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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28-day
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accord with established guideline

Data source

Referenceopen allclose all

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Study Plan target ranges for temperature and relative humidity were 22 ± 3ºC and 50 ± 20% respectively. Achieved ranges were 20-22ºC and 44-77% RH; the deviations from the target range for relative humidity were transient in na
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Remarks:
migrated information: paste
Details on test material:
Sponsor's identification : Bis(2,4-dichlorobenzoyl) peroxide (CAS 133-14-2), paste, 50% in silicone oil
Supplier : Akzo Nobel Polymer Chemicals, b.v.
Product name : Perkadox PD-50S-PS
EC name : Bis(2,4-dichlorobenzoyl) peroxide
Product number : 66190
CAS number : 133-14-2
Description : White paste
Chemical name : Di(2,4-dichlorobenzoyl) peroxide
Purity/Concentration : Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste (48.7%). The paste typically contains silicone oil
(polydimethylsiloxanes, CAS# 63148-62-9) at 50.7%, but the typical average concentrations do not constitute product specifications.
Batch number : 1012011395
Label : PERKADOX PD-50S-PS Batch number 1012011395
Date received : 08 November 2011
Storage conditions : Approximately -20ºC in the dark
Expiry date : 01 January 2013

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
3.1 Animals and Animal Husbandry
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Harlan Laboratories U.K. Ltd., Oxon, UK. On receipt the animals were
examined for signs of ill-health or injury. The animals were acclimatised for five days
during which time their health status was assessed. A total of forty animals (twenty
males and twenty females) were accepted into the study. At the start of treatment the
males weighed 155 to 175g, the females weighed 129 to 155g, and were approximately
six to eight weeks old.

The animals were housed in groups of five by sex in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C
Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. A
certificate of analysis of the batch of diet used is given in Addendum 1. Mains drinking
water was supplied from polycarbonate bottles attached to the cage. The diet and
drinking water were considered not to contain any contaminant at a level that might have
affected the purpose or integrity of the study. Environmental enrichment was provided in
the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire,
UK).

The animals were housed in a single air-conditioned room within the Harlan Laboratories
Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at
least fifteen air changes per hour and the low intensity fluorescent lighting was controlled
to give twelve hours continuous light and twelve hours darkness. Environmental
conditions were continuously monitored by a computerised system, and print-outs of
hourly temperatures and humidities are included in the study records. Study Plan target
ranges for temperature and relative humidity were 22 ± 3ºC and 50 ± 20% respectively.
Achieved ranges were 20-22ºC and 44-77% RH; the deviations from the target range for
relative humidity were transient in nature and were considered to have had no impact on
the purpose or integrity of the study.

The animals were randomly allocated to treatment groups using a stratified body weight
randomisation procedure and the group mean body weights were then determined to
ensure similarity between the treatment groups. The cage distribution within the holding
rack was also randomised. The animals were uniquely identified within the study by an
ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
3.2 Procedure
Dosages were selected in collaboration with the sponsor based on available toxicity data
including a preliminary seven day range-finder investigation (Harlan Study No.:
41103871) in the rat. In the preliminary study, a dosage of 1000 mg/kg bw/day was
associated with lower body weight gain and food intake for males but at a level that did
not preclude this dosage from further investigation of toxicity and it was therefore
selected as a high dosage in this twenty eight day toxicity study. The low and
intermediate dosages are selected to comply with the Globally Harmonised System of
Classification and Labelling of Chemicals.

The vehicle used for the preliminary study was Arachis Oil and as dosing formulations
proved satisfactory in that study the same vehicle was employed for this main 28 Day
Toxicity Study using a treatment volume of 4 ml/kg bw.

The test item was administered daily, for twenty-eight consecutive days, by gavage using
a stainless steel cannula attached to a disposable plastic syringe. Control animals were
treated in an identical manner with 4 ml/kg of Arachis oil BP.

The volume of test and control item administered to each animal was based on the most
recent body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
2.2 Preparation of Test Item
For the purpose of this study, the test item was prepared at the appropriate
concentrations in Arachis oil BP. The stability and homogeneity of the test item
formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical
Services. Results show the formulations to be stable for at least four hours but was
shown to be unstable after storage for 48 hours. Formulations were therefore prepared
on a daily basis and used within four hours of preparation.

Samples of test item formulations were taken on occasions during the study and
analysed for concentration of Bis(2,4-dichlorobenzoyl) peroxide (CAS 133-14-2), paste,
50% in silicone oil at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The
method used for analysis of formulations and the results obtained are given in Appendix
16.

METHOD OF ANALYSIS
1.1 Summary
The concentration of Bis(2,4-dichlorobenzoyl) peroxide (CAS 133-14-2) in the test item
formulations was determined by high performance liquid chromatography (HPLC) using
an external standard technique.
1.2 Samples
The test item formulations were initially diluted with tetrahydrofuran to aid dissolution
then further extracted with methanol to give a final, theoretical test item concentration of
approximately 0.1 mg/ml.
1.3 Standards
Standard solutions of test item were prepared in methanol after an initial dilution in
tetrahydrofuran to aid dissolution at a nominal concentration of 0.1 mg/ml. The standard
solutions contained the equivalent amount of vehicle to that of the relevant samples.
1.4 Procedure
The standard and sample solutions were analysed by HPLC using the following
conditions:
HPLC : Agilent Technologies 1200, incorporating autosampler
and workstation
Column : Prodigy 5μ C8 (250 x 4.6 mm id) at 40°C
Mobile phase : Methanol: water (90:10 v/v)
Flow-rate : 1 ml/min
UV detector
wavelength : 250 nm
Injection volume : 25 μl
Retention time : ~ 4 and 5 mins


With the exception of one occasion, formulations analysed during the study were
within 89-114% of nominal concentration confirming the accuracy of the formulation
procedure. On one occasion, the measured concentration at 7.5 mg/ml was only 62 % of
nominal, additional analyses at this concentration were within acceptable limits.
Formulation records indicated that the correct formulation procedure and quantity of test
item and vehicle had been used and it is considered that this atypical low value
represented an error during sampling rather than indicating low concentration in the
dosing formulation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
30 mg/kg bw; 300 mg/kg bw; 1000 mg/kg bw
Basis:
other: nominal as formulated; verified by analysis
No. of animals per sex per dose:
5 males; 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: top dose based on 7-day range finding study; low and intermediate doses selected to be complimentary to GHS classification scheme
- Rationale for animal assignment (if not random): random
Positive control:
Not Applicable

Examinations

Observations and examinations performed and frequency:
3.3 Observations
3.3.1 Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioural change
immediately before dosing, up to thirty minutes post dosing and one and five hours after
dosing during the working week. Animals were observed immediately before and after
dosing and one hour after dosing at weekends. All observations were recorded.

3.3.2 Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 25, all animals were observed
for signs of functional/behavioural toxicity. Functional performance tests were also
performed on all animals during Week 4, together with an assessment of sensory
reactivity to different stimuli. Observations were carried out from approximately two
hours after dosing on each occasion.

3.3.2.1 Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose
built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin colour
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988).
The scoring system used is outlined in The Key to Scoring System and Explanation for
Behavioural Assessments and Sensory Reactivity Tests.

3.3.2.2 Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors
were used to assess motor activity. Animals of one sex were tested at each occasion
and were randomly allocated to the activity monitors. The evaluation period was one
hour for each animal. The time in seconds each animal was active and mobile was
recorded for the overall one hour period and also during the final 20% of the period
(considered to be the asymptotic period, Reiter and Macphail, 1979).
Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each
animal was allowed to grip the proximal metal bar of the meter with its forepaws. The
animal was pulled by the base of the tail until its grip was broken. The animal was drawn
along the trough of the meter by the tail until its hind paws gripped the distal metal bar.
The animal was pulled by the base of the tail until its grip was broken. A record of the
force required to break the grip for each animal was made. Three consecutive trials
were performed for each animal. The assessment was developed from the method
employed by Meyer et al (1979).

3.3.2.3 Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and
proprioceptive stimuli. This assessment was developed from the methods employed by
Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to
Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity
Tests.
The following parameters were observed:
Grasp response Touch escape
Vocalisation Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

3.3.3 Body Weight
Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body
weights were also performed prior to terminal kill.

3.3.4 Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the
study. Food conversion efficiency was calculated retrospectively.

3.3.5 Water Consumption
Water intake was measured and recorded daily for each cage group.

3.3.6 Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from
each test and control group at the end of the study (Day 28). Blood samples were
obtained from the lateral tail vein. Where necessary repeat samples were obtained by
cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.
The methods used for haematological and blood chemical investigations are given in
Addendum 2 and normal ranges are shown in Addendum 4.

3.3.6.1 Haematology
The following parameters were measured on blood collected into tubes containing
potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin 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)
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).

3.3.6.2 Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes
containing lithium heparin anti-coagulant:
Urea Calcium (Ca++)
Glucose Inorganic phosphorus (P)
Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT)
Albumin Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation) Alkaline phosphatase (AP)
Sodium (Na+) Creatinine (Creat)
Potassium (K+) Total cholesterol (Chol)
Chloride (Cl-) Total bilirubin (Bili)
Bile acids


Sacrifice and pathology:
3.3.7 Pathology
On completion of the dosing period all animals were killed by intravenous overdose of
sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any
macroscopic abnormalities were recorded.
3.3.7.1 Organ Weights

The following organs, removed from animals that were killed at the end of the study,
were dissected free from fat and weighed before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus
Pituitary (post-fixation) Thyroid/Parathyroid
Prostate and Seminal Vesicles
(with coagulating glands and fluids)
Uterus with Cervix
Normal ranges for these organ weights are given in Addendum 5.

3.3.7.2 Histopathology
Samples of the following tissues were removed from all animals and preserved in
buffered 10% formalin except where stated:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint) Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and Rectum
pons) Salivary glands (submaxillary)
Caecum Sciatic nerve
Colon Seminal vesicles (with
Duodenum coagulating glands and fluids)
Epididymides ♦ Skin (hind limb)
Eyes * Spinal cord (cervical, mid-thoracic
Gross lesions and lumbar)
Heart Spleen
Ileum Stomach
Jejunum Testes ♦
Kidneys Thymus
Liver Thyroid/Parathyroid
Lungs (with bronchi)# Trachea
Lymph nodes (mandibular and mesenteric) Urinary bladder
Mammary gland Uterus & Cervix
Muscle (skeletal) Vagina
Oesophagus
All tissues were despatched to the histology processing Test Site (Harlan Laboratories
Ltd. Switzerland) for processing (Principal Investigator: S Gaehler). Initially the tissues
shown in bold from all control and 1000 mg/kg bw/day dose group animals were
prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with
Haematoxylin and Eosin for subsequent microscopic examination. Any macroscopically
observed lesions were also processed, together with the liver and spleen from all 30 and
300 mg/kg bw/day dose group animals. In addition, sections of testes and epididymides
♦ = preserved in Bouin’s fluid then transferred to Industrial Methylated Spirits (IMS) approximately
48 hours later
* = eyes fixed in Davidson’s fluid
# = Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative from all Control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff
(PAS) stain and examined.

Since there were indications of treatment-related changes, examination was
subsequently extended to include similarly prepared sections of thyroids, testes and
epididymides from all 30 and 300 mg/kg bw/day dose group animals and including
additional Periodic Acid-Schiff (PAS) staining for the testes and epididymides.
Microscopic examination was conducted by the Study Pathologist (Principal Investigator:
Dr. Yoshimasa Okazaki) at AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten,
Switzerland. A complete histopathology phase report, including methods, is presented in
Appendix 15.
Statistics:
3.4 Evaluation of Data
Data were processed to give summary incidence or group mean and standard deviation
values where appropriate. All data were summarised in tabular form.
Where considered appropriate, quantitative data was subjected to statistical analysis to
detect the significance of intergroup differences from control; statistical significance was
achieved at a level of p<0.05. Statistical analysis was performed on the following
parameters:
Grip Strength, Motor Activity, Body Weight Change, Haematology, Blood Chemistry,
Absolute Organ Weights, Body Weight-Relative Organ Weights
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics
Module as detailed below:
Where appropriate, data transformations were performed using the most suitable
method. The homogeneity of variance from mean values was analysed using Bartlett’s
test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA
with appropriate covariates. Any transformed data were analysed to find the lowest
treatment level that showed a significant effect, using the Williams Test for parametric
data or the Shirley Test for non-parametric data. If no dose response was found, but the
data shows non-homogeneity of means, the data were analysed 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 (nonparametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mortality. There were no unscheduled deaths on the study. Clinical Observations. Clinical signs were restricted to transient post-dosing salivation for three males receiving 1000 mg/kg bw/day on isolated occasions towards the end of the treatment period.
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality. There were no unscheduled deaths on the study. Clinical Observations. Clinical signs were restricted to transient post-dosing salivation for three males receiving 1000 mg/kg bw/day on isolated occasions towards the end of the treatment period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, body weight gain for females was lower than control during the first week of treatment; no overall body weight gain was clear
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, food conversion efficiency for females during the first week of treatment was slightly lower than control and a negative value for food conversion efficiency was observed during the last week of treatment,
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, water consumption was generally higher than control throughout most of the treatment period.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no adverse effects of treatment on haematology parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, MF elevated alanine aminotransferase and alkaline phosphatase levels; M/F 1000 mg/kg bw/day and M at 300 mg/kg bw/day, mean albumin/globulin ratio elevated; M/F 1000 mg/kg bw/day mean chloride levels elevated
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, increased absolute and body weight relative liver weights for both sexes, compared to control, were considered to be associated with the adaptive histopathological liver changes.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: cytoplasmic eosinophilic change of hepatocytes centrilobular region of the liver; 1000 mg/kg bw/day: thyroid follicular cell hypertrophy; minor testicular effects
Histopathological findings: neoplastic:
no effects observed
Details on results:
6. RESULTS
6.1 Mortality
There were no unscheduled deaths on the study.
6.2 Clinical Observations
A summary incidence of daily clinical observations is given in Table 1. Individual data is
presented in Appendix 1.
Clinical signs observed on the study were restricted to transient post-dosing salivation for
three males receiving 1000 mg/kg bw/day on one or two occasions towards the end of
the treatment period.
6.3 Functional Observations
A summary incidence of behavioural assessments is given in Table 2 and group mean
behavioural assessment scores are given in Table 3. Group mean functional
performance test values and standard deviations are given in Table 4 (statistically
significant differences are indicated). Individual values are given in Appendix 2 and
Appendix 3. Group mean sensory reactivity assessments are given in Table 5. Individual
responses are given in Appendix 4.
6.3.1 Behavioural Assessments
Assessment of the animals in a standard arena did not reveal any obvious effects of
treatment at 30, 300 or 1000 mg/kg bw/day.
6.3.2 Functional Performance Tests
Assessment of grip strength or motor activity did not reveal any consistent pattern of
results that indicated an adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.
During the assessment of motor activity, higher overall activity for females at 300 and
1000 mg/kg bw/day, compared to control, attained statistical significance; in the absence
of any dosage relationship this finding was considered to be incidental and unrelated to
treatment.

6.3.3 Sensory Reactivity Assessments
Sensory reactivity to different stimuli did not reveal any adverse effect of treatment at 30,
300 or 1000 mg/kg bw/day.
6.4 Body Weight
Group mean weekly body weights and standard deviations are given in Table 6 and are
presented graphically in Figure Error! Bookmark not defined. and Figure 2. Group
mean weekly body weight gains and standard deviations are given in Table 7
(statistically significant differences are indicated). Individual data are given in Appendix 5
and Appendix 6.
At 1000 mg/kg bw/day, mean body weight gain for females during the first week of
treatment was lower than control, with differences attaining statistical significance.
Thereafter, there were no statistically significant differences from control although a slight
mean body weight loss was observed during the last week of dosing. Overall body
weight gain for the treatment period was clearly lower than control.
For males at 1000 mg/kg bw/day, there were no statistically significant differences in
body weight gain during the first three weeks of treatment. Lower body weight gain
during the final week of treatment did attain statistical significance but there was little
impact on overall body weight gain, which was only slightly lower than control.
Body weight gain for either sex at 30 or 300 mg/kg bw/day was not obviously affected by
treatment.
6.5 Food Consumption and Food Efficiency
Group mean weekly food consumptions are given in Table 8 and are presented
graphically in Figure Error! Bookmark not defined. and Figure 4. Weekly food
efficiencies are given in Table 9.
There was no obvious effect of treatment on food consumption for either sex at 30, 300
or 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, food conversion efficiency for females during the first week of
treatment was slightly lower than control, reflecting the lower body weight gain observed
for these females during this time. A negative value for food conversion efficiency was
observed for these females during the last week of treatment, reflecting the slight mean
body weight loss observed for these females at this time.

There was no obvious effect on food utilisation for either sex at 30 and 300 mg/kg
bw/day or for males receiving 1000 mg/kg bw/day.
6.6 Water Consumption
Group mean daily water consumptions are given in Table 10.
At 1000 mg/kg bw/day, water consumption for both sexes was generally higher than
control, from the first or second day of treatment, throughout most of the treatment
period.
At 30 and 300 mg/kg bw/day there was no obvious adverse effect of treatment on water
intake for either sex.
6.7 Laboratory Investigations
6.7.1 Haematology
Group mean values and standard deviations for test and control group animals are given
in Table 11 (statistically significant differences are indicated). Individual data are given in
Appendices 7 to 9.
There were no differences from control for haematology parameters that were
considered to indicate an adverse effect of treatment.
For males at 1000 mg/kg bw/day, mean platelet count was statistically significantly
higher than control, however only two values exceeded the historical control range and,
in the absence of any histopathological change in the bone marrow, this finding was
considered to be of no toxicological significance.
For females at all dosages, mean prothrombin times were statistically significantly longer
than control but there was no consistent dosage relationship. With only one individual
value at 1000 mg/kg bw/day exceeding the historical control range, this finding was
considered to be incidental and unrelated to treatment.
For males at all dosages, mean cell haemoglobin concentration was lower than control
with differences attaining statistical significance; however, there was no dosage
relationship and all individual values for treated animals were within the historical control
range. In the absence of any other statistically significant differences for other
erythrocyte parameters, the differences observed for mean cell haemoglobin
concentration were considered to reflect unusually high control values (three of the five
PROJECT NUMBER: 41103872 PAGE 37
individual control values exceeded the historical control range) and were considered to
be unrelated to treatment.
6.7.2 Blood Chemistry
Group mean values and standard deviations for test and control group animals are given
in Table 12 (statistically significant differences are indicated). Individual data are given in
Appendix 10 and Appendix 11.
At 1000 mg/kg bw/day, higher alanine aminotransferase and alkaline phosphatase levels
for both sexes attained statistical significance when compared with control. The majority
of individual values for these treated animals exceeded the historical control range
compared to only a few isolated incidences for their control counterparts.
For both sexes at 1000 mg/kg bw/day and males at 300 mg/kg bw/day, higher mean
albumin/globulin ratio attained statistical significance when compared with control. All
individual values for these treated animals exceeded the historical control range but so
also did values for majority of control animals. The higher values for albumin/globulin
ratio were not accompanied by any statistically significant differences from control for
total protein or albumin levels and, in isolation, this finding was considered unlikely to
represent an effect of treatment.
For both sexes at 1000 mg/kg bw/day higher mean chloride levels were statistically
significantly higher than control, however only two individual male values exceeded the
historical control range. This finding was considered not to be of any great toxicological
significance.
For males at 300 and 1000 mg/kg bw/day, lower total cholesterol level also attained
statistical significance when compared with control, but again, all individual values for
treated animals were within the historical control range and this finding was considered
to be of no toxicological significance.
For males at all dosages, total bilirubin was lower than control with differences attaining
statistical significance, but all individual values for treated animals were within the
historical control range. In the absence of any histopathological correlates or effects on
erythrocyte parameters this finding was considered to be incidental and unrelated to
treatment.
For males at all dosages, lower blood urea levels attained statistical significance when
compared with control; there was no dosage relationship and only one individual control
value and one individual value at 1000 mg/kg bw/day exceeded the historical control
range. This finding was considered to be incidental and unrelated to treatment.
No statistically significant differences from control were apparent for blood chemistry
parameters for females at 30 and 300 mg/kg bw/day.
6.8 Pathology
6.8.1 Necropsy
A summary incidence of necropsy findings is given in Table 13. Individual data are given
in Appendix 12.
No macroscopic abnormalities were apparent at terminal necropsy.
6.8.2 Organ Weights
Group mean absolute and body weight-relative organ weights and standard deviations
for test and control group animals are presented in Table 14 (statistically significant
differences are indicated). Individual data are given in Appendix 13 and Appendix 14.
At 1000 mg/kg bw/day, increased absolute and body weight relative liver weights for both
sexes attained statistical significance when compared with control. Individual absolute
liver values for these treated animals exceeded the historical control range for two males
and two females. For body weight relative liver values, which are considered to be a
better indicator of toxicological effects, all individual values at 1000 mg/kg bw/day
exceeded the historical control range; however the majority of control values also
exceeded this historical range, albeit to a lesser extent. These findings were considered
likely to be associated with the adaptive histopathological liver changes observed at this
dosage and, therefore, while treatment-related they were considered not to represent an
adverse effect of treatment.
Additionally, for males at 1000 mg/kg bw/day, lower absolute and body weight relative
prostate/seminal vesicle weights attained statistical significance compared with control.
For these treated animals all absolute values exceeded the historical control range,
compared to only one control value, but when adjusted for body weight, only two values
remained outside the historical control range. In the absence of any histopathological
change for this tissue this finding was considered to be of no toxicological significance.
For males at 1000 mg/kg bw/day, higher absolute and body weight relative kidney
weights attained statistical significance when compared with control. For these treated
animals, all individual absolute values were within the historical control range (with one
control value being below this range) but, when adjusted for body weight, two individual
kidney values exceeded the historical control range. In the absence of any
histopathological change this finding was considered to be of no toxicological
significance.
For males at 1000 mg/kg bw/day, lower spleen weights attained statistical significance
when compared with control. Although, two absolute values for these treated animals
were below the historical control range, when adjusted for body weight, the individual
values were all within this historical range, while two body weight relative control values
exceeded the historical control range.
For females at 1000 mg/kg bw/day, lower absolute and body weight relative pituitary
weights attained statistical significance when compared to control. While three absolute
values for these treated animals were lower than the historical control range, when
adjusted for body weight all values were within the historical range. In the absence of
any histopathological change for this organ this finding was considered to be of no
toxicological significance.
For females at 300 and 1000 mg/kg bw/day, lower absolute and body weight relative
uterus weights attained statistical significance compared with control, but there was no
dosage relationship. All values for these treated animals were within the historical
control range, while values for two control females exceeded this historical range. This
finding appears to represent unusually high values for the control animals and was
considered to be unrelated to treatment.
For both sexes at 30 mg/kg bw/day and males at 300 mg/kg bw/day, there were no
differences in organ weights that indicated an effect of treatment.
For females at 30 mg/kg bw/day lower absolute and body weight-relative heart weights
attained statistical significance compared to control. In the absence of any similar
decrease in heart weights at higher dosages, these differences were considered
incidental and unrelated to treatment.
6.8.3 Histopathology
A complete histopathology report is presented in Appendix 15, the following is made in
summary.
For the liver, cytoplasmic eosinophilic change (increased cytoplasmic eosinophilic
stainability) of hepatocytes in the centrilobular region was observed at a minimal severity
in three males and three females at 1000 mg/kg bw/day. This hepatocytic cytoplasmic
change was considered to be of metabolic nature and of adaptive character, and hence,
deemed not to be adverse.
Thyroid follicular cell hypertrophy was recorded at minimal severity in one male and three
females of group 4 (1000 mg/kg bw/day). This change is deemed to be associated with
the increased hepatic metabolization of thyroid hormones (T3/T4) due to the above
mentioned hepatocellular cytoplasmic change. Such thyroidal change is deemed to
represent a secondary effect, and hence, deemed not to be adverse.
Increased Sertoli-cell vacuolation with focal, segmental tubular degeneration was
recorded in testes of three animals at 1000 mg/kg bw/day. In two of these males,
spermatid retention in stage X tubule was also observed at a minimal severity. In
addition, interstitial edema with inflammatory cell or mononuclear cell infiltration as well
as oligospermia and/or increased intraductal cellular debris were observed in the
epididymis of the same animals at 1000 mg/kg bw/day showing the histological findings
in the testis.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
No Observed Adverse Effect Level (NOAEL) for this study was 300 mg/kg bw/day
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: For both sexes at 1000 mg/kg bw/day and males at 300 mg/kg bw/day, mean albumin/globulin ratio was statistical significantly higher than control.
Dose descriptor:
NOEL
Effect level:
ca. 0 - ca. 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: lack of observed effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
It was considered that the No Observed Adverse Effect Level (NOAEL) for this study was 300 mg/kg bw/day and the No Observed Effect Level (NOEL) was 30 mg/kg bw/day.
Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity of the test item and follows the test method described in Commission Directive 96/54/EC (Method B7) and the OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity in Rodents" (adopted 03 October 2008) and was designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration,Evaluation, Authorisation and Restriction of Chemicals (REACH). Methods. The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP) over the same treatment period. Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Histopathological evaluation was extended to low and intermediate animals for those tissues considered to show treatment-related findings. Results. Mortality. There were no unscheduled deaths on the study. Clinical Observations. Clinical signs were restricted to transient post-dosing salivation for three males receiving 1000 mg/kg bw/day on isolated occasions towards the end of the treatment period. Behavioural Assessment. No obvious neurological effects of treatment at 30, 300 or 1000 mg/kg bw/day were apparent. PAGE 20 PROJECT NUMBER: 41103872 Functional Performance Tests. There was no consistent pattern of results that indicated an adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day. Sensory Reactivity Assessments. No adverse effect of treatment was apparent at 30, 300 or 1000 mg/kg bw/day. Body Weight. At 1000 mg/kg bw/day, body weight gain for females was lower than control during the first week of treatment and, although there were no subsequent statistically significant differences for body weight gain, overall body weight gain was clearly lower than control. For males at 1000 mg/kg bw/day, lower body weight gain was apparent during the final week of treatment and did attain statistical significance but overall body weight gain was only slightly lower than control. Body weight gain at 30 or 300 mg/kg bw/day appeared unaffected by treatment. Food Consumption. Food consumption was unaffected by treatment at 30, 300 or 1000 mg/kg bw/day. Food Efficiency. At 1000 mg/kg bw/day, food conversion efficiency for females during the first week of treatment was slightly lower than control and a negative value for food conversion efficiency was observed during the last week of treatment, reflecting slight mean body weight loss. Food conversion efficiency was unaffected for either sex at 30 and 300 mg/kg bw/day or for males receiving 1000 mg/kg bw/day. Water Consumption. At 1000 mg/kg bw/day, water consumption was generally higher than control throughout most of the treatment period. Water intake at 30 or 300 mg/kg bw/day appeared unaffected by treatment. Haematology. There were no adverse effects of treatment on haematology parameters. Blood Chemistry. At 1000 mg/kg bw/day, alanine aminotransferase and alkaline phosphatase levels for both sexes were statistically significantly higher than control. For both sexes at 1000 mg/kg bw/day and males at 300 mg/kg bw/day, mean albumin/globulin ratio was statistical significantly higher than control. For both sexes at 1000 mg/kg bw/day mean chloride levels were statistically significantly higher than control. PROJECT NUMBER: 41103872 PAGE 21 Necropsy. No macroscopic abnormalities were apparent at terminal necropsy. Organ Weights. At 1000 mg/kg bw/day, increased absolute and body weight relative liver weights for both sexes, compared to control, were considered to be associated with the adaptive histopathological liver changes. Histopathology. At 1000 mg/kg bw/day, cytoplasmic eosinophilic change of hepatocytes in the centrilobular region of the liver was observed; this was considered to be an adaptive metabolic response and did not represent an adverse effect. At 1000 mg/kg bw/day thyroid follicular cell hypertrophy was observed and was considered to reflect a secondary effect of the adaptive hepatocellular cytoplasmic change in the liver. For some males at 1000 mg/kg bw/day, increased Sertoli-cell vacuolation with focal, segmental tubular degeneration was observed in the testes accompanied, in some cases, by spermatid retention in stage X tubule. In addition, interstitial edema with inflammatory cell or mononuclear cell infiltration as well as oligospermia and/or increased intraductal cellular debris were observed in the epididymis of the males showing testicular change.

Conclusion. It was considered that the No Observed Adverse Effect Level (NOAEL) for this study was 300 mg/kg bw/day and the No Observed Effect Level (NOEL) was 30 mg/kg bw/day.