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Toxicological information

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:
11 July 2007 to 29 August 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Reactive Yellow 176 Ester
Reactive Yellow 176 Sulfato

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Crl:(WI) BR rats
Source: TOXI COOP Ltd. 1103 Budapest, Cserkesz u. 90.
Justification of strain: The Wistar rat as a rodent is one of the preferred species of toxicity studies.
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Number of animals: 30 male and 30 female (nulliparous, non-pregnant) rats
Age of animals: Young adult rats, 6 - 8 weeks old
Body weight range at randomisation: Male: 190 - 209 g
Female: 170 - 190 g
Acclimatisation period: 7 days
Animal health: Only healthy animals were used for the study. The veterinarian ascertained healthy status.
Room: 242/2
Housing: Five animals /cage
Cage type: Type III polypropylene/polycarbonate
Bedding: Laboratory bedding, changed together with the cages twice a week
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 8-12 air exchanges/hour by central air-condition system.
Food and Water Supply: The animals received ssniff® SM R/M-Z+H autoclavable complete feed for rats and mice breeding and maintenance produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water from municipal supply from 500 ml bottle, ad libitum.
The diet and drinking water are routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animal Identification: The individual identification was performed by tattoo numbers on the tail.
The boxes were marked by identity cards, with information about study code, sex, dose group, cage number and individual animal numbers.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on oral exposure:
The test item was formulated in concentrations of 6.25 mg/ml, 25 mg/ml and 100 mg/ml prepared with distilled water.
Animals were dosed with the single dose of the test item by gavage daily seven days each week for a period of 28 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of concentration of dosing formulations was conducted on days 5 and 22. All the measured concentrations varied in the range of 100 % and 107 % of the nominal concentrations.
Duration of treatment / exposure:
Duration of treatment: 28 days
Frequency of treatment:
Single dose of the test item daily seven days each week for a period of 28 days.
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 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:
GROUP Dose mg/kg bw/day Concentration mg/ml No. of Animals
Male Female
Group 1 Control 0 5+5* 5+5*
Group 2 62.5 6.25 5 5
Group 3 250 25 5 5
Group 5 1000 100 5+5* 5+5*
* animals for recovery groups.
Control animals:
yes
Details on study design:
The doses were chosen on the basis of the results of repeated dose toxicity studies of compounds with similar chemical structure.

Animals were dosed with the single dose of the test item by gavage daily seven days each week for a period of 28 days. Animals in the control group were treated with the vehicle and handled in an identical manner to those in the test groups. Treatment was carried out at similar times (± 2 hours) in the morning each day. The first treatment day was considered as day 0. Animals were not treated on the day of necropsy.

Duration of treatment: 28 days

Animals of the recovery groups were not treated from day 28. Daily and detailed weekly clinical observation, weekly body weight and food consumption measurements were conducted for a 14 days period. All recovery animals were processed on the same way as animals at termination of the treatment.
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
Clinical Observations: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time.
Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once a week thereafter.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on all animals in the fourth exposure week (day 27). General physical condition and behaviour of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).

Body Weight Measurement: Body weight was measured on day 0 (beginning of treatment), then weekly with a precision of 1 g.

Food Consumption Measurement: The food consumption was determined weekly by re-weighing the non-consumed diet with a precision of 1 g.

LABORATORY EXAMINATIONS
Blood sampling for laboratory examinations including haematology and clinical chemistry were conducted one day after the last treatment and at the end of the recovery period. After an overnight food deprivation (approximately 16 hours), animals were anaesthetised with Euthanyl (Batch No.: 42WG; Expiry date: July 2009; Bimeda-MTC, Animal Health Inc., Cambridge, ON N3C 2W4) and blood samples were collected by heart puncture. Three samples were taken from each animal: one for haematology (tubes contained K3-EDTA as anticoagulant), one for determination of blood clotting times (APTT and PT; tubes contained sodium citrate as anticoagulant) and the third one to obtain serum samples (tubes did not contain any anticoagulant) for clinical chemistry. Clinical chemistry parameters were measured after one day storage of serum at 2-8 ⁰C because of a transient failure of Vitros 250 equipment.

Haematology: parameters listed in table format submitted under any other information.

Clinical Chemistry: parameters listed in table format submitted under any other information.
Sacrifice and pathology:
Necropsy: A gross necropsy was performed on each animal just after the blood harvesting for clinical pathology examinations. One day after the last treatment, animals were sacrificed by exsanguination under pentobarbital anaesthesia (Euthanyl; Batch No.: 42WG; Expiry date: July 2009; Bimeda-MTC, Animal Health Inc., Cambridge, ON N3C 2W4). After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed. Any abnormality was recorded with details of the location, colour, shape and size.

The following organs and tissues were preserved in 10 % formaldehyde solution for histological evaluation:
Gross lesions, lymph nodes (submandibular, mesenteric) sternum, skin and female mammary gland, salivary glands (submandibular), femur + bone marrow, spinal cord (cervical, lumbar, thoracic level), pituitary, thymus, trachea, lungs (with main stem bronchi), heart, thyroid + parathyroid, oesophagus, stomach, caecum, duodenum, ileum, jejunum, colon, rectum, urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, testes with epididymides, ovaries, uterus with vagina, brain (including cerebrum, cerebellum, pons and medulla oblongata), eyes with optic nerve, lachrymal gland with Harderian gland, seminal vesicle, muscle (quadriceps), sciatic nerve and aorta.

Organ Weight Measurement
The following organs:
The following organ weights were weighed and recorded (paired organs were weighed together).
With precision of 0.01g: Liver, kidneys, testes, epididymides, thymus, spleen, brain and heart.
With precision of 0.001g: Adrenals

Histopathology: Histological examinations were performed on the preserved organs or tissues of the animals of the control and high dose groups including animals of recovery period. In groups 2 and 3, liver and kidneys were also processed histologically. The listed organs or their specimens were embedded into paraffin after dehydration. Slides were stained with haematoxylin eosin and examined by a light microscope.
Statistics:
Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- haematology
- clinical chemistry
- organ weight
The heterogeneity of variance between the groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a oneway analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of the inter-group differences. Where significant heterogeneity was found, the normal distribution of data by Kolmogorov-Smirnov test was used. In case of not-normal distribution, the nonparametric method of Kruskal-Wallis One-Way analysis of variance was applied. If positive results were obtained, the inter-group comparisons were performed using Mann-Whitney U-test.
Frequency of clinical symptoms, necropsy and histopathological findings and daily mean food consumption were calculated. At the end of recovery period, the homogeneity of variance between groups was determined by F-test. Depending on the result Poled or Separate variance estimate of the Two-Sample t-test was performed. For data, which was not normally distributed, the data were compared between groups according to Mann-Whitney U-test.

Results and discussion

Results of examinations

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:
not examined
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:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY: There was no mortality during the study.

CLINICAL OBSERVATIONS
Daily and Detailed Weekly Clinical Observations: Decreased activity, piloerection, hunched back, wryneck, tremor and stereotype movement were noted for one female rat (No.: 6764) at 62.5 mg/kg bw/day. The first signs were observed on day 3 (decreased activity, piloerection and hunched back) followed wryneck, tremor and stereotype movement from day 9 up to the end of the treatment. These symptoms were noted at the weekly observations, as well.
There were no clinical symptoms related to the test item in the other animals in the control, 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day groups in the course of the observation period. The physiological state and behaviour of animals were considered to be normal at the daily and at the detailed weekly observation.
In summary: No test item related clinical signs appeared. Neuro-behavioral symptoms of female animal No.: 6764 were considered to be individual disorder and not related to the test item.

Functional Observation Battery
Group 1 - Control: In the male animals, reduced (6/10) or increased (1/10) transfer arousal, lack of finger approach (4/10) or finger withdrawal (1/10), increased touch escape response (2/10), positional struggle to a marked degree (5/10) and vocalisation (5/10) were found. Negative tail pinch reaction was found in one rat (1/10). In the female animals, increased (2/10) or reduced transfer arousal (2/10), reduced finger approach (2/10), lack of finger withdrawal (1/10), increased touch escape response (2/10) and vocalisation (3/10) were noted.

Group 2 - 62.5 mg/kg bw/day: In the male animals, reduced transfer arousal (2/5) lack of finger approach (2/5) or elongated finger approach (1/5) and positional struggle to a marked degree (1/5), no tail pinch reaction (1/5) and vocalisation (1/5) were noted. In the female animals, head searching (1/5), circling (1/5), tremor (1/5), decreased transfer arousal (2/5) and finger approach (2/5) or lack of finger withdrawal (1/5), marked touch escape response (2/5) or positional struggle (2/5) and vocalisation (1/5) were observed.

Group 3 - 250 mg/kg bw/day: In the male animals, reduced (1/5) or increased (1/5) transfer arousal, lack of finger approach (1/5), marked touch escape response (1/5), increased positional struggle (2/5) and vocalisation (1/5) were found. In the female animals, reduced (1/5) or increased (1/5) transfer arousal, reduced finger approach (2/5) or marked finger withdrawal (2/5) positional struggle to a marked degree (2/5) and vocalisation (2/5) were observed.

Group 4 - 1000 mg/kg bw/day: In the male animals, reduced (3/10) or marked (3/10) transfer arousal, lack of finger withdrawal (4/10) marked finger withdrawal (3/10), marked touch escape response (3/10), positional struggle to a marked degree (3/10) and vocalisation (3/10) were observed. In the female animals, reduced (3/10) or increased (2/10) transfer arousal, lack of finger withdrawal (3/10), or marked finger withdrawal (4/10), increased touch escape response (5/10), increased positional struggle (1/10) and vocalisation (1/10) were noted.

In summary: There were no test item effects identified in the functional observation battery. Variations in transfer arousal, finger approach and finger withdrawal, in touch escape response, positional struggle, tail pinch reaction and vocalisation were observed with similar incidences in each group representing inter individual differences in animals behaviour and reactions to different type of stimuli or manipulations. Neuro-behavioural symptoms of female animal at 62.5 mg/kg bw/day were considered to be individual changes because of the singular occurrence and in the lack of any signs in the higher dose groups.

BODY WEIGHT AND BODY WEIGHT GAIN: There were no significant differences in the mean body weight or mean body weight gain between the control and 62.5, 250 or 1000 mg/kg bw/day of Gelb Sulfato groups during the entire treatment period. The mean body weight and mean body weight gain remained similar to the control value in the male and female animals during the recovery period. A significant body weight loss was noted in female animal No.: 6764, (62.5 mg/kg bw/day) during the first week as individual alteration corresponding with clinical signs.

FOOD CONSUMPTION: No test item related differences were observed in the daily food consumption between the control and Gelb Sulfato treated groups at 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day. The mean daily food intake was also similar in the control and high dose animals during the recovery period.

HAEMATOLOGY
Group 2 - 62.5 mg/kg bw/day: In the male animals, the mean platelet (thrombocyte) count (PLT) was slightly less and the percent of large unstained cells (LUC) was higher than the control value. In the female animals, the red blood cell count (RBC) was slightly less than the control.

Group 3 - 250 mg/kg bw/day: In the male animals, the percent of large unstained cells (LUC) was higher than the control value. In the female animals, the red blood cell count was slightly less than the control.

Group 4 - 1000 mg/kg bw/day: There were no statistically significant differences in the examined parameters when compared to the control in the male or in the female animals. At the end of the recovery period, the red blood cell distribution width (RDW) and percent of basophile cells (BA) were less, the percent of lymphocytes (LY) was higher than the control value in the male group of 1000 mg/kg bw/day. The haemoglobin content of plasma (HGB), the haematocrite (HTC), the mean corpuscular haemoglobin content of erythrocytes (MCHC) and the percent of monocytes (MO) were slightly less than the control value in the female animals.

In summary: There was no test item related adverse effect on the examined haematological parameters. Slight statistically significant differences from the control were noted for some parameters (PLT, LUC, RBC) only at 62.5 and 250 mg/kg bw/day, which were of low magnitude and within the normal physiological range (historical control) and were independent from dose. The differences of some parameters at the end of the recovery period represent also the biological variations of these parameters (RDW, BA, LY, HGB, HTC, MCHC, MO). So these were not considered to be toxicologically significant.

CLINICAL CHEMISTRY
Group 2 - 62.5 mg/kg bw/day: All examined clinical chemistry parameters were similar to the control in the male the female animals.

Group 3 - 250 mg/kg bw/day: In the male animals, the activity of alkaline phosphatase (ALKP) was slightly higher than the control value. In the female animals, there were no differences from the control in the examined parameters.

Group 4 - 1000 mg/kg bw/day: In the male animals, the ALKP activity was less than the control. In the female animals, the concentration of chloride (Cl-) was less than the control. In the recovery period, no significant differences were found in the examined parameters between the control and high dose animals.

In summary: There were no test item related changes in the examined clinical chemistry parameters. The slight differences in ALKP activity and chloride concentrations terminally represented physiologic variation. These were considered to be normal values independent from the treatment.

NECROPSY
Group 1 – Control: In the male animals, in the lungs pinprick-sized haemorrhages (2/5) and in the kidney one side pyelectasis (1/5) were observed. In the female animals, pinprick-sized (1/5) or point like (1/5) haemorrhages were found in the lungs and the liver was pale (1/5). At termination of the recovery period, pinprick-sized haemorrhages in the lungs (1/5, male) and hydrometra (1/5, female) were detected.

Group 2 - 62.5 mg/kg bw/day: In the male animals, pale raised areas in the lungs (1/5) and one side pyelectasis in the kidney (2/5) occurred. In the female animals, reddish mottled lungs (1/5) and hydrometra (1/5) were detected.

Group 3 - 250 mg/kg bw/day: In the male animals, in the lungs pinprick-sized haemorrhages (1/5) and pale kidneys (both sides, 2/5) and one side pyelectasis (1/5) were observed. In the female group, the liver (1/5) and kidneys (1/5) were pale in one rat.

Group 4 - 1000 mg/kg bw/day: In the male animals, pinprick-sized (1/5) or point like (1/5) haemorrhages were noted. In one female animal hydrometra (1/5) were observed. In the male recovery group, in the lungs pinprick-sized haemorrhages (1/5) were observed. Pinprick sized haemorrhages (2/5) and hydrometra (1/5) were noted for female recovery group.

In summary: No test item related macroscopic findings were detected at the necropsy. Kidney alterations occurred at the control and lower doses only (one side pyelectasis; male control, 62.5 and 250 mg/kg bw/day, pale kidneys at 250 mg/kg bw/day male and female). Pale liver occurred in single female animals of the control and 250 mg/kg bw/day groups but not in high dose group. Thus both kidneys and liver findings were considered to be independent from the test item. The haemorrhages, pale raised areas and reddish mottled colour in the lungs of animals were caused by the euthanasia procedures. Hydrometra indicative of the sexual cycle of animals was present in some female animals of the control and treated groups.

ORGAN WEIGHT
62.5 mg/kg bw/day, 250 mg/kg bw/day, 1000 mg/kg bw/day
The only statistically significant difference from the control was noted for male animals at 1000 mg/kg bw/day in the spleen weight relative to the body weight at the termination of the treatment. There were no differences in other examined organ weights (absolute and relative to the body and brain weights) between the control and any test item treated group.
At the end of the recovery phase, the heart and epididymides weights both relative to the body weights were slightly higher than the control value in male animals. There were no differences in the examined organs between the control and treated female animals.

In summary: There was no test item effect on the examined organ weights. The difference in spleen weight at 1000 mg/kg bw/day was low and not considered to be significant because of singular occurrence (there were no related haematological or histopathological findings). Heart and epididymides weights were not influenced at the termination of the treatment, so the small differences at the end of the recovery period were representative of biological variations and were not considered to have toxicological significance.

HISTOPATHOLOGY
Histopathological examinations revealed focal alveolar emphysema or haemorrhages in the lungs as follows:

Dose mg/kg bw/day Focal alveolar emphysema Focal haemorrhages
Male Female Male Female
Control 1/5 2/5 2/5 3/5
Control Recovery 1/5 1/5 1/5 0/5
1000 1/5 0/5 2/5 2/5
1000 Recovery 0/5 0/5 1/5 1/5

For male animals, in the kidneys one side pyelectasia was noted in the control (1/5), 62.5 mg/kg bw/day (2/5) and 250 mg/kg bw/day (1/5) groups.
In female groups, uterus dilatation was observed in some animal (1/5 in control recovery group; 1/5 in 1000 mg/kg bw/day terminal group, 1/5 in 1000 mg/kg bw/day recovery group).

No morphological evidence of acute or subacute injury of the alimentary tract, the pancreas, the cardiovascular system, the haematopoietic system, the immune system, the skeleton, the muscular system, the male and female reproductive system, or the central or peripheral nervous system was detected. The structure and the cell morphology of the endocrine glands was the same at the control and treated animals.

In summary: Histopathological examination did not reveal any toxic or other test item related findings. The focal alveolar emphysema and haemorrhages in the lungs were caused by hypoxia, dyspnoea and circulatory disturbance developed during exsanguination. The incidence and severity of these lesions were similar in the control and high dose treated groups. The pyelectasia (unilateral) in the kidney was slight individual disorders without toxicological significance. The dilatation of uterine horns in one female animal was a slight neuro-hormonal phenomenon in connection with the sexual function of the inner genital organs.

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Test substance caused no toxic effects at 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day doses in CRL:(W) BR rats after 28-day continuous oral (by gavage) administration.
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

Conclusions:
Gelb Sulfato caused no toxic effects at 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day doses in CRL:(W) BR rats after 28-day continuous oral (by gavage) administration.
The no observed effect level (NOEL) of Gelb Sulfato was 1000 mg/kg bw/day.
Executive summary:

The study was performed in accordance with the study plan agreed upon by Sponsor, the OECD Guidelines for Testing of Chemicals No. 407, Directive 96/54/EC B.7 and the Principles of Good Laboratory Practice (GLP) and reported with a GLP certificate.

 

A 28-day repeated dose oral (by gavage) toxicity study was performed in CRL:(WI) BR rats (n= 5 animals/sex/group and 5 animals/sex in the control and Group 4 for recovery) on Gelb Sulfato with dose levels of 0 (vehicle only), 62.5, 250 and 1000 mg/kg bw/day for 28 days. Animals were treated by daily oral gavage with test item or vehicle (distilled water) at a constant dosing volume of 10 ml/kg body weight. Recovery animals were retained for further 14-day observation without treatment.

The stability of test item in this vehicle was found to be adequate (at least 72 hours at room temperature). The concentration of dosing solutions varied in the range of 100 % to 107 % of the nominal concentrations.

General clinical observations were made daily and detailed clinical observations were performed weekly. A functional observation battery was conducted in week 4. Body weight and food consumption were measured weekly. Clinical pathology and gross pathology were conducted one day after the last treatment. Selected organs were weighed. A full histological examination was performed on the preserved organs and tissues of the animals of the Control group and Group 4 (1000 mg/kg bw/day). The liver and kidneys were evaluated histologically in Group 2 and Group 3. Animals of recovery groups were processed on the same way as animals at termination of the treatment.

 

Results

Mortality:There was no mortality.

Clinical observations:No test item related clinical symptoms were noted. There were no test item effect on the behaviour and reaction of animals to different type of stimuli (Functional Observation Battery).

Body weight:No test item influence on the body weight development was found.

Food consumption:There were no test item related differences in the mean food consumption between any dose levels.

Clinical pathology:Haematological and clinical chemistry investigations did not reveal any toxic changes related to test item in the examined parameters.

Organ pathology:There were no pathological findings related to the test item at gross necropsy, organ weight or histopathological examinations.

 

Conclusions

Gelb Sulfato caused no toxic effects at 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day doses in CRL:(W) BR rats after 28-day continuous oral (by gavage) administration.

 

The no observed effect level (NOEL) for Gelb Sulfato was 1000 mg/kg bw/day.