Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
other: read across from analogue substance
Adequacy of study:
key study
Study period:
From October 15,2012 to April 30 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant with international guideline.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: SPF breeding, VELAZ - Age at study initiation: 10 weeks on arrival- Weight at study initiation: body weight - group mean ± standard deviation (g)MALEGroup code 0 15 mg/kg bw 60 mg/kg bw 240 mg/kg bw 248.58 ± 10.33246.23 ± 13.77255.17 ± 11.91243.32 ± 6.97FEMALEGroup code 0 15 mg/kg bw 60 mg/kg bw 240 mg/kg bw 201.92 ± 6.38200.73 ± 9.63 205.87 ± 5.15199.53 ± 8.19 - Housing: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage.- Bedding: sterilized soft wood fibers (Lignocel)- Diet: complete pelleted diet for rats and mice in SPF breeding- Water: drinking water ad libitum, quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry - Acclimation period:6 days- Health chech: During the acclimatisation period the health condition of all animals was controlled daily. Then the animals were randomly divided into the control and test groups and they were marked individually. ENVIRONMENTAL CONDITIONS- Temperature (°C):22 ± 3°C- Humidity (%): 30-70%- Air changes (per hr):- Photoperiod: 12 hours cycle dark/light
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua pro injectione
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:Concentration Level 50 mg/10mLCa. 0.5 g of the test substance was weighted with wider end of glass Pasteur pipette into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle. The solution was stirred by magnetic stirrer (800 rpm) for 60 minutes. Concentration Level 1000 mg/10 mLCa. 10 g of the test substance was weighted with wider end of glass Pasteur into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle and disslolved in ultrasonic bath for a 15 min. This solution was stirred by magnetic stirrer (800 rpm) for 30 minutes.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and the homogeneity were determined by means of measuring of a peak area of the test substance by high-performance liquid chromatography based on the method developed at the test facility.The stability of the application form The stability of the application form was checked by determination of a concentration of the test substance within 120 min (at the time 0 min, 30 min, 60 min and 120 min). The samples were taken from the middle of the beaker content after required time intervals.The homogeneity of the application formThe homogeneity of the application form was checked by determination of a concentration of the test substance in three places of solution (at the bottom, in the middle and at the surface). Results of analysisFrom the results of analyses (homogeneity and stability) follows that the solution of the test substance in vehicle in concentration level 50 mg/10 ml prepared at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 120 minutes starting with preparation of the application form.From the results of analyses (homogeneity and stability) follows that the solution of the test substance in vehicle in concentration level 1000 mg/10 ml prepared at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 60 minutes from starting with analysis of the application form.
Duration of treatment / exposure:
Parental males:1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study Satellite males:1st day → 42nd day (administration) → 56th day (observation)Parental females:1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 4 post partum Satellite females:1st day → 42nd day (administration) → 56th day (observation)Non-pregnant females (without evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study Non-pregnant females (with evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study)
Frequency of treatment:
7 days per week at the same time (8.00 – 10.00 am). The vehicle control group was administered by aqua pro injectione in the same volume.
Remarks:
Doses / Concentrations:0, 15, 60 and 240 mg/kg/day Basis:actual ingested
No. of animals per sex per dose:
1. Control 012 males + 12 females2. Low dose 15 mg/kg/day12 males + 12 females3. Intermediate dose60 mg/kg/day 12 males + 12 females4. High dose 240 mg/kg/day12 males + 12 femalesSatellite groups:5. Control – vehicle – satellite: 06 males + 6 females 6. High dose – satellite:240 mg/kg/day6 males + 6 females
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yesmales and females - daily during the administration periodpups - as soon as possible after delivery and then dailyAll rats were observed daily during the administration period.This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (11.00 – 13.00 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.DETAILED CLINICAL OBSERVATIONS: Yes before the first application and then weekly (except the mating period) Functional observation: at the end of administration/observation periodThis observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, position of eyelids, breathing, tonic or clonic movements, stereotypes or bizarre behaviour.The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina. Functional Observation This observation was done at the end of administration period (only in 6 males and 6 females of each group) and recovery period.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 grip strength meter. Measurements were made on: 1) pectoral legs, 2) pelvis legs. Grip power was expressed in Newtons.BODY WEIGHT: Yesmales - weekly females - weekly in premating and mating period, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 0. or 1st, 3rd and 4th day;pups (litters) – 0. or 1st, 3rd and 4th day;satellite males and females - weeklyThe body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too.Weight increment was computed as an mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period. FOOD CONSUMPTION:males - weekly (except the mating period) females - weekly during premating period during pregnancy and lactation – on the same days as body weight satellite males and females – weeklyIn a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed. In males mean values were calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period. Food conversion in % (weight increment/food consumption x 100) was calculated for animals of repeated dose toxicity part of study. In pre-mating period the food consumption and conversion of females was calculated from values of all females. (Numbers of females for repeated dose toxicity part of study were chosen at the end of study.)WATER CONSUMPTION A: Yessatellite males and females – twice a weekThe drinking water consumption was recorded in satellite males and females. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study. OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data- Time schedule for examinations:- Dose groups that were examined:HAEMATOLOGY: Yes at the end of administration/observation periodThis examination was performed only in 6 males and 6 females of each group and in satellite males and females. The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation systems. Haematology analysers Coulter  AC.T diffTM, Celltac alfa and Coagulometer ACL 200 were used for examination and the following parameters were determined. Total erythrocyte count Mean corpuscular volume Haematocrit Haemoglobin concentration Total leucocyte count Total platelets count Partial thromboplastin time Prothrombin time Fibrinogen Granulocytes Lymphocytes MonocytesCLINICAL CHEMISTRY: Yes at the end of administration/observation periodThis examination was performed only in 6 males and 6 females of each group and in satellite males and females. The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.The following parameters were determined by automatic biochemical analysers SPOTCHEMTM EZ SP-4430 and SPOTCHEMTM EL SE-1520 (Arkray, Inc., Japan). Glucose Cholesterol, total Urea Bilirubin, total Aspartate aminotransferase Alanine amonitransferase Alkaline phosphatase Calcium Phosphorus Protein, total Protein, albumin Creatinine Sodium Potassium ChlorideURINALYSIS: Yesthe last day of administration/observation period – only malesThis examination was performed only in 6 males of each group and in satellite males. In females this examination was not performed (dams should not be removed from the pups for long time). The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100 g of body weight by gavage to the stomach.The following parameters were determined by analyser PocketChem PU-4210 (Arkray, Inc., Japan). Volume Colour Cloud Odour Glucose Protein Bilirubin Urobilinogen pH Specific gravity Blood Ketones Nitrite LeucocytesNEUROBEHAVIOURAL EXAMINATION: Yes / No / No data- Time schedule for examinations:- Dose groups that were examined:- Battery of functions tested: sensory activity / grip strength / motor activity / other:OTHER:- Mortality control: twice dailyAll rats during the treatment periods were examined for vitality or mortality changes twice daily.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes males and nonpregnant females – at the end of administration periodparental females and pups - on the 4th day of lactationsatellite males and females - at the end of observation periodDuring the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.HISTOPATHOLOGY: Yesafter necropsySamples of the following tissues and organs were collected at necropsy and fixed:Repeated dose toxicity part of study (6 males and 6 females from each main group + 6 males and 6 females from satellite groups)Adrenal glandsAortaBrain (incl. cerebellum and med. oblongata)CaecumCoagulating glandColonDuodenumPancreasRectumSalivary glands Sciatic nerveSeminal vesiclesSkeletal muscleSkinSpinal cord – thoracicSpleenStomachThymusThyroid gland incl. parathyroidTracheaUrinary bladderFemale mammary gland areaFemur HeartIleum Jejunum KidneysLiverLungsLymph nodes – mesenteric, paraaortalOesophagusAll gross lesionsReproduction part of study (12 males and 12 females from each main group)Pituitary glandOvaries UterusCervix of uterus VaginaEpididymis/EpididymidesProstate glandSeminal vesicles and coagulating glandTestesAll gross lesionsThe mentioned tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4% formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used. In Repeated Dose Toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Organs demonstrating treatment-related changes: brain (incl. cerebellum), spinal cord (thoracic), kidneys, liver, spleen, stomach, small and large intestine, lymph nodes (mesenterial), pancreas and organs with macroscopical changes were examined at the lowest and middle dose level groups used for Repeated Dose Toxicity part of study.
Other examinations:
- weight of organs: during necropsyAt 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, epididymis/epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart were recorded (repeated dose toxicity part of study – 6 males and females from each group + satellite groups); testes or ovaries, epididymis/epididymides or uterus, prostate gland, pituitary gland (reproduction part of study – all animals). Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis (the raw data were used for statistical analysis). This statistical analysis was used for the results of body weight, results of haematology, blood biochemistry, urinalysis, biometry of organs and selected reproduction parameters – number of live born pups, number of corpora lutea, number of implantations, mean weight of pup on the 0./1st day and mean weight of pup on the 4th day. Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
highest dose
Mortality:
mortality observed, treatment-related
Description (incidence):
highest dose
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
highest dose
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
highest dose
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
highest dose
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
middle and high dose
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
middle and high dose
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITYMales At the lowest and the middle dose level no clinical changes after application of the test substance were recorded. The following clinical changes after application of the test substance were detected in all males of the highest dose level: test substance-coloured excrements and orange-brown coloured shavings (probably urine stained bedding) from the 1st week to the 6th week of application period.Satellite malesIn satellite treated males test substance-coloured excrements and red-coloured shavings (probably urine stained bedding) from the 1st week to the 6th week of application period were found out during clinical examination performed after application.Females At the lowest and the middle dose level no clinical changes after application of the test substance were observed. The following clinical changes after application of the test substance were noted in all exposed females of the highest dose level: test substance-coloured excrements and orange-brown shavings (probably urine stained bedding). Satellite femalesIn satellite treated females clinical changes - test substance-coloured excrements and orange-brown shavings (probably urine stained bedding) were observed after application.Detailed Clinical Observation Males Excrements coloured by the test substance and orange-brown coloured shavings (bedding) were found out at the highest dose level from the 1st to the 6th week of the study. Satellite males Excrements coloured by the test substance and orange-brown coloured shavings (bedding) were observed in satellite treated males from the 1st to the 6th week of the studyFemales Excrements coloured by the test substance and orange-brown coloured shavings (bedding) were detected in females of the highest dose level from the 1st to the 6th week of the study. Satellite females Excrements coloured by the test substance and orange-brown coloured shavings (bedding) were recorded in satellite treated females from the 1st to the 6th week of the study. Functional ObservationMalesReactions to touch, noise, pain and pupillary reflex of treated males were the same as in the control group. Numbers of defecations and emictions were similar in treated males and control males. The activity – number of upstanding was also similar in treated and control males. The values of grip strength of pectoral legs and pelvic legs did not show any differences between control and treated males.Change of colour of urine was observed in males of the middle and the highest dose level.Satellite males No significant differences were detected in examined parameters. FemalesReactions to touch, noise, pain and pupillary reflex of treated females were the same as in the control females. Numbers of emictions and defecations were similar in treated and control females. The activity – number of upstanding was similar in females of the lowest and the middle dose levels and in control females. In females of the highest dose level the activity was slightly decreased compared to control. The values of grip strength of pectoral and pelvic legs were well-balanced in control and treated females. Change of colour of urine was observed in females of the highest dose level.Satellite females No significant differences were detected in examined parameters. BODY WEIGHT AND WEIGHT GAINMalesBody weight of control males and males of the lowest and the middle dose level was quite balanced during the application period. Body weight of males at the highest dose level was lower than control during the whole of application period. Statistically significant differences were not found in treated males. Satellite malesBody weight of satellite treated males was markedly decreased in comparison with satellite control males for the whole time of application and during recovery period. Statistically significant differences were not found in satellite treated males. FemalesBody weight of control females and females of the lowest and the middle dose level was quite balanced during the pre-mating period vice versa the body weight of females at the highest dose level was decreased in this period. During pregnancy and lactation period the body weight of control and females of the lowest and the middle dose level was similar. The body weight of females at the highest dose level was again decreased.Statistically significantly decreased body weight was found in females of the highest dose level in the 2nd week of application. Satellite femalesBody weight of satellite treated females was decreased in comparison with control animals for the whole time of application and recovery period. Statistically significant decrease of body weight was found in the 2nd and 8th week (necropsy weight) in satellite treated females. - AVERAGE BODY WEIGHT INCREMENTMalesWeight increments in control and treated males were comparable for the whole time of application period. Satellite malesWeight increments of satellite treated males in application period were mostly decreased compared to satellite control. In recovery period weight increments of satellite treated males were slightly increased against satellite control.FemalesWeight increments in females of the highest dose level were markedly lower compared to control in the 1st and 2nd week application period. The fall of weight was detected in females of the highest dose level in the 1st week of application. The evaluation of weight increments during pregnancy and lactation period is included in reproduction part of study. Satellite femalesWeight increments of satellite treated females were very variable in comparison with control: decreased in the 1st, 3rd, 4th, 6th week and increased in the 2nd, 7th and 8th week. The fall of body weight was recorded in satellite treated females in the 1st week and in satellite control and satellite treated females in the 5th week.FOOD CONSUMPTION AND CONVERSIONFood ConsumptionMalesThe food consumption of control and treated males were quite balanced for the whole time of application period (except slight decrease in males of the highest dose level in the 1st week). Satellite malesThe food consumption of satellite treated males was slightly decreased in the 1st and the 2nd week of application period. In observation period the food consumption of satellite treated males increased.FemalesIn pre-mating period the food consumption of females of the highest dose level was decreased compared to control. During pregnancy period the food consumptions of females of the lowest and the middle dose level and control females were similar. In females of the highest dose level the food consumption in pregnancy period was decreased. During lactation period the food consumption of females at the lowest and the highest dose level was decreased compared to control.Satellite femalesThe food consumption of satellite treated females was decreased for the whole time of application period. In observation period the food consumption of satellite treated females and satellite control females were balanced. FOOD CONVERSIONMalesThe food conversion of treated males was not changed in a dose dependent manner. Satellite malesThe food conversion of satellite treated males was lower compared to satellite control males – from the 1st to the 5th week. In the 6th week and in observation period the food conversion of satellite treated and satellite control males was well-balanced.FemalesThe food conversion of females at the lowest and the middle dose levels in the 1st week of pre-mating period were decreased compared to control females, in the 2nd week of pre-mating period it was analogous to control females. The food conversion of females at the highest dose level was decreased compared to control in pre-mating period. In the first week the minus value of food conversion at the highest dose level was calculated.During pregnancy period: the food conversions of treated and control females were similar in the 1st and the 2nd week, in the 3rd week the food conversion of females at the highest dose level was decreased. Vice versa during lactation period the food conversion of females at the lowest and the middle dose levels was decreased and at the highest dose level it was increased in comparison with control.Satellite femalesThe food conversion of satellite treated females was very variable in comparison with control: decreased in the 1st, 3rd, 4th and 6th and increased in the 2nd. In observation period marked differences were not recorded between satellite control and treated females. WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)Satellite malesThe water consumption of satellite treated males was higher compared to satellite control in application period – from the 1st to the 4th week. In the 5th and 6th week and in observation period the water consumptions of satellite treated males and satellite control males were quite balanced. Satellite femalesIn application period the water consumption of satellite treated females was higher compared to satellite control. In the 1st week of observation period the water consumption of satellite treated females was also increased and in the 2nd week it was similar to control.HAEMATOLOGYMales Statistically significant differences were not detected in treated males. Parameters of red and white blood component were similar in control and treated males. Most of haemocoagulation parameters (platelet count, PT) were not influenced in treated males. Only the value of APTT was slightly increased in treated males – without dose dependence. Values of APTT of treated males were within the limits of historic control. Satellite malesStatistically significant increase of APTT value was found out in satellite treated males (within the limits of historic control). Other parameters of red and white component and haemocoagulation were similar in satellite treated and control males. Females Statistically significant differences were not detected in treated females. Parameters of red and white blood component were similar in control and treated females. Percentual portion of granulocytes was increased in all females – in both treated and control groups (connection with previous pregnancy). Slight increase of APTT value (without dose dependence) was recorded at the highest dose level. Satellite femalesStatistically significant increase of red blood parameters values – mean corpuscular volume, haematocrite and haemoglobin was noted in satellite treated females. The values of MCV and HCT were slightly higher than historic control. Statistically significant increase of APTTCLINICAL CHEMISTRYMalesThe concentration of total cholesterol of 6-6-6-6 males was lower than 1.29 mmol/L (the measurable limit of analyser). The activity of ALT of 1-0-0-0 male was lower than 0.17 kat/L (the measurable limit of analyser). Statistically significantly increased ALT activity was recorded in males of the highest dose level (within the historical control). Increase of ALP activity was detected at the highest dose  level (without statistical significance and within the historical control). These changes were not dependent on dose level.Values of other biochemical parameters of treated males were similar to the control group.Satellite malesThe concentration of total cholesterol of 6-6 males was lower than 1.29 mmol/L (the measurable limit of analyser). The concentration of total bilirubin of 6-0 males was lower than 3.00 µmol/L (the measurable limit of analyser). Statistically significant increase of total builirubin and decrease of calcium and creatinine concentrations were recorded in satellite treated males. All these differences were within the limits of historic control. Values of other biochemical parameters of satellite treated males were similar to the satellite control group.FemalesThe concentration of total cholesterol of 6-6-6-4 females was lower than 1.29 mmol/L (the measurable limit of analyser). The activity of ALT of 0-0-0-1 female was lower than 0.17 kat/L (the measurable limit of analyser). Statistically significant differences were not found out in treated females. Increase of ALP activity and creatinine concentration was detected at the highest dose level (without statistical significance and within the historical control). These changes were not dependent on dose level.Values of other biochemical parameters of treated females were similar to the control group.Satellite femalesDifferences in values of some biochemical parameters between females of basic and satellite group related to previous pregnancy and lactation of basic females. The concentration of total cholesterol of 2-2 females was lower than 1.29 mmol/L (the measurable limit of analyser). The concentration of total bilirubin of 1-0 female was lower than 3.00 µmol/L (the measurable limit of analyser). The activity of ALT of 1-4 satellite females was lower than 0.17 kat/L (the measurable limit of analyser). Statistically significant increase of total bilirubin concentration and ALP activity and decrease of calcium and creatinine concentration were recorded in satellite treated females. Values of all these parameters were within the limits of historic control.Values of other biochemical parameters of satellite treated females were similar to the satellite control females.URINALYSISUrinalysis was performed only in males (six of each group) during the last week of the study. Statistical evaluation was performed for pH and volume of urine. Males Statistically significant differences were not recorded in treated males. The change of urine colour was found out in all males of the middle and the highest dose level and in some males of the lowest dose level. Presence of leucocytes in urine was sporadically noted in males of the lowest and the middle dose level and increased occurrence of leucocytes was found out in males of the highest dose level. Satellite malesStatistically significant decrease of urine volume was detected in satellite treated males. Urine colour was analogous in satellite treated and satellite control males. Leucocytes were sporadically found in urine of satellite treated males.NEUROBEHAVIOURORGAN WEIGHTSMales Statistically significant dose dependent increase of kidneys weight was detected at the highest dose level. Increase of absolute weight of liver without statistical significance was detected at highest dose level. Absolute weight of prostate gland of males at the highest dose level was decreased in comparison with control males (without statistical significance). Weight of other organs was similar in treated and control males. Satellite malesStatistically significant differences were not found out in satellite treated males.Absolute weight of thymus, adrenal glands and spleen was slightly decreased in satellite treated males. Weight of other organs was similar in satellite treated and satellite control males.Females Statistically significant difference was not detected in treated females. Slight dose dependent increase of absolute weight of spleen was found out in treated females. Weights of other organs were similar in treated and control females. Satellite femalesStatistically significant differences were not recorded in satellite treated females. Absolute weight of thymus, heart, liver and uterus were slightly decreased in satellite treated females. Weights of other organs were similar in satellite treated and control females. Relative Organ Weight Males Statistically significant and dose dependent differences were detected in males of the highest dose level – increase of relative weight of heart, kidneys and liver. Relative weights of other organs of treated males were similar to control males. Satellite malesStatistically significant increase of relative weights of brain, kidneys and liver was recorded in satellite treated males. Relative weights of other organs were similar in satellite treated and control males. Females Relative weight of ovaries was statistically significantly increased in females of the middle and the highest dose level. This difference was not dependent on dose level. Statistically significant increase of relative weight of spleen (dose dependent) and liver was detected in females of the highest dose level. Insignificant increase of relative weight of brain and kidneys was observed in females of the highest dose level.Weights of other organs of treated males were quite well-balanced in treated and control females. Satellite femalesStatistically significant differences were not detected in satellite treated females. Relative weight of thymus was insignificantly decreased satellite treated females. Relative weights of other organs were similar in satellite treated and control females.GROSS PATHOLOGYHISTOPATHOLOGY: NON-NEOPLASTICMales No macroscopical findings were recorded in 5-5-5-0 males. Change of colour of organs and tissues (or contents of organs) were observed in males of the middle and the highest dose level: brown colour of stomach mucosa membrane and chyme in 0-0-0-5 males, brown colour of stomach chyme in 0-0-1-1 males, dark colour of subcutis and skeletal muscle in 0-0-0-2 males. Other macroscopical changes were described sporadically. Females No macroscopical findings were recorded in 6-6-4-1 females.Change of colour of organs and tissues (or contents of organs) were observed in females of the middle and the highest dose level: dark or brown colour of stomach chyme in 0-0-2-5 females, brown colour of mammary gland in 0-0-2-4 females, yellow or brown colour of fat tissue in 0-0-2-1 females, yellow or brown colour of subcutis in 0-0-2-5 females, yellow or brown colour of skeletal muscle in 0-0-0-5 females, brown colour of heart in 0-0-0-2 females, dark red or brown-red colour of spleen in 0-0-0-5 females, dark brown colour of liver and brown-red colour of kidneys in 0-0-0-3 females.Satellite femalesNo macroscopical findings were recorded in 6-0 females. Change of colour of organs and tissues were observed in satellite treated females: yellow or brown colour of subcutis and skeletal muscle in 0-6 females, dark red or brown-red colour of spleen in 0-6 females and dark brown colour of liver only in 0-1 female. Satellite malesIn 6-5 satellite males no macroscopical findings were recordedHISTOPATHOLOGY: NEOPLASTICMalesIn 1-1-1-0 males no histological findings were diagnosed.Microscopical examination revealed presence of the following possible treatment related changes. Presence of brown pigment was sporadically detected in neurons of brain: in 0-0-0-1 male. In urinary system presence of pigment in epithelium of cortical tubules in kidneys of 0-0-0-6 males was found out, basophilic tubules was found out in renal cortex of 0-3-0-5 males. Deposits of brown pigment occurred in digestive system: in lumen of gastric glands in 0-0-1-5 males, in mucosa of small intestines in 0-0-0-4 males and in mucosa or/and submucosa of large intestines in 0-0-0-6 males. In lymphatic system presence of brown pigment in mesenteric lymph nodes in 0-0-0-5 males was registered. Other microscopical changes were observed for both control and treated groups or they occurred only sporadically.Satellite males In 0-0 satellite male no histological findings were diagnosed.The following microscopical changes probably caused by the test substance treatment were diagnosed in satellite treated males. Presence of brown pigment was detected in central nervous system: in neurons of brain in 0-4 satellite males, in neurons of thoracic spinal cord in 0-3 satellite males. In urinary system presence of pigment in epithelium of cortical tubules in kidneys of 0-6 satellite males was found out. Deposits of brown pigment occurred in digestive system: in lumen of gastric glands in 0-6 satellite males, in mucosa of small intestines in 0-6 satellite males and in mucosa or/and submucosa of large intestines in 0-6 satellite males. In lymphatic system presence of brown pigment in mesenteric lymph nodes and in red pulp of spleen in 0-5 satellite males was registered. Other histopathological changes were recorded for both satellite control and satellite treated groups or they were diagnosed only sporadicallyFemales In 1-0-0-0 female no histological findings were diagnosed.During microscopical examination the following possible treatment related changes were registered. Presence of brown pigment was recorded in central nervous system: in neurons of brain in 0-0-0-5 females and in neurons of thoracic spinal cord in 0-0-0-4 females. In urinary system presence of pigment in epithelium of renal cortical tubules of 0-4-6-6 females was noted. Deposits of brown pigment occurred in digestive system: in lumen of gastric glands or/and in stomach mucosa in 0-0-0-3 females, in mucosa of small intestines in 0-0-0-5 females and in mucosa or/and submucosa of large intestines in 0-0-0-5 females and in hepatic sinuses of 0-0-0-5 females. In lymphatic system presence of brown pigment in mesenteric lymph nodes in 0-0-0-6 females and in red pulp of spleen in 0-1-3-6 females was registered. Presence of brown pigment was found out also in reproductive organs: in ovaries of 0-0-0-6 females, in uterus of 0-0-1-5 females. In uterus the changes related to previous pregnancy were found: accumulation of siderophages in mesometrium or/and in endometrium in 5-5-4-3 females and haemorrhages or haematoma in mesometrium in 1-2-0-1 females.Other microscopical changes were observed only sporadically or they did not relate to test substance treatment – see the table No. 37.Satellite females In 2-0 females no histological findings were diagnosed.During microscopical examination of satellite treated females the following changes probably related to the test substance treatment were recorded. Presence of brown pigment was detected in central nervous system: in neurons of brain in 0-6 satellite females and in neurons of thoracic spinal cord in 0-6 satellite females. In urinary system presence of pigment in epithelium of renal cortical tubules of 0-6 satellite females was described. Deposits of brown pigment occurred in digestive system: in lumen of gastric glands or/and in stomach mucosa in 0-5 satellite females, in mucosa of small intestines in 0-6 satellite females and in mucosa or/and submucosa of large intestines in 0-5 satellite females and in hepatic sinuses of 0-5 satellite females. In lymphatic system presence of brown pigment in mesenteric lymph nodes in 0-5 satellite females and in red pulp of spleen in 0-6 satellite females was found out. Presence of brown pigment was found out also in reproductive organs: in ovaries of 0-5 satellite females, in uterus of 0-6 satellite females. In uterus of 1-1 satellite females hydrometra - the change associated with oestrous cycle was diagnosed. Other microscopical changes were not connected with the test substance treatment or they were observed only sporadically OTHER FINDINGSMortality ControlMalesThere were no unscheduled deaths during the main study. FemalesThere were no unscheduled deaths during the main study.
Dose descriptor:
NOAEL
Effect level:
ca. 60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight gains in both sexes and irreversible accumulation of pigment in neurons of central nervous system , brain and spinal cord.
Critical effects observed:
not specified
Conclusions:
The analogue substance 2 treatment produced no overt adverse significant changes in general health condition, clinical and functional observation in all treated groups. The main changes observed were related to the increased pigmentations of tissues and organs. To note, these very similar pigmentations, have also been observed for other dyes as well. Other modulations (observed especially at the highest testing dose in both genders) such as haematological ex., biochemical ex., and biometry of organs ranged from minimal to moderate. Few modulation have been described as statistically significant, however the most of them were still within the limits of historical controls. Other modulations might be considered as incidental (one female of the middle dose terminated before scheduled death) and not related to the exposure. As cited above, the main modulation observed was the increased pigmentation in tissues and organs as the dose increased. Though, as correctly described in the report the increased pigmentation might be related to the increased accumulation of dye on the target organs, such modulation alone cannot be considered as "adverse"; other modulations (either inhibiting or stimulating) possibly originated by that pigmentation might be observed in order to describe the pigmentation as adverse. Yet, this is not the case. However, as the severity of such modulations along with observations on organ weights, haematology etc. at the dose of 240mg/kg/day are to be considered as biologically significant increases in frequency or severity of possibly adverse effects, the highest dose might be put forward as the lowest exposure adverse level (LOAEL) for the combined repeated dose toxicity study.The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 60 mg/kg/day body weight/day. The value was determined mainly on the basis of irreversible reduction in body weight gains in both sexes and irreversible accumulation of pigment in neurons of central nervous system – brain and spinal cord.
Executive summary:

Introduction

The test substance, Direct Brown 44, was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22nd 1996.

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 15, 60, 240 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (240 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment.

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females – during pregnancy and till the 3rd day of lactation,

males – after mating period – totally for 42 days,

nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating,

non-mated females – to the 54th day of study.

    After the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.   

During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in the specified time intervals. Detailed clinical observation was carried out weekly. The functional observation was performed at the end of application and observation period. Vaginal smears were prepared daily during the mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, weight, sex and vitality of pups) were also recorded.

The study was finished by urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of main groups the sperm parameters, sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

Results

 Repeated oral administration of Direct Brown 44 to rats by gavage at the dose levels 15, 60 and 240 mg/kg/day did not cause any mortality. One female of the dose level 60 mg/kg/day was euthanasied before the scheduled time of necropsy by the reason of bad health status (abortion on the 24th day of pregnancy)

Repeated dose toxicity part of study:

Change of colour of urine in males (sporadically) and occurrence of pigment in kidneys of females at the dose level 15 mg/kg/day were related to the test substance administration.

Change of colour of urine in males, increase of relative weight of ovaries, change of colour of organs (mammary gland, fat tissue, subcutis) in females and stomach chyme in males and females, deposits of pigment in kidneys and spleen of females at the dose level 60 mg/kg/day can be considered as changes related to the test substance administration.

     Growth of males and females (irreversible decrease of body weight), water consumption (reversible increase in males), clinical status of males and females (reversible change of urine and excrements colour), haematological (delayed increase of red blood parameters value in females and irreversible increase of APTT value in males and females) and biochemical parameters (irreversible increase of blood ALP activity in females, reversible increase of blood ALT activity in males, delayed increase of total bilirubin concentration in blood of males and females, delayed decrease of calcium and creatinine concentration in blood of males and females), properties of urine (reversible change of urine colour, occurrence of leucocytes in urine and delayed decrease of urine volume in males), biometry of organs (irreversible and dose dependent increase of relative weight of kidneys and liver in males, reversible and dose dependent increase of absolute weight of kidneys in males, reversible and dose dependent increase of relative weight of heart in males, reversible increase of relative weight of ovaries, spleen and liver in females, delayed increase of relative weight of brain in males), macroscopical appearance of organs (change of colour of subcutis, skeletal muscle and stomach mucosa and chyme in males, change of colour of subcutis, skeletal muscle, spleen, stomach chyme, mammary gland, fat tissue, heart and liver in females) and microscopical structure of organs (irreversible occurrence of pigment in brain, spinal cord, kidneys, stomach and intestines mucosa, mesenteric lymph nodes in males and females and in spleen, ovaries and uterus of females) at the dose level 240 mg/kg/day were influenced by administration of the test substance.

Conclusion

The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 60 mg/kg/day body weight/day. The value was determined mainly on the basis of irreversible reduction in body weight gains in both sexes and irreversible accumulation of pigment in neurons of central nervous system , brain and spinal cord.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to Regulation EC 1272/2008 (CLP) classification in Category 2 for repeated dose is applicable, when significant toxic effects observed in a 90-day repeated-dose study conducted in experimental animals are seen to occur within the following guidance value :

 

Oral (rat) mg/kg body weight/day 10 < C100

 

Based on available data no classification for repeated dose is warrated for analogue substance 2 and therefore based on the read across considerations, the same applies for Basic brown 1 acetate.