Benzenesulfonic acid, 4-chloro-2-[2-[4,5-dihydro-3-methyl-5-oxo-1-(3-sulfophenyl)-1H-pyrazol-4-yl]diazenyl]-5-methyl-, ammonium salt (1:2)

Endpoint summary

Description of key information

A seven day study (dose-range finder) and a 28d study in rats (EU B.7, OECD 407, GLP) were performed to evaluate the subacute repeated dose toxicity of the test substance. The test substance did not induce any mortalities, abnormalities or clinical symptoms. Based on the results of these studies, the NOEL and the NOAEL of the test substance is 1000 mg/kg bw per 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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

12 May 1981

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KL384
- Expiration date of the lot/batch: not specified
- Purity test date: not specified


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under storage conditions: not specified
- Stability under test conditions: The chemical stability and homogeneity of test substance formulations were assessed prior to the start of treatment and concentration analyses of formulations prepared for administration on Days 1 and 22 were performed by the testing facility. The results confirm that specimen formulations were homogeneous and stable for a 4-hour period representing the maximum time from preparation to completion of dosing.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: mixed with the vehicle (1% w/v aqueous methylcellulose)
- Final dilution of a dissolved solid, stock liquid or gel: not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material): mixed with the vehicle. Formulations were prepared freshly each day.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent, England
- Age at study initiation: 28 ± 1 d (on arrival)
- Weight at study initiation: males: 181 g (mean); females: 165.5 g (mean)
- Fasting period before study: no
- Housing: 5 rats of the same sex per cage
- Diet: standard pelleted laboratory rodent diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 13 days
- Microbiological status when known: not specified
- Method of randomisation in assigning animals to test and control groups: Four days prior to the start of treatment each animal was weighed and sixty rats were randomly allocated to four groups, two groups consisting of ten males and ten females and two groups consisting of five males and five females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.5 - 25
- Humidity (%): 39 - 71
- Air changes (per hr): approx. 19
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 13 July 1994 (arrival date) To: not specified

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed out and mixed with the vehicle (1% w/v aqueous methylcellulose) using a high shear homogeniser. For each concentration this process was repeated. Formulations were prepared freshly each day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chemical stability and, homogeneity of test substance formulations were assessed prior to the start of treatment and, concentration analyses of formulations prepared for administration on Days 1 and 22 were performed by the testing facility.

The analytical results confirm that the doses were accurately formulated for Day 1 and Day 22 of the toxicity study. The results also confirm that specimen formulations were homogeneous and stable for a 4-hour period representing the maximum time from preparation to completion of dosing.

Duration of treatment / exposure:

28 d

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

150 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 for high dose and control
5 for low and mid dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dosage was selected on the basis of available toxicity data and a preliminary oral toxicity investigation. A dosage of 1000 mg/kg/day is the limit level for this study design.
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: Following the 4 week treatment period, five male and five female animals from Groups 1 and 4 (lowest animal numbers were selected) and surviving animals of Groups 2 and 3 were killed on Day 32 (post-treatment sacrifice). These rats were dosed until the day prior to sacrifice. The remaining animals of Groups 1 and 4 were retained for a 2 week post-treatment observation period and were killed on Day 46 (recovery sacrifice); these rats received their last dose on Day 28.
- Section schedule rationale (if not random): not specified

Positive control:

none

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 3 x daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- The quantity of food consumed in each cage was measured at weekly intervals throughout the study

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period (day 30); for remaining rats after the 2 week recovery period (Day 44)
- Anaesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes (overnight)
- How many animals: five male and five female rats selected from vehicle control and high dose group and all rats of low and mid dose groups
- Parameters examined: Packed cell volume (PCV), Haemoglobin (Hb), Red blood cell count (RBC), Mean corpuscular haemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Platelet count (Plts), Total white blood cell count (WBC), Thrombotest (TT), Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period (day 30); for remaining rats after the 2 week recovery period (Day 44)
- Animals fasted: Yes (overnight)
- How many animals: five male and five female rats selected from vehicle control and high dose group and all rats of low and mid dose groups
- Parameters examined: Glucose, Total protein, Albumin (Alb), Globulin (Glob), Albumin/Globulin ratio (A/G), Urea nitrogen (Urea Nitr), Creatinine, Alkaline phosphatase (AP), Glutamic-pyruvic transaminase (GPT), Glutamic-oxaloacetic transaminase (GOT), Gamma-glutamyltransferase (γ-GT), Total bilirubin (Bilirubin), Sodium (Na), Potassium (K), Calcium (Ca), Chloride (Cl), Inorganic phosphorus (P), Cholesterol (Chol), Triglycerides (Tri-glyc)

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of treatment period (day 30); for remaining rats after the 2 week recovery period (Day 44)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- Parameters examined: Volume (Vol), pH, protein (not determined for recovery male animals), Total reducing substances (TRS), Glucose, Ketones, Bile pigments, Urobilinogen, Haem pigments

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
adrenals*, aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), femur (for bone and marrow sections) with joint Harderian gland, heart*, head (to preserve nasal cavity, paranasal, inuses, oral cavity, nasopharynx, middle ear, teeth, lachrymal gland and Zymbal's gland), ileum, jejunum, kidneys, larynx, liver*, lungs, lymph nodes (cervical and mesenteric*), mammary gland, oesophagus, ovaries, pancreas, pharynx, pimitary, prostate, rectum
salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical level), spleen*, stomach, sternum, testes including epididymides* (Bouins as fixative), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus (with cervix), vagina, any macroscopically abnormal tissue*

* Tissues required for histopathological examination for rats from vehicle group and high dose group

HISTOPATHOLOGY: Yes

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. Different sequence of statistical tests was used for bodyweight gains, food consumption (using weekly cage totals), organ weight and clinical pathology data.

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:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

The only clinical observation during the treatment period of the study was yellow-coloured faeces on the cage tray paper under the cages of rats receiving 1000 mg/kg/day. This incidental finding was considered to be due to the presence of the test substance in the faeces and not a manifestation of toxicity.

After the recovery period, kidney weight (bodyweight adjusted) was statistically significantly lower than the control for male rats treated at 1000 mg/kg/day and higher than control adrenal weights were recorded for female rats treated at 1000 mg/kg/day. In the absence of any treatment-related effects on these organs at Week 5 these small differences from control were considered to reflect variation.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No effects observed up to and including the highest tested dose.

Critical effects observed:

no

Conclusions:

It was concluded that 1000 mg/kg/day represents the no-observed effect level (NOEL) for the test substance in the rat.

Executive summary:

The substance was administered by oral gavage, once daily, to three groups of rats for a minimiun of twenty eight consecutive days, at dosage levels of 15, 150 or 1000 mg/kg/day. The test material was prepared as suspensions in 1% w/v aqueous methylcellulose (1% MC) at concentrations of 0.15, 1.5 or 10% w/v and was administered at a dosage volume of 10 ml/kg/day. Control animals received the vehicle (1% MC) alone at the same dose volume (10 ml/kg/day).

All rats of Groups 2 and 3 (15 and 150 mg/kg/day respectively) and five males and five females from each of Groups 1 and 4 (Control and 1000 mg/kg/day respectively) were killed following the fourweek treatment period (Day 32). The remaining animals (five males and five females from Groups 1 and 4) were retained for a two-week recovery period, following which, they were also killed (Day 46).

Bodyweights, food consumption and clinical observations were recorded during the study. Blood and urine samples were taken from all rats shortly prior to termination following the four-week treatment and two-week recovery periods. All animals were killed and subsequently examined macroscopically; specified tissues were then prepared for histopathological examination.

There were no changes seen in the parameters measured in this study namely clinical signs, bodyweights, food consumption, clinical pathology, organ weight analysis, macroscopic or microscopic pathology.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

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 pre-test and a main study were performed to evaluate the subacute repeated dose toxicity of the test substance in rats. In the pre-study, the test substance was administered orally to a group of three male and three female rats at 1000 mg/kg/day (at a dosage volume of 10 ml/kg/day), formulated as a 10% w/v suspension in 1 % w/v aqueous methylcellulose (1 % MC). A further group of three male and three female rats received the vehicle (1 % MC) alone as a control. The study started on 14 June 1994 and following seven consecutive days of treatment all animals were killed on Day 8 (21 June 1994). Clinical signs, bodyweight, food consumption, organ weight and macroscopic examinations were recorded for the study.

There were no mortalities. No treatment-related clinical signs were noted for any of the animals throughout the study. The only clinical observation during the study was yellow-coloured faeces on the tray paper for all rats receiving 1000 mg/kg/day. No treatment related effects on organ weight, water consumption or food consumption were observed. There were no macroscopic abnormalities observed in any rats.

In the main study, the substance was administered by oral gavage, once daily, to three groups of rats for a minimum for twenty eight consecutive days, at dosage levels of 15, 150 or 1000 mg/kg/day. The test material was prepared as suspensions in 1% w/v aqueous methylcellulose (1% MC) at concentrations of 0.15, 1.5 or 10% w/v and was administered at a dosage volume of 10 ml/kg/day. Control animals received the vehicle (1% MC) alone at the same dose volume (10 ml/kg/day).

All rats of Groups 2 and 3 (15 and 150 mg/kg/day respectively) and five males and five females from each of Groups 1 and 4 (Control and 1000 mg/kg/day respectively) were killed following the fourweek treatment period (Day 32). The remaining animals (five males and five females from Groups 1 and 4) were retained for a two-week recovery period, following which, they were also killed (Day 46).

Bodyweights, food consumption and clinical observations were recorded during the study. Blood and urine samples were taken from all rats shortly prior to termination following the four-week treatment and two-week recovery periods. All animals were killed and subsequently examined macroscopically; specified tissues were then prepared for histopathological examination.

There were no changes seen in the parameters measured in this study namely clinical signs, bodyweights, food consumption, clinical pathology, organ weight analysis, macroscopic or microscopic pathology, that were considered to be related to treatment with the test substance.

The test substance did not induce any mortalities, abnormalities or clinical symptoms. The only remarkable finding in the pre-study was a yellow discoloration of faeces. This incidental finding was considered to be due to the presence of the test substance in the faeces and not a manifestation of toxicity. Based on the results of these studies, the NOEL and the NOAEL of the test substance is 1000 mg/kg bw per day.

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 1272/2008. The NOEL and the NOAEL of the test substance was 1000 mg/kg bw per day in an oral subacute repeated dose study. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.