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Description of key information

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:
key study
Study period:
The study was performed between 09 September 2009 and 26 November 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 05 to 09 September 2007 and 26 to 30 November 2007 Date of Signature on GLP certificate: 30 April 2008
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™:HsdRccHan™:WIST strain rat
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Source:
Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon

- Age at study initiation:
Five to six weeks old

- Weight at study initiation:
males weighed 167 to 199g, the females weighed 149 to 180g,

- Fasting period before study:
Animals were not fasted prior to sampling.

- Housing:
The animals were housed in groups of five in polpropylene grid floor cages suspended over trays lined with absorbent paper.

- Diet:
A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K., Oxon, UK) was used (ad libitum).

- Water (e.g. ad libitum):
ad libitum mains water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water was considered not to have any level of contaminant.

- Acclimation period:
Minimum of six days


ENVIRONMENTAL CONDITIONS

- Temperature (°C):
21˚C +/- 2˚C

- Humidity (%):
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.

IN-LIFE DATES:
From: Day1 To:Day 28
Route of administration:
oral: gavage
Vehicle:
other: Polyethylene glycol 400
Details on oral exposure:
Method of administration:
Gavage
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations
as a solution in Polyethylene glycol 400


DIET PREPARATION
- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable.

- Storage temperature of food:
Not applicable.


VEHICLE
- Justification for use and choice of vehicle(if other than water): Most suitable

- Concentration in vehicle:
7.5, 75 and 250 mg/ml.

- Amount of vehicle (if gavage):
4 ml/kg bodyweight

- Lot/batch no. (if required):
Unknown

- Purity:
Unknown
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of test material formulations was determined by gas chromatography. The test material formulations were sampled and analysis showed stability for up to four hours. The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study. Dose formulations were prepared daily
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
Remarks:
Doses / Concentrations:
Dose levels of 30, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Male and Female: 5 animals per sex at 0 mg/kg/day
Male and Female: 5 animals per sex at 30 mg/kg/day
Male and Female: 5 animals per sex at 300 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on available toxicity information and compliance with regulatory hazard classification.

- Rationale for animal assignment (if not random):
Random.

- Rationale for selecting satellite groups:
Not applicable

- Post-exposure recovery period in satellite groups:
Not applicable

- Section schedule rationale (if not random):
Not applicable
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

- Cage side observations checked in table [1] please see attached tables

DETAILED CLINICAL OBSERVATIONS: Yes see above
- Time schedule: As above

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual bodyweights were recorded on Day 1 (prior to start of treatment) and on Days 8, 15, 22 and 29. Terminal bodyweights were also recorded at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not applicable

FOOD EFFICIENCY:
- Weekly food efficiency (bodyweight gain/food intake) was calculated.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:
Water intake was visually observed daily except for Week 3 when gravimetric measurement was employed.

OPHTHALMOSCOPIC EXAMINATION: No data:
Not applicable
- Time schedule for examinations:
Not applicable
- Dose groups that were examined:
Not applicable


HAEMATOLOGY: Yes
- Time schedule for collection of blood:
At the end of the treatment period (Day 28).
- Anaesthetic used for blood collection: No
Not applicable
- Animals fasted: No
- How many animals:
Five males and five females
- Parameters checked in table [11] please see attached Tables.
Routine haematological parameters were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
At the end of the treatment period (Day 28).
- Anaesthetic used for blood collection: No
Not applicable
- Animals fasted: No
- How many animals:
Five males and five females
- Parameters checked in table [12] please see attached Tables


URINALYSIS: No
- Time schedule for collection of urine: Not applicable
- Metabolism cages used for collection of urine: Not applicable
- Animals fasted: Not applicable
- Parameters checked in table [No.?]. Not applicable

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during final week of study.
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity
- Parameters checked in table [No.3 to 5].

Behavioural Assessments were undertaken for all animals at weekly intervals throughout the study. Motor Activity, Forelimb/Hindlimb Grip Strength and Sensory Reactivity were undertaken for all animals during the final week of treatment.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes please see attached Table 14

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

HISTOPATHOLOGY: Yes please see attached Table 15

All control and high dose animals were subjected to a full histological examination and low and intermediate group animals were routinely subjected to examination of liver and spleen.

In addition: Since there were indications of treatment-related kidney and thyroid gland changes, examination was subsequently extended to include similarly prepared sections of these tissues from all animals in the other treatment groups.

Other examinations:
MORTALITY DATA
ORGAN WEIGHTS
Yes please see attached Table 13
Statistics:
All data was summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data.
If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
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:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality:

There were no unscheduled deaths.

Clinical Observations:

No clinically observable signs of toxicity were detected.

Behavioural Assessment:

No treatment-related effects were detected.

Functional Performance Tests:

No treatment-related effects were detected in grip strength or motor activity.

Sensory Reactivity Assessments:

No treatment-related effects were detected in sensory reactivity scores.

Bodyweight:

No adverse effects on bodyweight change were detected.

Food Consumption:

No adverse effects on dietary intake or food conversion efficiency were detected.

Water Consumption:

No adverse effects on water intake were evident for treated animals when compared to controls.

Haematology:

A possible slight anaemia, consisting of reductions in haemoglobin, erythrocyte and haematocrit counts with lower mean cell haemoglobin and mean cell volume were evident for animals of either sex treated with 1000 mg/kg/day when compared to controls. Slight increases in leucocyte counts, specifically in the neutrophil and lymphocyte fractions were also evident for animals of either sex treated with 1000 mg/kg/day when compared to controls. Elevated neutrophil counts were also evident for males treated with 300 mg/kg/day when compared to controls.
No such effects were detected for females treated with 300 mg/kg/day or for animals of either sex treated with 30 mg/kg/day.

Blood Chemistry:

Increases in alkaline phosphatase levels were evident for males treated with 1000 and 300 mg/kg/day, and a reduction in alanine aminotransferase levels observed for males treated with 1000 mg/kg/day, when compared to controls.
No such effects were detected for females treated with 1000 and 300 mg/kg/day or for animals of either sex treated with 30 mg/kg/day.

Organ Weights:

Animals of either sex treated with 1000 mg/kg/day showed an increase in absolute and bodyweight-relative liver weight, when compared to controls, with the effects extending into the 300 mg/kg/day dose group. Thyroid/parathyroid weights were also higher for animals of either sex treated with 1000 mg/kg/day in comparison to controls values, with the effect extending into the female 300 mg/kg/day dose group.
Males treated with 1000 and 300 mg/kg/day showed an increase in kidney and spleen weights, both absolute and relative to terminal bodyweights when compared to controls.

No treatment-related effects were detected for animals of either sex treated with 30 mg/kg/day.

Necropsy:

No treatment-related macroscopic abnormalities were detected.

Stage of Oestrus:

No treatment-related effects were detected.

Histopathology:

Histopathology examinations revealed the following treatment-related effects:

LIVER: Centrilobular hepatocyte enlargement was seen for animals of either sex treated with 1000 mg/kg/day.

KIDNEYS: A greater incidence of higher grades of severity of globular accumulations of eosinophilic material in the tubular epithelium was observed for males treated with 1000 and 300 mg/kg/day. Associated tubular basophilia and tubular necrosis were also observed for males treated with 1000 and 300 mg/kg/day.

THYROID GLAND: A greater incidence of higher grades of severity of follicular cell hypertrophy was seen for males treated with 1000 and 300 mg/kg/day.
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: . overall effects clinical signs; mortality; functional observations; body weight; food consumption; food efficiency; water consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology; other:; no data
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

A Form 1 notification summary for Japanese Chemical Substance Control Law was completed please attached “Form 1”

JUSTIFICATION OF NO OBSERVED EFFECT LEVEL (for Japanese Chemical Substance Control Law)

The oral administration of E0286P/040A to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment-related changes at 1000 and 300 mg/kg/day.The effects at 300 mg/kg/day for females were not considered to represent an adverse effect, therefore a ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 300 mg/kg/day for females. A NOAEL was not established for males at this level. No treatment-related changes were evident at 30 mg/kg/day, therefore a ‘No Observed Effect Level’ (NOEL) was considered to be 30 mg/kg/day.

The following differences between treated and control animals were considered not to be indicative of test material toxicity: 

Clinical Observations

Post-dose increased salivation was detected for two males treated with 1000 mg/kg/day between Days 13 to 23, one of which also showed staining around the mouth following dosing on Day 15.Increased salivation and associated staining around the mouth is commonly observed following the oral administration of an unpleasant tasting and/or locally irritant test material formulation and, in isolation, is considered not to be indicative of systemic toxicity.

Noisy respiration was evident for one female treated with 1000 mg/kg/day on Day 25. This was an isolated incident and as such, was not considered to represent systemic toxicity.

Functional Observations

Behavioural Assessments

All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Sensory Reactivity Assessments

All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Water Consumption

Increases in water intake were evident during Week 3 for treated animals when compared to controls. No overt differences were evident during the daily visual inspections and a convincing dose-related response was not evident. The slight differences detected during Week 3 were therefore considered not to be attributable to test material toxicity.

Haematology

Males treated with 1000 mg/kg/day showed a statistically significant increase in platelet counts when compared to controls (P<0.01). No such effect was observed for females treated at this dose level, and all individual values for the treated males were within the normally expected ranges for this parameter. One control value however was lower than the historical ranges therefore, this increase was not considered to represent a true effect of treatment and of no toxicological importance.

Blood Chemistry

A statistically significant increase in albumin/globulin ratio was observed for males treated with 1000 mg/kg/day when compared to controls. The significance achieved was minimal (P<0.05) and all individual values were within the normally expected ranges for this parameter. In the absence of any further blood chemistry changes which may suggest an effect of treatment, (e.g. changes in total protein or albumin), this increase was considered to have arisen incidentally and was of no toxicological importance.

Organ Weights

Females treated with 1000 and 300 mg/kg/day showed an increase in absolute and bodyweight-relative pituitary weights in comparison to controls values (P<0.05). A convincing dose-related response was not observed and in the absence of any histopathological correlates, these minimal increases were considered to have arisen incidentally and were unrelated to treatment with the test material.

Necropsy

A reddened thymus was recorded for one control male during thepost-mortemprocedure and one control female displayed a small left kidney. These were isolated findings, and in the absence of treatment at this dose level, were considered to be of no toxicological importance.

Histopathology

 

ADRENAL GLANDS: Cortical vacuolation is a commonly observed spontaneous condition.

 

BONE MARROW: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study there was no difference between control and treated groups.

 

LIVER: Scattered mononuclear cell foci were observed for many animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered. The absence of the condition among control rats was considered to be purely fortuitous.

 

SPLEEN: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits.

 

KIDNEY: Isolated groups of basophilic tubules are frequently encountered in the renal cortex of laboratory maintained rats and have no pathological significance at the severities or frequencies reported in this study. Similarly focal corticomedullary mineralisation is a commonly observed background condition among female rats.

 

LUNGS: A minimal severity of bronchus associated lymphoid tissue was reported for all animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are similarly not suggestive of significant respiratory disease.

 

EPIDIDYMIDES: Spermatocoel granuloma are not uncommonly seen as a background finding in laboratory maintained rats.

 

UTERUS: Dilatation of the uterine horns and keratinisation of the cervical epithelium are commonly observed cyclical conditions in laboratory maintained female rats.

 

VAGINA: Keratinisation of the vaginal epithelium is a normal cyclical change in the female rat.

 

All other morphological changes in the above and remaining tissues 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.

 

 

Conclusions:
The oral administration of E0286P/040A to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment-related changes at 1000 and 300 mg/kg/day. The effects at 300 mg/kg/day for females were considered not to represent an adverse effect, therefore a ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 300 mg/kg/day for females, based on haematological changes observed at 1000 mg/kg/day.
No treatment-related changes were evident at 30 mg/kg/day, therefore the overall ‘No Observed Effect Level’ (NOEL) was considered to be 30 mg/kg/day.
Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity of the test material. It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

Methods.

The test material was administered by gavage to three groups, each of five male and five female Wistar Han™:HsdRccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, functional observations, bodyweight development, dietary intake and waterconsumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Mortality.There were no unscheduled deaths.

Clinical Observations.No clinically observable signs of toxicity were detected.

Behavioural Assessment.No treatment-related effects were detected.

Functional Performance Tests.No treatment-related effects were detected in grip strength or motor activity.

Sensory Reactivity Assessments.No treatment-related effects were detected in sensory reactivity scores.

Bodyweight.No adverse effects on bodyweight change were detected.

Food Consumption. No adverse effects on dietary intake or food conversion efficiency were detected.

Water Consumption.No adverse effects on water intake were evident for treated animals when compared to controls.

Haematology.A possible slight anaemia, consisting of reductions in haemoglobin, erythrocyte and haematocrit counts with lower mean cell haemoglobin and mean cell volume were evident for animals of either sex treated with 1000 mg/kg/day when compared to controls. Slight increases in leucocyte counts, specifically in the neutrophil and lymphocyte fractions were also evident for animals of either sex treated with 1000 mg/kg/day when compared to controls. Elevated neutrophil counts were also evident for males treated with 300 mg/kg/day when compared to controls.

No such effects were detected for females treated with 300 mg/kg/day or for animals of either sex treated with 30 mg/kg/day.

Blood Chemistry.Increases in alkaline phosphatase levels were evident for males treated with 1000 and 300 mg/kg/day, and a reduction in alanine aminotransferase levels observed for males treated with 1000 mg/kg/day, when compared to controls.

No such effects were detected for females treated with 1000 and 300 mg/kg/day or for animals of either sex treated with 30 mg/kg/day.

Organ Weights. 

Animals of either sex treated with 1000 mg/kg/day showed an increase in absolute and bodyweight-relative liver weight, when compared to controls, with the effects extending into the 300 mg/kg/day dose group. Thyroid/parathyroid weights were also higher for animals of either sex treated with 1000 mg/kg/day in comparison to controls values, with the effect extending into the female 300 mg/kg/day dose group.

Males treated with 1000 and 300 mg/kg/day showed an increase in kidney and spleen weights, both absolute and relative to terminal bodyweights when compared to controls.

No treatment-related effects were detected for animals of either sex treated with 30 mg/kg/day.

Necropsy.

No treatment-related macroscopic abnormalities were detected.

Stage of Oestrus.

No treatment-related effects were detected.

Histopathology.

Histopathology examinations revealed the following treatment-related effects:

LIVER:

Centrilobular hepatocyte enlargement was seen for animals of either sex treated with 1000 mg/kg/day.

KIDNEYS:

A greater incidence of higher grades of severity of globular accumulations of eosinophilic material in the tubular epithelium was observed for males treated with 1000 and 300 mg/kg/day. Associated tubular basophilia and tubular necrosis were also observed for males treated with 1000 and 300 mg/kg/day. 

THYROID GLAND:

A greater incidence of higher grades of severity of follicular cell hypertrophy was seen for males treated with 1000 and 300 mg/kg/day.

Conclusion.

The oral administration of E0286P/040A to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment-related changes at 1000 and 300 mg/kg/day. The effects at 300 mg/kg/day for females were considered not to represent an adverse effect, therefore a ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 300 mg/kg/day for females, based on haematological changes observed at 1000 mg/kg/day.

No treatment-related changes were evident at 30 mg/kg/day, therefore the overall ‘No Observed Effect Level’ (NOEL) was considered to be 30 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is GLP compliant with a klimisch score = 1

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

The study was designed to investigate the systemic toxicity of the test substance and was conducted in accordance with OECD TG 407 Repeated Dose 28 Day Oral Toxicity Study in Rodents.

The oral administration of the test substance to rats for a period of 28 consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment related changes at 1000 and 300 mg/kg/day. The effects at 300 mg/kg/day for females were considered not to represent an adverse effect, therefore a 'No Observed Adverse Effect Level' (NOAEL) was considered to be 300 mg/kg/day for females, based on haematological changes observed at 1000 mg/kg/day.

No treatment-related changes were evident at 30 mg/kg/day, therefore the overall 'No Observed Effect Level' (NOEL) was considered to be 30mg/kg/day


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

Justification for classification or non-classification

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