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Administrative data

Description of key information

A modern 90-day rat study performed with the substance reports a NOAEL of 1000 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23rd April - 24th July 2013 (in-life)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed in accordance with official guidelines, and in accordance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
: Several minor deviations from the protocol were identified. These deviations were considered not to have impact on the integrity or outcome of this study.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Edinburgh stock, Charles River UK Ltd, Margate, Kent, UK
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 188-237 g for males and 128-180 g for females
- Fasting period before study: No
- Housing: 2 or 3 per cage by sex in suspended polycarbonate cages with stainless steel grid tops, solid bottoms and a separate stainless steel food hopper
- Diet (e.g. ad libitum): Rat and Mouse (modified) No. 1 Diet SQC Expanded ad libitum
- Water (e.g. ad libitum): water taken from the public water supply ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24°C
- Humidity (%): 44-76%
- Air changes (per hr): minimum of 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From: 23 Apr 2013 To: 24 Jul 2013
Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) methyl cellulose in water for irrigation
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 0, 20, 50, 100 mg/ml
- Amount of vehicle: 10 ml/kg
- Lot/batch no. : 021M0067V and G27R031
- Purity: 0.5% (w/v) methyl cellulose in water for irrigation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis from test item formulations on Day 1, Week 6 and Week 12 for all groups. Concentration and homogeneity were analysed. Analyses were performed by reverse phase HPLC with ELSD Detection using a validated analytical procedure.
Duration of treatment / exposure:
91 days
Frequency of treatment:
single, daily oral gavage dose
Remarks:
Doses / Concentrations:
0 mg/kg bw/day (vehicle control)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were agreed with the Sponsor following examination of a previous toxicity study in which animals received 200, 500 or 1000 BIS-MPA ™ /kg/day for up to 28 days. In that study, 1000 mg/kg/day was considered to be well tolerated with no effects recorded on clinical observations, body weight or food consumption profiles, organ weights or macroscopic investigations and therefore a suitable high dose level for this 13 Week Toxicity study.

- Section schedule rationale (if not random): The animals were euthanised rotating across dose groups such that similar numbers of animals from each group, including controls were necropsied at similar times throughout the day.
Positive control:
Not relevant for this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Animals were checked for viability. All animals were examined at frequent intervals throughout the day for signs of ill health or reaction to treatment, staring immediately post dose, with particular attention paid to the first hour after dosing.
- Posture/condition on first approach (animal undisturbed), checked for: Prostration, Lethargy, Writhing, Circling, Breathing abnormalities, Gait abnormalities, Tremor, Fasciculation, Convulsions, Biting (of cage components or self mutilating), Vocalisations, Piloerection.
Ease of removal from the cage.
Condition of the eyes, checking for: Pupillary function, Miosis, Mydriasis, Exophthalmos, Encrustation, Lacrimation.
Condition of the coat. Presence of salivation. Overall ease of handling.
- Observations in a Standardised Arena (2 Min Observation Period): Latency (time to first locomotory movement), Level of mobility, Rearing, Grooming, Urination/defecation, Arousal (level of alertness), Posture, tremor/convulsions, vocalisation, piloerection, Palpebral closure, Gait abnormalities, Stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week
- Animals received a detailed clinical examination including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta

BODY WEIGHT: Yes
- Time schedule for examinations: once during the pretrial period and daily from the first day of dosing (Day 1) until the end of then observation period (reported weekly). Body weighs recorded immediately before dosing were classified as Day 0 body weights.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each cage of animals was recorded once during pretrial, and weekly during the dosing period

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

WATER CONSUMPTION: Yes
- Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the pretrial and during Week 13
- Dose groups that were examined: The eyes of all animals (including extras) were examined during the pretrial period. All animals in the control and high dose group (1000 mg/kg/day) were also examined during Week 13 of treatment

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 92 - 93
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Red blood cell count, Haemoglobin, Haematocrit, Mean cell volume, Mean cell haemoglobin concentration, Mean cell haemoglobin, Reticulocytes, Reticulocyte count (absolute), Red blood cell distribution width, Platelets, White blood cell count, Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Large unstained cells, coagulation.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 92 - 93
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatise, Creatine phosphokinase, Lactate dehydrogenase, Sodium, Potassium, Chloride, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium, Inorganic phosphate.

URINALYSIS: Yes
- Time schedule for collection of urine: Day 87 - 88
- Metabolism cages used for collection of urine: Yes over a period of 16-18 hours
- Animals fasted: Yes, animal had access to water only while in the metabolism cages
- Parameters checked: Microscopic evaluation of spun deposit, Colour, Turbidity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Leukocytes, Blood pigments, Urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during pretrial and once during the treatment period (Week 13)
- Dose groups that were examined: all animals
- Battery of functions tested: Reaction to sudden sound, Reaction to touch on the rump with a blunt probe, Grip strength, Pain perception, Landing foot splay, Motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were subject to necropsy including unsceduled deaths
- All animals were euthanised by exposure to a rising concentration of carbon dioxide, weighed, and exsanguinated by severance of major blood vessels. Animals were not fasted before their scheduled necropsy.
- Complete necropsy examination: evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
- The following organs were weighed at necropsy for all scheduled euthanasia animals: Brain, Epididymis, Adrenal gland, Pituitary gland, Prostate gland, Thyroid gland, Heart, Kidney, Liver, Lung, Ovary, Spleen, Testis, Thymus, Uterus. Paired organs were weighed separately and reported together. Organs were weighed before fixation unless otherwise noted. Organ to body weight ratio/percentage (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes
- Representative samples of the following tissues were collected from all animals: Animal identification, Aorta artery, Bone marrow smear, Bone marrow from femur, Bone marrow from sternum, Femur bone, Sternum bone, Brain, Cervix, Epididymis, Eye, Adrenal gland, Harderian gland, Lacrimal gland, Mammary gland, Parathyroid gland, Pituitary gland, Prostate gland, Salivary gland, Seminal vesicle gland, Thyroid gland, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine (caecum, colon, rectum), Larynx, Liver, Lung, Mandibular lymph node, Mesenteric lymph node, Skeletal muscle, Nasal cavity, Optic nerve, Sciatic nerve, Oesophagus, Ovary, Oviduct, Pancreas, Pharynx, Skin, Small intestine (duodenum, ileum, jejunum), Spinal cord, Spleen, Stomach, Testis, Thymus, Tongue, Trachea, Ureter, Urinary bladder, Uterus, Vagina
Other examinations:
None
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately. Body weight, food consumption, selected functional observational battery and motor activitydata, haematology, coagulation, clinical chemistry and selected urinalysis data were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student’s t test; i.e. pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student’s t test).
In circumstances were it was not possible to perform the F-Max test due to zero standard deviation in at least one group, the non-parametric ANOVA results were reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above as an exploratory analysis.
In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data were subjected to a rank transformation prior to analysis. Where it was not possible to perform the F-Max test due to the small sample size (i.e. less than 3 animals in any group), the untransformed parametric ANCOVA results are reported.
Clinical signs:
no effects observed
Description (incidence and severity):
Two unscheduled deaths were not attributable to treatment
Mortality:
no mortality observed
Description (incidence):
Two unscheduled deaths were not attributable to treatment
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
Urinary pH was statistically significantly lower in both males and females that received 500 or 1000 mg/kg/day
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased absolute and relative liver weight in males at 1000 mg/kg bw/day
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 two unscheduled deaths during the course of this study. A 200 mg/kg/day female was euthanized on Day 35 due to an accidental transfer within the testing facility and another 200 mg/kg/day female was euthanized moribund on Day 29. The moribund condition of this animal was considered related to a gavage procedural error based on gross and microscopic findings in the thoracic cavity, lungs and thymus.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment-related differences in group mean body weight or body weight gain.

FOOD CONSUMPTION
There were no treatment-related differences in food consumption.

WATER CONSUMPTION
Visual inspection of the water bottles indicated no observable differences in water consumption.

OPHTHALMOSCOPIC EXAMINATION
There were no findings during ophthalmoscopic examinations that could be attributed to the test substance.

HAEMATOLOGY
There were no notable inter-group differences in haematological parameters.

CLINICAL CHEMISTRY
There were no notable inter-group differences in clinical chemistry parameters.

URINALYSIS
Urinary pH was statistically significantly lower in both males and females that received 500 or 1000 mg/kg/day.

NEUROBEHAVIOUR
All of the behaviours exhibited and observations were considered to be typical for rats of this age and strain on this type of study. There were no treatment-related differences in the quantitative functional observation parameters. There were no notable inter-group differences in motor activity

ORGAN WEIGHTS
Absolute and relative liver weights were increased in the 1000 mg/kg/day Bis-MPA™ males

GROSS PATHOLOGY
No treatment related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No treatment related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals.
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 toxicologically significant effects of treatment were observed at the highest dose level.
Critical effects observed:
not specified

Effects of treatment in this study were limited to increased absolute and relative liver weights in 1000 mg/kg bw/day males. Findings occurred in the absence of any clinical chemistry or histopathological correlates and are not considered to be of toxicological significance.

Conclusions:
Daily oral gavage administration of Bis-MPA to rats for at least 91 consecutive days was well tolerated and produced no toxicologically relevant effects on at dose levels up to and including 1000 mg/kg/day when compared to the vehicle control. The NOAEL for this study is therefore 1000 mg kg/bw/day.
Executive summary:
A study was conducted to determine the potential toxicity of Bis-MPA when given orally by gavage for at least 91 days to rats. Ten rats per sex per dose were administered Bis-MPA at dose levels of 0, 200, 500, or 1000 mg/kg bw/d.

The following were assessed at predetermined intervals from pre-trial until study completion for all animals: viability, clinical observations, body weights, food consumption, a visual assessment of water consumption and ophthalmoscopy examinations. All animals also received a detailed functional observation battery (including motor activity) during pre-trial and Week 13 of treatment. Blood samples for haematology, coagulation and blood chemistry investigations and urine samples were collected from all animals during Week 13 of treatment. All surviving animals were terminated after completion of at least 91 days of treatment and underwent a detailed necropsy examination with selected organs weighed. Tissues from all control and high dose (1000 mg/kg/day) animals were subjected to a comprehensive histological examination, with gross lesions (where appropriate) examined from low and intermediate dose animals.

There were two unscheduled deaths; a 200 mg/kg bw/d female was killed prematurely due to a possible gavage procedural error and another female in this group was killed prematurely after being accidentally transferred out of the animal room. There were no treatment-related clinical observations noted during treatment. Body weight and food consumption profiles of all treated animals were similar to concurrent controls. There were no ophthalmoscopy findings noted. There were no differences in qualitative or quantitative functional observations or motor activity assessments noted during the neurotoxicity assessment. There were no treatment-related differences in haematological, coagulation or blood chemistry. Urinalysis findings were limited to reduced urine pH in both sexes at 500 and 1000 mg/kg bw/d. This finding is likely to reflect urinary excretion of the test material and is not considered to be of toxicological significance. Gross necropsy and histopathology did not reveal any treatment-related findings. Mean absolute and relative liver weights were significantly increased in males only at the highest dose level; this finding (in the absence of clinical chemistry or histopathological correlates) is considered to be an adaptive effect and not of toxicological significance.

A NOAEL of 1000 mg/kg bw/d is therefore derived for this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A high-quality 90-day rat study is available for the substance

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

Findings in a 90-day rat toxicity study performed with bis-MPA were limited to reduced urinary pH in both sexes at dose levels of 500 and 1000 mg/kg bw/d and significantly increased absolute and relative liver weight in male rats at the highest dose level of 1000 mg/kg bw/d. Urinalysis findings are likley to reflect urinary excretion of the test substance and are therefore not considered to be of toxicological significance. The effect on liver weight in the absence of clinical chemistry or histopathological correlates is considered to be an adaptive effect and not of toxicological significance. A NOAEL of 1000 mg/kg bw/d is therefore determined for this study. Bis-MPA shows very low toxicity following repeated oral administration to the rat.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only study available for this endpoint

Justification for classification or non-classification

Based on the data available, no classification is required under Directive 67/548/EEC or under the CLP Regulation.