Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 May 2012 and 02 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study done under GLP and OECD guidelines

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
other: OPPTS 870.3050
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Sponsor's identification : F-3014
Description : off white powder
Chemical name : End capped brominated epoxy
Chemical formula : C6H2OBr3(C18H16O3Br4)nC9H8O2Br3
CAS number : 534584-61-7
Purity : Bromine content 59.5%
Batch number : 290110574
Date received : 10 November 2011
Storage conditions : room temperature in the dark
Expiry date : 04 September 2014

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: six to eight weeks old.
- Weight at study initiation: males weighed 208 to 236g, the females weighed 142 to 171g
- Housing:The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%):55 ±15%
- Air changes (per hr):The rate of air exchange was at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light):12hr/12 hr

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
B.P.
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): prepared twice during the tretment period.
- Mixing appropriate amounts with : appropriate concentration in Arachis oil BP
- Storage temperature of food: room temperature

VEHICLE
- Concentration in vehicle (mg/ml): 0 (Control); 0 (recovery period), 7.5 (low), 75 (intermediate), 250 (High) and 250 recovery high
- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
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). Two recovery groups,
each of five males and five females, were treated with the high dose (1000 mg/kg
bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained
without treatment for a further fourteen days.
Frequency of treatment:
Diet provided provided through out the study
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
low
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
intermediate
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
high
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control and recovery control
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
recovery high
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on previus study: SEVEN DAY REPEATED DOSE ORAL (GAVAGE) RANGE-FINDING TOXICITY
STUDY IN THE RAT
- Rationale for animal assignment (if not random): random
Positive control:
N/A

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes. Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body
weights were also performed prior to terminal kill and, in the case of recovery group
animals, on Days 36 and 43 prior to terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: Yes.
Food consumption was recorded for each cage group at weekly intervals throughout the
study. Food conversion efficiency was calculated retrospectively.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes.
Water intake was observed daily, for each cage group, by visual inspection of the water
bottles for any overt changes except during Week 3 where water intake was measured
gravimetrically. Where possible intergroup difference was detected during Week 3,
water consumption was continued to be measured and recorded for each cage group
until the termination of the study.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological and blood chemical investigations were performed on all non-recovery
test and control group animals at the end of the treatment period (Day 28) and on all
recovery group animals at the end of the treatment-free period (Day 42). Blood samples
were obtained from the lateral tail vein. Where necessary repeat samples were obtained
by cardiac puncture prior to necropsy on Days 29 and 43. Animals were not fasted prior
to sampling.

CLINICAL CHEMISTRY: Yes; Blood chemistry
- Parameters checked:
Urea Inorganic phosphorus (P)
Glucose Gamma glutamyltranspeptidase (γGT)
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+) Triglycerides (Tri)
Chloride (Cl-) Total cholesterol (Chol)
Calcium (Ca++) Total bilirubin (Bili)
Bile acids
URINALYSIS: Yes
- Parameters checked:
Volume Ketones
Specific Gravity Bilirubin
pH Urobilinogen
Protein Blood
Glucose
NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observations
Prior to the start of treatment and on Days 6, 14, 21 and 27, 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.
Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose
built arena. The following parameters were observed:
Gait and co-ordination 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
The 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.
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 tests were performed at
approximately the same time each day, 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 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).
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
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

see below
Statistics:
Data were processed to give summary incidence or group mean and standard deviation
values were 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,
Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative
Organ Weights
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics
Module as detailed below:
Where appropriate, data transformations were performed using the most suitable
method. The homogeneity of variance from mean values was analysed using Bartlett’s
test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA
with appropriate covariates. Any transformed data were analysed to find the lowest
treatment level that showed a significant effect, using the Williams Test for parametric
data or the Shirley Test for non-parametric data. If no dose response was found, but the
data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s
(parametric) or Steel (non-parametric) test to determine significant difference from the
control group. Where the data were unsuitable for these analyses, pair-wise tests was
performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinically observable signs of toxicity and mortality were detected
Mortality:
no mortality observed
Description (incidence):
No clinically observable signs of toxicity and mortality were detected
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No adverse effects on body weight change were detected for treated animals when compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effects were detected
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effects were detected.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was increased in males treated with 1000 or 300 mg/kg bw/day and during recovery period
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant haematological changes were detected.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically significant blood chemical changes were detected
Urinalysis findings:
no effects observed
Description (incidence and severity):
No toxicological important changes were detected in the urine parameters investigated.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant changes in organ weights were detected.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see attached data on results
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
see attached data on results

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of F-3014 by gavage for a period of twenty-eight consecutive
days at dose levels of 30, 300 and 1000 mg/kg bw/day resulted in treatment-related
findings in the liver, thyroid and pituitary glands.The findings were considered not to represent an adverse health effect; therefore a ‘No
Observed Adverse Effect Level’ (NOAEL) was established at 1000 mg/kg bw/day.
Executive summary:

The oral administration of F-3014 by gavage for a period of twenty-eight days resulted in

treatment-related changes in liver, thyroid and pituitary glands.

Centrilobular hepatocellular hypertrophy of the liver was found at minimal severity in

most of the males treated with 1000 mg/kg bw/day. The finding mostly resolved after the

treatment free period. The finding was not paralleled by increased degenerative or

inflammatory lesions and, therefore, is likely to represent an adaptive change due to

increased metabolism of the test item.

Follicular hypertrophy of the thyroid gland was found at minimal severity degrees in

males treated with 1000 mg/kg bw/day. After the treatment free period no increased

incidence of follicular hypertrophy was observed. Since no clear dose relationship could

be established after the investigation of intermediate dose groups and since the

background incidences in the recovery animals reached levels that were comparable to

the main study males treated with 1000 mg/kg bw/day, a treatment related effect is

unlikely but cannot completely be excluded.

Activated basophilic/chromophobe pituicytes were found in the pituitary gland in a dose

dependent manner in males treated with 30, 300 and 1000 mg/kg bw/day. The reason

for this finding is unclear. Since no increased incidence could be found after the

treatment free period, the finding is deemed to represent a non-adverse finding.

A ‘No Observed Effect Level’ (NOEL) could not be established; however a ‘No Observed

Adverse Effect Level’ (NOAEL) was established at 1000 mg/kg bw/day.