Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29.03.2016 – 15.03.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted by the Council on 29th July 2016
Deviations:
yes
Remarks:
see Any other information ...
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: 7031/2007Stability/Expiration: Feb 2018Storage: The test substance should be stored in dry room in dark in supplied container at the room temperature.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: by Sponsor, Batch No. 7031/2007- Expiration date of the lot/batch: 02/2018- Storage condition of test material: The test substance was stored in dry and dark place in closed container at room temperature- Stability under test conditions:testing laboratory analysis of homogeneity and stability: the both application forms (1000 mg/10 mL and 10 mg /10 mL) of the test substance, Direct Blue 85, at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) are homogenous and stable at least for 120 minutes from the finalization of application form preparation.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: see below Details on oral exposureFORM AS APPLIED IN THE TEST (if different from that of starting material)solution in water for injection

Test animals

Species:
rat
Strain:
Wistar
Remarks:
CRL (SPF quality - guaranteed)
Details on species / strain selection:
- according to guideline- random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed ± 20% of the mean weight
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500- Females (if applicable) nulliparous and non-pregnant: yes- Age at study initiation: sexually adult, 7-9 weeks on arrival- Weight at study initiation: males 404.8 - 405.5 g, females 250.7 - 252.5 g- Fasting period before study: no- Housing: SPF conditions according to internal SOP No.12; sterilized soft wood fibers Lignocel;Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – 1 M + 1 F in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage;- Diet (e.g. ad libitum): maintenance pelleted diet for rats and mice - Altromin for Rats/Mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany- Water (e.g. ad libitum): ad libitum; quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law- Acclimation period: at least 5 days (DRFE 5 days, main study 6 days)ENVIRONMENTAL CONDITIONS- Temperature (°C): 22±3- Humidity (%): 30-70- Air changes (per hr): approximately 15 air changes per hour- Photoperiod (hrs dark / hrs light): 12 /12STUDY TIME SCHEDULEAdministration (from 04.10.2016)Parental males (totally 49 days of administration):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day ( mating ) → 43rd day – 63rd day (administration period) → 64th day (necropsy) Satellite males (totally 49 days of administration + 14 days of observation):1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy)Parental females:1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day (mating)→ gestation → lactation → day 12 post partum Satellite females (totally 49 days of administration + 14 days of observation):1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy)Non-pregnant females (without evidence of copulation):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day (mating) → 25 days after the end of mating period Non-pregnant females (with evidence of copulation):1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day (mating) → 25th day after confirmed matingObservationsUrinalysis: only males – 63rd and 77th day of studyBlood collection for haematology and biochemistry: parental males – 64th day of study satellite males – 78th day of studyparental females - 13th day of lactation periodpups – 2 pups per litter – 4th day of lactation; 2 pups per litter – 13th day of lactationsatellite females – 78th day of studyNecropsy: parental males – 64th day of studysatellite males – 78th day of studyparental females - 13th day of lactation periodpups – 2 pups per litter – 4th day of lactation, other pups - 13th day of lactationsatellite females – 78th day of studynon-pregnant females – 26th day after the end of mating period or confirmed matingEnd of histopathological examination: 15.03.2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:The test substance was weighted on analytical balances into glass beaker and the beaker was gradually replenished by water for injections. During that the sample was intensively stirred with a glass rod. The test substance was dissolved in vehicle in ultrasonic bath for 30 min; the solution was stirred by magnetic stirrer (800 rpm) for 30 minutes. The application forms were prepared daily just before administration.The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. For each dose level concentration, the solution was prepared separately. The administration of the test substance to animals was performed during two hours after preparation of application form. VEHICLE - Lot/batch no.: 1606130339; exp. 06/2018 - Concentration in vehicle:The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Stability and homogeneity were determined by means of measuring of a peak area of the test substance by a liquid chromatography based on a method developed at the test facility.
Duration of treatment / exposure:
The treated groups were administered daily for the following periods: males and females – 2 weeks prior to the mating period and during the mating periodpregnant females – during pregnancy and till the 12th day of lactationmales – after mating period – totally for 49 daysnonpregnant females (mated females without parturition) – for 25 days after the confirmed matingnon-mated females – for 25 days after the end of mating period
Frequency of treatment:
7 days per week at the same time
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
(vehicle only)
No. of animals per sex per dose:
12 females and 12 males per group, 6 males and 6 females per satellite group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study were determined on the basis of results of a dose-range finding experiment. - Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
HEALTH CONDITION CONTROL: Yes - Time schedule: daily - during the acclimatization and the experimental partMORTALITY CONTROL: - Time schedule: twice dailyCLINICAL OBSERVATIONS: Yes males and females - daily during the administration period in natural conditions in cagesDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: before the first application and then weekly (except the mating period)FUNCTIONAL OBSERVATIONS: Yes - Time schedule: at the end of administration / observation periodNEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: at the end of administration / observation period- Dose groups that were examined: 6 males and 6 females of each group and in satellite males and females- Battery of functions tested: sensory activity / grip strength / motor activityBODY WEIGHT: Yes- Time schedule for examinations:males - the first day of administration and then weekly,females - the first day of administration and then weekly in premating and mating period, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 1st, 4th, 12th and 13th day,satellite males and females - the first day of administration and then weekly.FOOD CONSUMPTION: - Food consumption determined and group mean daily diet consumption calculated as g food/animal/day: Yes- Time schedule: weekly and on the same days as body weight (except the mating period); satellite males and females – weeklyFOOD EFFICIENCY:- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: YesTime schedule: 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 – weeklyWATER CONSUMPTION: Yes- Time schedule for examinations: twice a week (only in satellite animals)HAEMATOLOGY: Yes- Time schedule for collection of blood: at the end of administration (M) / observation period (satellite M,F)- Anaesthetic used for blood collection: Yes, light ether narcosis- Animals fasted: Yes- How many animals: 6 males and 6 females of each group and in satellite males and females- Parameters checked in table [No.3; tables in the attached document] were examined.CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: at the end of administration (M, non-pregnant F) / observation period (satellite M,F)- Animals fasted: Yes- How many animals: 6 males and 6 females of each group and in satellite males and females- Parameters checked in table [No.4] were examined.URINALYSIS: Yes- Time schedule for collection of urine: the last day of administration / observation period – only males- Metabolism cages used for collection of urine: Yes- Animals fasted: Not specified- Parameters checked in table [No.2] were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities and biometry of organsHISTOPATHOLOGY: Yes (see table [No.5] Organs for histopathological examination). In Repeated dose toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and satellite animals.
Statistics:
For statistical evaluation the software Statgraphic ® Centurion (version XVII, USA) was used. 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. The results statistically significant on probability level 0.05 were indicated in the summary tables in report.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
coloured faeces were recorded (at the dose level 1000 mg/kg/day: from the 3rd week of study)
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological examination of males was without changes and females showed sporadic findings only; all findings were without dose dependence. Only differential percentage counts of lymphocytes and granulocytes were significantly changed without dose dependency at the dose level 500 mg/kg/day. The concentration of fibrinogen (FIB) was statistically significantly increased in the satellite treated females. All findings observed in both sexes (but in absence of a treatment-related clinical signs of toxicity) were considered to be of no toxicological significance.All haematological parameters were in range of historical control.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical examination of treated males and females revealed minimal changes. In males delayed changes were recorded. Increased value of Potassium at the dose level 250 mg/kg/day and statistically significantly decreased of ALP in satellite treated males was measured. Statistically significantly changed parameters were recorded only in satellite treated females (increase of ALP, decrease of Phosphorus).All biochemical parameters were in range of historical control.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase was recorded in volume of urine in males at the dose level 250 mg/kg/day. Statistically significant differences were recorded in pH of urine in males at the dose levels 500 and 1000 mg/kg/day. The pH was increased dose-dependently.Presence of proteins and leucocytes were recorded in treated males as well as in control males that is why these findings were not associated with the application of the test substance.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
During biometry of organs, significant changes related to administration of the test substance were not detected.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopical findings related to the colour of the test substance treatment were recorded during the pathological examination of treated males and females (coloured content of stomach and intestines).
Neuropathological findings:
no effects observed
Description (incidence and severity):
Microscopical evaluation showed that the test substance orally administered at the dose level of 1000 mg/kg/day did not cause any histopathological changes in any examined organs.Effect of the test substance treatment was not observed during histopathological examination. All stated changes was considered as biologically insignificant.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopical evaluation showed that the test substance orally administered at the dose level of 1000 mg/kg/day did not cause any histopathological changes in any examined organs.Effect of the test substance treatment was not observed during histopathological examination. All stated changes was considered as biologically insignificant.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All stated changes was considered as biologically insignificant.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Deviations:

During the study short decreasing below 30% (27.4 - 29.6%, 8 hours) and increasing upon 70% (76.5%, 1h) of relative humidity in animal room was recorded.

These microclimatic deviations did not affect the welfare of animals and had no impact on the results of the study.

One deviation from the Study Plan occurred. The study was performedaccording to the updated OECD guideline No. 422:Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on the 29thJuly 2016 instead of version given in Study Plan.

No other deviations from study plan or test guidelines were found out during the study performance.

Applicant's summary and conclusion

Conclusions:
The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 1000 mg/kg body weight/day. The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 1000 mg/kg body weight/day.
Executive summary:

Introduction

The test substance, Direct Blue 85, 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 29th July 2016

Methods

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 250, 500, 1000 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 (1000 mg/kg/day). The dose levels for study were determined on the basis of results of a dose-range finding experiment (see the Annex 2) and approved by Sponsor.

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 12th day of lactation,

males – after mating period – totally for 49 days,

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

non-mated females – for 25 days after the end of mating period.

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 controls 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, 2 weeks before start of administration period (oestrous cycle monitoring), during the mating period (until the presence of spermatozoa) and at necropsy day. Reproduction parameters relevant to pups (number of pups, weight of litters, weight, sex and vitality of pups, measurement of anogenital distance, nipple retention) 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 adult animals were removed for weighing and histopathological examination. The thyroid gland was taken out, weighed and histologically examined in selected pups.

 

Results

Repeated oral administration of the test substance, Direct Blue 85, to rats by gavage at the dose levels of 250, 500 and 1000 mg/kg/day did not cause any mortality.

Repeated Dose Toxicity part of study:

The test substance did not interfere with normal growth of treated animals. Body weight and body weight increment, food consumption and conversion and water consumption of treated animals at all dose levels were not affected by the test substance administration.

The test substance treatment did not produce changes detected in functional observation of animals. Changes connected with colour of test substance (colouration of faeces) were found out during clinical observations in animals at the dose level 1000 mg/kg/day.

Urinalysis of treated males did not manifest negative effect of the test substance treatment at all dose levels. Statistically significant increases were recorded in volume of urine in males at the dose level 250 mg/kg/day. Statistically significant differences were recorded in pH of urine in males at the dose levels 500 and 1000 mg/kg/day. The pH was increased dose-dependently.

Haematological examination of males was without any statistically significant changes at the all dose levels.

Haematological examination of females was without any statistically significant changes at the dose level 250 and 1000 mg/kg/day. Only differential percentage counts of lymphocytes and granulocytes were significantly changed without dose dependency at the dose level 500 mg/kg/day. The concentration of fibrinogen (FIB) was statistically significantly increased in the satellite treated females.

Biochemical examination was without any statistically significant changes at the dose level 500 and 1000 mg/kg/day in males. Statistically significantly increased concentration of Pottasium was recorded only at the dose level 250 mg/kg/day and statistically significantly decreased of ALP was observed in satellite treated males.

No significant changes in biochemical examination were observed in females at all dose levels. Statistically significantly changed parameters were recorded only in satellite treated females (increased ALP value, decreased value of Phosphorus).

All haematological and biochemical parameters were in range of historical control.

Statistically significantly decreased absolute weight of epididymis a was recorded in males at the dose level 250 and 1000 mg/kg/day nevertheless the relative weight of this organ was similar to the control group.

Statistically significantly increased relative weight of kidneys and pituitary gland was recorded in males at the dose level 1000 mg/kg/day and in satellite females was recorded statistically significantly decreased relative weight of liver was recorded.

Changes connected with colour of the test substance were found out during pathological examination. Coloured content of stomach or stomach and intestines were recorded at all dose levels in males and in one female at the dose level 1000 mg/kg/day.

Effect of the test substance treatment was not observed during histopathological examination.

All stated changes was considered as biologically insignificant.

 

Reproduction part of study:

The test substance treatment did not affect physical growth of parental animals (body weight, body weight increment, food consumption) in any phase of the study (before mating, during mating period, pregnancy and lactation period).

In males statistically significantly decreased absolute weight of thyroid gland at the dose level 1000 mg/kg/day was observed nevertheless the relative weight of this organ was not changed.

In females at the dose level 500 and 1000 mg/kg/day statistically significantly decreased absolute weight of thyroid gland was observed nevertheless the relative weight of this organ was changed only at the dose level 1000 mg/kg/day.

Slightly increased percentage of affected sperms was recorded at the dose level 500 mg/kg/day and increased presence of sperms with changed motility in all treated males was recorded but male ability to produce sperm that can fertilise eggs was not affected.

Numbers of corpora lutea, implantations and pups were not influenced by the test substance treatment at any dose level too. No adverse effect of the test substance treatment was observed during biochemical examination of parental males (concentration of thyroxine hormone) at the dose level 250 and 1000 mg/kg/day not even during pathological and histopathological examination of reproductive organs. Statistically significantly decreased T4 value was recorded in parental males at the dose level 500 mg/kg/day.

Further evaluation of body weight of pups, development of pups, concentration of thyroid hormone (T4) in pups and macroscopical examination of pups did not reveal any influence of the test substance treatment at any dose level.

 

Conclusion

The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 1000 mg/kg body weight/day.

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 1000 mg/kg body weight/day.