Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and appropriate guidelines
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
n/a
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
SOL-DP (Batch No. 869090084), a white powder. Stored at room temp, dry, closed container.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Animals' age was 41 to 47 days at the begining of the study
Males: 210-263g
Females: 160-202g

Housing and Husbandary:
5 of one sex animals per cage
Temp: protocol 19-23 Deg C, actual 24 Deg C at four occasions during the test. This deviation was minor and was not considered to have influenced the health of the animals or the outcome the study
RH: protocol 40-70%
Air flow: 15 air changes/hr
12 light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: methylcellulose, in water for formulation
Details on oral exposure:
The test substance was prepared for administration as a series of graded concentrations in the vehicle. The required amounts of test material were wighed out. Starting with the lowest concentration, the test material was transferred to a mortar and ground to a fine powder. Small amounts of vehicle were added and mixed with the test material to form a smooth paste. The suspension was poured into a measuring cylinder. This was then made to the required volume to provide the correct concentration. The suspension was then mixed using a high shear homogeniser and
transferred to the final issue containers, via syringe, whilst magnetically stirring. The remaining concentrations were made in ascending concentration order. Dosing formulations were prepared weekly and stored at room temperature, protected from light, prior to administration.

The exposure of the animals to the test material was by gavage and was not mixed with the food.

VEHICLE: A similarly constituted Control group received the vehicle 1% MC (methylcellulose, in water for formulation), at the same volume-dose throughout the same period.

The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet), except overnight before blood sampling for haematology and blood chemistry. 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration during the first and last weeks of treatment (i.e first week in the preliminary phase and last week in the main phase) were analysed for achieved concentration of the test substance. Four samples (nominally 1 mL accurately weighed) were taken from all formulations; two samples were assayed from each formulation. The remaining samples from each formulation were retained frozen (nominally -20°C) as contingency for analysis if required.
The samples were analysed in accordance with the validated Huntingdon Life Sciences Analytical Procedure FIA/M093/10. The analytical method involved dissolution in acetonitrile and further dilution using acetonitrile/water 50/50 v/v followed by reverse phase high performance liquid chromatographic analysis with ultra-violet detection (261 nm). Sample concentrations were determined with reference to external standards prepared in the concentration range 2.0 - 10.0 μg/mL.
At Week 1 and the last week of treatment, freshly prepared test formulations were sampled (4 × 1 mL, accurately weighed) by Pharmacy personnel and submitted for analysis.Duplicate samples were analysed in accordance with the analytical procedure, and the remaining samples were retained for contingency. The contingency samples for Week 1 Group 2 were analysed as the coefficient of variation (CV) was outside the applied limit of 5%.
Duration of treatment / exposure:
The test substance was administreded over a period of 13 concsecutive weeks. Animals assigned to the recovery phase completed a further four weeks without treatment.
Frequency of treatment:
All animals were dosed once each day at approximately the same time eachday, seven days per week.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 250 and 1000 mg/kg/day. Volume dose: 5 ml/kg
Basis:
actual ingested
No. of animals per sex per dose:
The study consisted of one Control and three treated groups of rats.
Control animals:
yes, concurrent vehicle
Details on study design:
The doses used in this study were selected in conjunction with the Sponsor based on reactions seen in the previous combined oral toxicity and reproductive/developmental toxicity study in rats. In that study, doses of 50, 250 and 1000 mg/kg/day were administered to CD rats. No treatment-related effect was observed. Therefore, NOAEL was considered in rats to be greater than 1000 mg/kg/day and as suche the same doses were considered to be appropriate for this 13 week study. The animals were assigned randomly.
Three groups, each comprising ten males and ten female rats received Sol-DP at the relevant dose. A similarly constituted Control group received the vehicle, 1% MC, at the same volume-dose (10ml/kg). A further five males and five female rats were assigned to each of the Control and high dose groups. These animals were treated for 13 weeks, followed by a 4 weeks period without treatment to assess recovery from any treatment related effect.

Positive control:
no

Examinations

Observations and examinations performed and frequency:
During the study the following observations and examinations were performed: clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight food consumption, water consumption (vissual assessment only), opthalmic examination, haematology, blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

CAGE SIDE OBSERVATIONS:
Time schedule: At least twice a day/ seven days a week
Cage side observations checked: Morbidity, mortality, signs of injury, availability of food and water

DETAILED CLINICAL OBSERVATIONS:
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 and weekly thereafter, 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 the doseing of all groups, as late as possible in the working day. In addition, a more detailed physical examination was performed on each animal to monitor general health. During the acclimation and recovery periods, observations of the animals and their cages were recorded at least once per day.

DETAILED PHYSICAL EXAMINATION and ARENA OBSERVATIONS:
Before treatment comenced ans during each week of treatment and recovery, all animals were examined. Finding were reported as "present" or assigned a sevirity grade - slight, moderate or marked.

BODY WEIGHT:
One week before treatment comenced (Week-1), on the day that treatment commenced (Week-0), and weekly throughout the treatment and recovery periods, and before the necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for each week throughout the treatment and recovery periods. From these records the mean weekly conssumption per animal (g/rat/week) was calculated for each cage.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation. As no treatment-related changes were suspected no precise measurments were recorded.

OPHTHALMOSCOPIC EXAMINATION:
Before treatment comenced , the eyes of all animals were examined. During Week 12 of treatment the eyes of all animals of Group 1 (Control) and 4 (1000 mg/kg/day) weresimilarly examined.


HAEMATOLOGY, PERIPHERAL BLOOD:
During Week 13 of treatment only, blood samples were obtained from all serviving animals and about 15 different haematologic parameters were examined (see below). During Weeks 13 of treatment (before dosing) and Week 4 of recover, blood samples were obtained from all surviving animals and two parameters were examined.
Anaesthetic used for blood collection: Yes, Isoflurane
Animals fasted: Yes, overnight
Parameters checked: For the Week 13 treatment rats only the following parameters were examined: Het, Hb, RBC, Retic, MCH, MCHC, MCV, WBC, Differencial WBC (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monicytes and LUC), Plt. For the Week 13 of treatment and Week 4 of recovery two additional parameters were examined as follows: Prothrombin time (PTP) & Activated partial thromboplastin time (APTT).

BLOOD CHEMISTRY:
At the same time and using the same animals as for the peripheral hematology, further blood samples were collected for examination of the following parameters:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)~
Triglycerides (Trig)~
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)


SENSORY REACTIVITY and GRIP STRENGTH:
All animals during Week 12 of treatment (before dosing) were examined. The following measurements, reflexes and responses were recorded:Approach response, Touch response, Auditory startle reflex, Tail pinch response and grip strength.

MOTOR ACTIVITY:
During Week 12 of treatment, all animals were examined.


HAEMATOLOGY:
During Week 13 of treatment only, blood samples were obtained from all serviving animals and about 15 different haematologic parameters were examined (see below). During Weeks 13 of treatment (before dosing) and Week 4 of recover, blood samples were obtained from all surviving animals and additional two parameters were examined.
Anaesthetic used for blood collection: Yes, Isoflurane
Animals fasted: Yes, overnight
Parameters checked: For the Week 13 treatment rats only the following parameters were examined: Het, Hb, RBC, Retic, MCH, MCHC, MCV, WBC, Differencial WBC (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monicytes and LUC), Plt. For the Week 13 of treatment and Week 4 of recovery two additional parameters were examined as follows: Prothrombin time (PTP) & Activated partial thromboplastin time (APTT).


NEUROBEHAVIOURAL EXAMINATION:
Sensory reactivity and grip strength assessments were performed (before dosing) on all
animals during Week 12 of treatment.
The following measurements, reflexes and responses were recorded: Approach response, touch response, auditory startle reflex, tail pinch response, grip strength and motor activity.

ORGAN WEIGHTS:
The following organs, taken from each animal killed after 13 weeks of treatment or 4 weeks of recovery, were dissected free of adjacent fat and other contiguous tissue and the weights recorded:
Adrenals Ovaries
Brain Spleen
Epididymides Testes
Heart Thymus
Kidneys Uterus with cervix
Liver
Sacrifice and pathology:
All animals surviving until the end of the scheduled treatment or recovery period were subject to a detailed necropsy.

MACROSCOPIC PATHOLOGY:
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormal position, morphology or interaction was recorded.

PATHOLOGY:
Microscopic examination was performed for all tissues preserved for examination (as specified above). The examination was taken care for all animals of Groups 1 (Control) and 4 (1000 mg/kg/day) sacrificed on completion of the scheduled treatment period and for the animal found dead during the study. Tissues reported at macroscopic examination as being grossly abnormal were examined for main and recovery animals in line with current practice. Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

HISTOLOGY:
For those animals specified in the Pathology section, the relevant tissues were subject to histological processing.
Statistics:
Parametric analysis, Bartlett's test, t-tests, Williams’ test, Dunnett's test, Wilcoxon rank sum tests, Shirley's test, Steel's test, Fisher's Exact tests
For a detailed Statistics, see attached.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no deaths considered to be related to treatment during the treatment or recovery periods. There was one death during the treatment period.

GENERAL APPEARANCE & BEHAVIOR: General appearance and behaviour, sensory activity, grip strength and motor activity were not affected by treatment and there were no post-dose signs or any treatment-related death.

BODY WEIGHT AND WEIGHT GAIN: There were no abnormalities in weight gain considered to be related to treatment during the treatment or recovery periods.

FOOD CONSUMPTION AND COMPOUND INTAKE: Mean food consumption was considered to be unaffected by treatment.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): A visual assessment of water intake did not reveal any treatment-related change.

OPHTHALMOSCOPIC EXAMINATION: There were no treatment-related ophthalmic findings reported in Week 12 of treatment.

HAEMATOLOGY & CLINICAL CHEMISTRY: There were no toxicologically significant haematological or biochemical changes in the blood at the Week 13 investigation.

NEUROBEHAVIOUR: Sensory reactivity observations and grip strength values were considered to be unaffected by treatment.

ORGAN WEIGHT, MACROSCOPY & HISTOPATHOLOGY: There were no treatment-related organ weight, macroscopic or histopathological findings.



Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were found.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of SOL-DP to Sprague-Dawley (Crl:CD(SD)) rats at doses up to 1000 mg/kg/day for 13-weeks was well tolerated and did not result in any toxicologically significant change. Consequently, the no observed adverse effect level (NOAEL) in this study was considered to be 1000 mg/kg/day.
Executive summary:

The systemic toxic potential of SOL-DP to Crl:CD(SD) rats by oral gavage administration was assessed over a period of 13 weeks, followed by a 4 week recovery period. Three groups, each comprising ten male and ten female rats received SOL-DP at doses of 50, 250 or 1000 mg/kg/day. A similarly constituted Control group received the vehicle, 1% MC (methylcellulose, in water for formulation), at the same volume-dose (10 mL/kg). A further five male and five female rats were assigned to each of the Control and high dose groups. These animals were treated for 13 weeks, followed by a 4 week period without treatment to assess recovery from any treatment related effect. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual assessment only), ophthalmic examination, haematology, blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

The results showed no effect of the test item on any of the endpoints tested; general appearance and behavior, sensory activity, grip strength and motor activity were not affected by treatment and there were no post-dose signs or any treatment-related death. There was no effect of treatment on bodyweight gain or food consumption. A visual assessment of water intake did not reveal any treatment-related change. There were no treatment-related ophthalmic findings. There were no toxicologically significant haematological or biochemical changes in the blood at the Week 13 investigation. There were no treatment-related organ weight, macroscopic or histopathological findings. Based on the results it was concluded that the oral administration of SOL-DP, a flame retardant, to Sprague-Dawley (Crl:CD(SD)) rats at doses up to 1000 mg/kg/day for 13-weeks was well tolerated and did not result in any toxicologically significant change. Consequently, the no observed adverse effect level (NOAEL) in this study was considered to be 1000 mg/kg/day.