Reaction mass of tetrasodium 3-[[4-[[4-[(6-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-6-sulphonato-1-naphthyl]azo]-1-naphthyl]azo]naphthalene-1,5-disulphonate and tetrasodium 3-[[4-[[4-[(6-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-7-sulphonato-1-naphthyl]azo]-1-naphthyl]azo]naphthalene-1,5-disulphonate

Endpoint summary

Description of key information

NOAEL, oral, rast, 28 days = 1000 mg/kg bw /day

NOAEL, oral, rats, 9 weeks (OECD 422) )= 500 mg/kg bw/day

NOAEL, oral, rats, 90da = 500 mg/kg bw /day

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Remarks:

the perfomed OECD 422 last from 7 to 9 weeks and can be considered as sub chronic test

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Study period:

2016

Reliability:

2 (reliable with restrictions)

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 28th July 2015

Deviations:

yes

Remarks:

see Any other information ...

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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. 5008
- Expiration date of the lot/batch: 09/2017
- Storage condition of test material: The test substance was stored in dry and dark place 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, Acid Black 26, 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 412 - 426 g, females 248 - 357 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: 12 days (DRFE 5 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 22.03.2016)
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. 10. 2016

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 (750 rpm) for 40 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.

VEHICLE
- Lot/batch no.: 1505050287; exp. 5/2017
- 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 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 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 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.

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):

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.

Mortality:

no mortality observed

Description (incidence):

Only non-treatment-related accident: 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:

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):

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.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

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.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Deviations: During the study, short increases of temperature (22.05.2016 - for ten hour, temperature from 25.2 to 26.4°C and 23.05.2016 for two hours, temperature from 25.2 to 25.4°C) in animal room was recorded. This microclimatic deviation did not affect the welfare of animals and had no impact on the results of the study.No other deviations from study plan or test guidelines were observed during the study performance.

Conclusions:

The substance was tested for lon term toxicity following OECD 422, with 7 to 9 weeks dosing. Under the exoerimental conditions the value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 500 mg/kg body weight/day both for MALES and FEMALES. The value of NOAEL was determined on the basis of increased intensity of chronic progressive nephropathy in kidneys in both sexes (dose dependent). In females also delayed increased value of creatinine was recorded (out historical control limits).

Executive summary:

Introduction

The test substance 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 28th July 2015.

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. 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 the tested substance 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 dose level 250 mg/kg/day – No changes during health condition control and clinical observation were observed (except thinner consistency of excrements and coloured of excrements). Body weight and body weight increments of treated animals were not significantly affected by the test substance administration.

In females increased value of total erythrocyte count was recorded during the haematological examination (but in range of historical control).

Decreased value of albumin in males and decreased value of urea in females was detected during the biochemical examination but all values were in range of historical control.

No significant changes of organs were recorded during the biometry of organs, macroscopic examination and histopathological examination.

 

The dose level 500 mg/kg/day – No changes during health condition control and clinical observation were observed (except thinner consistency of excrements and coloured of excrements). Body weight and body weight increments of treated animals were not significantly affected by the test substance administration.

During examination of urine in males significant increased pH of urine was recorded.

During the haematological examination the following statistically significant changes were detected: increased value of reticulocytes in both sexes and prolonged APTT in males. These parametres were in range of historical control limits.

In females increased value of cholesterol total and decreased value of urea was recorded during the biochemical examination (but in range of historical control).

No significant changes of organs were recorded during the biometry of organs, macroscopic examination and histopathological examination.

 

The dose level 1000 mg/kg/day - No significant changes during health condition control and clinical observation were observed (except thinner consistency of excrements and coloured of excrements).

Body weight and body weight increments of treated animals were adequate to species, sex and age of animals in experiment.

During examination of urine in males significant increased pH of urine was recorded.

Haematological examination showed irreversible prolonged APTT, reversible increased value of reticulocytes in males and increased value of monocytes, increased value of reticulocytes, delayed decreased value of reticulocytes and prolonged APTT in females.

Reversible increased value of albumin and potassium ions in males and reversible decreased activity of ALP, increased value of cholesterol total, delayed increased value of creatinine in females (above historical control limits) were recorded during the biochemical examination.

During the macroscopic examination coloured content in stomach and intestines was detected but these after end of administration of the test substance disappeared.

Histopathological examination showed eosinophil infiltration of mucosa and/or lamina propria in stomach and intestines in males. These related with place of application (digestive tract) and disappear during recovery period. In kidneys higher intensity of chronic progressive nephropathy in both sexes was found out – the influence of the test substance could not be excluded.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

One study for subacute oral toxicity in rats for the target substance is available with a NOAEL of 100 mg/kg bw/day. Other two studies are availale for lont term toxicity oral in rats on similar substances where the NOAEL are equal to 500 mg/kg bw/day. Althugh, therefore one single study on the substance itself is available, the test on analogue substance 3 is considered to be the key study for NOAEL setting for CSR due to the reliability of the study, the longest exposure of the animals to the substance and the similarity with the target substance.

The third study, even though with a low reliability due to deficiencies of the study desgin, is used as to support the value o he set NOAEL.

Justification for classification or non-classification

Regulation EC 1272/2008 regarding classification criteria for substances (Annex I, table 3.9.1) states that "Substances are classified in category 2 for target organ toxicity (repeated exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations".

Moreover, at paragraph 3.9.2.9.2 it reads:" In order to help reach a decision about whether a substance shall be classified or not, and to what degree it shall be classified, dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects.

Repeated-dose studies conducted in experimental animals are designed to produce toxicity at the highest dose used in order to optimise the test objective and so most studies will reveal some toxic effect at least at this highest dose. What is therefore to be decided is not only what effects have been produced, but also at what dose/concentration they were produced and how relevant is that for humans".

 

These "guidance values" are provided in table 3.9.3, and refers to effects observed in a standard 90 day repeated dose study in which classification is not applicable when "significant toxic effects" are detected over a dose of 100 mg/kg/day.

 

Then by using dose/exposure extrapolation similar to Haber's rule for inhalation which states that "the effective dose is directly proportional to the exposure concentration and the duration of exposure" we might put forward the hypothesis that classification is not applicable in a 28 day repeated toxicity study if significant toxic effects are observed over a dose of about 300 mg/kg/day. For an OECD422 spanning about 9 weeks (ca. 60 days), a correction factor of 1.5 can be applied and the threshold established ca. 150 mg/kg bw /day.

 

The repeated oral toxicity test of the present study on analogue substance 3 did not exert any toxic effect related to the test item in death, FOB, morphology, hematological analysis and biochemistry, urinalysis, gross pathology of animals up to the dose of 1000 mg/kg/day for both genders. However, some findings at the histopathological level for kidney nephropaty was recorded, setting the NOAEL for repetead dose toxicity (subchronic) at 500 mg/kg bw /day.

Therefore, no classification for repeated dose toxicity is warranted under Regulation 1272/2008.