Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-05-06 to 2008-10-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Pigment Yellow 93
Specific details on test material used for the study:
- Lot/batch No.: 7218
- Expiration date of the lot/batch: 02/2023
- Stability under test conditions: 15 years
- Storage condition of test material: the test substance was stored at the labotarory temperature during the study

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:SPF breeding, VELAZ s.r.o.., Kolec u Kladna, Czech Republic, RCH CZ 21760152
- Age at study initiation: 6 -7 weeks
- Weight at study initiation: ca. 130 g (females) and 170 g (males)
- Fasting period before study: no information
- Housing: 2-3 rats of the same sex in one plastic cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16.09.2008 To: 16.10.2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
The application form (test substance suspension in olive oil) was prepared daily just before administration.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test substance is insoluble in many solvents. Low solubility of the test substance is declared by the sponsor and it was also checked up within the substance identity verification. In this study the substance was found also insoluble in olive oil. Therefore the test substance was administered as an olive oil suspension. A suitable analytical method was not found for homogeneity and stability testing. Since undissolved particles of the test substance are easily visible in the application form, homogeneity was checked by eye (suspension were mixed for 15 minutes by magnetic stirrer).
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Range-finder study
- Rationale for selecting satellite groups: Reversibility of potential effects at the top dose.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Mortality, general health condition and general clinical symptoms were observed daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
At the first part of observation behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behaviour. The second part was the observation during removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption: weekly

WATER CONSUMPTION : Yes
- Time schedule for examinations: twice per week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28 and day 42 (for recovery animals)
- Anaesthetic used for blood collection: Yes (light ether narcosis)
- Animals fasted: 18h
- How many animals: all
- Parameters: total erythrocytes count, mean corpuscular volume, haematocrit, haemoglobin concentration, total leucocytes count, total platelets count, partial thromboplastin time, prothrombin time, fibrinogen, granulocytes, lymphocytes, monocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28 and day 42 (for recovery animals)
- Animals fasted: 18h
- How many animals: all
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: day 28 and day 42 (for recovery animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the last week of treatment and the last week of the recovery time.
- Dose groups that were examined: all
- Battery of functions tested: During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using dynamometer. Measurements were made on: 1) pectoral legs, 2) pelvis legs, 3) all four legs. Grip power was expressed in Newtons.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, thymus, spleen, brain, pituitary gland and heart were recorded.

HISTOPATHOLOGY: Yes (see table 4)

Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with satellite treated group.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the tables of medians or averages.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinically observed signs of toxicity were detected. The animal' s health condition was very good during whole study and functional observation evidenced no effect of the test substance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the test.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No adverse effect on body weight development was detected. The body weight of all treated and control groups were well-balanced without statistically significant difference during the whole application and recovery period. The intergroup differences were minimal and isolated, therefore they were regarded as fortuitous. No significant differences of average body weight increment were recorded in treated groups of males and females (in comparison with control).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on dietary intake or food conversion was detected. Food consumption of all groups was well-balanced. Sporadic slight intergroup reduction or increasing of food consumption and food conversion of males were considered to be a result of normal variation, which could not be related with administration of the test substance. Inspection of water consumption revealed no significant intergroup differences during the application period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The haemocoagulation examination detected statistically significant changes only in males. Shortening of protrombine time and prolongation of partial thromboplastin time were recorded at all dose levels but without dose-response relationship. These changes were not recorded in satellite treated males after 14-day recovery period - the effect of the test substance on this blood component was reversible. All changed parameters were within historical control range. These intergroup differences were considered to be adaptative response of a living organism to stress without toxicological significance.
The haematology parameters of red blood cells were without significant changes. Decreased value of haematocrite was measured in males of the lowest dose level only at the end of application period, but expected related changes of total erythrocyte count, mean corpuscular volume or haemoglobin volume were not detected. Changes of differential leucocyte count were recorded in both sexes but with statistical significance only in males at the middle dose level at the end of application period. Increased total leucocyte count was found out at the end of recovery period. These differences of haematological parameters were considered to be adaptative response to stress without toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis detected no treatment-related effects in males and females.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
unctional observation evidenced no effect of the test substance. All inter and intra group differences in behavioural scores (upstanding, emiction, defecation) and sensory reactivity were considered to be a result of normal variation for rats of the strain and age used and were no toxicological importance. There were no treatment-related changes in functional performance parameters measured (grip strength).
Description (incidence and severity):
Biometry of organs detected intergroup difference in absolute and relative weight of thymus in males but statistically significant increase was found only in absolute weight in the middle dose level. Slight intergroup difference of uterus weight was recorded at treated females. These morphological changes are commonly observed in employed strain of females and are considered to be not of toxicological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Pathology examination revealed yellowish colour of chyme and coloured mucous membrane in digestive system at the end of application period with dose-related response. Other macroscopic findings were found out in stomach of treated males and females: congestion of mucous membrane, dilatation or focal changes on mucous membrane. These changes were not recorded at the end of recovery period - the effect of the test substance was reversible. Application of the test substance caused temporary colouring of the chyme but without marked effect on function of digestive tract.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Metastatic mineralization in kidneys of females at the middle and highest dose levels were found out at the end of the application period. Due to absence of changed urinary parameters, this was considered to be no toxicological importance. Further sporadic pathologic changes were recorded: inflammation in prostate gland in males and hydrometra of uterus or cyst on ovaries in females. Frequencies of these microscopic findings in genital tract were similar in treated and control groups. This was considered to be of no toxicological importance.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic findings were recorded by histopathological examination.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Other findings: Yellowish colour of excrement in the treated animals was recorded from the 1st week of application to the 1st week of the recovery period. As the pigment is of yellow color, this intakes passage of the test item through the gastrointestinal tract.

Biochemical Examination:
Statistical analysis of parameters revealed one significant difference in males (decreased calcium concentration against control at the lowest dose level). This variable factor was not important toxicologically because its fluctuation was within historical control range.
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no unscheduled deaths during the test. No clinically observed signs of toxicity were detected. The animal' s health condition was very good during whole study and functional observation evidenced no effect of the test substance.

BODY WEIGHT AND WEIGHT GAIN: No adverse effect on body weight development was detected. The body weight of all treated and control groups were well-balanced without statistically significant difference during the whole application and recovery period. The intergroup differences were minimal and isolated, therefore they were regarded as fortuitous. No significant differences of average body weight increment were recorded in treated groups of males and females (in comparison with control).

FOOD/WATER CONSUMPTION AND COMPOUND INTAKE: No adverse effect on dietary intake or food conversion was detected. Food consumption of all groups was well-balanced. Sporadic slight intergroup reduction or increasing of food consumption and food conversion of males were considered to be a result of normal variation, which could not be related with administration of the test substance. Inspection of water consumption revealed no significant intergroup differences during the application period.

HAEMATOLOGY: The haemocoagulation examination detected statistically significant changes only in males. Shortening of protrombine time and prolongation of partial thromboplastin time were recorded at all dose levels but without dose-response relationship. These changes were not recorded in satellite treated males after 14-day recovery period - the effect of the test substance on this blood component was reversible. All changed parameters were within historical control range. These intergroup differences were considered to be adaptative response of a living organism to stress without toxicological significance.
The haematology parameters of red blood cells were without significant changes. Decreased value of haematocrite was measured in males of the lowest dose level only at the end of application period, but expected related changes of total erythrocyte count, mean corpuscular volume or haemoglobin volume were not detected. Changes of differential leucocyte count were recorded in both sexes but with statistical significance only in males at the middle dose level at the end of application period. Increased total leucocyte count was found out at the end of recovery period. These differences of haematological parameters were considered to be adaptative response to stress without toxicological significance.

CLINICAL CHEMISTRY: Statistical analysis of parameters revealed one significant difference in males (decreased calcium concentration against control at the lowest dose level). This variable factor was not important toxicologically because its fluctuation was within historical control range.

URINALYSIS: Urinalysis detected no treatment-related effects in males and females.

NEUROBEHAVIOUR: Functional observation evidenced no effect of the test substance. All inter and intra group differences in behavioural scores (upstanding, emiction, defecation) and sensory reactivity were considered to be a result of normal variation for rats of the strain and age used and were no toxicological importance.There were no treatment-related changes in functional performance parameters measured (grip strength).

ORGAN WEIGHTS: Biometry of organs detected intergroup difference in absolute and relative weight of thymus in males but statistically significant increase was found only in absolute weight in the middle dose level. Slight intergroup difference of uterus weight was recorded at treated females. These morphological changes are commonly observed in employed strain of females and are considered to be not of toxicological significance.

GROSS PATHOLOGY: Pathology examination revealed yellowish colour of chyme and coloured mucous membrane in digestive system at the end of application period with dose-related response. Other macroscopic findings were found out in stomach of treated males and females: congestion of mucous membrane, dilatation or focal changes on mucous membrane. These changes were not recorded at the end of recovery period - the effect of the test substance was reversible. Application of the test substance caused temporary colouring of the chyme but without marked effect on function of digestive tract.

HISTOPATHOLOGY: Metastatic mineralization in kidneys of females at the middle and highest dose levels were found out at the end of the application period. Due to absence of changed urinary parameters, this was considered to be no toxicological importance. Further sporadic pathologic changes were recorded: inflammation in prostate gland in males and hydrometra of uterus or cyst on ovaries in females. Frequencies of these microscopic findings in genital tract were similar in treated and control groups. This was considered to be of no toxicological importance. No neoplastic findings were recorded by histopathological examination.

OTHER FINDINGS: Yellowish colour of excrement in the treated animals was recorded from the 1st week of application to the 1st week of the recovery period. As the pigment is of yellow color, this intakes passage of the test item through the gastrointestinal tract.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion