriate. Any abnormality in the appearance or size of any organ and
tissue was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
2.5.3 Organ weights
The following organs, taken from each animal killed after 13 weeks of treatment, were
dissected free of adjacent fat and other contiguous tissue and the weights recorded:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
* Weighed after partial fixation
Spleen
Testes
Thymus
Thyroid with parathyroids^
Uterus with cervix
Bilateral organs were weighed together. Organ weights were also adjusted for terminal
bodyweight, using the weight recorded before necropsy.

Histology
For those animals specified in the Pathology section, the relevant tissues were subject to
histological processing.
Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four
to five micron thickness and stained with haematoxylin and eosin.
Those tissues subject to histological processing included the following regions:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Femur with joint - longitudinal section including articular surface, epiphysial
plate and bone marrow
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Liver - section from all main lobes
Lungs - section from two major lobes, to include bronchi
Spinal cord - transverse and longitudinal section at the cervical, lumbar and
thoracic levels
Sternum - included bone marrow
Stomach - included keratinised, glandular and antrum in sections
Thyroid - included parathyroids in section where possible
Uterus - uterine body with cervix section
For bilateral organs, sections of both organs were prepared. A single section was prepared
from each of the remaining tissues required for microscopic pathology.

Pathology
2.6.1 Light microscopy
Microscopic examination was performed as follows:
All tissues preserved for examination (as specified above) were examined for all
animals of Groups 1 (Sham control) and 4 (1000 mg/kg/day) sacrificed on
completion of the scheduled treatment period and for all animals killed during the
study. Additionally, the lungs were examined for all animals of Groups 2
(125 mg/kg/day) and 3 (500 mg/kg/day) sacrificed on completion of the scheduled
treatment period.
Tissues reported at macroscopic examination as being grossly abnormal were
examined in line with current practice.
Findings were either reported as "present" or assigned a severity grade. In the latter case one
of the following five grades was used - minimal, slight, moderate, marked or severe. A
reviewing pathologist undertook a peer review of the microscopic findings.
Other examinations:
2.4.6 Haematology, peripheral blood
During Week 13 of treatment, blood samples were obtained from all animals after overnight
withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane
and blood samples were withdrawn from the sublingual vein.
Blood samples (nominally 0.5 mL) were collected into tubes containing EDTA as
anticoagulant and examined for the following characteristics using a Bayer Advia 120
haematology analyser:
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Reticulocyte count (Retic)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total white cell count (WBC)
Differential WBC count
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Pit)
Morphology flags were generated by the Advia 120 analyser. The most common
morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were
categorised as follows:
= no abnormalities detected
+ = slight
++ = moderate
+++ = marked
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by
light microscopy, in the case of flags from the Advia 120 analyser. Confirmation or a written
description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate
anticoagulant and examined in respect of:
Prothrombin time (PTP) - using an ACL 3000 Plus analyser and IL PT-Fibrinogen
reagent
Activated partial thromboplastin time (APTT) - using an ACL 3000 Plus Analyser
and IL APTT reagent

2.4.7 Blood chemistry
At the same time and using the same animals as for peripheral haematology, lurther blood
samples (nominally 0.7 mL) were collected into tubes containing lithium heparin as
anticoagulant. All tubes were mechanically agitated for at least two minutes and the sample
subsequently centrifuged at 2000 g for 10 minutes in order to separate the plasma. After
separation, the plasma was examined in respect of:
Using a Roche P Modular Analyser:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Urea
Creatinine (Great)
Glucose (Glue)
Total cholesterol (Choi)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and
analysed albumin concentration.
Statistics:
See below

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
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 examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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:
no effects observed

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
dermal irritation
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No adverse effect level is the maximum dose tested, 1,000 mg/kg bw/day
Executive summary:

The systemic toxic potential of MCP2484 (a base oil), to Crl:CD(SD) (Sprague-Dawley) rats

by dermal (semi-occluded) administration, on five days of each week, to Sprague-Dawley

rats for 13 weeks was assessed at doses of 125, 500 or 1000 mg/kg/day.

One animal was killed for welfare reasons following six weeks of treatment as, following

attempts to remove the semi-occlusive bandage, it had caused physical trauma to its paws and

head, which was considered to be an exaggerated individual response to the restrictive

semi-occlusive dressing and not a result of treatment with MCP2484.

There were no clinical signs or signs related to administration and there were no dermal

reactions attributable to the test substance. Some animals in the Sham Control or treated

groups showed evidence of trauma, limited to the edges of the test site, including cuts,

encrustations, abrasions or red staining to feet (forelimbs and hindlimbs/feet) and a few had

scratches on the test site, which were considered attributable to attempts by the animal to

remove the bandage. In cases where the semi-occlusive bandage was removed, it was reapplied.

Data shows that there was a higher incidence of bandage removal in animals treated

with MCP2484 than in the Sham Control group; however, as there was no relationship with

dose and no evidence of a dermal response caused by the test substance, it was considered

that the physical nature of the test substance aided attempts to remove the bandage.

Bodyweight gain was considered to be unaffected by treatment and there was no effect of

treatment on food consumption and no ophthalmic change. It was considered that there was

no toxicologically important effect of treatment on haematology or chemical constituents of

the blood in Week 13, or organ weights following 13 weeks of treatment.

There were no histopathological findings related to treatment and assessment of the lungs of

all animals revealed no evidence of infection.