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Administrative data

Description of key information

Repeated dose toxicity - oral route:
In an OECD 407 study 28-day repeated dose oral study in Sprague-Dawley rats, the NOAEL of Molyvan 855 is 150 mg/kg/day. Treatment-related renal changes were observed.
In a 14-day range finding study in rats (gavage) to support the 28-day OECD 407 repeated dose study, no clinical signs were detected at the 150 or 400 mg/kg/day dose levels. Clinical signs associated at 1000 mg/kg/day were attributed to unpleasant taste and slight irritant, the NOAEL of Molyvan 855 was found to be >1000 mg/kg/day.
In a 14-day, and 21-day range-finding study (from OECD 416 - Dietary two generation reproduction study in the rat with evaluation of subchronic toxicity), the NOAEL of Molyvan 855 in male and female rats is 12000 ppm(600 mg/kg) (SPL, 2006).

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted under EU Guideline under GLP conditions.
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River (UK) Limited, Manston, Kent.

- Age at study initiation:
Five to six weeks old.

- Weight at study initiation:
The males weighed 128 to 157g, and the females weighed 117 to 141g.

- Fasting period before study:

- Housing:
The animals were housed in a single air-conditioned room in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.

- Diet:
ad libitum

- Water:
ad libitum

- Acclimation period:
The animals were acclimated for seven days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
The temperature was controlled to remain within target ranges of 21 +/- 2 deg C.

- Humidity (%):
The humidity was controlled to remain with target ranges of 55 +/- 15%.

- Air changes (per hr):
The rate of air exchange was at least fifteen air changes per hour.

- Photoperiod (hrs dark / hrs light):
The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Stability and homogeneity of the test material formulations were determined by Safepharm. Formulations were stable for 14-days and were therefore, prepared weekly and stored at approximately 4 degrees C in the dark.

VEHICLE
- Amount of vehicle (if gavage):
4ml/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test material formulation and were analysed for concentration of Molyvan 855 (Oil Free) at Safepharm. The results indicate that the prepared formulations were within + or - 10% of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
15
Basis:
other: mg/kg/day
Remarks:
Doses / Concentrations:
150
Basis:
other: mg/kg/day
Remarks:
Doses / Concentrations:
1000
Basis:
other: mg/kg/day
No. of animals per sex per dose:
Dose level 15 mg/kg/day
5 male
5 female

Dose level 150 mg/kg/day
5 male
5 female

Dose level 1000 mg/kg/day
5 male
5 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose selection was based on results from the range finding 14-day study in rats following Oral (gavage) administration. The test material was tested up to the maximum tolerated dose level (1000 mg/kg/day). This information was used to determine the dose levels for this study.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.



Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Animals were observed immediately before dosing and one hour after dosing at weekends and public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual bodyweights were recorded on Day 0 (the day before the start of the treatment) and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION:
Daily visual inspection of water bottles from Week 2 onwards revealed possible intergroup differences. Water consumption was therefore, measured and recorded for each cage group from Day 22 onwards.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Day 28.
- Animals fasted: No

- Parameters checked:
Haemoglobin (HB)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)

Clotting (prothrombin) time (CT) was assessed by 'Hepato Quick' using samples collected into sodium cirate solution (0.11 mol/l).

BLOOD CHEMISTRY: Yes
- Parameters checked:
Urea
Lucose
Total protein (Tot.Prot)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total bilirubin (Bili)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On completion of the dosing period all animals were killed by intravenous sodium pentobarbitone (Sagatal, 60 mg/ml) followed by exsanguination.

All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:

Adrenals, Gonads, Kidneys, Brain, Heart, Liver, Spleen.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain
Caecum
Colon
Duodenum
Eyes
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)
Lymph nodes (cervical and mesenteric)
Muscle (skeletal)
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles
Skin (hind limb)
Spleen
Stomach
Testes (with epididymides)
Thymus
Thyroid/parathyroid
Trachea
Urinary bladder
Uterus

All tissues were despatched to Finn International UK for processing. The following preserved tissues from all control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 micro m and stained with haematoxylin and easin for subsequent microscopic examination:

Adrenals, Heart, Kidneys, Liver, Spleen, Testes

The liver and spleen from all 15 and 150 mg/kg/day dose group animals were also processed.

Since there were indications of treatment-related renal changes examination was subsequently extended to include similarly prepared sections of kidney from all animals in the remaining groups.
Microscopic examination was conducted by the Study Pathologist, All finding were entered into the ROELEE 84 pathology computerisation system for tabulation and report production.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Bodyweight gain, haematological, blood chemical and organ weight (absolute and relative to terminal bodyweight) data, were assessed for dose response relationships using linear regression analysis. Data showing a significant dose response were analysed for homogeneity of variance using Levene's test. Data showing homogeneous variances were analysed by one way analysis of variance (ANOVA) and pairwise comparisons were made using Dunnett's test. Data showing heterogeneous variances were re-analysed using Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney U-test.

The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Probability values (p) are presented as follows:
p <0.001***
p <0.01**
p <0.05*
p>= 0.05 (not significant)
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 examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no deaths during the study. No clinically observable signs of toxicity were detected in test or control animals throughout the study period.

Animals of either sex treated with 1000 mg/kg/day showed increased salivation approximately two minutes after dosing from Day 8 onwards, followed by occasional incidents of increased salivation noted up to one hour after dosing from Day 15. Red/brown staining and/or wetting of the external body surface were also apparent in 1000 mg/kg/day animals from Day 5 onwards together with an isolated incidence of noisy respiration detected on Day 8 only. Such observations are often reported when the test material formulation has an unpleasant taste or is slightly irritant and, in isolation, were considered not to be indicative of systemic toxicity.

Animals of either sex treated with 150 or 15 mg/kg/day showed no such clinical signs throughout the study.

Isolated incidents of fur staining were observed but these were confined to two control males and, as such, were incidental.

BODY WEIGHT AND WEIGHT GAIN

Males treated with 1000 mg/kg/day showed a statistically significant reduction in bodyweight gain from Week 2 onwards, with one male showing a bodyweight loss at Day 28.

No adverse effect on bodyweight development was detected for females treated with 1000 mg/kg/day or for animals of either sex treated with 150 or 15 mg/kg/day throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

FOOD EFFICIENCY:
Males treated with 1000 mg/kg/day showed a slight reduction in dietary intake and food efficiency (the ratio of weight gain to food consumption) from Week 2 onwards when compared with that of controls. No such effects were detected for females treated with 1000 mg/kg/day or for animals of either sex treated with 150 or 15 mg/kg/day throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
Quantitative measurement revealed an increase in water consumption for animals of either sex treated with 1000 mg/kg/day compared with controls. The effect was more pronounced amongst males than females. No adverse effect on water intake was detected at 150 or 15 mg/kg/day throughout the study.

HAEMATOLOGY

There were no toxicologically significant changes in the haematological parameters measured.

A slight, but significant reduction (
BLOOD CHEMISTRY

There were no toxicologically significant changes in the blood chemical parameters measured.

Males from all treatment groups showed a slight but statistically significant (p <0.05) increase in aspartate aminotransferase compared with controls. All individual values were within the normal range for rats of the age and strain employed and, in the absence of a convincing dose-relationship, the increase was considered to be irrelevant.

The remaining statistically significant intergroup differences (an increase in 1000 mg/kg/day male plasma creatinine and a reduction in 1000 mg/kg/day female bilirubin) were confined to isolated, minimal (p <0.05) changes where all individual values were within the respective normal ranges. Such changes were, therefore, considered to be without toxicological importance.

ORGAN WEIGHTS

Animals of either sex treated with 1000 mg/kg/day showed a substantial increase in group mean kidney weight, both absolute and relative to terminal bodyweight compared with controls, with one male absolute weights not achieving statistically significance. Nine out of the ten individual relative kidney weights were outside the respective normal ranges for rats of the strain and age employed.

No such effects were detected at 150 or 15 mg/kg/day.

GROSS PATHOLOGY
Three animals of either sex treated with 1000 mg/kg/day showed pale kidneys at terminal kill.

No macroscopic abnormalities were observed at 150 or 15 mg/kg/day.

One male treated with 1000 mg/kg/day showed hydronephrosis of the left kidney at terminal kill but this finding represents a common, spontaneously-arising congenital conditions amongst laboratory maintained rats of the strain and age used in the study and was of no toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC

Treatment-related renal changes were observed. The incidence and severity of groups of basophilic cortical tubules were increased in relation to treatment for rats of either sex dosed at 1000 mg/kg/day.

Treated animals from the remaining dose groups were unaffected.

All remaining morphological changes 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.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically significant effects were detected at 150 or 15 mg/kg/day and the NOAEL was, therefore, considered to be 150 mg/kg/day.
Critical effects observed:
not specified
Conclusions:
In an EU Method B.7 study 28-day repeated dose oral study in Sprague-Dawley rats, the NOAEL of Molyvan 855 is 150 mg/kg/day.
Executive summary:

Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day, resulted in toxicologically significant effects at the highest dose level. No such effects were detected at 150 or 15 mg/kg/day and the No Observed Effect Level (NOEL) and No Observed Adverse Effect Level (NOAEL) was, therefore, considered to be 150 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

Oral route:

Key study:

In an OECD 407 oral repeated dose toxicity study, conducted under GLP conditions, administered to male and female Sprague-Dawley rats at dose levels of up to 1000 mg/kg/day, the No Observed Effect Level (NOEL) of Molyvan 855 is 150 mg/kg/day. (SPL, 1997)

Supporting study

In a 14-day range finding study, using male and female Sprague-Dawley rats, the NOAEL of Molyvan 855 is >1000 mg/kg day. (SPL, 1997)

Supporting study:

In a 14 and 21-day range finding study (from Dietary two generation reproduction study in the rat with evaluation of subchronic toxicity), using male and female Sprague-Dawley rats, reduced food consumption was observed at all dose levels, reduced body weight gain was noted at the highest dose level. The NOAEL of Molyvan 855 in male and female rats is 12000 ppm (600 mg/kg). (SPL, 2006)

Inhalation route:

The oral exposure route was chosen in the absence of indications from the acute toxicity studies, physico-chemical properties and likely human exposure that alternative exposure routes are not appropriate. Additional testing for repeat dose is not justified as the route of exposure is not relevant nor does the existing data set indicate any triggers or concerns that would warrant additional testing.

Dermal route:

The oral exposure route was chosen in the absence of indications from the acute toxicity studies, physico-chemical properties and likely human exposure that alternative exposure routes are not appropriate. Additional testing for repeat dose is not justified as the route of exposure is not relevant nor does the existing data set indicate any triggers or concerns that would warrant additional testing.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Well conducted study in accordance with EU method B.7 and GLP.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: kidneys

Justification for classification or non-classification

In accordance with Regulation No 1272/2008 Table 3.9.3 Molyvan 855 is not classified for repeated dose toxicity (oral), based on the NOEL (male and female rats) of 150 mg/kg b.w./day.