3-Pyridinemethanesulfonic acid, 5-[2-[5-[[4-chloro-6-[(4-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-1-ethyl-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-, potassium sodium salt (1:?:?)

Endpoint summary

Description of key information

Based on the results obtained in the 28-day repeated toxicity study by gavage, the NOAEL was determined to be 1000 mg/kg bw  for male and female.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf, Switzerland
- Age at pretest: 7-8 weeks
- Weight at pretest: males: 174-194 g, females: 164 - 187 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding ('Lignocel', Schill AG, 4132 Muttenz, Switzerland).
- Diet (e.g. ad libitum): Pelleted standard Kliba 343, Batch 72/87 and 73/87 rat maintenance diet ('Kliba', Klingentalmuehle AG, 4303 Kaiseraugst, Switzerland) available ad libitum
- Water (e.g. ad libitum): Community water from Itingen, available ad libitum
- Acclimation period: Seven days under laboratory conditions, after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

4%

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 10mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test article/vehicle mixtures were performed prior to the first application (pretest) and at week 3 of treatment. Analysis were performed in the RCC Analytical Laboratory, according to a method supplied by the sponsor.

Duration of treatment / exposure:

up to 28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
50, 200, 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based upon data received from acute studies and a five-day range finding study (RCC Project 083204).

Observations and examinations performed and frequency:

- Viability/mortality: Observations for mortality were recorded daily.
- Clinical signs: Signs of toxicity were assessed daily. Descriptions of all abnormalities were recorded and the subsequent progress was monitored.
- Food consumption: The food consumption was recorded once during the acclimatization period and weekly thereafter.
- Body weight: The body weight of each animal was recorded weekly during the acclimatization and treatment period.
- Ophthalmoscopic examinations: Ophthalmoscopic examinations were performed on all animals. A description of any abnormality was recorded. Examinations were performed at termination of treatment. Ten minutes after the application of a mydriatic solution (Dispersa AG, Winterthur / Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet. AG, Allschwil / Switzerland).
- Clinical laboratory investigations: Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for 18 hours before blood sampling but water was provided. Blood samples were collected from each animal between the hours of 7.00 and 9.30 a.m. to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retroorbital plexus. Urine was collected over an 18-hour period into a specimen vial using a metabolism cage, during which time the animals were deprived of food but allowed access to water ad libitum. Parameters being measured for hematology: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes normoblasts, total leukocyte count, differential leukocyte count, red cell morphology, thromboplastin time, partial thromboplastin time. Parameters being measured for clinical biochemistry: glucose, urea, creatinine, bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total. Parameters being measured for urinalysis: volume (18 hour), specific gravity, pH, protein, glucose, ketone, bilirubin, blood, nitrite, urobilinogen, urine sediment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were necropsied and descriptions of all macroscopic abnormalities were recorded. The following organ weights were recorded on the scheduled date of necropsy: adrenals, heart, kidneys, liver, testes.

HISTOPATHOLOGY: Yes
- Slides of adrenals, heart, kidneys, liver, spleen, stomach and gross lesions (all animals) were collected at terminal sacrifice and from the animals of the control and high-dose groups examined by a pathologist. Upon detection of treatment-related morphologic changes in the organs of any high-dose animal, histologic evaluation of the same organs in all dose groups were performed.

Statistics:

Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups. The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.Group means were calculated for continuous data and medians were calculated for discrete data (scores).

Details on results:

- Mortality: No death occurred.
- Clinical signs: Starting with day 3 of the treatment period, all animals of the 200 and 1000 mg/kg bw groups showed yellowish discolored feces. This discoloration was observed in all animals of these groups until termination of treatment. No other signs or symptoms were observed in the animals of the control and test article-treated groups.
- Food consumption: No significant differences in food consumption were observed between the animals of the control and test article-treated groups throughout the treatment period.
- Body weights: No significant differences in body weight gain were observed between the animals of the control and test article-treated groups throughout the treatment period.
- Ophthamlnoscopic examinations: No differences between the control and test article-treated animals were observed.
- Clinical laboratory investigations: The assessment of hematological, biochemical and urinalysis data indicated no changes of toxicological significance at the end of the treatment. All differences in the results of the hematology, clinical biochemistry and urinalysis parameters were considered to be incidental and of normal biological variation
- Organ weights: No significant differences which were related to test article treatment were observed in absolute and relative organ weights between the animals of the test article-treated and control groups. All differences observed were considered to be incidental and of normal biological variation.
- Macroscopic findings: Treatment related macroscopic findings took the form of yellowish discoloration of the stomach mucosa in three rats at the intermediate dose and in eight rats of the high dose group. In one case (No. 17, male group 4), this was accompanied by a red focus. Other gross findings were unremarkable.
- Microscopic findings: There were no microscopic correlates of the grossly observed discoloration of the stomach mucosa, nor could dyestuff particles be identified in the lumen of the organ. In the one case identified above, the gross finding in the stomach was correlated with a focus of minimal erosive gastritis. This finding is considered to be the result of an accidental gavage injury. Other microscopic findings recorded were spontaneous in nature and are within the normal range observed in this age and strain of rat. They may be attributed to subclinical illness, spontaneous congenital abnormalities or physiological status.

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: Effect level based on clinical symptomps, body weight, food consumption, ophthalmoscopic examination, clinical laboratory investigations, and pathology

Critical effects observed:

not specified

Conclusions:

NOAEL was determined to be 1000 mg/kg/bw.

Executive summary:

In a GLP compliant repeated toxicity study, performed according to OECD guideline 407, Wistar rats were treated with the test substance (0, 50, 200, and 1000 mg/kg bw) by repeated oral gavage, for a period of 28 days. The study was comprised of four groups, each containing five male and five female rats. No treatment related effects were observed on mortality, clinical signs, food consumption, body weight, ophthalmoscopic examinations, clinical laboratory investigations, organ weights, macroscopic and microscopic findings. The only test substance related finding was yellowish discoloration of the feces and gastric mucosa at the high and intermediate doses. Therefore, the NOAEL was determined to be 1000 mg/kg/bw.

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 compliant guideline study, klimisch 1.

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

A 5-day range finding study was performed to select the dosages for a 28-day oral toxicity study (RCC 1987). It was recommended to expose the animals to 0, 50, 200, or 1000 mg/kg bw.

In the following GLP compliant repeated toxicity study, performed according to OECD guideline 407, Wistar rats were treated with the test substance (0, 50, 200, and 1000 mg/kg bw) by repeated oral gavage, for a period of 28 days (RCC 1988). The study was comprised of four groups, each containing five male and five female rats. No treatment related effects were observed on mortality, clinical signs, food consumption, body weight, ophthalmoscopic examinations, clinical laboratory investigations, organ weights, macroscopic and microscopic findings. The only test substance related finding was yellowish discoloration of the feces and gastric mucosa at the high and intermediate doses. Therefore, the NOAEL was determined to be 1000 mg/kg/bw.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only 28-day subacute study available.

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study, the test substance does meet the criteria of the Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and therefore does not need to be classified.