Registration Dossier

Administrative data

Description of key information

Repeated dose oral:
A repeated dose oral study (Dhinsa N K and Brooks P, 2008) was available which was of limited duration and hence is considered to be a supporting study. This study showed that Lowest Observed Adverse Effect Level (LOAEL) for the test substance is 150 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 02 July to 02 October 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Extensive study summary, no guideline but there were detailed information about mortality, haematology, blood chemistry, necropsy, histopathology and the other observations. However the study is only of 7 days duration, hence considered as reliable with restrictions.
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test material was administered by gavage to three groups, each of three male and three female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to seven consecutive days, at dose levels of 150, 500 and 1000 mg/lg/day. A control group of three males and three females was dosed with vehicle alone (Distilled water).
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD® (SD) IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approximately nine weeks old
- Weight at study initiation: The males weighted 335 to 385g, the females weighted 203 to 237g.
- Fasting period before study:
- Housing: The animals were housed in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flakes. The animals were housed in a single air-conditioned room within the test facility Barrier Maintained Rodent Facility.
- Diet (e.g. ad libitum): Free access to food, a pelleted diet used.
- Water (e.g. ad libitum): Free access to water, mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2℃
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fiffteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
seven days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 150, 500 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
3 males and 3 females per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted accordingly.
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
Positive control:
none stated
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately post dosing and one hour after dosing. Due to welfare concerns, additional observations were undertaken when necessary.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, 4 and prior to termination whenever possible

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 7
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 12
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 7
- Animals fasted: No
- How many animals: 12
- Parameters checked in table [No.?] were examined.

URINALYSIS: No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, liver and spleen
HISTOPATHOLOGY: Yes, spleen
Other examinations:
Organ weights
Statistics:
none stated
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
Mortality:
Two females treated with 1000 mg/kg/day, were killed in extremis on Day 1 and one female was killed in extremis on Day 2, following the severity of the clinical signs detected. All males treated with 1000 mg/kg/day, were found deaded on Day 2.
Animals of either sex treated with 500 mg/kg/day were killed in extremis on Day 3 due to the onset of clinical signs of toxicity similar to those observed at 1000 mg/kg/day.
There were no unscheduled deaths at 150 mg/kg/day.

Body weight:
At 1000 mg/kg/day, bodyweight losses were evident for all females prior to their early termination. All males showed bodyweight loss at 1000 mg/kg/day prior to early termination.
At 500 mg/kg/day, one female showed bodyweight loss before early termination and all remaining animals showed a reduction in bodyweight gain when compared to controls.
Slight reductions in bodyweight gains were evident for animals of either sex treated with 150 mg/kg/day, when compared to controls during treatment period.

Food consumption:
Slight reductions in food intake were evident for animals of either sex treated with 150 mg/kg/day when compared to controls.
Due to the early termination of the 1000 and 500 mg/kg/day dose groups, comparisons of dietary intake to control values could not be accurately measured.

Water consumption:
Daily visual inspection of water bottles did not reveal any overt differences in water consumption.

Haematology:
Males treated with 150 mg/kg/day displayed a statistically significant reduction in haemoglobin and haematocrit counts when compared to controls (p<0.01). Reductions in erythrocyte counts, mean cell haemoglobin and mean cell volume were also detected, although statistical significance was not achieved. Similar effects were also evident for females treated with 150 mg/kg/day, although statistical significance was not achieved for these parameters. A slight increase in mean cell haemoglobin was detected for animals of either sex treated with 150 mg/kg/day in comparison to controls. Furthermore, assessment of reticulocyte counts revealed a statistically significant increase for 150 mg/kg/day males when compared to controls (P<0.05).
A reduction in white blood cell counts was evident for 150 mg/kg/day females in comparison to controls, specifically in the lymphocyte and neutrophil fractions. Males treated with 150 mg/kg/day also showed slight reductions in lymphocyte counts; however neutrophil counts were slightly higher than control values.
Slight redutions in clotting times were detected for animals of either sex treated with 150 mg/kg/day in comparison to controls. Treated females also displayed a slight reduction in activated partial prothrombin times and platelet counts (p<0.05) and 150 mg/kg/day males showed an increase in platelet counts when compared to their respective controls.

Clinical chemistry:
Although statistical significance was never achieved, slight reduction in plasma urea levels were detected for animals of either sex treated with 150 mg/kg/day when compared to controls. Slighted increases in glucose and total protein leveals were also detected animals in comparison to controls.
Slight reductions in alanine aminotransferase and alkaline phosphatase levels were detected for animals of either sex treated with 150 mg/kg/day when compared to controls. Females treated with 150 mg/kg/day also showed a slight increase in aspartate aminotransferase levels in comparison to controls. Males also showed slight increases in cholesterol and bilirubin levels.Animals of either sex treated with 150 mg/kg/day showed lower inorganic phosphate levels in comparison to the concurrent controls.

Organ weights:
Statistically significant increases in thymus and liver weights, both absolute and relative to terminal bodyweights were detected for 150 mg/kg/day males, when compared to controls. Males from this dose group also showed higher spleen weights, both absolute and relative to terminal bodyweights in comparison to controls, although statistical significance was not achieved. Animals of either sex treated with 150 mg/kg/day showed higher absolute and relative heart and kidney weights when compared to controls, and slight increase in adrenal weights. Ovaries and uterus weights were higher for 150 mg/kg/day females and testis and epididymis were higher for 150 mg/kg/day males, when compared to controls.

Gross pathology:
Males treated with 1000 mg/kg/day showed enlarged liver and small spleen at necropsy.
No macroscopic abnormalities were detected for anminals treated with 500 and 150 mg/kg/day.

Histopathology:
SPLEEN: Generally higher grades of severity of extramedullary haemopoiesis were seen among males treated with 150 mg/kg/day. Although this is a normal background condition in the rat spleen the severities observed were considered higher than might normally be expected and, bearing in mind the small group size, a relationship to treated cannot be excluded. Instances of higher grades of severity of extramedullary haemopoiesis seen among other treatment groups are not reliably related to treatment since the affected animals were found dead early in the investigation.
All remaining morphological groups that were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.
No cause of death or moribundity was seen for any of the animals found dead or killed in extremis prior to scheduled termination.
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Critical effects observed:
not specified
Conclusions:
The LOAEL was considered to be 150 mg/kg/day.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
2 (reliable with restrictions)

Mode of Action Analysis / Human Relevance Framework

Additional information

Repeated dose oral:

A repeated dose oral study was conducted according to the method which was described in Dhinsa N K and Brooks P, 2008. The study period was seven days. Supporting study. This study showed that LOAEL for the test substance is 150 mg/kg/day, though due to the brief study duration this is not being utilised for derivation of DNELs.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Extensive study summary, no guideline but there were detailed information about mortality, haematology, blood chemistry, necropsy, histopathology and the other observations. However the study is only of 7 days duration, hence considered as reliable with restrictions.

Justification for classification or non-classification

The existing data (Dhinsa N K and Brooks P, 2008) is of insufficient duration to make a judgement on whether classification is appropriate for repeat dose endpoint.