Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Endpoint summary

Description of key information

In a combined repeated dose oral gavage toxicity study with the reproduction/developmental toxicity screening with the test item the No-Observed-Adverse-Effect Level (NOAEL) for general toxicity was considered to be 1000 mg/kg bw/day.

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

Remarks:

subacute

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-23 to 2018-01-24

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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number: #13298465
- Expiration date of the lot/batch: Mar 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

FORM AS APPLIED IN THE TEST
Suspension

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available on these Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at supply: males: about 10- 11 weeks, females: about 9 weeks
- Weight at study initiation: (P) Males: ca 356.1 - 386.9 g; Females: ca. 203.2 - 221.6 g
- Housing: During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany;
During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III; For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material
- Diet: ad libitum, Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water: ad libitum
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: water containing 0.5 % sodium carboxymethyl cellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, 0.5 % sodium carboxymethyl cellulose in drinking water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced weekly, at least.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, one sample from the mid concentration was additionally taken for concentration control analysis. The samples collected at the beginning of the administration period and during the lactation period were analyzed.
The concentrations of the test item in the samples were calculated by means of their nitrogen content (determination of nitrogen after Kjeldahl digestion by titration with sulfuric acid). The results demonstrated the correctness of the concentrations of the test item in 0.5 % sodium carboxymethyl cellulose in drinking water. The concentrations of the samples were in compliance with the expected concentrations (recovery of 103-114 %).

Duration of treatment / exposure:

The duration of treatment covered a 2 weeks premating period and mating period in both sexes (mating pairs were from the same dose group), approximately 2 days post-mating in males, the entire gestation period as well as up to 13 days of the lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days and once daily on Saturdays, Sundays and public holidays
- Parameters checked: dead or moribund animals

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
- Parameters checked: signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity, parturition and lactation behavior of the dams

BODY WEIGHT: Yes
- Time schedule for examinations: During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week.

FOOD CONSUMPTION AND COMPOUND INTAKE
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
• Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning, samples were taken from the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning, samples were taken from the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was analysed during clinical observations in all animals prior to the administration period and thereafter at weekly intervals. Besides that, a functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period, during which the urine excreted within 2 minutes was examined for its amount and colour. For both observations, the animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes.
- To determine urine constituents semiquantitatively the dry chemical reactions on test strips (Combur-Test 10 M; Sysmex, Norderstedt, Germany) were evaluated with a reflection photometer (Miditron M; Sysmex, Norderstedt, Germany).

OTHER:
Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for determination of thyroid hormone concentrations.
Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement.
At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
All parental animals were sacrificed by decapitation under isoflura. On PND 13, one selected male and one female pup per litter were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4 % formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing.

HISTOPATHOLOGY: Yes (see table 1)
Fixation was followed by histotechnical processing, examination by light microscopy. Special attention was given on the stages of spermatogenesis in the testes.

Statistics:

see table 4

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All male and female animals of test group 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) showed yellowish discolored feces from study day 4 onwards until the end of the treatment period. This finding was considered treatment-related but not adverse.

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:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the administration period in males of test group 1 (100 mg/kg bw/d) alanine aminotransferase (ALT) activities and potassium levels were significantly decreased, but the changes were not dose-dependent. In males of test group 3 (1000 mg/kg bw/d) ALT was also significantly decreased, but the mean was within the historical control ranges (males, ALT 0.55-0.92 µkat/L). Therefore, the mentioned alterations were regarded as incidental and not treatment-related.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to control group 0 (set to 100 %), the mean absolute weight of the epididymes was significantly decreased in the 1000 mg/kg bw/day dose group. When compared to control group 0 (set to 100 %), the mean relative weight of the adrenal glands was significantly increased in the 1000 mg/kg bw dose group.
The weak significant decrease of the absolute weight of the epididymides (1.091 g) was within the historical control range (1.224 – 1.037 g), and occurred with neither relative weight changes nor histopathological findings. Therefore, it was regarded as incidental and not treatment-related.
The significant weight increase of the adrenal glands (0.019 %) was within the historical control range (0.016 – 0.022 %) and had no histopathological correlate. Therefore, it was considered not treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A yellow discoloration of the contents was found in the glandular stomach of males and females, and in the jejunum, colon and cecum of males only, at 100, 300 and 1000 mg/kg bw/d, with higher incidences in general at the highest dose level. This finding was consistent with the color of the test substance and had no histopathological correlate. Therefore, this change was regarded as treatment-related but not adverse. All other findings occurred individually and were considered incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings (e.g. reduced epididymes size, focal constriction in the liver, reduced size of testes, eosinophi lic infiltrate in jejunem) occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings (e.g. basal cell metaplasia in glandular stomach, cyst formation in ovaries) occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and Guideline study

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

In a combined repeated dose oral gavage toxicity study with the reproduction/developmental toxicity screening (OECD 422) the test item was administered by gavage to groups of 10 male and 10 female Wistar rats (F0 generation) at dose levels of 0 mg/kg bw/d (test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Drinking water containing 0.5 % sodium carboxymethyl cellulose served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 during the entire study period. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of of 1000 mg/kg bw/d. There were neither treatment-related organ weight changes nor histopathological lesions. Macroscopically, a yellow discoloration of the contents was found at 100, 300 and 1000 mg/kg bw/d in the glandular stomach of males and females, and in the jejunum, colon and cecum of males only. This finding was consistent with the color of the test substance and had no histopathological correlate. Therefore, it was considered treatment-related but not adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008 

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776