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-)

Endpoint summary

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.03.2016 – 11.02.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Specific details on test material used for the study:

- Stability under test conditions:
It follows from the results of analyses (homogeneity and stability) that the both application forms (1000 mg/ 10 mL and 10 mg/ 10 mL) of the test substance, Reactive Blue 234, 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 exposure

FORM AS APPLIED IN THE TEST (if different from that of starting material)
solution in water for injection

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 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 /12

STUDY TIME SCHEDULE
Administration (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 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) → 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 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 mating

Observations
Urinalysis: only males – 63rd and 77th day of study
Blood collection for haematology and biochemistry:
parental males – 64th day of study
satellite males – 78th day of study
parental females - 13th day of lactation period
pups – 2 pups per litter – 4th day of lactation; 2 pups per litter – 13th day of lactation
satellite females – 78th day of study
Necropsy:
parental males – 64th day of study
satellite males – 78th day of study
parental females - 13th day of lactation period
pups – 2 pups per litter – 4th day of lactation, other pups - 13th day of lactation
satellite females – 78th day of study
non-pregnant females – 26th day after the end of mating period or confirmed mating
End of histopathological examination: till 11. 2. 2017

Route of administration:

oral: gavage

Vehicle:

water

Details on oral 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.
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.

VEHICLE
- Lot/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.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Only 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 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

Frequency of treatment:

7 days per week at the same time

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

Observations and examinations performed and frequency:

HEALTH CONDITION CONTROL: Yes
- Time schedule: daily - during the acclimatization and the experimental part

MORTALITY CONTROL:
- Time schedule: twice daily

CLINICAL OBSERVATIONS: Yes
males and females - daily during the administration period in natural conditions in cages

DETAILED 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 period

NEUROBEHAVIOURAL 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 activity

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 – weekly

FOOD 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: Yes
Time 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

WATER 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 / observation period
- 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 mentioned there are 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
a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities and biometry of organs

HISTOPATHOLOGY: 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 control animals and satellite animals. Organs with macroscopical changes were examined also at the middle dose level groups.

Statistics:

For statistical evaluation the software Statgraphic ® Centurion (version XV, 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 are indicated in the summary tables in report.

Clinical signs:

no effects observed

Description (incidence and severity):

(except coloured faeces)

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, treatment-related

Description (incidence and severity):

Only the value of reticulocytes was statistically significantly increased in males at the dose level 1000 mg/kg/day (out of historical range). This change was reversible, so it can be considered as adaptive response of organism to chemical stress.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant changes in biochemical examination were observed in males at all treated groups. Decreased values of total protein and albumin concentration in comparison with the control group were recorded in males at the dose level 1000 mg/kg. Increased values of concentration of triglycerides, cholinesterase were recorded in males at the dose level 500 mg/kg. Increased values of concentration of cholinesterase were recorded in males at the dose level 250 mg/kg. Decreased concentration of chloride ions was recorded in males at all dose levels - statistically significant and dose-dependent. Decreased concentration of natrium ions was recorded in males at all dose levels - statistically significant. Increased concentration of kalium ions was recorded in males at all dose levels – dose-dependent. All changes were reversible.
Statistically significant differences were also found in satellite treated males – increased concentration of calcium, decreased concentration of phosphorus and cholinesterase. Other parameters of treated animals were well-balanced with control animals.
No statistically significant changes in biochemical examination were observed in females at all treated groups. Slightly and insignificantly decreased concentration of chloride ions was recorded in females at all dose levels. Decreased concentration of natrium ions was recorded only in females at dose level 1000 mg/kg. Other parameters of treated females were similar to the control females. Statistically significant difference was found only in satellite treated females – concentration of natrium was decreased. Other parameters were well-balanced.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis manifested the influence of the test substance administration on value of pH in males at the dose level 1000 mg/kg. The pH was decreased and this change persisted into the end of recovery period. The volume of urine was significantly changed in males at the dose level 1000 mg/kg – decreased at the end of application period and increased at the end of recovery period.

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):

Statistically significant changes in absolute weights of prostate gland with seminal vesicles and thyroid gland in males were recorded. Decreased weight of prostate gland with seminal vesicles was noted in males at the dose level 250 and 1000 mg/kg. Decreased weight of thyroid gland was recorded in males at the highest dose level only. Vice versa absolute weight of thyroid gland in treated satellite males was statistically significantly increased. Absolute weight of kidneys was increased in males at all treated groups (without statistical significance but dose-dependently). Statistically significantly increased relative weight of kidneys was recorded in males at all dose levels (without dose dependency). In satellite treated males no significantly changed the weight of organs was reported.
During biometry of organs, significant changes in absolute weights of kidneys and thyroid gland in females were recorded. Increased weight of kidneys was noted in females at the dose level 1000 mg/kg. Increased weight of thyroid gland was recorded in females at the dose level 250 and 500 mg/kg and simultaneously decreased in females at the highest dose level. Statistically significantly increased relative weight of thyroid gland was recorded in females at the dose levels 250 and 500 mg/kg. Relative weight of kidneys in females at the dose level 1000 mg/kg. In satellite treated females significantly decreased absolute and relative weight of thymus and decreased absolute weight of heart was reported.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Colored content of small, large intestine, caecum and colored kidneys were observed. Other findings were probably of spontaneous character.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The test substance orally administered at the dose of 1000 mg/kg/day (the highest dose level) did not cause histopathological changes indicative of a toxic effect in any examined organs. Mild hydronephrosis of kidneys was reported in dosed animals as well as in a control animals.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No biologically or statistically significant changes were observed.

Critical effects observed:

no

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY in MALES was established as lower than 250 mg/kg body weight/day. This judgement is based predominantly on significant changes in serum electrolytes concentrations - decreased value of chloride ions concentrations in males (dose-dependently and statistically significantly), natrium ions (statistically significantly) and increased absolute (dose-dependently) and relative weight (statistically significantly) of kidneys in males. The target organ seems to be kidneys.
The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY in FEMALES was established as 1000 mg/kg body weight/day. No biologically or statistically significant changes 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 234to 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 treatment did not produce changes detected in health condition control, daily clinical observation after application (except coloured faeces) and functional observation of animals.

The test substance did not interfere with normal growth of treated parental animals. Body weight and body weight increments of treated animals were not significantly affected by the test substance administration. 

Haematological examination of males did not show negative effect of the test substance on blood and coagulation parameters. Only the value of reticulocytes was statistically significantly increased in males at the dose level 1000 mg/kg/day (out of historical range). This change was reversible, so it can be considered as adaptive response of organism to chemical stress. 

Haematological examination of females showed significant changes only in females at the dose level 500 mg/kg (increased concentration of haemoglobin and increased APTT – still in range of historical control). Haematological parameters of high-dosed females in comparison with the control females were not influenced by the test substance administration.  

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.

Statistically significant changes in biochemical examination were observed in males at all treated groups. Decreased values of total protein and albumin concentration in comparison with the control group were recorded in males at the dose level 1000 mg/kg. Increased values of concentration of triglycerides, cholinesterase were recorded in males at the dose level 500 mg/kg. Increased values of concentration of cholinesterase was recorded in males at the dose level 250 mg/kg. Decreased concentration of chloride ions was recorded in males at all dose levels - statistically significant and dose-dependent. Decreased concentration of natrium ions was recorded in males at all dose levels - statistically significant. Increased concentration of kalium ions was recorded in males at all dose levels – dose-dependent.

All changes were reversible.

Statistically significant differences were also found in satellite treated males – increased concentration of calcium, decreased concentration of phosphorus and cholinesterase. Other parameters of treated animals were well-balanced with control animals.

No statistically significant changes in biochemical examination were observed in females at all treated groups. Slightly and insignificantly decreased concentration of chloride ions was recorded in females at all dose levels. Decreased concentration of natrium ions was recorded only in females at dose level 1000 mg/kg. Other parameters of treated females were similar to the control females. Statistically significant difference was found only in satellite treated females – concentration of natrium was decreased. Other parameters were well-balanced.

Urinalysis manifested the influence of the test substance administration on value of pH in males at the dose level 1000 mg/kg. The pH was decreased and this change persisted into the end of recovery period. The volume of urine was significantly changed in males at the dose level 1000 mg/kg – decreased at the end of application period and increased at the end of recovery period.

During biometry of organs, statistically significant changes in absolute weights of prostate gland with seminal vesicles and thyroid gland in males were recorded. Decreased weight of prostate gland with seminal vesicles was noted in males at the dose level 250 and 1000 mg/kg. Decreased weight of thyroid gland was recorded in males at the highest dose level only. Vice versa absolute weight of thyroid gland in treated satellite males was statistically significantly increased. Absolute weight of kidneys was increased in males at all treated groups (without statistical significance but dose-dependently). Statistically significantly increased relative weight of kidneys was recorded in males at all dose levels (without dose dependency). In satellite treated males no significantly changed the weight of organs was reported. 

During biometry of organs, significant changes in absolute weights of kidneys and thyroid gland in females were recorded. Increased weight of kidneys was noted in females at the dose level 1000 mg/kg. Increased weight of thyroid gland was recorded in females at the dose level 250 and 500 mg/kg and simultaneously decreased in females at the highest dose level. Statistically significantly increased relative weight of thyroid gland was recorded in females at the dose levels 250 and 500 mg/kg. Relative weight of kidneys in females at the dose level 1000 mg/kg. In satellite treated females significantly decreased absolute and relative weight of thymus and decreased absolute weight of heart was reported. 

Macroscopical changes related to test substance color were recorded in males and females of the dose level 500 and 1000 mg/kg. Colored content of small, large intestine, caecum and colored kidneys were observed. Other findings were probably of spontaneous character.

Microscopical evaluation showed that the test substance orally administered at the dose of 1000 mg/kg/day (the highest dose level) did not cause histopathological changes indicative of a toxic effect in any examined organs. Mild hydronephrosis of kidneys was reported in dosed animals as well as in a control animals.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliability 1

System:

urinary

Organ:

kidney

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