Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.03.2016 - 11.02.17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
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:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)
EC Number:
304-149-5
EC Name:
Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)
Cas Number:
94246-75-0
Molecular formula:
C23H11ClCuN7O11S3.3K
IUPAC Name:
tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)
impurity 1
Chemical structure
Reference substance name:
Potassium chloride
EC Number:
231-211-8
EC Name:
Potassium chloride
Cas Number:
7447-40-7
Molecular formula:
ClK
IUPAC Name:
potassium chloride
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Reactive Blue 234 - Other name: Ostazin Blue H-5R- Lot/batch No.: 8002- Expiration date of the lot/batch: unlisted
Specific details on test material used for the study:
- Stability under test conditions:Testing laboratory analysis of homogeneity and stabilityThe solution of the test substance in vehicle at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 120 minutes from a finalization of the application form preparation.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: see below Details on 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 or test system 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 377 - 381 g, females 248 - 251 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): complete pelleted diet for rats and mice in SPF breeding - 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 6 daysENVIRONMENTAL 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 30.08.2016)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) → 64th 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) → 64th 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: till 11. 2. 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:The test substance was weighted into glass beaker and the beaker was replenished by water for injections. The test solution was dissolved in ultrasonic bath for a 30 minutes and then the solution was stirred by magnetic stirrer (500 rpm) for 20 minutes. Taking of the test substance was performed at reduced speed - 300 rpm.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 application forms were prepared daily just before administration.The administration of the test substance to animals was performed during one hour after preparation of application form. The stirring of solutions continued during administration.VEHICLEwater for injection; batch no.:1508310535, expiration: 08/2017 (DRFE)1511090667, expiration: 11/2017 (DRFE)1602150079, expiration: 2/2018 1603110144, expiration: 3/2018 1605100264, expiration: 5/2018 - 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: males - 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 – 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 were examined also at the middle dose level groups. In Reproductive Toxicity part of study the full histopathology of the preserved organs and tissues was performed for all high dose and control animals. Organs with macroscopical changes were examined at the middle dose level groups. Treatment-related changes were not observed at the high dose group therefore detailed histological examination of testes was performed only for all high dose and control males 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 WEIGHTS The 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. Macroscopical findings were not observed.Control: 127 pups were examined – no macroscopical findings were recorded.250 mg/kg/day: 156 pups were examined – 1♀ from one litter (female No. 131) was not examined due to cannibalism. No macroscopical findings were recorded in all others pups.500 mg/kg/day: 125 pups were examined – 1♀ from one litter (female No. 144) was found dead on the 4th day (without cause). No macroscopical findings were recorded in all others pups.1000 mg/kg: 162 pups were examined - 1♀ from one litter (female No. 162) was not examined due to cannibalism. No macroscopical findings were recorded in others 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 WEIGHTS The 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
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female No.166 (at the dose level 1000 mg/kg/day) died on the 39th day of application due to intubation error.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Females: During lactation decreased mean body weight at the dose levels 250 and 500 mg/kg/day was detected (in the dose level 250 mg/kg/day at the 1st and 4th day of lactation period and in the dose level 500 mg/kg/day at the 4th day of lactation period). Disbalance of mean body weight increment was recorded during lactation period. Males:Body weight was slightly decreased in males at the dose level 1000 mg/kg/day. Body weight increment of all treated males and control males was similar.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Increase of mean food consumption in females at the dose level 1000 mg/kg/day was recorded during the whole pregnancy and lactation period.The mean food consumption of treated males was slightly higher than the mean food consumption of control group.
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, treatment-related
Description (incidence and severity):
Haematological examination of males and females showed altered value of reticulocytes. This is probably related to adaptation organism to the test substance treatment. Others haematological parameters of red blood line were not changed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In satellite females the value of creatinine found during the biochemical examination was changed. This value of creatinine in satellite females were out in historical control limit and can be related with histopathological findings of kidneys.Others changes of biochemical parameters observed during biochemical examination were without toxicological importance and they are related with adaptation of organism to the test substance treatment.Reproduction part:The concentration of thyroid hormone thyroxine (T4) in males was decreased at the middle and highest dose levels but these is values were in physiological range for rats (without toxicological importance).
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, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A serious kidneys chronic progressive nephropathy was found out. The influence of the test substance on kidneys could not be excluded because the intensity of chronic progressive nephropathy in kidneys irreversibly increased with dose level.Occurrence microscopic findings of stomach and/or intestines (eosinophile infiltration of mucosa and/or lamina propria) related with place of application (digestive tract) and disappear during recovery period.Reproduction part:Histopathological changes in ovaria, uterus and vagina related with reproduction cycle in females or previous pregnancy.Examination of sperm in males was without significant changes. Only slight increase in number of affected sperm at the lowest and highest dose levels was detected but without toxicological importance. Number of females showing evidence of copulation and number of females achieving pregnancy was not seriously affected. In females, number of corpora lutea, number of implantations was not significantly changed changes. Male and female fertility index at the highest dose level was decreased.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS (PARENTAL ANIMALS)Excrements in treated animals were thinner consistency and coloured by the test substance. After the end of application of the test substance this clinical symptom disappeared.REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)Female with abortion was not recorded. Number of females showing evidence of copulation and number of females achieving pregnancy was not seriously affected. Number of corpora lutea, number of implantations was not significant changed. The number of females with pups was reduced at the highest dose level but one female died during the study and this unrelated with the test substance treatment. Male and female fertility index at the highest dose level was insignificantly decreased. Gestation and survival index was without toxicological significant changes.ORGAN WEIGHTS (PARENTAL ANIMALS)250 mg/kg/day: Macroscopic examination not showed findings of reproductive organs. Biometry of organs shows decreased relative and absolute weight of prostate gland and seminal vesicles in males, differences from control were insignificant. In females no changes of weight of organs were recorded.500 mg/kg/day: During biometry of organs and macroscopic examination of organs no significant changes related to the test substance treatment were not detected.1000 mg/kg/day: Biometry of organs showed significant decrease relative and absolute weight of prostate gland and seminal vesicles in males.During the examination of microscopical structure of reproductive organs no toxicological significant changes appeared. HISTOPATHOLOGY (PARENTAL ANIMALS)A serious kidneys chronic progressive nephropathy was found out. The influence of the test substance on kidneys could not be excluded because the intensity of chronic progressive nephropathy in kidneys irreversibly increased with dose level.Occurrence microscopic findings of stomach and/or intestines (eosinophile infiltration of mucosa and/or lamina propria) related with place of application (digestive tract) and disappear during recovery period.Reproduction part:Histopathological changes in ovaria, uterus and vagina related with reproduction cycle in females or previous pregnancy.Examination of sperm in males was without significant changes. Only slight increase in number of affected sperm at the lowest and highest dose levels was detected but without toxicological importance. Number of females showing evidence of copulation and number of females achieving pregnancy was not seriously affected. In females, number of corpora lutea, number of implantations was not significantly changed changes. Male and female fertility index at the highest dose level was decreased.

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 biologically or statistically significant changes of reproduction parameters were observed.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Mean number of pups per litter and sex ration were not affected by the test substance treatment. No differences in postnatal developmental 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.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The total number of pups was lower at the higher dose level and on the contrary at the middle and lowest dose levels the total number of pups was higher. This variation is due to differences in the number of mothers in each group. Stomach empty (without milk) in 16 pups from one mother at the highest dose level was recorded during the macroscopic examination – these pups died after parturition. These findings were probably not related with the test substance treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In pups at the highest dose level the mean body weight was insignificantly decreased.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Concentration of thyroxine hormone T4 in pups was similar with the control group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Stomach empty (without milk) in 16 pups from one mother at the highest dose level was recorded during the macroscopic examination – these pups died after parturition. These findings were probably not related with the test substance treatment. Other pups in all dose level were without macroscopic findings.
Histopathological findings:
no effects observed
Description (incidence and severity):
During the microscopical evaluation of thyroid gland in pups no findings were found out.

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
Basis for effect level:
other: No biologically or statistically significant changes of reproduction parameters were observed.

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

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. No biologically or statistically significant changes of reproduction parameters were observed.
Executive summary:

Introduction

The test substance, Reactive Blue 234, 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,

non-pregnant 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 parental animals and pups were removed for weighing and histopathological examination.

Results

Repeated oral administration of Reactive Blue 234 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:

Parental males and females

The number of females achieving pregnancy and microscopical structure of reproductive organs in both parental males and females seem to be not affected by the substance administration.

The mean body weight of parental animals was not significantly affected by the test substance treatment.

Observation of sperm in parental males did not show negative effect. Male ability to produce sperm that can fertilise eggs was not affected by the test substance administration.

All parental males were assessed for serum levels of thyroid hormone thyroxine (T4). Significantly decreased concentration of hormone was recorded in all treated groups of males (in historical control range). Histopathological examination showed thyroid gland of males without pathological findings. Slightly decreased concentration of hormone T4 in males can be considered to be of no toxicological significance.

Statistically significant changes of absolute and relative weights of genital organs in males and females were not recorded. Statistically significantly decreased absolute weight only of thyroid gland in parental males was recorded at the dose level 1000 mg/kg. In females absolute and relative weight of thyroid gland was reported at the dose level 1000 mg/kg. 

Macroscopical findings related to the test substance colour only were recorded during the pathological examination of treated males and females.

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. No findings related to the test substance treatment were recorded, that is why only organs with macroscopical changes were examined at the other dose level groups.

Pups

Number and sex ratio of pups were not significantly affected by the test substance administration. No differences in postnatal developmental 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. Concentration of thyroxine hormone T4 in day 13 pups from the treated groups was not significantly influenced by the test substance treatment. .

Microscopical evaluation did not show any findings related to the test substance treatment.

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.

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