REAKTIV-GELB F-68 072 FW

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2006 to 14 September 2006

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

lithium sodium 2-amino-4-{[4-(cyanoazanidyl)-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl]amino}-5-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)benzene-1-sulfonate

EC Number:

413-090-5

Cas Number:

189574-45-6

Molecular formula:

Hill formula: C24H18LixN10NayO12S4

IUPAC Name:

lithium sodium 2-amino-4-{[4-(cyanoazanidyl)-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl]amino}-5-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)benzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

See below

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat has proved to be a suitable species for repeat-dose oral toxicity testing providing ample historical data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Crl:(WI) BR rats
Source: TOXI COOP Ltd. 1032 Budapest, Cserkesz u. 91.
Justification of strain: The Wistar rat as a rodent is one of the preferred species of toxicity studies
Hygienic level at arrival: SPF
Number of groups: 4 groups: one control and three groups treated with test item
Number of animals: 50 male and 50 female rats
15 animals/sex in the control and high dose groups (including 5 animals/sex for recovery groups) 10 animals/sex in the low
and middle dose groups
Age of animals: Young adult rats, 6 weeks old
Body weight range at randomisation: Male: 170- 194 g; Female: 120 - 152 g
Acclimatisation period: 9 days
Animal health: Only healthy animals were used for the study. The veterinarian ascertained healthy status.
Room: 242-3; 242-4
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

The temperature and relative humidity were checked and recorded twice daily during the study

Food and Water Supply
The animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiaten GmbH, D-59494 Soest Germany and tap water from municipal supply from 500 ml and 1000 ml bottles, 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 numbers were given on the basis of LAB International Research Centre Hungary Ltd.'s master file, for each animal allocated to the study.
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

Details on route of administration:

The oral route is a likely exposure route for humans

Vehicle:

water

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. Formulations were prepared daily during working days and beforehand for weekends by Central Dispensary Unit of LAB International Hungary Ltd.
Reaktiv Gelb F68072 FW was stable at the applied concentrations at room temperature for 24 hours and in refrigerator (5 ± 3 °C) for 4 days. Analytical control of concentration of dosing formulations was conducted on first and last weeks of the treatment. All the measured concentrations varied in the range of 92 % and 109 % of the nominal concentrations.

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 first and last weeks of the treatment (22 May 2006 and 11 August 2006). All the measured concentrations varied in the range of 92 % and 109 % of the nominal concentrations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

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 10+5* 10+5*
Group 2 62.5 6.25 10 10
Group 3 250 25 10 10
Group 4 1000 100 10+5* 10+5*
* Animals for recovery groups.

Control animals:

yes, concurrent no treatment

Details on study design:

Justification of doses
The doses were chosen on the basis of the results of the acute oral toxicity study with the test item.

Treatment Volume
A constant treatment volume of 10 ml/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent body weight of the animals.

Procedure
Animals were dosed with the single dose of the test item by gavage daily seven days each week for a period of three month. 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: 90 days for male animals
91 days for female animals

Animals of the recovery groups were not treated from day 90. Daily and detailed weekly clinical observation, weekly body weight and food consumption measurements were conducted for a 28-day period. All recovery animals were processed on the same way as animals at termination of the treatment.

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 13th exposure week (day 89). General physical condition and behaviour of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: la. 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.

Ophthalmoscopy
The fundus of eyes of all animals was examined before treatment and in the control and high dose groups (Groups 1 and 4) on week 13. Mydriasis was produced after instillation of eye drops "Mydrum" into the conjunctival sac. The examination was performed using an ophthalmoscope

Laboratory Examinations
Laboratory examinations were conducted at termination of the treatment (including haematology, clinical chemistry and urinalysis) and at the end of the recovery period (including haematology and clinical chemistry). After an overnight food deprivation (approximately 16 hours), animals were anaesthetised with Euthanyl 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. After blood sampling animals were sacrificed by exsanguination.

Haematology-see table attached under any other information.

Clinical Chemistry-see table attached under any other information

Urinalysis
Urine samples were collected for 16 hours at the termination of the treatment. The evaluation of the urine samples was performed by test strips. Table of analysis attached under any other information.

Necropsy
Dead animal was subjected to gross pathology just after he was found dead. Terminally and at the end of the recovery period, 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 (Euthanyl) anaesthesia. 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:
Gross lesions, lymph nodes (submandibular, mesenteric) sternum, skin and female mammary gland, salivary glands (submandibular), larynx, 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, Harderian glands and lachrymal gland, seminal vesicle, muscle (quadriceps), sciatic nerve, aorta

Sacrifice and pathology:

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

Histopathology
Full histological examinations were performed on the organs or tissues of the animals of the control and high dose groups. In groups 2 and 3, liver, kidneys and organs with macroscopic alteration 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

Other examinations:

A functional observation battery was conducted on week 13. This considered of the following evaluations:
Body position, Locomotor Activity, Respiration rate, Respiration Type, Piloerection, Head searching, Compulsive Biting or Licking, Circling, Upright Walking, Retropulsion, Jumping, Exophthalmos, Twitches, Clonic convulsions, Tonic convulsions, Tremor, Startle, Transfer arousal, Spatial locomotion, Gait, Posture (hunched back), Limb position, Finger approach, Finger withdrawal, Touch escape response, Diarrhoea, Diuresis, Visual placing, Grip Strength, Body tone, Corneal reflex, Toe pinch, Positional struggle, skin, mucous membrane colour, salivation, Palperbral closure, Lachymation, Limb tone, Abdominal tone, Tail pinch, Righting reflex, Vocalisation.

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
body weight
food consumption
haematology
clinical chemistry
urinalysis
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 one way 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 non-parametric 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 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:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, polyuria and increased water consumption were noted from day 2 up to the end of the treatment period. The drinking of water and diuresis of animals became normal on the 10th day of the recovery period.
The water consumption and polyurea was ascribed to the high salt level in the test item (lithium and sodium), This physiological effect is not considered to reflect an adverse toxic response.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal (No.: 52; 1000 mg/kg bw/day) was found dead on day 66. There were no preceding clinical signs. Gross necropsy revealed signs of para gastric treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Occasionally lower the body weight gains of male animals dosed at 1000 mg/kg bw/day resulted in body weight differences when compared to the control from day 56 onwards. The recovery group showed increased weight gain after cessation of treatment. This is considered to be a secondary effect of the higher water uptake; however, all weights were within the normal range of body weight for this strain and age of rats.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A slightly reduced daily mean food consumption was noted at 1000 mg/kg bw/day dose in males in parallel to body weight changes, which reversed in the recovery period. This is considered to be a secondary effect of the higher water uptake.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, increased water consumption was noted from day 2 up to the end of the treatment period. The drinking of water of animals became normal on the 10th day of the recovery period. The increased water consumption was ascribed to the high salt level in the test item (lithium and sodium). This physiological effect is not considered to reflect an adverse toxic response.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Higher urine excretion was observed in all groups, which manifested in an elevated volume of urine at 62.5 mg/kg bw/day (no statistical significance in male group), 250 mg/kg bw/day and 1000 mg/kg bw/day and in a decreased specific gravity at 250 mg/kg bw/day male and at 1000 mg/kg bw/day (male and female animals). Although the effect was related to treatment, it was considered to be a physiological effect, related to the high salt level in the test item and resulting higher water intake, and not an adverse effect of treatment.

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
One male animal (No.: 52; 1000 mg/kg bw/day) was found dead on day 66. There were no preceding clinical signs. Gross necropsy revealed signs of paragastric treatment, and was not considered to be attributed to test material toxicity.

CLINICAL OBSERVATIONS
Daily and detailed weekly clinical observations

Control, 62.5 mg/kg bw/day and 250 mg/kg bw/day
There were no clinical signs in the control, 62.5 mg/kg bw/day or 250 mg/kg bw/day groups in the course of the 3-month observation period. The physiological state and behaviour of animals were considered to be normal in the control and in all Reaktiv Gelb F68072 FW treated groups at the daily and at the detailed weekly observation.

Control animals were symptom free in the recovery period.

1000 mg/kg bw/day
Polyuria and increased water consumption were noted from day 2 up to the end of the treatment period. The drinking of water and diuresis of animals became normal on the 10th day of the recovery period.

The water consumption and polyurea was ascribed to the high salt level in the test item (lithium and sodium). This physiological effect is not considered to reflect an adverse toxic response.

Functional observation battery
Group 1 - Control
In the male animals, lack of startle reaction (2/15) or extreme startle reaction (1/15), dulled (2/15) or hyperalert (1/15) transfer arousal, vigour spatial locomotion (1/15) marked positional struggle (4/15) and vocalisation (4/15) were noted. In the female animals, increased locomotor activity (4/15), startle reaction (2/15), transfer arousal (2/15) and spatial locomotion (3/15), extreme positional struggle (2/15) and vocalisation (2/15) were observed.

Group 2-62.5 mg/kg bw/day
In the male animals, lack of startle reaction (1/10) and reduced transfer arousal (2/10), rapid spatial locomotion (1/10) marked positional struggle (2/10) and vocalisation (2/10) were found.
In the female animals, increased (2/10) or less (2/10) locomotor activity, hperalert transfer arousal (2/10) and spatial locomotion (2/10), extreme positional struggle (2/10) and vocalisation (2/10) were observed.

Group 3-250 mg/kg bw/day
In the male animals, an increased locomotor activity (1/10), dulled (2/10) or hyperalert (2/10) transfer arousal, vigour (1/10) or less than normal (1/10) spatial locomotion, positional struggle to a marked degree (4/10) and vocalisation (4/10) were observed.
In the female animals, rapid locomotor activity (3/10), lack of startle reaction (2/10) and marked startle reaction (2/10), extreme transfer arousal (2/10), vigour spatial locomotion (2/10), an extreme touch escape response (1/10) or lack of touch escape response (1/10), marked positional struggle (2/10) and vocalisation (2/10) were observed.

Group 4 - 1000 mg/kg bw/day
In the male animals, an extreme (2/14) or decreased (1/14) startle reaction hyperalert (2/14) or dulled (1/14) transfer arousal, rapid (1/14) or less (1/14) spatial locomotion, extreme positional struggle (4/14) and vocalisation (4/14) were found. In the female animals, increased locomotor activity (5/15), lack of startle reaction (3/15) or marked startle reaction (3/15) increased (4/15) or decreased (1/15) transfer arousal and vigour spatial locomotion (4/15), increased positional struggle (3/15) and vocalisation (3/15) were noted.

In summary: There were no differences related to the test item in the examined parameters of sensory reaction to different type of stimuli or motoric activity. The changes in locomotor activity, startle reaction, transfer arousal, spatial locomotion, touch escape response, positional struggle and vocalisation of several rats in the control and test item treated groups revealed inter-individual differences independent from the test item but indicative of different emotional, behavioural features of rats.

BODY WEIGHT AND BODY WEIGHT GAIN

Group 2-62.5 mg/kg bw/day
In the male animals, there were no significant differences in the mean body weight during the treatment period. The mean body weight gain was less than the control on weeks 4, 5 and 11.
In the female animals, the mean body weight was similar to the control during the entire observation period. The mean body weight gain was slightly below the control on week 12.

Group 3-250 mg/kg bw/day
In the male animals, there were no significant differences in the mean body weight or mean body weight gain during the three-month.
In the female animals, the mean body weight was slightly above (7-12 %) the control value with statistical significances in several cases (days 21, 28, 35 and 63, 77, 84 and 89) from day 21 up to termination of the treatment. The mean body weight gain was higher than the control on each week except three weeks, with statistical significance on weeks 1 and 11. These changes resulted in an approximately 20 % higher summarised body weight gain at the termination of the treatment.

Group 4 - 1000 mg/kg bw/day
In the male animals, there were no significant differences in the mean body weight during the first half part of the study (from day 0 to day 49), however it was significantly less than the control (between minus 9 % and minus 24 %) from day 56 up to end of the treatment. The mean body weight gain was less than the control on weeks 2, 5 and from week 7 up to week 11 and exceeded the control value on week 12. Consequently, the summarised body weight gain was also less than in the control group (-28 %) at the termination of the treatment.
In the female animals, the mean body weight was higher (8-12 %) from day 14 up to 77. The body weight gain was higher than that in the control group during the first three weeks (26-41 %), on week 9 (96 %) and it was less on week 12 (-90 %). The summarised body weight gain was slightly higher (14 %) at termination of the treatment.

In the recovery period, the body weight remained less in male animals from day 96 up to day 117, even though the body weight gain exceeded the control (100 % when summarised).
There were no differences during the recovery period in the female group.

In summary: The lower the body weight gain of male animals dosed at 1000 mg/kg bw/day resulted in body weight differences when compared to the control. The reversibility groups showed much increased weight gain after cessation of treatment, this is a typical response to non-specific toxicity. The body weight change in males at 1000 mg/kg was considered to be an effect of treatment, although all weights were within the normal range.

The elevated body weight gain and related higher body weight independent from doses observed in female rats at 250 mg/kg bw/day and 1000 mg/kg bw/day. Thus differences in female groups were considered as physiological variations and not related to treatment. The historical control data support this statement: Mean = 126.4 ± 25.8 g; (min = 77 g; max = 205 g; n= 48) vs.103. 93 ± 19.81 g, n= 15 in the control group and 131.13 ± 21.53 g, n= 15 in the high dose group. It is evident, that both values are within the background limits, but in opposite directions, resulting in statistical significant differences in the body weight gain data, of no toxicological significance.

FOOD CONSUMPTION
There were no differences in the daily food consumption between the control and test item treated groups at the 62.5 mg/kg bw/day (male and female) and 250 mg/kg bw/day (male) doses.

In the female 250 mg/kg bw/day group, the daily food intake was higher than the control on weeks 4 and 5.

In the 1000 mg/kg bw/day group, a slightly less food consumption was noted for male group on weeks 9 and 10, while the daily food consumption slightly exceeded the control value in female animals during the entire treatment period with statistical significances on weeks 2, 3, 4, 5 and 7.
In the recovery period, the food consumption of 1000 mg/kg bw/day group (male and female) exceeded the control value in the most occasions.

In summary: The mean daily food consumption correlated with the body weight differences in male and female groups. A slight test item influence on the daily mean food consumption was noted at 1000 mg/kg bw/day dose in male groups, which reversed in the recovery period. In female animals, the differences were related with the body weight differences at this time period and were not considered to be of toxicological significance.

HAEMATOLOGY

62.5 mg/kg bw/day
In the male animals, the mean prothrombin time (PT) was slightly less than the control value.
In the female animals, the partial thromboplastin time (APPT) was slightly longer than in the control group.

250 mg/kg bw/day
In the male group, there were no differences in the examined haematological parameters.
In the female animals, ratio of segmented white blood cells (SE) and monocytes (MO) exceeded the control and the lymphocyte count (LY) was less than the control.

1000 mg/kg bw/day
In the male animals, the red blood cell count (RBC), the haemoglobin concentration (HGB) and the haematocrit value (HCT) were less, while the mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were higher than the appropriate control. The platelet count (PLT) was also below the control. In the female animals, the mean corpuscular haemoglobin concentration (MCHC) and white blood cell count (WBC) was higher and the prothrombin time was shorter than the control. At the end of the recovery period, no differences were noted for male and female groups.

In summary: There were no toxicologically significant test item induced alterations in the examined haematological parameters at termination of the treatment. At 62.5 and 250 mg/kg/d there were no differences that could be related to treatment. At 1000 mg/kg/d there were statistical differences in male values, which may have been influenced by treatment, but all values were well within the historical control range. None of the statistical differences were indicative of a biologically significant change in haematological parameters.

CLINICAL CHEMISTRY

Group 2-62.5 mg/kg bw/day
In the male group, the urea and calcium (Ca2+) concentration were higher than the control value.
In the female animals, creatinine (Creat.) level and activity of aspartate aminotranspherase (AST) were slightly less, while the calcium (Ca2+), cholesterol (Choi.) and albumin level (Alb) and albumimglobulin ratio (A/G) was higher than the control.

Group 3 - 250 mg/kg bw/day
In the male animals, sodium (Na+) and chloride (CI-) level were less and calcium (Ca2+) concentration were higher than the control.
In the female animals, urea, calcium, phosphorous (Phos.) concentrations were higher the sodium and chloride concentration were slightly less than the control.

Group 4 - 1000 mg/kg bw/day
In the male animals, the glucose, urea, creatinine (Creat.) and potassium (K+) levels were slightly less, while the calcium (Ca2+) concentration was slightly above the control value.
In the female animals, an elevated level of calcium and phosphorous was observed. The concentrations of urea, creatinine, sodium, potassium and chloride, albumin, total bilirubin (T-BIL) and activity of AST were slightly less.

In the recovery group, the creatinine concentration was less, the sodium and chloride concentration were higher than the control in male animals. In the female group, the activity of AST was below the control value.

In summary: There were no test item related marked differences in the examined biochemical parameters. The statistically significant differences in several parameters (glucose, urea, creatinine, sodium, potassium and chloride, calcium and phosphorous, cholesterol, albumin, total bilirubin and A/G ratio as well as activity of AST) were considered not related to the treatment because of the singular occurrence or slight magnitude of the alteration or lack of any dose relevance or any related pathology changes.

URINALYSIS

Group 2-62.5 mg/kg bw/day
In the male group, there were no differences in the examined urine parameters when compared to the control.
In the female group, the volume of urine (VOL) was slightly higher.

Group 3 -250 mg/kg bw/day
In the male animals, the specific gravity (SG) was slightly less the volume of urine was higher than the control.
In the female animals, the volume of urine exceeded the control value.

Group 4 - 1000 mg/kg bw/day
In the male animals, the specific gravity was slightly less and the volume of the urine was higher than the control.
In the female animals, the specific gravity was slightly below the control, the pH and the volume of the urine was higher than the control.

In summary, a test item influence on the urine excretion was observed in all groups, which manifested in an elevated volume of urine at 62.5 mg/kg bw/day (no statistical significance in male group) 250 mg/kg bw/day and 1000 mg/kg bw/day and in a decreased specific gravity at 250 mg/kg bw/day male and at 1000 mg/kg bw/day (male and female animals). Although the effect was related to treatment, it was considered to be a physiological effect, related to the high salt level in the test item, and not an adverse effect of treatment.

NECROPSY

Dead animal
Dark red lungs, blood filled thoracic cavity and yellow colour of the organs were noted for a single dead rat (No.: 52, 1000 mg/kg bw/day).

Group 1 - Control
In the male animals, pinprick-sized haemorrhages in the lungs (2/10), one side pyelectasis (1/10) and haematoma in the thymus (1/10) were noted.
In the female animals, in the lungs swollen white eminences (1/10) and pinprick-sized haemorrhages (2/10) were present and hydrometra was observed in three animals (3/10).

In the recovery control group, pinprick-sized (1/5 male) or point-like (2/5 male) haemorrhages in the lungs, one side pyelectasis (1/5 male) and compact formation in the liver (1/5 female) occurred.

Group 2-62.5 mg/kg bw/day
In the male animals, tumour-like formation was found in the thymus (1/10) and seminal vesicle was enlarged (1/10).
In the female animals, pinprick-sized haemorrhages in the lungs (2/10) as well as hydrometra in the uterus (2/10) were found.

Group 3 - 250 mg/kg bw/day
In the male animals pinprick-sized haemorrhages were found in the lungs (2/10). Pyelectasis (one side) was observed in one animal (1/10).
In the female animals, pinprick-sized haemorrhages were found in the lungs (2/10), and hydrometra in one rat (1/10).

Group 4 - 1000 mg/kg bw/day
In the male animals, pinprick-sized haemorrhages were observed in the lungs (1/10). In the female animals, in the lungs swollen white eminences (1/10) were present. Hydrometra was observed in two animals (2/10).

In the recovery 1000 mg/kg bw/day group, pinprick-sized haemorrhages in the lungs (2/5 female), one side pyelectasis (1/4 male) and hydrometra (1/5) were found.

In summary: No test item related macroscopic findings were detected at the necropsy. The haemorrhages in the lungs of animals were caused by the euthanasia procedures and were present in the control and in the test item treated groups. Pyelectasis as common necropsy findings in experimental rats occurred in the control and test item treated groups with similar incidence. Hydrometra due to the sexual cycle of animals occurred in some female animals of the control and dose groups. Some animals showed individual macroscopic findings (haematoma or tumour-like formation in the thymus, swollen and white eminences in the lungs, compact formation in the liver, enlarged seminal vesicle), which occur also in untreated experimental rats. These observations have no toxicological importance in this study.

ORGAN WEIGHT

Group 2 - 62.5 mg/kg bw/day and Group 3 - 250 mg/kg bw/day

There were no differences in the weight (absolute and relative to the body and brain weight) of examined organs in male and female animals. The fasted body weight of female animals exceeded the control value at 250 mg/kg bw/day, consequently there was a slight difference in the brain weight relative to the body weight and body weight relative to the brain weight when compared to the control.

Group 4 - 1000 mg/kg bw/day.
In the male animals, the mean adrenal weights (absolute and relative to the body and brain weight) were higher than the control value. The weights of brain, liver, heart, spleen, kidneys and epididymides relative to the body weight were higher and body weight relative to the brain weight was less.
In the female animals, the liver, heart, spleen, kidneys and adrenal weights were higher than the control. The brain weight relative to the body weight was less, while the liver, heart, kidneys and adrenal weights relative to the body weight and liver, heart, spleen, kidneys and adrenal weights relative to the brain weight exceeded the control value.

At the end of the recovery phase, the body weight, testes and epididymides weights were below the control. The weights of brain, liver, heart and kidneys relative to the body weight were higher, while the body weight relative to the brain weight was less than the control in the male animals.
In the female group, the brain weight relative to the body weight was less, the body weight and heart and kidney weights relative to the brain weight were higher than the control.

In summary: No test item related differences were noted in the weighed organs of test groups. The differences in relative weights of organs of male animals except adrenals were due to the body weight change terminally and at the end of the recovery period. There were no related macroscopic and histopathological findings, thus the higher adrenal weight probably was due to an increased physiologic function. Likewise, the testes and epididymides weight at the end of recovery period were indicative of a reduced function. The weight differences of brain, liver, heart and kidneys, spleen and adrenals of female animals were not verified by relevant pathologic findings (clinical or histopathological) thus these were not considered indicative of toxic signs, but as physiological differences, which might be influenced by the body weight differences, too. These statistical significances were not representative of adverse effects.

HISTOPATHOLOGY

Dead animal
Histological examination of the organs revealed congestion and alveolar oedema in the lung as the probably cause of death (animal No.: 52, male 1000 mg/kg bw/day recovery). Para-gastric treatment resulted in peripheral circulatory and breathing disturbance manifested in congestion and alveolar oedema in the lungs. No degenerative or other - possible toxic - lesion was detectable in the investigated organs.

Surviving animals Control
In the male animals, in the lungs alveolar emphysema (3/10) and focal haemorrhages (3/10), in the kidneys one side pyelectasis (1/10) and in the thymus haematoma (1/10) were observed.
In the female animals, alveolar emphysema (3/10) and focal haemorrhages (2/10) and foamy cells (1/10) were observed in the lungs. Dilatation of uterus was noted for three animals (3/10).
In the control recovery group, alveolar emphysema (1/5 male and 1/5 female) and focal haemorrhages (3/5 male) were found in the lungs. One side pyelectasis was present in one male rat (1/5 male).

62.5 mg/kg bw/day
In the male animals, thymoma (1/1) and dilatation of seminal vesicle (1/1) were observed. There were no histopathological findings in the examined organs (liver and kidneys) of female animals.

250 mg/kg bw/day
In the male animals, one side pyelectasis (1/10) was recorded.
There were no histopathological findings in the examined organs (liver and kidneys) of female animals.

1000 mg/kg bw/day
In the male animals, no histological lesions were observed.
In the female animals, alveolar emphysema (2/10) and foamy cells (1/10) in the lungs and uterus dilatation (2/10) were found.
In the recovery 1000 mg/kg bw/day group, in the lungs alveolar emphysema (1/5 male; 1/5 female) and focal haemorrhages (2/5 female) were found. One side pyelectasis (1/5 male) and uterus dilatation (1/5) were observed.

In summary: There were no test item related lesions in the organs of animals subjected to histopathological examination. The alveolar emphysema and focal haemorrhage in the lungs was considered as consequence of hypoxia, dyspnoea and circulatory disturbance developed during exsanguination.

The foamy cells in the lungs (alveolar histiocytosis) found in single animals of the control and high dose group was an incidental finding occurring commonly in experimental rats. The pyelectasis (one side) in the kidney, the dilatation in the seminal vesicle, the thymoma or the haematoma in the thymus occurred sporadically and were considered as individual disorder.

The dilatation of the uterine homs in some female animals is a slight neuro-hormonal phenomenon in connection with the sexual function of the inner genital organs. No morphological evidence of acute or subacute injury of alimentary tract, the cardiovascular system, the immune system, the haematopoietic system, the skeleton, the muscular system, or the central or peripheral nervous system was detectable.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: All effects observed are secondary effects due to the lithium content of the test material. Lithium is known to cause polydipsia and polyuria, as seen in the present study in the high-dose animals and to a lesser degree in intermediate dose levels.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Reaktiv Gelb F68072 FW caused an increased water consumption and urine excretion in the course of 90-day consecutive oral administration at 1000 mg/kg bw/day dose level to CRL:(WI) BR rats, which recovered within 10 days after cessation of the treatment. This effect was ascribed to high levels of salts in the test item (lithium and sodium) and is considered to be a physiological response and not an adverse toxic effect. Especially lithium is known to cause polydipsia and polyuria, as seen in the present study in the high-dose animals and to a lesser degree in intermediate dose levels.

As this effect is not relevant for humans and not related to toxicity of the test substance, the NOAEL is considered to be 1000 mg/kg bodyweight/day.

Executive summary:

This study has been performed in accordance with the OECD Guidelines for Testing of Chemicals (OECD No.: 408), Directive 2001/59/EC B.26, and the Principles of Good Laboratory Practice (GLP).

A 90-day repeated dose oral (gavage) toxicity study was performed in CRL:(WI) BR rats (n= 10 animals/sex/group and further 5 animals/sex in the control and Group 4 for recovery) on Reaktiv Gelb F68072 FW with dose levels of 0 (vehicle only), 62.5, 250 and 1000 mg/kg bw/day. 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 28-day observation without treatment.  

General clinical observations were made daily and detailed clinical observations were performed weekly. A functional observation battery was conducted on week 13. Body weight and food consumption were measured weekly. Ophthalmoscopy was made on all animals before starting of the treatment and in the control and high dose groups on week 13. Urinalysis was conducted on week 12, clinical pathology and gross pathology 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, kidneys and organs with macroscopic findings were evaluated histologically in Group 2 and Group 3. Animals of recovery groups were processed in the same manner at termination of the recovery period.

 

There was no test item related mortality. One male rat of 1000 mg/kg bw/day dose was found dead without preceding signs on day 66 due to. Para-gastric treatment resulted in peripheral circulatory and breathing disturbance, which is in line with the histological observation: congestion and alveolar oedema causing death of animal.

At 1000 mg/kg bw/day polyuria in male and female animals from day 2 up to the end of the treatment was observed, which recovered 10 days after the cessation of test item administration. There were no test item related effects on animal's behaviour or reaction to different type of stimuli at the functional observation battery.

The mean body weight gain of male animals at 1000 mg/kg bw/day was less than the control value during the treatment period resulting in a statistically significant difference in the body weight, although the body weight remained within the normal range. Body weight gain increased in the recovery period, however the terminal body weight at the end of the recovery period was still slightly lower.

 There were no differences in the mean food consumption between any dose levels except weeks 9 and 10 in male 1000 mg/kg bw/day dose, where significantly less food intake was measured.

There were no test item related adverse effects of treatment in the examined haematological or clinical chemistry parameters. A higher volume of the urine was noted for 62.5 mg/kg bw/day, 250 mg/kg bw/day and 1000 mg/kg bw/day doses at the terminal urinalysis.

No test item related macroscopic finding were found at necropsy. There were no relevant changes in organ weights (male and female). No histopathological changes related to the test item were detected.

 

In conclusion, Reaktiv Gelb F68072 FW caused an increased water consumption and urine excretion in the course of 90-Day consecutive oral administration at 1000 mg/kg bw/day dose level to CRL:(WI) BR rats, which recovered within 10 days after cessation of the treatment. This resulted in a secondary lower body weight and food consumption  in males dosed at 1000 mg/kg bw/day, which reversed in the recovery phase. Furthermore, the higher water uptake caused a higher volume of urine at 62.5 mg/kg bw/day, 250 mg/kg bw/day and 1000 mg/kg bw/day after 16 hours collection at termination of the treatment. The increased volume of urine indicates an increased physiological function without any related pathologic (clinical or histopathology) alterations. These effects were ascribed to high levels of salts in the test item (lithium and sodium) and is considered to be a physiological response and not an adverse toxic effect. Especially lithium is known to cause polydipsia and polyuria, as seen in the present study in the high-dose animals and to a lesser degree in intermediate dose levels.

The changes observed were minor in nature and reversible, and are considered due to the effects of lithium salts rather than the substance itself. There were no signs of serious irreversible toxicity at 1000 mg/kg bw/day.

As this effect is not relevant for humans and not related to toxicity of the test substance, the NOAEL is considered to be 1000 mg/kg bodyweight/day.