Bis(2,4-dichlorobenzoyl) peroxide

Endpoint summary

Description of key information

The oral (gavage) administration of Bis(2,4 -dichlorobenzoyl) peroxide (CAS# 133 -14 -2), paste, 50% in silicone oil, for ninety consecutive days, to Wistar HanTM rats (10/sex) of either sex at dose levels of 100, 300 or 1000 mg/kg bw/day resulted in treatment-related effects in body weight development, hematology parameters (anemia), sperm analysis, reduced testes, epididymides and cauda epididymis weight and microscopic changes in the bone marrow (fatty vacuolation and subsequent decreased cellularity), testes/ epididymides (tubular atrophy with an accompanying aspermia in the epididymis) and thymus (atrophy) at 1000 mg/kg bw/day and similar microscopic changes in the testes and epididymides in males treated with 300 mg/kg bw/day. At necropsy, nine high dose group males showed small testes and epididymides. Five of these also had flaccid testes and epididymides. One male treated with 300 mg/kg bw/day also showed small and flaccid testes and epididymides.  No such macroscopic effects were seen at 100 mg/kg bw/day. Sperm motility assessment: All of the high dose males showed no/very little alive sperm .The sperm concentration and % mobility in the 300 mg/kg bw/day group was also slightly reduced as compared to controls and one of which was the male that also had the macroscopic findings. These changes were considered to represent an adverse effect of treatment. No toxicologically significant effects were evident in females treated with 300 mg/kg bw/day or in animals of either sex treated with 100 mg/kg bw/day. There were also subgroups (4 week recovery period) at 0 and 1000 mg/kg bw/d. The 'No Observed Adverse Effect Level (NOAEL) from this OECD 408 study was therefore considered to be 300 mg/kg bw/day for females and 100 mg/kg bw/day for males.

Previously, in a 28 day oral gavage study in rats, histopathologic changes were noted in some organs at 1000 mg/kg/day. The change observed in the liver (cytoplasmic eosinophilic change of hepatocytes in the centrilobular region) was considered to be an adaptive metabolic response. 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, increased Sertoli-cell vacuolation with focal, segmental tubular degeneration was observed in the testes accompanied, in some cases, by spermatid retention in tubule. Additionally, 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: The NOAEL and NOEL for the 28day study were 300 and 30 mg/kg bw/day, respectively.

As the worst case scenario, the NOAEL of 100 mg/kg bw/d from the 90 -day study was chosen for this substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-day

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in accord with established guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study 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. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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.

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

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

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)

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.

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

Critical effects observed:

not specified

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 07 January 2016 and 29 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 September 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: see remarks

Version / remarks:

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).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST strain

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: Males: 205 - 241g, Females: 159 - 199g
- Fasting period before study: None
- Housing: in groups of three or four by sex in solid floor polypropylene cages
with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) was used. Certificates of analysis of the batches of diet used are in the raw data; expiry date for the batch number 070715MA was extended to 07 July 2016.
Mains drinking water was supplied from polycarbonate bottles attached to the cage.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3”C
- Humidity (%): 50 ± 20%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 February 2016 To: 03 June 2016

Route of administration:

oral: gavage

Details on route of administration:

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

Vehicle:

arachis oil

Details on oral exposure:

Dose Administration
Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL) Animal Numbers
Male Female
Control 0 4 0 10 (1-10) 10 (11-20)
Recovery Control 0 4 0 10 (81-90) 10 (91-100)
Low 100 4 25 10 (21-30) 10 (31-40)
Intermediate 300 4 75 10 (41-50) 10 (51-60)
High 1000 4 250 10 (61-70) 10 (71-80)
Recovery High 1000 4 250 10 (101-110) 10 (111-120)

For clarification, Animal No. 1 – 80 are referred to as non-recovery animals and Animals 81 - 120 are referred to as recovery animals.
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

The test item was administered daily, for ninety 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. Recovery group animals were maintained for a further twenty-eight days following termination of treatment.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of two peaks.

Preparation of Calibration Standards
Stock solutions of test item in extract solvent (see Section 2.2.2) were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with extract solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL. The standard solutions contained the equivalent amount of vehicle to that of the relevant samples.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument parameters below.
To assess the calibration range of the method, a range of standard solutions were prepared in extract solvent from a stock solution of 1.122 mg/mL by serial dilution using dilution solvent covering the concentration range 0.0561 mg/mL to 0.2244 mg/mL.

Preparation of Test Samples
The formulations received were diluted with extract solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with extract solvent this was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration. See Table 1 for preparation details.

Preparation of Accuracy and Precision Samples
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the test samples in section 2.2.4.
The concentration of Test Item in the final solution was quantified by HPLC using UV detection as detailed in the instrument parameters section 2.2.6.

Instrumentation Parameters

HPLC : Agilent Technologies 1100 and 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 : ~ 3.8 and 4.8 mins

Data Evaluation and Calculations
The peak area response for Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms was measured.
Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The linearity of detector response over the calibration standard concentration range.
The method accuracy (recovery) and precision, by analyzing five recovery samples at nominal concentrations of 25 mg/mL and 250 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study which was 0.0561 mg/mL.

Homogeneity and Stability in Vehicle Formulations
The homogeneity and stability of Test Item in Arachis Oil BP formulations was assessed at nominal concentrations of 25 mg/mL and 250 mg/mL during refrigerated storage.

Refrigerated storage (nominally +4ºC)
The formulations were analysed and refrigerated on receipt. On Day 21; the formulations were removed from storage and equilibrated to ambient temperature. The formulations were mixed as stated in the mixing procedure (described in the main part of this study) and single samples were removed for analysis from the top, middle and bottom of the mixed formulation.

Concentration of Dose Formulations
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved.

RESULTS
Method Validation
The analytical procedure was successfully validated for Test Item in Arachis Oil BP with respect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision. Results are summarized below.

The specificity of the analytical method was demonstrated by the absence of a peak at the characteristic retention time for Test Item in the control sample chromatogram.
The data was found to have a linear correlation within the calibration range of 0.0561 mg/mL to 0.2244 mg/mL. The R² fit of the calibration curve to the data was 0.9991, and was c onsidered to be acceptable.
Method accuracy (recovery) and precision were confirmed. A mean recovery value of 96% (CV=3.00%, n=5) was obtained for 25 mg/mL and 95% (CV=1.97%, n=5) was obtained for 250 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study which was 0.0561 mg/mL.


Homogeneity and Stability of Dose Formulations
The homogeneity and stability of Test Item in Arachis Oil BP formulations was assessed with respect to the level of concentration at nominal concentrations of 25 mg/mL and 250 mg/mL.
Homogeneity was confirmed at the initial stability time point. The mean analyzed concentration for the nine samples remained within 10% of the initial time zero value and the variation was less than 10%.

Concentration of Dose Formulations
The mean concentrations of Test Item in test formulations were within applied limits ±16%, confirming accurate formulation.

Duration of treatment / exposure:

90 Days

Frequency of treatment:

once daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The study was performed according to the study plan and was designed to investigate the systemic toxicity of the test item, by repeated oral administration to the Wistar Han™:RccHan™:WIST strain rat for a period of ninety consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of ten males and ten females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further twenty-eight days.
The dose levels were chosen in collaboration with the Sponsor and based on the results of previous toxicity work (Harlan Laboratories Ltd Study Number: 41103872). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Animal Welfare
The study was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Envigo Research Limited, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012. The conduct of the study may be reviewed, as part of the Envigo Research Limited, Shardlow,
UK Ethical Review Process.
The study was conducted in accordance with the UK Home Office Guidance document on Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation

Positive control:

No

Observations and examinations performed and frequency:

Serial Observations
General Observations/Measurements
Clinical Observations
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.
During the treatment-free period, animals were observed once daily. All observations were recorded.

Body Weight
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
Food consumption was recorded for each cage group at weekly intervals throughout the study. Weekly food efficiency was calculated retrospectively.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all non-recovery animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed:

Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior 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 Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. 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 nonrecovery 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. 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).

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 Behavioral Assessments and Sensory Reactivity Tests.

The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Ophthalmoscopic Examination
The eyes of all control and treated animals were examined pre-treatment and all non-recovery control and high dose animals before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of
the internal structure of the eye using an ophthalmoscope was performed.

Estrous Cycle Assessment
Vaginal smears were taken daily for 21 days, on all non-recovery test and control group females, during the final three weeks of dosing. The stage of estrous was recorded for each day.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all non-recovery animals from each test and control group at the end of the study (Day 90) and on all recovery group animals at the end of the treatment-free period (Day 118). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 91 and 119. Animals were not fasted prior to sampling.
Urinalytical investigations were performed on all non-recovery test and control group animals during Week 12 and on all recovery group animals during Week 16. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.
The methods used for hematological and blood chemical investigations and normal ranges are shown in the raw data.

Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
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).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

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++)

Urinalysis
The following parameters were measured on collected urine:

Volume Ketones
Specific Gravity Bilirubin
pH Urobilinogen
Protein Blood
Glucose Appearance

Sacrifice and pathology:

Terminal Investigations
Necropsy
On completion of the dosing period or in the case of recovery group animals, at the end of the treatment-free period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Sperm Analysis
At necropsy, the left testis and epididymis were removed from all males, dissected from connective tissue and weighed separately.
For the epididymis, the distal region was incised and a sample of the luminal fluid was collected and transferred to a buffer solution for analysis of sperm motility. The semen sample was assessed using an automated semen analyzer to determine the numbers of motile, progressively motile and non-motile sperm.
For the testis, the tunica albuginea was removed and the testicular tissue was stored frozen at approximately -20°C. The cauda epididymis was separated from the body of the epididymis and weighed. The cauda epididymis was frozen at approximately -20°C.
Morphological assessment was performed for all males on a sample of a minimum of 200 sperm, where possible, to determine the number with apparent structural anomalies.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:

Adrenals Ovaries
Brain Spleen
Right Epididymis Right Testis
Heart Thymus
Kidneys Uterus
Liver
Normal ranges for organ weights are given in the report and raw data.

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 pons) Rectum
Caecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Right Epididymis ♦ Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes * and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum (including Peyer’s patches) Right Testis ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Tongue•
Lungs (with bronchi)# Trachea
Lymph nodes (mandibular and mesenteric) Urinary bladder
Mammary gland Uterus (with cervix)
Muscle (skeletal) Vagina

All tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX)for processing. All tissues from non-recovery 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 Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of testis was stained with Periodic Acid-Schiff (PAS) from non-recovery control and 1000 mg/kg bw/day males and immunohistochemical staining of the kidneys for α-2u-globulin was also performed from additional sections from both kidneys for nonrecovery males from the control and high dose groups. Full details of staining reagents and equipment used is included in the raw data/study records.
Since there were indications of treatment-related testis, epididymis, thymus (both sexes) and bone marrow (both sexes) changes, examination was subsequently extended to include similarly prepared sections of the testis, epididymis, thymus (both sexes) and bone marrow (both sexes) from animals in the low, intermediate and recovery dose groups.

Pathology
Microscopic examination was conducted by the Study Pathologist. A peer review of the findings observed was conducted at the histopathology peer review test site. A complete histopathology phase report is presented in
Annex 1 and represents the consensus view of both pathologists.

Statistics:

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, Hematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights and Sperm analysis parameters.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased salivation was evident in animals of either sex treated with 1000 mg/kg bw/day between Days 13 and 89 (males) and Days 31 and 88 (females). Increased salivation was also evident to a lesser extent in animals of either sex treated with 300 mg/kg bw/day between Days 44 and 80 (males) and Days 49 to 50 (females).
No toxicologically significant effects were detected in animals of either sex treated with 100 mg/kg bw/day.
One female treated with 100 mg/kg bw/day had chromodacryorrhea on Day 41 and a further female from this treatment group had a stained snout on Day 91. Due to the isolated nature of these observations, and in the absence of a dose related response they were deemed to be unrelated to treatment with the test item. One control male showed increased salivation on Day 52 only. This was considered to be the result of the dosing procedure.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 mg/kg bw/day showed a statistically significant reduction (p<0.01) in body weight gain during the first week of treatment. Recovery was evident in females thereafter, however, males continued to show a statistically significant (p<0.01) reduction in body weight gain up to Week 10. Following the twenty-eight day treatment free period males that were previously given 1000 mg/kg bw/day showed statistically significant increases (p<0.05-0.01) in body weight gain.
No toxicologically significant effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.
Fluctuations in weekly group mean body weight gains were observed in males treated with 300 mg/kg bw/day during Weeks 7 and 9, in males treated with 100 mg/kg bw/day during Week 12 and in females from all treatment groups during Week 12, achieving statistical significance (p<0.05-0.01). Body weight gain during the remainder of the study for these animals was comparable to controls and there was generally no dose dependence therefore these findings were considered to represent normal biological variation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 mg/kg bw/day showed a slight reduction in food consumption during the first week of treatment. Recovery was generally evident thereafter however overall food consumption for these males was slightly lower (7%) than controls. Incidences of reduced food conversion efficiency was also evident in these males during the treatment period and generally followed the fluctuations seen in body weight gain.
No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Visual inspection of water bottles did not reveal any inter-group differences.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Opthalmoscopic examination of animals of both sexes from the control and 1000 mg/kg bw/day dose groups did not indicate any treatment-related differences.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the dosing period, anemia was evident in animals of either sex treated with 1000 mg/kg bw/day; with statistically significant reductions in hemoglobin (p<0.01), erythrocyte count (p<0.01) and hematocrit (p<0.01) being evident. Females from this treatment group also showed statistically significant reductions in mean corpuscular hemoglobin (p<0.01) and mean corpuscular volume (p<0.01). Following the treatment free period, males previously given 1000 mg/kg bw/day showed statistically significant reductions in hemoglobin (p<0.05), erythrocyte count (p<0.05) and hematocrit (p<0.05). A number of individual values for the non-recovery animals were below historical control ranges. These intergroup differences were considered to be associated with the microscopic effects evident in the bone marrow of these animals and as such were considered to be an adverse effect of treatment.
No toxicologically significant effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.
Males treated with 300 mg/kg bw/day showed a statistically significant reduction in hemoglobin (p<0.05), erythrocyte count (p<0.01) and hematocrit (p<0.05). Males treated with 100 mg/kg bw/day also showed a statistically significant reduction in erythrocyte count (p<0.01) and hematocrit (p<0.05). Females treated with 300 mg/kg bw/day showed a statistically significant reduction in mean corpuscular hemoglobin (p<0.01) and mean corpuscular volume (p<0.01). Although similar effects were evident at 1000 mg/kg bw/day, all but one individual value for erythrocyte count in 100 mg/kg bw/day males were within historical control ranges for these parameters. Also there were no associated microscopic changes in the bone marrow, therefore the intergroup differences were considered not to be of toxicological significance. At the end of the treatment period, prothrombin time in males treated with 1000 mg/kg bw/day was statistically significantly (p<0.01) longer than controls.
This change was not observed in any of the female dose groups and all but one individual value was within historical control range, therefore it was deemed not to be of an adverse nature.
At the end of the treatment period, females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in lymphocytes (p<0.05). A dose relationship was not evident and the majority of individual values were within historical control range. This change was not observed in any of the male dose groups and was completely reversible in recovery females therefore the intergroup difference was considered not to be of toxicological importance. Group mean total leukocyte count in females from all treatment groups was statistically significantly higher than controls (p<0.05-0.01) which was considered primarily to be due to a higher neutrophil count in these animals (p<0.01). A dose-relationship was apparent in these females and most of the individual values for treated females exceeded the historical control data ranges. However, all of the individual control values for neutrophil count and four individual control values for total leukocyte also exceeded historical control ranges. There were no corresponding intergroup differences for males or in recovery females at the end of the treatment-free period and in the absence of any associated histopathological findings, these observations were considered to be of no toxicological relevance.
At the end of the treatment-free period, males previously given 1000 mg/kg bw/day showed a statistically significant increase in platelet count (p<0.05) whilst females previously given 1000 mg/kg bw/day showed a statistically significantly shorter activated partial thromboplastin time (p<0.05). Statistical significance was minimal and in the absence of similar effects in animals at the end of the treatment period, the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.
At the end of the treatment period, animals of either sex treated with 1000 mg/kg bw/day showed a statistically significant reduction in total protein (p<0.05-0.01) and statistically significant increases in aspartate aminotransferase (p<0.01) and alanine aminotransferase (p<0.01). Animals of either sex treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in albumin/globulin ratio (p<0.01) with males from these treatment groups also showing a statistically significant increase in alkaline phosphatase (p<0.01). These intergroup differences may indicate minor perturbation in metabolism and the majority of individual values did exceed historical control ranges in a number of these parameters, however, in the absence of any associated microscopic hepatic changes evident, the intergroup differences were considered not to represent an adverse effect of treatment.
Animals of either sex from all treatment groups showed a statistically significant reduction in cholesterol (p<0.05-0.01) and females treated with 1000 mg/kg bw/day and males from all treatment groups also showed a statistically significant reduction in glucose (p<0.05-0.01). Females from all treatment groups and males treated with 1000 and 300 mg/kg bw/day also showed a statistically significant reduction in bilirubin (p<0.01). Whilst a number of control animals showed values which exceeded the historical data ranges and which may have contributed to these differences, the corresponding values from most test item treated animals were within these ranges. Although these intergroup differences may again indicate minor perturbation in metabolism, in the absence of any associated microscopic hepatic changes evident these differences were considered not to represent an adverse effect of treatment.
Non-recovery males treated with 1000 mg/kg bw/day showed a statistically significant increase in albumin (p<0.05) and phosphorus (p<0.05). The effect on albumin also extended to males treated with 300 mg/kg bw/day and these males also showed a statistically significant reduction in urea (p<0.05). Following the treatment-free period males that were previously given 1000 mg/kg bw/day continued to show a statistically significant increase in phosphorus. The majority of the individual values for the non-recovery animals were within historical control ranges and in the absence of a similar effect in females or a true dose related effect, the intergroup differences were considered not to be of toxicological significance.
At the end of the treatment-free period, males previously given 1000 mg/kg bw/day showed a statistically significant reduction in bile acids (p<0.05). Statistical significance was minimalnand in the absence of similar effects in animals at the end of the treatment period, the intergroup difference was considered not to be of toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no adverse effect of treatment with the test item at any dose level in animals of either sex on urinalysis parameters evaluated towards the end of the treatment or treatment free periods.
In non-recovery animals of either sex treated with 1000 mg/kg bw/day, urine volume was statistically significantly higher than controls (p<0.05-0.01). The remaining urinalysis parameters for these animals were similar to controls and in the absence any microscopic renal changes these findings were considered to be of no toxicological significance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioral Assessments
There were no changes in the behavioral parameters considered to be related to treatment with the test item at any dose level.

Functional Performance Tests
There were no intergroup differences at any dose level considered to be related to treatment with the test item.
Statistical analysis of the data did not reveal any significant intergroup difference.

Sensory Reactivity Assessments
Sensory reactivity scores across all test item-treated dose groups were similar to controls.

Immunological findings:

not examined

Description (incidence and severity):

At the end of the treatment period, males treated with 1000 mg/kg bw/day showed a statistically significant reduction in testes (p<0.01), epididymides (p<0.01) and cauda epididymis (p<0.05) weight both absolute and relative to terminal body weight. Animals of either sex treated with 1000 mg/kg bw/day showed a statistically significant reduction in absolute and relative thymus weight (p<0.05). Following the treatment free period, males that were previously given 1000 mg/kg bw/day showed a statistically significant reduction in testes (p<0.01), epididymides (p<0.01) and cauda (p<0.01) weight both absolute and relative to terminal body weight.
No toxicologically significant effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.
At the end of treatment period, males treated with 1000 mg/kg bw/day showed a statistically significant reduction in spleen weight (p<0.01) both absolute and relative to terminal body weight. Although the majority of individual values for absolute weights were below historical control range, the majority of individual values for relative weights were within historical control range. Relative weights are considered to be a better indicator of a true effect of treatment and in the absence of any associated micropscopic spleen changes in these animals, the intergroup difference was considered not to be of toxicological significance. Animals of either sex treated with 1000 and 300 mg/kg bw/day and females treated with 100 mg/kg bw/day showed a statistically significant increase in liver weight (p<0.05-0.01) both absolute and relative to terminal body weight. Although the majority of individual values exceeded historical control ranges a true dose related response in males was not
evident. The increase in liver weights may also be associated with the blood chemical changes identified which again may indicate minor perturbation in metabolism, however, in the absence of any associated microscopic hepatic changes these differences were considered not to represent an adverse effect of treatment. At the end of treatment period, females from all treatment groups and males treated with 300 and 100 mg/kg bw/day showed a statistically significant increase in absolute and relative kidney weights (p<0.05-0.01). Males treated with 300 mg/kg bw/day also showed a statistically significant increase in absolute and relative adrenal weights (p<0.01) and males treated with 100 mg/kg bw/day also showed statistically significant increases in absolute and relative brain (p<0.01) and adrenal (p<0.05) weights. Although there were a number of individual values outside of historical control ranges for these organs a true dose related response was not evident and in the absence of any histopathology correlates, these findings were considered not to be of any toxicological significance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period nine males treated with 1000 mg/kg bw/day had small testes and epididymides at necropsy. Five of these males also had flaccid testes. Following the treatment free period, nine males that were previously given 1000 mg/kg bw/day had small testes and epididymides and eight of these males also had flaccid testes. One male treated with 300 mg/kg bw/day also had small and flaccid testes and small epididymides. These observations were associated with the microscopic changes identified in the testes and epididymides. No such effects were detected in males treated with 100 mg/kg bw/day.
No toxicologically significant macroscopic abnormalities were detected in treated females.
A number of animals across most dose groups including controls showed reddened lungs. Such findings are common in this type of study and were considered unrelated to treatment with the test item. A non-recovery female treated with 1000 mg/kg bw/day had dark and mottled kidneys and one female treated with 300 mg/kg bw/day had reddened mandibular lymph nodes. Following the treatment free period, one female that was previously given 1000 mg/kg bw/day had a small left ovary and one male that was previously given 1000 mg/kg bw/day had enlarged fluid filled kidneys with increased pelvic space. In the absence of any associated treatment-related microscopic changes, these findings were considered to be incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related microscopic abnormalities were detected:
Testis and Epididymis: Tubular atrophy, marked or moderate was present in all males treated with 1000 mg/kg bw/day with an accompanying aspermia in the epididymis. This persisted in recovery animals with only one male showing any evidence of sperm production (mild degeneration with cellular debris in the epididymis). In males treated with 300 mg/kg bw/day, minimal degeneration in the seminiferous tubules (scattered necrotic cells) was present in three animals with minimal debris present in the epididymis of seven animals. No such effects were detected in males treated with 100 mg/kg bw/day.

Thymus: Thymic atrophy minimal or mild was present in nine males and seven females treated with 1000 mg/kg bw/day. This was also evident in one male treated with 300 mg/kg bw/day however this occurrence in one animal is considered to be incidental. The thymic changes had completely resolved after the recovery period.

Bone Marrow: Increased fatty vacuolation (and subsequent decreased cellularity) was present at a mild or minimal level in all males and at a mild level in three females treated with 1000 mg/kg bw/day. At 300 and 100 mg/kg bw/day, a minimal increase was also evident in two males from each dose level. The low incidence and minimal severity at 300 and 100 mg/kg bw/day along with the lack of a dose dependant response suggests that this is incidental. After the recovery period this was present in six males and two females, all at a minimum severity. This indicates evidence of recovery although reversibility was not complete after the recovery period.
Zona glomerulosa hypertrophy was present in the adrenal glands, at a mild level in control and treated animals and whilst there was an increase in incidence in males treated with 1000 mg/kg bw/day, the change was not out-with control limits and was considered to be incidental and part of normal variation within this study.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Estrous Cycling
There was no adverse effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the last three weeks of dosing.
One control female and one female treated with 300 mg/kg bw/day was in diestrus during the first two weeks of the estrous cycling assessment phase. Both of these females then showed stages of estrus during the final week therefore the extended period of diestrus was considered to be the result of the females being in pseudo pregnancy. These were incidental findings and of no toxicological significance.

Sperm Analysis
At the end of the dosing period, males treated with 1000 and 300 mg/kg bw/day showed reduced sperm concentration, motility and progressive motility in relation to controls. Statistical significance was achieved at 1000 mg/kg bw/day (p<0.01) and 3/10 males treated with 1000 mg/kg bw/day did not show any sperm. At 1000 mg/kg bw/day, all males with sperm showed a marked increase in the number of abnormal sperm with 4/10 males from the 300 mg/kg bw/day dose group also showing a slight increase. The sperm abnormalities included misshapen sperm, sperm with abnormal hook, head only, no head and short tail. At the end of the treatment-free period, recovery males previously given 1000 mg/kg bw/day showed a reduction in sperm concentration, motility and progressive motility with 8/9 males showing no motile sperm and a marked increase in the number of abnormal sperm; these abnormalities were confined to misshapen sperm, sperm with two tails, head only, no head and short tail. These effects were associated with macroscopic findings identified as small and/or flaccid testes and small epididmides and treatment-related histopathological findings in these tissues from males treated with 1000 and 300 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

haematology

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

haematology

histopathology: non-neoplastic

other: Reproductive system changes and microscopic changes in the bone marrow) and thymus (atrophy) at 1000 mg/kg bw/day and similar microscopic changes in the testes and epididymides in males treated with 300 mg/kg bw/day.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

cauda epididymis

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

bone marrow

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The oral (gavage) administration of Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil, for ninety consecutive days, to Wistar rats of either sex at dose levels of 100, 300 or 1000 mg/kg bw/day resulted in treatment-related effects in body weight development, hematology parameters (anemia), sperm analysis, reduced testes, epididymides and cauda epididymis weight and microscopic changes in the bone marrow (fatty vacuolation and subsequent decreased cellularity), testes/epididymides (tubular atrophy with an accompanying aspermia in the epididymis) and thymus (atrophy) at 1000 mg/kg bw/day and similar microscopic changes in the testes and epididymides in males treated with 300 mg/kg bw/day. These changes were considered to represent an adverse effect of treatment. No toxicologically significant effects were evident in females treated with 300 mg/kg bw/day or in animals of either sex treated with 100 mg/kg bw/day. The 'No Observed Adverse Effect Level (NOAEL) was therefore considered to be 300 mg/kg bw/day for females and 100 mg/kg bw/day for males.

Executive summary:

The study was designed to investigate the systemic toxicity of Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil and is compatible with the following regulatory guideline:

The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

This study was also 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

Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of ten males and ten females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. Ophthalmoscopic examination was also performed on control and treated groups prior to the start of treatment and on non-recovery control and high dose animals during Week 12.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

Increased salivation was evident in animals of either sex treated with 1000 mg/kg bw/day and to a lesser extent animals of either sex treated with 300 mg/kg bw/day during the treatment period. No such effects were detected in animals of either sex treated with 100 mg/kg bw/day or for animals previously treated with 1000 mg/kg bw/day during the recovery period.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests

There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Animals of either sex treated with 1000 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Recovery was evident in females thereafter, however, males continued to show a reduction in body weight gain up to Week 10 of treatment. No toxicologically significant effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.

Food Consumption

Animals of either sex treated with 1000 mg/kg bw/day showed a slight reduction in food consumption during the first week of treatment. Recovery was generally evident thereafter however overall food consumption for these males was slightly lower than controls. Incidences of reduced food conversion efficiency was also evident in these males during the treatment period. No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.

Water Consumption

Visual inspection of water bottles did not reveal any inter-group differences.

Estrous Cycling

There was no adverse effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the last three weeks of dosing.

Ophthalmoscopy

Opthalmoscopic examination of animals of both sexes from the control and 1000 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment related differences.

Hematology

Non-recovery animals of either sex treated with 1000 mg/kg bw/day showed reductions in hemoglobin, erythrocyte count and hematocrit. Females from this treatment group also showed reductions in mean corpuscular hemoglobin and mean corpuscular volume. Following the treatment free period, males previously given 1000 mg/kg bwday showed reductions in hemoglobin, erythrocyte count and hematocrit. No toxicologically significant effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.

Blood Chemistry

Blood chemistry evaluations at the end of the treatment or treatment-free periods did not indicate any effects of toxicological relevance in animals of either sex resulting from test item administration.

Urinalysis

Urinalysis evaluations towards the end of the treatment or treatment-free periods did not identify any effects of toxicological importance in males or females receiving the test item up to a dose level of 1000 mg/kg bw/day.

Necropsy

Nine non-recovery males treated with 1000 mg/kg bw/day had small testes and epididymides at necropsy. Five of these males also had flaccid testes. Following the treatment free period, nine males that were previously given 1000 mg/kg bw/day had small testes and epididymides and eight of these males also had flaccid testes. One male treated with 300 mg/kg bw/day also had small and flaccid testes and small epididymides. No such effects were detected in males treated with 100 mg/kg bw/day. No toxicologically significant macroscopic abnormalities were detected in treated females.

Sperm Analysis

Sperm evaluation at the end of the treatment period identified reduced sperm concentration and motility which was associated with an increase in sperm morphological abnormalities in males treated with 1000 and 300 mg/kg bw/day. These observations persisted in recovery group males previously given 1000 mg/kg bw/day after a treatment-free period of twentyeight days. Microscopic examination of the right testis and the right epididymis identified treatment-related alterations at both 1000 and 300 mg/kg bw/day and the effect on sperm parameters was considered to be of an adverse nature.

Organ Weights

Non-recovery males treated with 1000 mg/kg bw/day showed a reduction in testes, epididymides and cauda epididymis weight both absolute and relative to terminal body weight. Non-recovery animals of either sex treated with 1000 mg/kg bw/day showed a reduction in absolute and relative thymus weight. Following the treatment free period, males that were previously given 1000 mg/kg bw/day showed a reduction in testes, epididymides and cauda epididymis weight both absolute and relative to terminal body weight. No toxicologically significant effects were detected in animals of either sex treated with 300 or

100 mg/kg bw/day.

Histopathology

The following treatment-related microscopic abnormalities were detected:

Testis and Epididymis: Tubular atrophy, marked or moderate was present in all males treated with 1000 mg/kg bw/day with an accompanying aspermia in the epididymis. This persisted in recovery animals with only one male showing any evidence of sperm production (mild degeneration with cellular debris in the epididymis). In males treated with 300 mg/kg bw/day, minimal degeneration in the seminiferous tubules (scattered necrotic cells) was present in three animals with minimal debris present in the epididymis of seven animals. No such effects were detected in males treated with 100 mg/kg bw/day.

Thymus: Thymic atrophy minimal or mild was present in nine males and seven females treated with 1000 mg/kg bw/day. This was also evident in one male treated with 300 mg/kg bw/day however this occurrence in one animal is considered to be incidental. The thymic changes had completely resolved after the recovery period.

Bone Marrow: Increased fatty vacuolation (and subsequent decreased cellularity) was present at a mild or minimal level in all males and at a mild level in three females treated with 1000 mg/kg bw/day. At 300 and 100 mg/kg bw/day, a minimal increase was also evident in two males from each dose level. The low incidence and minimal severity at 300 and 100 mg/kg bw/day along with the lack of a dose dependant response suggests that this is incidental. After the recovery period this was present in six males and two females, all at a minimum severity. This indicates evidence of recovery although reversibility was not

complete after the recovery period.

Conclusion

The oral (gavage) administration of Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil, for ninety consecutive days, to Wistar rats of either sex at dose levels of 100, 300 or 1000 mg/kg bw/day resulted in treatment-related effects in body weight development, hematology parameters (anemia), sperm analysis parameters, reduced testes, epididymides and cauda epididymis weight and microscopic changes in the bone marrow (fatty vacuolation and subsequent decreased cellularity), testes/ epididymides (tubular atrophy with an accompanying aspermia in the epididymis) and thymus (atrophy) at 1000 mg/kg bw/day and similar microscopic changes in the testes and epididymides in males treated with 300 mg/kg bw/day. These changes were considered to represent an adverse effect of treatment. No toxicologically significant effects were evident in females treated with 300 mg/kg bw/day or in animals of either sex treated with 100 mg/kg bw/day. The 'No Observed Adverse Effect Level (NOAEL) was therefore considered to be 300 mg/kg bw/day for females and 100 mg/kg bw/day for males.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Very good, performed according to OECD test guidelien and GLP guidelines.

System:

male reproductive system

Organ:

testes

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Data from a 90 -day repeated dose oral gavage study (OECD 408; GLP) revealed that 100 and 300 mg/kg bw/d are NOAEL in males and females, respectively. The NOAEL for males was chosen as the most sensitive NOAEL for this substance. Data from a previously performed 28day repeated dose oral gavage study (OECD 407, GLP), showed 300 mg/kg bw/d as the NOAEL.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: testes

Justification for classification or non-classification

The effects (NOAEL = 100 mg/kg bw/d in the subchroinc 90day study males) revealed macroscopic (organ weight, size), microscopic findings (tubular atrophy, aspermia), and functional effects (spem concentration, motility, morphological abnormalities) on the male sex organs. Therefore, this substance is classifed as Category 1B reproductive toxicant. These data are conclusive and thus, no further animal testing is deemed to be necessary for this endpoint.