p-[4,5-dihydro-4-[[2-methoxy-5-methyl-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonic acid, sodium salt

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

subacute toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23. October 2019 to 10. July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

p-[4,5-dihydro-4-[[2-methoxy-5-methyl-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonic acid, sodium salt

EC Number:

263-417-9

EC Name:

p-[4,5-dihydro-4-[[2-methoxy-5-methyl-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonic acid, sodium salt

Cas Number:

62121-75-9

Molecular formula:

C20H22N4O11S3.xNa C20H(22-x)N4NaxO11S3; x<=2

IUPAC Name:

sodium 4-[4-(2-{2-methoxy-5-methyl-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl]benzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

Name of test substance: Reactive Yellow 15

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats are commonly used and recommended to assess toxicity. A large number of publications on the subject are available as well as historic data and firsthand experience at the test facility

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Germany
- Females: nulliparous and non-pregnant
- Age at study initiation: 11-12 weeks
- Weight at start of administration: Main: 380 -450 g (males), 205-.246 g (females); Recovery: 357-442 g (males), 212-258 g (females)

HOUSING
- During acclimatization, pre-exposure and pre-mating: male animals as well as female recovery animals were caged in groups of two or three animals per cage (open macrolon cages type 2000P, Techniplast (size slightly larger than GV-SOLAS Type IV)); Individually housed females (main groups), paired animals and single dams with their litters were housed in open macrolon cages Type III; throughout the mating period, animals were kept in pairs of one female and one male rat. The female was placed with the same male until pregnanacy occured ot two weeks have elapsed. After the mating period, male rats were housed in groups of three, which were originally formed during the pre-mating phase; female rats were housed individually.

- Diet (ad libitum): Maintenance diet for rats and mice, No. 1324 TPF; dams: breeding diet for rats and mice, No. 1314 TPF (both: Altromin Spezialfutter GmbH & Co. KG)
- Water (ad libitum): sterilized community tap water
- Acclimation period: 6-9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 11. November 2019 to 31. January 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Concentration in vehicle:
200 mg/ml (females 100 mg/ml from study day 38-41 onwards)
60 mg/ml
20 mg/ml
Application volume: 5 ml per kg bodyweight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the quantitative re-analysis, the recovered test item content in the test item/water solutions was compared with the target test item content in the preparations. Analysis was conducted by Liquid Chromatography-Diode Array Detector (LC-DAD).
The concentration of the test item in the respective solutions was determined once within pre-mating phase, within the gestation phase and at the end of gestation/ beginning of lactation phase.
Recovery rates between 90-110 % were set as acceptance limits. All criteria were fulfilled.

Duration of treatment / exposure:

Main groups: Males were dosed daily for 41-43 days (depending on cohort), including the day before the scheduled termination of the in-life phase. Females were dose daily for 53 to 66 days, depending on the female performance during mating and preganancy.

Recovery groups: Male and female animals were dosed daily for 49 days follwed by an observation period of 14 days post treatement.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose; for females reduced to 500 mg/kg bw/day from study day 38-41 onwards

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

medium dose

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Recovery high dose; for femlaes reduced to 500 mg/kg bw/day from study day 38-41 onwards

Doses / concentrations 6

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Recovery, vehicle

No. of animals per sex per dose:

12/sex/dose (reproductive toxicity)
--> thereof 5/sex/dose for the repeated dose toxicity evaluation (randomly selected)
5/sex/dose (recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: According to the available literature, no toxic effects of the test item has been observed at a dose of 500 mg/kg bw during a 20-day oral administration with a total of 14 administrations . In addition, the LD50 value (oral) in mice was reported to be >10.000 mg/kg bw, the LD50 for rats is specified as greater 2000 mg/kg bw. Based on this information, the Sponsor decided to test 1000 mg/kg bodyweight per day as the highest dose. An interval of approximately 3 has been chosen for the two subsequent doses resulting in 300 mg/kg bw/day for the medium dose and 100 mg/kg bw/day for the low dose group.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (IRWIN test) in a standard arena, on all animals (Main and recovery groups)

BODY WEIGHT: Yes
- Time schedule for examinations: At least once weekly (inlcuding once before beginning of application)

FOOD CONSUMPTION: Yes
- Time schedule for examinations: At least once weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: At least once weekly

HAEMATOLOGY: Yes
On randomly selected male and female animals per dose group (main groups):
- 5 females at the end of the pre-mating period
- 5 males at the end of the treatement period.
- on all recovery animals at the end of the recovery period
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: No
- Parameters examined: Leukocytes, Erythrocytes, Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular Haemoglobin (MCH), Mean corpuscular haemoglobin conc. (MCHC), Thrombocytes, Reticulocytes, Neutrophil granulocytes, Lymmphocytes, Monocytes, Eosinophils, Basophils, Blood clotting
time.
- How many animals: 5 per sex and dose group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On randomly selected male and female animals per dose group (main groups):
- 5 females at the end of the pre-mating period
- 5 males at the end of the treatement period.
- on all recovery animals at the end of the recovery period
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: No
- Parameters examined: Alkaline Phosphatase (AP), Aspartate
aminotransferase (AST); Alanine aminotransferase (ALT),
Gamma-glutamyl-transferase (GGT), Cholesterol, Urea, Sodium, Potassium, Calcium, Chloride, Glucose, Total protein, Albumin, Globulin, A/G ratio, Creatinine, Bile acids, T4-hormone levels
- How many animals: 5 per sex and dose group


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the in-life phase
- Dose groups that were examined: all
- Battery of functions tested: sgrip strength and beam-walking test

Sacrifice and pathology:

All adult animals were sacrificed humanely by asphyxiation in a CO2/O2 atmosphere and were examined macroscopically. All occurring lesions were recorded on checklists for each individual animal. Special attention was paid to the organs of the reproductive system. The number of implantation
sides was recorded. Organs were surgically extracted from adult male animals (testes, epididymis, prostate and seminal gland vesicles with coagulating gland as a whole, LABC muscle, Cowper’s gland,
glans penis) and from all adult female animals (uterus including cervix, ovaries) and were weighed as soon as possible after dissection. The thyroid glands were surgically extracted from both, male and female animals and weighed before fixation. Paired organs were weighed individually. The ovaries, testes, epididymis, accessory sex organs (cervix, prostate etc.), thyroid and all other organs showing macroscopic lesions of all adult animals were preserved in the appropriate fixative.
From all recovery animals and 5 randomly selected male and female animals per dose group (main groups), additional organs were preserved in the appropriate fixative/s (adrenal glands, bone marrow (sternum), brain, esophagus, eyes, heart, large and small intestine, kidneys, liver, lung, lymph nodes (axillary and mesenterial), mammary glands, sceletal muscle, peripheral nerve, pituitary gland, spinal cord, spleen, stomach, thymus, trachea, urinary bladder, vagina). Organs to be weighed were trimmed of any adherent tissue and their wet weight was taken as soon as possible after dissection. Paired organs were weighed individually.
Modified Davidson solution was used for testis and epididymis; Davidson solution was used for the eyes. All other organs/tissues were fixed in formalin.

ADDITIONAL ORGAN WEIGHTS
for 5 male and female animals per dose group:
brain, heart, liver, spleen, thymus, kideny, adrenal glands

HISTOPATHOLOGY (selected animals)
A detailed histological examination was performed on selected organs and tissues of the animals of the main high dose and the vehicle control group. The thyroid glands of the remaining animals were included in the histology panel, due to test item-related findings from the T4-analysis.
The tissues were embedded in paraffin wax, sectioned, and stained with hemalum and eosin.

Statistics:

Anova with Dunnett's t-test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation after test item administration as well as wiping the mouth in the cage bedding was observed for individual male animals of the high dose groups (main and recovery). Signs of salivation were considered related to the lack of palatability of the administered test item and were rated as neglectable or mild under consideration of intensity and frequency of incidence, and with no adversity associated.

Mortality:

mortality observed, treatment-related

Description (incidence):

Five female animals treated with the high dose of the test item (four of the main groups, one of the recovery group) and one animal of the medium dose group died soon after gavage, showing signs of reflux. The test item solutions showed viscous, not yet described physical characteristics. A gavage related reflux was assumed as most likely cause of death for those animals. A physicochemical relationship is therefore assumed rather than a toxicological property of the substance.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Male main group animals of the high dose as well as female animals of the recovery groups showed test-item related increase in water consumption during treatment period, when compared to the respective vehicle control group, but below amounts considered to be adverse. A secondary effect related to the increase in salivation and/or the high concentration of the formulation might be conceivable.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

T4-analysis of parental animals revealed significant increase of the T4 hormone level in the high dose groups (male and female) and the medium dose animals (males only) when compared to the respective vehicle control group. The values for the pups (day 4pp and day 13 pp) showed no difference between test item treated animals and controls. After 14 days recovery, no significant changes for the T4 hormone levels have been observed

Urinalysis findings:

not examined

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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL regarding the repeated dose toxicity was set to >= 1000 mg/kg body weight for the male and female animals when administered for at least 41 days.

Executive summary:

A daily oral administration of the test item Reactive Yellow 15 to male and female Wistar rats at dose levels of 1000 mg (500 mg from study day 38 to 41 onwards for the female animals), 300 mg and 100 mg/kg body weight over a time period of 41-43 days for males and 54-66 days for females resulted in some minor animal behavioral changes observed in male and female animals predominately of the high dose groups, alterations in water consumption in male (main) and female (recovery) high dose groups and changes in T4 hormone levels in the high dose group. Findings for T4 levels could not be seen after a 14-day recovery period. The findings were not considered adverse. As the findings could not be associated with a substantial impact on the animal’s health, the NOAEL regarding the repeated dose toxicity was set to 1000 mg/kg body weight for the male and female animals when administered for at least 41 days.
All results show that the physicochemical properties of the substance are responsible for the deaths within the female dose groups. Toxicological properties, which should have been taken into account for the determination of the NOAEL, have not been observed in those animals.
Furthermore, no pathological evidence for toxic effects on the reproduction performance of female and male rats was found. Regarding the overall time period of gestation/lactation and after birth until end of in-life phase no evidence for a toxic effect on the pup development could be detected. The NOAEL regarding reproduction and development of Reactive Yellow 15 under these study conditions was set to be >= 1000 mg/kg body weight for the female and male animals.