Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-06-29 to 2005-01-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 27 July 1995
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: Crl: CD® (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-7 weeks
- Weight at study initiation: 126-164 g (males); 123-151 g (females)
- Housing: in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in dried Arachis oil BP. After extensive method development it was found that only the high dose level could be analysed due to interference from the vehicle at lower levels. The formulations were found to be stable for at least four weeks. They were therefore prepared weekly and stored at approximately +4ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was found appropriate to obtain an applicable solution with the described concentrations.
- Concentration in vehicle: 0, 1.25, 3.75, 12.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test item in the test material formulations was determined by high performance liquid chromatography mass selective detection (HPLC/MSD) using an external standard technique. After extensive method development it was found that only the high dose level could be analysed due to interference from the vehicle.
The test material formulations were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 5 mg/L.
Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 5 mg/L.
The standard and sample solutions were analysed by HPLC.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
the sample results have not been corrected for recovery, because 99% of the nominal concentration was found after HPLC analysis
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
the sample results have not been corrected for recovery, because 99% of the nominal concentration was found after HPLC analysis
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
the sample results have not been corrected for recovery, because 99% of the nominal concentration was found after HPLC analysis
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results from a preliminary study

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before dosing, one hour after and 5 hours after dosing during working week and before dosing and one hour after dosing at weekends and on public holidays

BODY WEIGHT: Yes
- Time schedule for examinations: body weights 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:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

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
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 28
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 28
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 3, 10, 17 and 24 all animals were observed for signs of functional/behavioural toxicity.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Gait, Hyper/Hypothermia, Tremors ,Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation, Motor activity, For/Hindlimb Grip Strength,
Sensory Reactivity: Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see tables 2 & 3)
Statistics:
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):
Animals of either sex treated with 50 mg/kg/day showed increased salivation up to ten minutes after dosing from Day 10 onwards. Males from this treatment group showed prolonged incidents of salivation, up to one and five hours after dosing on Days 22 and 24 respectively. One female treated with 15 mg/kg/day developed a bulging left eye on Day 23, which continued until Day 28. This is a slight physical injury and of no toxicological significance. No such effects were detected in animals of either sex treated with 15 or 5 mg/kg/day.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No adverse effect on bodyweight development was detected.
Females treated with 50 or 5 mg/kg/day showed a statistically significant reduction in bodyweight gain during week 2 of the study. The dose response was unconvincing and, in isolation, the intergroup difference were considered to be without toxicological importance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on dietary intake was detected. Food efficiency (the ratio of bodyweight gain to dietary intake) in test animals was similar to that of controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles revealed no intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no adverse effects detected in the haematological parameters measured.
Statistical analysis of the data revealed no significant intergroup differences.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no adverse effects detected in the blood chemical parameters measured.
Females treated with 50, 15 or 5 mg/kg/day showed a statistically significant reduction in total plasma protein and plasma urea when compared to controls. All individual values were within the normal ranges for rats of the strain and age used and the intergroup differences were considered to be of no toxicological importance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the functional performance parameters measured.
Statistical analysis of the data revealed no significant intergroup differences.
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the organ weights measured.
Females treated with 50 mg/kg/day showed a statistically significant increase in adrenal weight relative to bodyweight. In the absence of histopathological correlates this minimal (p<0.05) increase was considered to be fortuitous and of no toxicological importance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related macroscopic abnormalities were detected.
One male treated with 50 mg/kg/day showed red lungs. In the absence of any histopathological correlates, this was considered incidental and of no toxicological importance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
STOMACH: Agglomeration of gastric secretion with associated mucosal atrophy and erosion were seen as an effect of treatment for animals of either sex treated with 50 mg/kg/day. Agglomeration of secretion was also seen for two males and for one female treated with 15 mg/kg/day and although this condition is occasionally encountered among control rats as a spontaneous entity, the possibility of a relationship to treatment in this instance cannot be excluded. 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.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Table 2: Summary Incidence of Histopathological Findings - Males

Histopathological Finding

Dose Level (mg/kg bw/day)

0

5

15

50

Number of animals examined at terminal kill

5

5

5

5

Bone marrow

Adipose infiltration

 

 

 

 

no data

0

5

5

0

(minimal)

4

0

0

5

(slight)

1

0

0

0

Heart

Focal myocarditis

no data

0

5

5

0

absent

2

0

0

2

(minimal)

3

0

0

3

Pericarditis

 

 

 

 

no data

0

5

5

0

absent

4

0

0

5

present

1

0

0

0

Kidneys

Groups of basophilic tubules

no data

0

5

5

0

absent

3

0

0

2

(minimal)

2

0

0

3

Hydronephrosis

no data

0

5

5

0

absent

4

0

0

5

(minimal)

1

0

0

0

Liver

Mononuclear cell foci

no data

0

5

5

0

absent

1

0

0

0

(minimal)

4

0

0

4

(slight)

0

0

0

1

Lungs

Perivascuolar/Peribronchiolar

lymphoid aggregations

 

 

 

 

no data

0

5

5

0

absent

1

0

0

0

(minimal)

4

0

0

5

Spleen

Extramedullary haemopoiesis

no data

0

5

5

0

(minimal)

5

0

0

4

(slight)

0

0

0

1

Stomach

Agglomeration secretion gastric mucosa

absent

5

5

3

0

(minimal)

0

0

2

4

(slight)

0

0

0

1

Mucosal atrophy/erosion

absent

5

5

5

2

(minimal)

0

0

2

4

(slight)

0

0

0

1

Thyroids

Follicular cell hypertrophy

no data

0

5

5

0

absent

4

0

0

4

(minimal)

1

0

0

1

Urinary Bladder

Epithelial hyperplasia

no data

0

5

5

0

absent

5

0

0

4

(minimal)

0

0

0

1

Statistical information

Mode of death

Terminal kill

5

5

5

5

Table 3:

Summary Incidence of Histopathological Findings - Females

Histopathological Finding

Dose Level (mg/kg bw/day)

0

5

15

50

Number of animals examined at terminal kill

5

5

5

5

Bone marrow

Adipose infiltration

no data

0

5

5

0

(minimal)

5

0

0

4

(slight)

0

0

0

1

Heart

Focal myocarditis

no data

0

5

5

0

absent

4

0

0

5

(minimal)

1

0

0

0

Kidneys

Groups of basophilic tubules

no data

0

5

5

0

absent

4

0

0

3

(minimal)

1

0

0

2

Corticomedullary mineralisation

no data

0

5

5

0

absent

2

0

0

4

(minimal)

3

0

0

1

Liver

Mononuclear cell foci

no data

0

5

5

0

(minimal)

5

0

0

5

Periportal lipid vacuolation

no data

0

5

5

0

absent

3

0

0

5

(minimal)

2

0

0

0

Lungs

Perivascuolar/Peribronchiolar

lymphoid aggregations

no data

0

5

5

0

absent

 

 

 

 

(minimal)

5

0

0

5

Spleen

Extramedullary haemopoiesis

no data

0

5

5

0

(minimal)

5

0

0

5

Stomach

Agglomeration secretion gastric mucosa

absent

5

5

4

1

(minimal)

0

0

1

2

(slight)

0

0

0

2

Mucosal atrophy/erosion

absent

5

5

5

1

(minimal)

0

0

1

2

(slight)

0

0

0

2

Urinary Bladder

Epithelial hyperplasia

no data

0

5

5

0

absent

5

0

0

4

(minimal)

0

0

0

1

Uterus/Cervix

Dilatation horn1

no data

0

5

5

0

absent

5

0

0

4

(slight)

0

0

0

1

Dilatation horn2

no data

0

5

5

0

absent

5

0

0

4

(minimal)

0

0

0

1

Applicant's summary and conclusion

Conclusions:
In the present study conducted according to OECD guideline 407 (adopted 27 July 1995), 5 female and 5 male Sprague-Dawley rats per group were daily administered 0, 5, 15 or 50 mg/kg bw of the test substance for an experimental time of 28 days. No Mortality occurred during the experimental time, clinical signs observed were mainly salivation. Histopathological examinations at the end of the study revealed an agglomeration of gastric secretion with associated mucosal atrophy and erosion in the high dose group and in the mid dose group. The possibility of a relationship to treatment in this instance could not be excluded. Based on the obtained results the NOAEL of the test substance was established to be 5 mg/kg bw/day.