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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Due to the preliminary nature of this study no specific regulations or guidelines were applicable.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
see attached document
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: Trinol
Identity Trinol or FR-513
Chemical name: Tribromoneopentyl alcohol
Action: Flame retardant
Description: White/off-white flakes
Storage conditions: Ambient temperature
Purity: 98.4%

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Crl:CD(SD) rats (a total of 25 male and 25 female) were received from Charles River (UK).
The rats were ordered at 29 to 35 days of age and within a weight range of 118 to 145 g for
males and 108 to 135 g for females.
On arrival, the animals were removed from the transit boxes and allocated to study cages.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with
the procedure being repeated until each cage held the appropriate number of animals. Each
sex was allocated separately.
The cages constituting each group were blocked together by sex and the groups were
dispersed in batteries so that possible environmental influences arising from their spatial
distribution were equilibrated, as far as was practicable. Additionally, batteries of cages were
rotated around the room at weekly intervals to further minimise possible spatial variations.
Each animal was assigned a number and identified uniquely within the study by a tail tattoo.
Each cage label was colour-coded according to group and was numbered uniquely with cage
and study number, as well as the identity of the occupants.
Before the start of treatment two females with bodyweights at the extreme of the weight
range were replaced with spare animals of suitable weight from the same batch.
The animals were allowed to acclimatise to the conditions described below for 12 days before
treatment commenced. For those animals selected for this study, their age at the start of
treatment was 41 to 47 days and their bodyweights were in the range of 209 to 272 g for
males and 161 to 197 g for females.
The spare animals were removed from the study room after treatment commenced.
Animals were housed inside a barriered rodent facility (Building 8, Room 0802). Before the
study the room was cleaned and disinfected.
Each animal room was kept at positive pressure with respect to the outside by its own supply
of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature
and relative humidity controls were maintained within the range of 19 to 23°C and 40 to 70%
respectively and monitored continuously. There were no deviations from these ranges during
the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and
12 hours continuous dark per 24 hours.
Alarms were activated if there was any failure of the ventilation system, or temperature limits
were exceeded. A stand-by electricity supply was available to be automatically brought into
operation should the public supply fail.
The animals were housed five of one sex per cage. The cages were made of a polycarbonate
body with a stainless steel mesh lid. Wood based material was used as bedding and was
sterilised by autoclaving and changed at appropriate intervals each week. Cages, food
hoppers and water bottles were changed at appropriate intervals.
The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1
Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or
prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles
fitted with sipper tubes.
Each cage of animals was provided with an Aspen chew block for environmental enrichment.
Chew blocks were provided throughout the study and were replaced when necessary. Each
cage of animals was provided with a plastic shelter for environmental enrichment, which was
replaced at the same time as the cages.
Each batch of diet was analysed routinely by the supplier for various nutritional components
and chemical and microbiological contaminants. Supplier’s analytical certificates were
scrutinised and approved before any batch of diet was released for use. The quality of the
water supply is governed by regulations published by the Department for Environment, Food
and Rural Affairs. Certificates of analysis were received routinely from the water supplier
and the suppliers of the bedding and Aspen chew blocks. Since the results of these various
analyses did not provide evidence of contamination that might have prejudiced the study,
they are not presented.
No other specific contaminants that were likely to have been present in the bedding, chew
blocks, diet or water were analysed, as none that may have interfered with or prejudiced the
outcome of the study was known.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
ml/kg
Details on oral exposure:
Animals received the test substance or vehicle control formulations orally at a volume-dose
of 5 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via
the mouth into the stomach.
All animals were dosed once each day at approximately the same time each day. Group 4
males were dosed for four consecutive days, whilst the remaining animals were dosed for
14 consecutive days. The volume administered to each animal was calculated from the most
recently recorded bodyweight.
A daily record of the weight of each formulation dispensed and the amount remaining after
dosing was made. The balance of these two weights was compared with the predicted usage
as a check that the doses had been administered correctly. The weight/mL for the
formulations on this study in corn oil was 0.920, 0.930, 0.948 and 1.015 for Groups 1, 2, 3
and 4, respectively. Huntingdon Life Sciences Standard Operating Procedures state that
“For formulations with a weight/mL (specific gravity) below 0.95g or above 1.05g the
following adjustment will be required: Expected Total Volume Dosed x Weight/mL
= Expected Weight used.” It was considered necessary for this adjustment to be made when
calculating the expected weight used for Groups 1, 2 and 3.
In addition, the normal acceptable deviations from acceptable volume were extended due to
the viscous nature of the corn oil formulations and low animal numbers to be dosed, which
may have resulted in a larger volume of wastage than a less viscous formulation. The
acceptable limits of dosing overage were 20% over and 1% under. On a few occasions these
limits were exceeded, however, this increase in usage was considered not to affect the
integrity of the study because it was most likely associated with wastage seen when wiping
the catheter prior to intubation.
Formulations were stirred using a magnetic stirrer before and throughout the dosing
procedure.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
The test substance, Trinol, was administered over a period of 14 consecutive days. The
necropsy procedures were completed on Day 15. Males receiving 1000 mg/kg/day were
killed early on Day 4 of treatment.
Frequency of treatment:
Animals received the test substance or vehicle control formulations orally at a volume-dose
of 5 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via
the mouth into the stomach.
All animals were dosed once each day at approximately the same time each day. Group 4
males were dosed for four consecutive days, whilst the remaining animals were dosed for
14 consecutive days.
Doses / concentrations
Remarks:
Doses / Concentrations:
The doses used in this study (0, 100, 300 and 1000 mg/kg/day). Concentrations used in this study (0, 20, 60, 200 mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
5 male and 5 female per dose
Control animals:
yes
Details on study design:
see attached document

Examinations

Observations and examinations performed and frequency:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Cages were inspected daily for evidence of ill-health amongst the occupants. Any
deviation from normal was recorded at the time in respect of nature and severity, date and
time of onset, duration and progress of the observed condition, as appropriate.
Daily detailed observations were recorded at the following times in relation to dose
administration:
Immediately before dosing
Immediately after dosing on return of the animal to its cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day
In addition, a more detailed weekly physical examination was performed on each animal to
monitor general health.
During the acclimatisation period, observations of the animals and their cages were recorded
at least once per day.
Sacrifice and pathology:

Animals were killed by carbon dioxide asphyxiation. The sequence in which the animals
were killed after completion of treatment was selected to allow satisfactory inter-group
comparison.

All animals were subject to a detailed necropsy.
After a review of the history of each animal, a macroscopic examination of the tissues was
performed. All external features and orifices were examined visually. After ventral mid-line
incision, the thoracic and abdominal cavities and their viscera were exposed and examined in
situ. Any abnormal position, morphology or interaction was recorded. If observations
indicated possible neurotoxic action the cranial roof was removed to allow observation of the
brain, pituitary gland and cranial nerves.
The requisite organs were weighed and external and cut surfaces of the organs and tissues
were examined as appropriate. Any abnormality in the appearance or size of any organ and
tissue was recorded and abnormalities were preserved in appropriate fixative (10% Neutral
Buffered Formalin).

The following organs, taken from each animal were dissected free of adjacent fat and other
contiguous tissue and the weights recorded:
Liver
Kidneys
Spleen
Bilateral organs were weighed together. Organ weights were also adjusted for terminal
bodyweight, using the weight recorded before necropsy.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All males receiving 1000 mg/kg/day were killed for welfare reasons on Day 4 of treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
All males receiving 1000 mg/kg/day were killed for welfare reasons on Day 4 of treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight for males receiving 1000 mg/kg/day was affected to varying degrees prior to their early termination, with some animals losing weight whilst others appeared unaffected.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was considered to be unaffected by treatment.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The organ weight variations were considered to be within background weight ranges and therefore considered to be unaffected by treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlargement of the liver with associated dark areas was seen in one female receiving 1000 mg/kg/day.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality:
All males receiving 1000 mg/kg/day were killed for welfare reasons on Day 4 of treatment.
Last in-life signs included, abnormal gait, unresponsive, underactive, flat posture, prostrate
posture and high levels of urine staining in all males at this dosage. Macroscopic
examination revealed abnormal contents and pallor of the jejunum in three of the five
animals, but there were no other consistent macroscopic observations recorded for these
animals.
Signs:
In addition to the post dosing and clinical observations seen in the males killed prematurely,
post dose salivation and chin rubbing was observed on occasion in the majority of animals at
all dose levels. However, this is a commonly observed finding in animals where the testmaterial
is administered by oral gavage and as such is considered to be of no toxicological
significance.
The clinical observations not associated with dosing revealed three out of five females
receiving 1000 mg/kg/day to show urine staining during the treatment period (Day 4, 11 and
15), with this sign also observed at macroscopic examination.
Bodyweight
Bodyweight and bodyweight gains for those animals surviving the treatment period were
considered to be unaffected by treatment.
Bodyweight for males receiving 1000 mg/kg/day was affected to varying degrees prior to
their early termination, with some animals losing weight whilst others appeared unaffected.
3.4 Food consumption

Food consumption was considered to be unaffected by treatment.
Organ weights
The organ weight variations were considered to be within background weight ranges and
therefore considered to be unaffected by treatment.
Huntingdon Life Sciences
Macropathology
The macroscopic examination performed after 14 days of treatment revealed the following
intergroup differences:
Liver
Enlargement of the liver with associated dark areas was seen in one female receiving
1000 mg/kg/day.
The nature and incidence of all other findings were consistent with the commonly seen
background of macroscopic changes.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Females well tolerated doses up to 1000 mg/kg bw/day
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Males well tolerated doses up to 300 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
It is concluded that daily oral gavage administration of Trinol to CD rats at
doses up to 1000 mg/kg/day in females and 300 mg/kg/day in males was well tolerated and
these doses are considered to be the no-observed-adverse-effect-level (NOAEL). However,
doses of 1000 mg/kg/day in males necessitated premature sacrifice of these animals on Day 4
and was considered to exceed the maximum tolerated dose.
Executive summary:

The systemic toxic potential of Trinol (a flame retardant), to Crl:CD(SD) rats by oral

administration was assessed over a period of 14 days. Three groups, each comprising five

male and five female rats received Trinol at doses of 100, 300 or 1000 mg/kg/day. A

similarly constituted Control group received the vehicle, corn oil, at the same volume-dose.

The males assigned to the high dose group (1000 mg/kg/day) were killed early, on Day 4 of

treatment, due to the signs observed.

During the study, clinical condition, bodyweight, food consumption, water consumption

(visual assessment only), organ weight and macropathology investigations were undertaken.