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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09. 09. 2019 – 15. 06. 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

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:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch no. 9A475
Purity >98
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 (10 mg and 1000 mg/10 mL) of the test item, Pigment Red 4, prepared at defined laboratory conditions (laboratory temperature. mixing. stirring) 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)
The test item was administered in the form of a suspension in olive oil.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
CRL (SPF quality - guaranteed)
Details on species / strain selection:
- laboratory rat has been chosen because testing laboratory has long experience with this species and because rat is recommended acc. to the test guideline
- random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed ± 20% of the mean weight
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: sexually adult, 9 weeks on arrival
- Weight at study initiation: males 454 - 472 g, females 260 - 268 g
- Fasting period before study: no
- Housing: SPF conditions according to internal SOP No.12; sterilized soft wood fibers Lignocel;
Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – 1 M + 1 F in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage;
- Diet (e.g. ad libitum): maintenance pelleted diet for rats and mice - Altromin for rats/mice; manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany
- Water: drinking water 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 14.01.2020)
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, administration ) → 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, administration)→ 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, administration) → 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, administration) → 25th day after confirmed mating

Observations
Urine collection: 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
Histopathological examination: 14. 05. - 15. 06. 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

Preparation of the application form was based on the result of the study Determination of homogeneity and stability of Pigment Red 4 in olive oil vehicle. The test item was weighted into a glass beaker and the beaker was replenished by olive oil. The suspension was stirred by magnetic stirrer (500 rpm) for 10 minutes.
The concentrations of suspension 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 protected from light and prepared daily just before administration. The administration of the test item to animals was performed during one hour after preparation of application form. The stirring of solutions continued during administration.

VEHICLE
Olive oil – Dr. Kulich Pharma,s.r.o, Czech Republic
Batch No. and expiration:
8003998001, exp. 01/2020
8004329001, exp. 06/2020
19K11-T12, exp. 10/2021


- Concentration in vehicle:
The concentrations of suspension at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Stability and homogeneity were determined by means of measuring of a peak area of the test item by a spectrophotometric method validated within the study: Pigment Red 4 – Validation of Spectrophotometric Method, Study No.: 157/19/48neSLP, Report No.: 19-350.
The results of stability and homogeneity of application form were summarized in Analytical Report 1 which constitutes the annex to the study final report.
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 – 49 days in total
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
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
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:
Satellite group (vehicle only)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Satellite group
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 the study were determined of the requirement of the Study Monitor after evaluation of the results of the DRFE (Annex 2).
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
HEALTH CONDITION CONTROL: Yes
- Time schedule: daily - during the acclimatization and the experimental 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: No

BODY WEIGHT: Yes
- Time schedule for examinations:
males and satellite animals - the first day of administration and then weekly,
females - the first day of administration and then weekly, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 1st, 4th day, 12th day and 13th day

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: satellite males and females – twice a week

Laboratory examinations:

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 "Tables with examined parametres_PR4.pdf"] 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.

Functional observations, haematology, biochemistry, full biometry and full pathology was performed only in 6 males + 6 females of each dose level and in satellite animals.
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 were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative.

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.

- pathological examination:
males and nonpregnant females – after the end of application period
2 pups per litter - on the 4th day of lactation
parental females and pups – on the 13th day of lactation
satellite animals – after the end of observation period

- weight of organs: during necropsy
- sperm observation: parental males – during and after necropsy (not performed in satellite males)
- histopathological examination: after necropsy
Statistics:
For statistical evaluation the software Statgraphic ® Centurion (version XVII, USA) was used.
Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group. The results statistically significant on probability level (p ≤0.05) were indicated in the summary tables.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical findings revealed influence of the test item on clinical status of treated animals and satellite treated animals were recorded. Only coloured excrements was recorded in all treated animals this changes related to the colour of the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening one female (at the dose level 1000 mg/kg/day) died during the study probably for pregnancy and delivery complications.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased mean of body weight was detected in females in all treated group during the pregnancy period. The body weight increment in treated females was variable. In some time weight period the body weight gain was decreased by more than 10% compared to control so this effect could be regarded as toxicologically significant. In males the body weight of males was relative balanced.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreased or variable the food conversion and food consumption were recorded during the study, in animals of both sexes.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Decreased or variable the food conversion and food consumption were recorded during the study, in animals of both sexes.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Satellite M: similar compared to water consumption of satellite control males during the whole application and recovery period
Satellite F: slightly higher compared to water consumption of satellite control females during the whole application and recovery period
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological examination showed statistically significantly decreased values of total erythrocyte count, haemoglobin and hematocrite in males of all treated groups (with dose dependence). Decrease of these parameters were recorded in females at the end of recovery period. Statistically insignificantly increased value of mean corpuscular volume was detected in males at the middle (500 mg/kg/day) and the highest (1000 mg/kg/day) dose levels.
In all treated females statistically significantly and dose dependently increased percentual portion of lymphocytes was recorded.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical examination in treated animals showed following statistically significant differences: reversible increased value of bilirubin total in animals of both sexes (dose dependently), reversible decreased values of urea and creatinine in all treated males, decreased value of chloride ions in males at the lowest (250 mg/kg/day) and the highest dose levels. Delayed statistically significantly increased value of inorganic phosphorus in both sexes, value of cholinesterase in males and activity of ALP in females were detected.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
During the urine examination of males presence of bilirubin and leucocytes in males of treated groups were recorded. Concentration of bilirubin in urine was higher than measurable limit in two males at the lowest dose level and in all examined males at the middle and the highest dose level. Change of colour of urine from light yellow to dark yellow was recorded.
The change in urine colour was probably due to the presence of bilirubin in the urine
Statistically significantly decreased volume of urine in males at the middle dose level was detected. At the highest dose level the volume of urine was decreased insignificantly.
Increased value of bilirubin total in serum and urine could be related with faster decomposition of the red blood cells detected during haematological examination.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Biometry of organs showed statistically significantly increased absolute and/or relative weight of liver and kidneys in treated males. Increasing of weight of kidneys in treated males was irreversible. On the contrary significantly decreased absolute weight of kidneys was detected in females at the end of recovery period.
Absolute or relative weight of spleen in males or females was reversible statistically significantly increased.
These changes of weight of organs related with changes detected during the hematological, biochemical examination and examination of urine. These probably related to the adaptation process of organism to the test item administration.
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:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No histopathological findings in gastrointestinal tract, kidneys, liver and hemopoietic organs, which related with the test item treatment, were detected during the histopathological examination of organs in animals at the highest dose level and satellite treated animals.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL (No Observed Adverse Effect Level) value for REPEATED DOSE TOXICITY in male and female rats for the test item Pigment Red 4 could not be determined (the value is less than 250 mg/kg/day).

The LOAEL (Lowest Observed Adverse Effect Level) value for the test item Pigment Red 4 for REPEATED DOSE TOXICITY in male and female rats was established as 250 mg/kg/day.

The values of NOAEL (No Observed Adverse Effect Level) and LOAEL (Lowest Observed Adverse Effect Level) were established on the basis of results of decreased body weight of females, haematological examination (decreased values of RBC, haemoglobin and haematocrite in males and in females at the end of recovery period, increased value of lymphocytes in females), biochemical examination (increased value of bilirubin total in both sexes and decreased values of creatinine and urea in males), examination in urine in males (presence of bilirubin in urine), biometry of organs (increased relative and/or absolute weight of liver, kidneys spleen).
Executive summary:

Introduction

The test item, Pigment Red 4, 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 (SPF quality) were used for testing. The test item was administered in the form of a suspension in olive oil. 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/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 the study were determined of the requirement of the Study Monitor after evaluation of the results of the DRFE (Annex 2).

The first six males and six mothers who delivered pups per group (as per internal SOP) and a satellite groups of animals (control and treated) are part of the repeated dose toxicity study and examined with respect to toxicity of the test item. Satellite animals were used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effects up to 14 days post treatment. All twelve males and females per group are a part of the reproduction study and examined with respect to reproduction parameters.

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 – 49 days in total

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 in the 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 at the specified time intervals. Detailed clinical observation was carried out weekly. Functional observations were performed at the end of the application and observation period. Vaginal smears were prepared daily, 2 weeks before start of the administration period (oestrous cycle monitoring), during the mating period (until the presence of spermatozoa) and at necropsy. Reproduction parameters relevant to pups (number of pups, weight of litters and weight of pups, sex and vitality of pups, measurement of anogenital distance, nipple retention, serum levels of thyroid hormones (T4 + TSH in pups) were also recorded. The study was completed with urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of the main groups, the sperm parameters, sperm motility and sperm morphology were examined. Selected organs from adult animals and pups were removed for weighing and histopathological examination.

 

Results

Repeated Dose Toxicity part of study:

The six males per group (Nos. 1-6, 21-26, 41-46, 61-66) and first six mothers per group that delivered pups were examined from the main group (control: Nos. 101, 102, 103, 104, 107, 111, the lowest dose: Nos. 121, 122, 123, 124, 127, 130, the middle dose: Nos. 141, 142, 143, 145, 147, 148 and the high dose: Nos. 161, 163, 164, 170, 171, 172). Also, satellite animals (control: males - Nos. 81-86, females – Nos. 181-186 and high dosed: males - Nos. 91-96 and females Nos. 191-196) were part of the examination of the repeated dose toxicity of the test item.

 

During the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening one female (at the dose level 1000 mg/kg/day) died during the study probably for pregnancy and delivery complications.

 

Significantly decreased mean of body weight was detected in females in all treated group during the pregnancy period. The body weight increment in treated females was variable. In males the body weight of males was relative balanced.

Decreased or variable the food conversion and food consumption were recorded during the study, in animals of both sexes.

 

No clinical findings revealed influence of the test item on clinical status of treated animals and satellite treated animals were recorded. Only coloured excrements was recorded in all treated animals; this changes related to the colour of the test item.

 

Haematological examination showed statistically significantly decreased values of total erythrocyte count, haemoglobin and hematocrite in males of all treated groups (with dose dependence). Decrease of these parameters were recorded in females at the end of recovery period. Statistically insignificantly increased value of mean corpuscular volume was detected in males at the middle (500 mg/kg/day) and the highest (1000 mg/kg/day) dose level.

In all treated females statistically significantly and dose dependently increased percentual portion of lymphocytes was recorded.

 

Biochemical examination in treated animals showed following statistically significant differences: reversible increased value of bilirubin total in animals of both sexes (dose dependently), reversible decreased values of urea and creatinine in all treated males, decreased value of chloride ions in males at the lowest (250 mg/kg/day) and the highest dose level. Delayed statistically significantly increased value of inorganic phosphorus in both sexes, value of cholinesterase in males and activity of ALP in females were detected.

 

During the urine examination of males presence of bilirubin and leucocytes in males of treated groups were recorded. Concentration of bilirubin in urine was higher than measurable limit in two males at the lowest dose level and in all examined males at the middle and the highest dose level. Change of colour of urine from light yellow to dark yellow was recorded. The change in urine colour was probably due to the presence of bilirubin in the urine

Statistically significantly decreased volume of urine in males at the middle dose level was detected. At the highest dose level the volume of urine was decreased insignificantly.

  

Biometry of organs showed statistically significantly increased absolute and/or relative weight of liver and kidneys in treated males. Increasing of weight of kidneys in treated males was irreversible. On the contrary significantly decreased absolute weight of kidneys was detected in females at the end of recovery period.

Absolute or relative weight of spleen in males or females was reversible statistically significantly increased.

These changes of weight of organs related with changes detected during the hematological, biochemical examination and examination of urine.

No histopathological findings in gastrointestinal tract, kidneys, liver and hemopoietic organs, which related with the test item treatment were detected during the histopathological examination of organs in animals at the highest dose level and satellite treated animals.