Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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 did 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 (by 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 partumSatellite 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 periodNon-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 studysatellite 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 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.VEHICLEwater for injection; 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.
Details on mating procedure:
Animals were mated from the 29th day of study.- M/F ratio per cage: 1:1- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
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.
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

Parental animals: Observations and examinations:
HEALTH CONDITION CONTROL: Yes- Time schedule: daily - during the acclimatization and the experimental partCLINICAL OBSERVATIONS: Yesmales and females - daily during the administration period in natural conditions in cagespups - as soon as possible after delivery and then dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: before the first application and then weekly (except the mating period)BODY 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-weightedaverages 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 – weekly
Oestrous cyclicity (parental animals):
yes, before beginning of treatment; vaginal smears of all females were monitored daily for two weeks
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:- sperm motility, sperm morphology- in all males (except the satellite group)
Litter observations:
CLINICAL OBSERVATION OF PUPS- Time schedule:All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 1 post-partum) and on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.Anogenital distance measurement: 4th day of lactationNipples examination (the presence and number of nipples in male pups): counted on day 13 of lactation. BODY WEIGHTpups (litters) - 1st , 4th day, 12th day and 13th daypups – individually – 4th day of lactation
Postmortem examinations (parental animals):
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 with macroscopical changes and kidneys, stomach, small intestine, large intestine and caecum were examined at the lowest and middle dose level groups used for Repeated Dose Toxicity part of study.In Reproduction Toxicity part of study the histopathology of the reproductive organs, pituitary gland and thyroid gland was performed for all high dose and control animals and organs with macroscopical changes were examined at the lowest and middle dose level groups. Detailed histological examination was performed on testes of all high dose and control animals from Reproduction Toxicity part of study (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). SACRIFICEAfter the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment. GROSS NECROPSYDuring 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 / ORGAN WEIGHTSThe tissues indicated in Table [5] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: The macroscopic examination was performed in all pups. No pathological findings were recorded at the dose levels 250 and 500 mg/kg/day and in control pups. Stomach – empty was recorded at the dose level 1000 mg/kg/day in the 16 pups from one female. These pups died after parturition.No histopathological findings were recorded during examination of thyroid gland in pups.GROSS NECROPSYDuring 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 / ORGAN WEIGHTSThe tissues indicated in Table [5] were prepared for microscopic examination and weighed, respectively.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Only coloured faeces were recorded from the 3rd week of study.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
M: Body weight of treated males was relatively well-balanced in comparison with control males and changes were without statistical significance. Body weight increment of all treated males was not adversely affected by the test substance. F: Pre-mating periodMean body weight of treated females was similar compared to the control group.The mean body weight increment was lower at the lowest dose level before mating in comparison with control group.PregnancyFemales without parturition (non-pregnant females) were not included in the evaluation of mean body weight increments during pregnancy. Mean body weight of all treated groups was comparable to the control group or even higher during whole pregnancy.The mean body weight increments of all treated pregnant females were similar to the control females. LactationOnly mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. The body weight of females was similar with control females during the rest of lactation period. The mean body weight increments of treated mothers were comparable to control animals only a slightly decline was observed at the lowest dose level during the lactation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
M: Food consumption of treated males was relatively well-balanced in comparison with control males. F: Pre-mating periodThe mean food consumption of treated females was slightly increased during 2nd week in pre-mating period in comparison with the control animals. PregnancyFemales without parturition (non-pregnant or aborted females) were not included in the evaluation of food consumption during pregnancy. The mean food consumption of pregnant females treated by the test substance was similar in comparison with the control group. LactationOnly mothers (females with live pups born) were included in evaluation of food consumption during lactation period.The mean food consumptions of treated mothers at the all dose levels were slightly lower during the first four days of the lactation. At the end of the lactation period treated mothers consumptions were quite well balanced compared to control mothers.
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, non-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).Significantly decreased concentration of hormone thyroxine (T4) was recorded in males at the dose level 500 mg/kg/day. This finding can be considered to be an incidental, because concentration of other dosed groups was similar to the control group.All biochemical parameters were in range of historical control.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopical evaluation showed that the test substance orally administered at the dose of 1000 mg/kg (the highest dose level) did not cause any pathological changes in the male and female genital organs, pituitary and thyroid gland.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
Oestrous cycles were monitored before treatment started to select for the study females with regular cyclicity daily for two weeks (all the females had regular oestrus cycle).
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Reproductive performance:
no effects observed
Description (incidence and severity):
The number of females achieving pregnancy, sperm parameters and microscopical structure of reproductive organs in both parental males and females seem to be not affected by the test substance administration.Reproduction performance of males and females was evaluated according to the male and female reproduction data and calculated reproduction parameters. Male ability to produce sperm that can fertilise eggs and female ability to achieve pregnancy were not affected by the test substance administration.

Effect levels (P0)

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: No significant changes: in growth, during examination of sperm or microscopical structure of reproductive organs. Reproduction parameters were not significantly affected.
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Number and sex ratio of pups were not significantly affected by the test substance administration. No differences in postnatal development were observed in pups at the treated groups – presence of nipples in male pups was not recorded and anogenital distance in treated male and female pups in comparison with the control pups was similar.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Increased mean body weight of pups with statistical significance was recorded at the lower dose level.The mean weight of litter was balanced in all groups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Concentration of thyroxine hormone T4 in day 13 pups from the treated groups was similar to the control group.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid gland weight: No significant differences were not recorded in pups from treated groups in comparison with the control pups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination was performed in all pups. Macroscopical findings were observed sporadically and did not relate to the test substance administration.Control: 158 pups were examined – cannibalism of 4 pups (3 litters) and malnutrition with flatulence in one dead pup (other litter) was observed. No macroscopical findings were recorded in other pups.250 mg/kg/day: 150 pups were examined – empty stomach in one pup. 1 pup was not examined because organs were autolysed. No macroscopical findings were recorded in other pups.500 mg/kg/day: 201 pups were examined – empty stomach in one pup and one died pup without pathological findings (from two litters) were observed. Cannibalism of 5 pups (from three litters) was observed.1000 mg/kg: 185 pups were examined - cannibalism of 5 pups (from two litters) was observed.
Histopathological findings:
no effects observed
Description (incidence and severity):
Microscopical evaluation of thyroid gland of pups did not show any findings related to the test substance treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive 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 performed according to the updated OECD guidelineNo. 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 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.

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 REPRODUCTION and DEVELOPMENT was established as 1000 mg/kg body weight/day.