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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2001-01-17 to 2001-07-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
other: draft study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
Annex V
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no

Test material

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, UK
- Age at study initiation: five to seven weeks
- Weight at study initiation: 178 to 229g for males, 151 to 186g for females
- Fasting period before study: no
- Housing: group of 5 by sex and by cage in grid-floor polypropylene cages.
- Diet: ad libitum
- Water : ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): 15
- Photoperiod: 12 hrs dark /12 hrs light

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was prepared at the appropriate concentrations as a solution in arachis oil. Test material formulations were showed to be stable for at least fourteen days.therefore, the dosing solutions were prepared on weekly basis and stored at 4°C in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility of the test material
- Concentration in vehicle: 0, 7.5, 75 and 500 mg/ml
- Amount of vehicle (if gavage): 2 ml/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of substance in the test material formulations was determined by atomic absorption spectroscopy (AAS) using an external standard technique.
The test material formulations were analysed as detailed hereinafter to give a final theoretical test material concentration of approximately 0.3 mg/ml. Standard solutions of test material containing the equivalent amounts of dried arachis oil to that of the test material formulations were also prepared at nominal concentrations in the range of 0.16 to 0.6 mg/ml.
Test material formulations and standard solutions were extracted with methanol, evaporated to dryness and the residue redissolved in sulphuric acid and water. Ammonium sulphate was then added prior to heating over a bunsen flame until a yellow solution was formed and allow to cool, before diluting 0.1% potassium chloride containing 10% hydrochloric acid. The test material formulations were sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days. The test material formulations were sampled and analysed within two days of preparation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 15, 150 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 rats/dose/sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results from the fourteen day preliminary study, where no effects were observed on clinical signs, body weight and necropsy at 1000 mg/kg bw/day dose. Therefore, the dose levels for the present study were selected as 15, 150 and 1000 mg/kg bw/day.

Examinations

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 and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and on Days 6, 13, 20 and 23, all animals were observed for signs of functional and behavioural toxicity. Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded. The animals were placed in arena to evaluate the locomotor activity.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: The quantity of food consumed by the animals in each cage was recorded once a week until the end of the study.
Food consumption was calculated per animals and per day.

FOOD EFFICIENCY: Yes
- 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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all animals
- Parameters examined: Erythrocytes, Hemoglobin, Mean cell volume, Packed cell volume, Mean cell hemoglobin concentration, Mean cell hemoglobin, Thrombocytes, Leucocytes, Differential white cell count with cell morphology, neutrophils, eosinophils, basophils, lymphocytes and monocytes, reticulocytes, Prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period.
- Animals fasted: No
- How many animals: all animals
- Parameters examined: Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Creatinine, Total bilirubin, Total proteins, Albumin, Albumin/globulin ratio, Total cholesterol, Alkaline phosphatase, Aspartate aminotransferase, Alanine aminotransferase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes : Functional Observation Battery (FOB)
Each animal was evaluated once at the end of the treatment period.
This evaluation included a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and motor activity.
The animals were randomized in order to ensure "blind" evaluation.

1- Detailed clinical observation
The following parameters were assessed and graded:
fur appearance, salivation, lacrimation, piloerection, exophthalmos, grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.

2- Reactivity to manipulation and different stimuli
The following measurements, reflexes and responses were recorded: touch response, forelimb /hindlimb grip strength, pupillary reflex, auditory startle reflex, tail pinch response, righting reflex, blink reflex, finger approach, grasp response and vocalisation.

3- Motor activity
For each animal, motor activity was measured once by automated infra-red sensor equipment.
Sacrifice and pathology:
At the end of the treatment period, the animals in each group were sacrificed.

GROSS PATHOLOGY: Yes
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. 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, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes and thymus.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

HISTOPATHOLOGY: Yes (See the "tissue procedure table")
A microscopic examination was performed on:
. all tissues listed in the Tissue Procedure Table for animals of the control and high-dose groups sacrificed at the end of the treatment period,
. all macroscopic lesions as well as the liver and spleen from all low- and intermediate-dose animals sacrificed on completion of the treatment period.

In view to the microscopic results of the high-dose group, the stomach from the low- and intermediate-dose animals sacrificed on completion of the treatment period was examined.
Other examinations:
No
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods:Kruskal-Wallis ANOVA and Mann-Whitney 'U' test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No mortality but clinical signs.
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality but clinical signs.
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):
not examined
Ophthalmological findings:
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
in the stomach
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One female treated with 1000 mg/kg/day was killed in extremis on Day 10 following a mal-dose the previous day.there were no further deaths during the study.
Hunched posture developed in 1000 mg/kg/day animals from Day 9 onwards, predominantly in the females, which increased in occurence and persisted for the duration of the treatment period. Isolated incidents of noisy respiration were also noted during the study. One 1000 mg/kg/day female showed clinical signs consistent with a mal- dose on Days 9 and 10 and was subsequently sacrificed.
No clinical signs were detected at 150 or 15 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN
All animals showed normal gains in bodyweight throughout the study period. Bodyweight development in test animals was similar to that of controls.

FOOD CONSUMPTION
There was no advers effect on food consumption during the study.

FOOD EFFICIENCY
Food efficiency was similar to that of controls.

HAEMATOLOGY
No relevant changes between controls and test item-treated animals were noted in the hematological parameters.

CLINICAL CHEMISTRY
No relevant changes between controls and test item-treated animals were noted in the blood biochemical parameters.

NEUROBEHAVIOUR
Open field assessments confirmed the clinical signs of hunched posture and noisy respiration detected in 1000mg/kg/day animals during the
study.No treatment-related effects were detected at 150 or 1 5mg/kg/day.
No treatment-related effects were observed in Functional Performance tests and Sensory Reactivity Assessments.

ORGAN WEIGHTS
There were no changes in the mean organ weights which were indicative of an effect of the test item.

GROSS PATHOLOGY
No toxicologically significant macroscopic abnormalities were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of tissue sections revealed treatment related stomach changes. Acanthosis, hyperkeratosis and subepithelial cell infiltrates were seen in relation to treatment in the non-glandular gastric epithelium of animals of either sex treated with 1000 mg/kg/day. In addition, treatment-related agglomeration of secretion and hyperplasia of mucus secreting cells in the gastric mucosa was seen in this treatment group. The gastric mucosa of one 150mg/kg/day female was slightly affected.
No treatment-related microscopic abnormalities were detected in 150 mg/kg/day males or animals of either sex treated with 15 mg/kg/day.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Marked histopathological evidence of gastric irritation associated with clinical signs at 1000 mg/kg bw/day
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Minor histopathological changes associated with the irritancy of the test material observed in one female treated at 150 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 7.5.1/1 Clinical signs and time of onset

Sex 

Males

Females

Doses (mg/kg bw /day)

0

15

150

1000

0

15

150

1000

Noisy respiration

-

-

-

1(D9)

-

-

-

1(D5-6, D8-9)

Hunched posture

-

-

-

1(From D21 to D28)

-

-

-

1(D8, D9, D15)

2(D16 to D18, D25 to D28)

3(D19)

4(D21 to 23)

Total of affected animals

0/5

0/5

0/5

1/5

0/5

0/5

0/5

4/4

- = not observed

Table 7.5.1/2 Mean bodyweight gains in g

Sex

Males

Females

Doses (mg/kg bw /day)

0

15

150

1000

0

15

150

1000

Week 1

57±6

60±8

59±5

58±6

25±4

24±9

26±4

24±3

Week 2

55± 3

54± 4

52± 3

50± 8

18± 4

19± 3

18± 1

21± 5

Week 3

40± 5

41± 6

35± 5

35± 7

15± 6

15± 4

16± 2

9± 2

Week 4

32± 2

28± 8

27± 9

33± 8

18± 6

10± 6

14± 2

14± 5

Table 7.5.1/3 Summary incidence of histopathological findings in the stomach

Sex

Males

Females

Doses (mg/kg bw/day)

0

15

150

1000

0

15

150

1000

Number of animals examined

5

5

5

5

5

5

5

4

Acanthosis

Absent

Minimal

Slight

Moderate

5

0

0

0

5

0

0

0

5

0

0

0

3

1

1

0

5

0

0

0

5

0

0

0

5

0

0

0

2

1

1

0

Hyperkeratosis

Absent

Minimal

Slight

5

0

0

5

0

0

5

0

0

4

1

0

5

0

0

5

0

0

5

0

0

3

1

0

Agglomeration secretion mucosa

Absent

Minimal

5

0

5

0

5

0

1

4

5

0

5

0

4

1

3

1

Hyperplasia mucus secreting cells

Absent

Minimal

Slight

5

0

0

5

0

0

5

0

0

0

1

4

5

0

0

5

0

0

4

1

0

2

1

1

Subepithelial inflammatory cell infiltrates

Absent

Minimal

5

0

5

0

5

0

4

1

5

0

5

0

5

0

2

2

Submucosal inflammatory cells

Absent

Present

5

0

5

0

5

0

4

1

5

0

5

0

5

0

5

0

Applicant's summary and conclusion

Conclusions:
Based on the experimental conditions of this study, the NOAEL was considered to be 150 mg/kg bw/day. Minor histopathological changes associated with the irritancy of the test material formulation was observed in one female only at 150 mg/kg bw/day .These types of changes are relatively common in rodent oral gavage studies when the test material is of an irritant nature and are not considered to represent a serious damage to health according to the criteria defined in Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration by gavage to rats for 4 weeks.

The test item was administered daily for 28 days by gavage to groups of 5 male and 5 female Sprague-Dawley rats at dose-levels of 15 or 150 ans 1000 mg/kg bw/day under a constant dosage-volume of 2 mL/kg/day. An additional group received the vehicle only, arachis oil, under the same experimental conditions and acted as a control group. On completion of the treatment period, the animals were sacrificed.

The test item was administered as a solution in the vehicle. Samples of the control and test item dosage forms were taken every week for determination of the test item concentration. The animals were checked daily for mortality and clinical signs. Detailed clinical signs were performed once before the treatment period and once a week throughout the study. A Functional Observation Battery (FOB), including detailed clinical observation, assessment of reactivity to manipulation and different stimuli and motor activity, was performed at the end of the treatment period. Body weight and food consumption was recorded once a week until the end of the study. Hematology and blood biochemistry were performed at the end of the treatment period. On completion of the treatment, the animals were subjected to a full macroscopic post-mortem examination. Designated organs were weighed and designated tissues specimens were preserved. A microscopic examination was performed on designated tissues from control- and high-dose animals and on all macroscopic lesions from low- and intermediate-dose animals sacrificed on completion of the treatment period. In addition, the stomach from the low- and intermediate-dose animals was examined.

One female treated with 1000 mg/kg bw/day was killed in extremis on day 10 following a mal-dose the previous day. There were no further deaths during the study. Hunched posture developed in 1000 mg/kg bw/day animals from Day 9 onwards, predominantly in the females, which increased in occurence and persisted for the duration of the treatment period. Isolated incidents of noisy respiration were also noted. No clinical signs were detected at 150 or 15 mg/kg bw/day.There were no signs of disturbance of neurobehavior in any of the test item-treated animals. No adverse effects on bodyweight or bodyweight gain were noted in treated animals when compared to controls. Hematological and blood biochemical parameters did not reveal any toxicologically significant effect of the test item. There were no treatment-related changes in the mean organ weights of animals sacrificed at the end of treatment and no test item-related changes were observed at necropsy. Microscopic examination revealed signs of pronounced irritation in the non-glandular gastric epithelium of animals given 1000 mg/kg bw/day. These consisted of acanthosis, hyperkeratosis, and subepithelial cell infiltrates observed in both sexes. In addition, treatment-related agglomeration of secretion and hyperplasia of mucus secreting cells in the gastric mucosa was seen in this treatment group. In the 150 mg/ kg bw /day group, one female showed minimal changes in the gastric mucosa. No treatment-related microscopic abnormalities were detected in 150 mg/kg/day males or animals of either sex treated with 15 mg/kg/day.

Based on the experimental conditions of this study, the NOAEL was considered to be 150 mg/kg bw/day. Minor histopathological changes associated with the irritancy of the test material formulation was observed in one female only at 150 mg/kg bw/day .These types of changes are relatively common in rodent oral gavage studies when the test material is of an irritant nature and are not considered to represent a serious damage to health.