Disodium 6-(4,6-dichloro-1,3,5-triazin-2-ylamino)-1-hydroxy-2-(4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-3-sulfonate

Endpoint summary

Description of key information

The NOEL and NOAEL of the test substance were determined to be 1000 mg/kg bw/d or above .

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

Study period:

From Oct. 01, 1997 to Mar. 03, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat has proved to be a suitable species for subacute oral toxicity testing with many different substances,

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HMR Deutschland Gmbh, Kastengrund, SPF breeding colony
- Age at study initiation: Approximately 5-6 wk
- Housing: Macrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet: ssniff R/M (V1534), ad libitum, except for the period in which the animals were kept in diuresis cages
- Water: Tap water in plastic bottles, ad libitum, except for the period in which the animals were kept In diuresis cages
- Acclimation period: At least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C
- Humidity (%): 50±20 °C
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

IN-LIFE DATES: From Oct. 01, 1997 to Oct. 29, 1997

Route of administration:

oral: gavage

Details on route of administration:

The oral route is considered to be a potential exposure route man

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance was suspended in the concentrations of 1.25, 5.0 and 20 % in deionized water. After each measurement of the body weight, the calculation of the application volume was repeated. The final dosing volume was 5 mL/kg bw

VEHICLE
- Concentration in vehicle: 1.25, 5.0 and 20 % w/v
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Remarks:

First and last week of treatment phase

Details on analytical verification of doses or concentrations:

HPLC

Duration of treatment / exposure:

29 d, 28 applications

Frequency of treatment:

Once a day for 28 d

Dose / conc.:

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

Dose / conc.:

250 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:

5/sex/dose in main groups
5/sex/dose in recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In an acute oral toxicity study, the LD50 in Wistar rats was >2000 mg/kg bw/d. Animals showed no signs of toxicity. Development of body weight was not impaired. The animals killed at the end of the observation period showed no macroscopically visible changes.
Based on these results test substance was tested in the present study at the dose levels of 0, 62.5, 250 and 1000 mg/kg bw/d.
- Rationale for animal assignment (if not random): Randomization using computer-generated algorithm
- Rationale for selecting satellite groups: To check the reversibility of the effects, if any
- Post-exposure recovery period in satellite groups: Yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily in all groups

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before start of the study and once a week during the study

BODY WEIGHT: Yes
- Time schedule for examinations: Before the start of the study and twice weekly throughout the study

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, 2 times/wk

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes (intraperitoneal injection of 67 + 6.7 mg/kg bw Ketamine HCl + Xylazine)
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes, killed by section of the vena cava cranialis in deep narcosis (intraperitoneal injection of 67 + 6.7 mg/kg bw Ketamine HCl + Xylazine) and exsanguinated

URINALYSIS: Yes
- Time schedule for collection of urine: Few days before termination of the study as well as before the end of the recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before start of the study and once a week during the study
- Dose groups that were examined: All groups
- Battery of functions tested: sensory reactivity, measurement of motor activity, rearings, forelimb and hindlimb grip strength and landing foot-spread

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals were necropsied and checked for macroscopically visible abnormalities. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.

HISTOPATHOLOGY: Yes, Following tissues/organs were preserved in a suitable fixative and processed for histopathological investigations: adrenal gland, bone marrow , brain, heart, colon, jejunum, kidneys, liver, lungs, lymph nodes (mandibular and iliac), ovaries, uterus, thyroid and parathyroid glands, prostate gland, spleen, stomach, testis, epididymides, thymus, trachea, urinary bladder, N. ischiadicus and spinal cord (cervical).

Other examinations:

Following organs were weighed and the organ to body weight ratios calculated:
Heart , liver, kidneys, adrenal glands, spleen, testes, epididymides, thymus and brain

Statistics:

Evaluation was performed by IS Research and Preclinical Devolopment; HMR Deutschland GmbH, with the aid of a program package for the evaluation of toxicological studies.

Clinical signs:

no effects observed

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No deaths occurred throughout the study. Male and female animals of the high dose group showed orange coloured faeces from Day 7 till the end of the study.

BEHAVIOUR AND STATE OF HEALTH: Remained unaffected by the administration of the test substance

BODY WEIGHT AND WEIGHT GAIN: Remained unaffected by the administration of the test substance in all dose groups.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption remained unaffected by the administration of the test substance throughout the study in all dose groups.

WATER CONSUMPTION AND COMPOUND INTAKE: Water consumption remained unaffected by the administration of the test substance

HAEMATOLOGY: No treatment-related changes observed in any of the dose groups

CLINICAL CHEMISTRY: Increases in urea, cholesterol, triglyceride, and albumin values as well as decreases in inorganic phosphor and serum glucose levels in males of the high dose group. In addition, male animals of the intermediate and high dose groups showed reduced ALAT levels. In female animals of the high dose group decreased uric acid values and in females of the intermediate and high dose groups decreased creatinine values were found. All these changes were within the physiological range of rats and these are not considered as treatment related.

URINALYSIS: No treatment-related changes were detected by urine analysis.

NEUROBEHAVIOUR: Neurotoxicological measurements including 'open field' observations, assessment, of sensory function, and forelimb and hindlimb grip strength were not influenced by the administration of the test substance in all groups.

ORGAN WEIGHTS: No treatment-related changes observed in any of the dose groups

GROSS PATHOLOGY: No treatment-related macroscopically visible changes were observed in all dose groups

HISTOPATHOLOGY: No treatment-related microscopic changes in the organs of the examined animals

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

Dose descriptor:

NOEL

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

Key result

Critical effects observed:

no

Conclusions:

Under the test conditions, both the NOEL and NOAEL of the test substance were determined to be 1000 mg/kg bw/d or above .

Executive summary:

A study was conducted to assess the sub acute repeated dose toxicity of the test substance in rats according EU Method B.7. and OECD guideline 407 in compliance with GLP

Groups of male and female rats received test substance by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day for a period of 28 d. On Day 29, 5 males and 5 females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 d.

Behavior and state of health were observed daily in all groups, Body weights and food consumption were recorded twice weekly, water consumption once weekly. Once before the first treatment and once a week thereafter detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa and impairment of dental growth.

Neurotoxicological measurements including assessment of sensory function, motor activity, rearings, forelimb and hind limb grip strength, landing foot-spread were conducted at the end of the treatment period. Hematological examinations, clinical chemistry and urine analysis were carried out at the end of the treatment period and after the recovery period.

During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, hematological and clinical chemistry data, urine data (pH, volume, specific weight), absolute and relative organ weights and neurotoxicological measurements (motor activity, forelimb and hind limb grip strength) were analyzed with the aid of a statistical program.

No deaths occurred throughout the study. Male and female animals of the high dose group showed orange coloured faeces from Day 7 till the end of the study. Behavior, state of health, neurotoxicological, body weight gains, food and water consumption remained unaffected by the administration of the test substance in all dose groups.

Hematological and clinical chemistry investigations and organ weights did not exhibit treatment related adverse effects. At necropsy of the final and/or recovery, no compound-related macroscopically visible changes were observed in all dose groups. Likewise, histopathological examinations revealed no treatment related changes in any dose group.

In conclusion, no treatment-related adverse effects were observed in any of the dose group animals.

Under the test conditions, both the NOEL and NOAEL of the test substance were determined to be 1000 mg/kg bw/d or above .

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

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

A study was conducted to assess the subacute repeated dose toxicity of the test substance in rats according OECD guideline 407 and EU Method B.7.

Groups of male and female rats received test substance by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day for a period of 28 d. On Day 29, 5 males and 5 females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 d.

Behavior and state of health were observed daily in all groups, Body weights and food consumption were recorded twice weekly, water consumption once weekly. Once before the first treatment and once a week thereafter detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa and impairment of dental growth.

Neurotoxicological measurements including assessment of sensory function, motor activity, rearings, forelimb and hind limb grip strength, landing foot-spread were conducted at the end of the treatment period. Hematological examinations, clinical chemistry and urine analysis were carried out at the end of the treatment period and after the recovery period.

During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, hematological and clinical chemistry data, urine data (pH, volume, and specific weight), absolute and relative organ weights and neurotoxicological measurements (motor activity, forelimb and hind limb grip strength) were analyzed with the aid of a statistical program.

 

No deaths occurred throughout the study. Male and female animals of the high dose group showed orange coloured faeces from Day 7 till the end of the study. Behavior, state of health, neurotoxicological, body weight gains, food and water consumption remained unaffected by the administration of the test substance in all dose groups.

Hematological and clinical chemistry investigations and organ weights did not exhibit treatment related adverse effects. At necropsy of the final and/or recovery, no compound-related macroscopically visible changes were observed in all dose groups. Likewise, histopathological examinations revealed no treatment related changes in any dose group.

In conclusion, repeated administration of test substance caused no adverse effects in any of the dose group animals. The NOAEL and NOEL of the test substance were determined to be 1000 mg/kg bw/day or above.

 

Justification for classification or non-classification

The NOAEL and NOEL of the test substance was determined to be 1000 mg/kg bw/day in the 28-d oral study in rats and therefore does not meet the requirement for classification according to according to EC criteria (67/548/EEC) and according to CLP criteria (EC 1272/2008).