6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.

Endpoint summary

Description of key information

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

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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment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

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River ltalia S.p.A. Calco, Como, Italy.
- Age at study initiation: 27-29g
- Weight at study initiation: 10g
- Housing: Animals were housed five of one sex to in clear polycarbonate cages measuring 59x38.5x20cm with a stainless steel mesh lid and floor (Type 4 Code 1354G Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent paper which was inspected and changed at least three times a week.
- Diet (e.g. ad libitum): via water bottles, ad libitum
- Water (e.g. ad libitum): commercially available laboratory rodent diet (Altromin MT pelleted diet, A. Rieper, Bolzano, Italy), ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±10
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on oral exposure:

1000 mg/kg body weight (10 mL/kg bw)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Remarks:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the sponsor
- Post-exposure recovery period in satellite groups: The control and high dose group also included 5 additional animals per sex sacrified after a 2 week recovery period.

Observations and examinations performed and frequency:

Clinical signs: All clinical signs were recorded for individual animals. Examination of individual animals for signs of reaction to treatment was carried out daily approx. 24 hours after dosing. Once per treatment week each animal was subjected to an additional physical examination and aany abnormality or sign of ill health was recorded.
Mortality: All animals were checked early in each working day and again in the afternoon to look for dead or moribund animal. At weekends and public holidays the second check was carried out at approx. mid-day.
Body weight: Animals were weighed at the day of allocation, on the day treatment commenced, weekly thereafter and just prior to necropsy.
Food consumption: The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation and the mean daily intake per rat was calculated.
Ophthalmoscopy: Both eyes of all animals initially assigned to the study were examined just prior to the commencement of treatment by means of an opthalmoscope and by a slit-lamp biomicroscope after the instillation of 0.5% tropicamide (Visumidriatic, Merck, Sharpe, and Dohme, Rome, Italy). The eyes of all surviving animals in all groups were re-examined during week 4 of treatment and week 2 of the recovery period.
Clinical pathology investigations: once during week 4 of treatment and once during week 2 of the recovery period samples of blood were withdrawn under light ether anaesthesia from the retro-orbital sinus of all males and females from each group under conditions of food and water deprivation. At the same time interval, individual urine samples were collected from the same animals under the same conditions.
Haematology: Parameters being measured: Hematocrit, Hemoglobin, Red blood cell count, Mean corpuscular hemoglobin, Mean red blood cell volume, Mean corpuscular hemoglobin concentration, white blood cell count, platelets, Differential leucocyte count (neutrophils, lymphocytes, eosinephils, basophils, monocytes), abnormalities of the blood film.
Clinical chemistry: Parameters being measured: alkaline phosphatase, alanine transferase, aspartate transferase, gamma glutamyltransferase, urea, creatinine, glucose, triglycerides, total billirubin, total cholesterol, total protein, sodium, potassium, calcium, chloride, albumin, globulin, phosphorus.
Urinalysis: Parameters being measured: appearance, volume, specific gravity, pH, protein, glucose, ketones, blood, sediment was examined for epithelial cells, polymorphonuclear leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components.

Sacrifice and pathology:

Gross pathology: The following organs were dissected free of fat and weighed: adrenal glands, brain, heart, kidneys, liver, ovaries, pituitary gland, spleen, testes, thyroid and parathyroid glands, uterus. The ratios of organ weight to body weight and organ weight to brain weight were calculated.
Histopathology: the following tissues were preserved in 10% buffered formol-saline and processed: abnormalities, adrenal glands, heart, kidneys, liver, spleen and testes.

Statistics:

For continuous variables the significance of the differences amongst group means were assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's Test using a pooled error variance. The homogeneity of the data was verified by Barlett's Test before Dunnett's test. If data were found to be inhomogenous a modified T-test (Cochran and Cox) was applied.

Details on results:

Mortality: No deaths occurred.
Clinical signs: There were no clinical signs of toxicological importance. Blue feces, observed in the litter tray of treated animals and recorded as cage
data, were considered to be due to the dark blue color of the test substance. For this reason, blue coloration of the skin/fur seen in treated animals, was not recorded as a clinical sign.
Body weight: There were no treatment related differences in body weights. Treated males of the 1000 mg/kg bw group showed a slight reduction in body weight towards the end of treatment when compared with controls. This was not apparent in females.
Food consumption: Not affected by treatment.
Ophthalmoscopy: No significant lesions were observed.
Hematology: Values in treated animals remained generally comparable to controls. The statistically significant variations observed were either confined to the low dose and intermediate groups and/or fell within the normal range of archive data.
Clinical chemistry: The statistically significant differences between control and treated groups were often inconsistent between groups and sexes. Both males and females in the high dose group (1000 mg/kg/day), showed statistically significant reductions in total protein and increases in chloride and inorganic phosphorus. However, those values fell within the normal range of archive data. The changes were not apparent at the end of the recovery period. The plasma of most treated animals showed a pale green to blue coloration, the intensity of which was related to the dose level of the test substance administered. Some coloration was also apparent in the plasma of three of the high dose recovery animals, but to a much lesser degree.
Urinalysis: The values for urinalysis were generally comparable to those of controls. The urine of all animals in the high dose group (1000 mg/kg/day) showed a blue/green coloration which, after approximately two weeks of recovery became light green.
Organ weights: Thyroid weights were statistically significantly lower than study control values in all treated groups and recovery females of the high dose group (1000 mg/kg/day). All these values were within the range of historical control data and no dose response relationship was evident, Furthermore, no evidence of histological change was seen in this organ. This appears to indicate that this change may be spontaneous in nature and not related to treatment.
Macroscopic observations: Blue staining was observed in several organs and tissues, particularly in animals of the high dose group (1000 mg/kg/day). This coloration, considered to be due to the blue color of the test substance, was still evident in recovery animals of this group.
Microscopic observations: Blue pigmentation, considered to be due to the test substance and/or it's metabolites, was observed in the cortical tubular cells of the kidneys and the interstitium of the testes of both test (receiving 1000 mg/kg/day) and recovery animals. Renal tubular cell vacuolation was noted in the female showing the highest degree of severity of blue pigmentation. The toxicological significance of this finding is not known in detail but is considered to be a sign of substance absorption, distribution and excretion. The remaining changes observed were considered to be an expression of spontaneous pathology.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs; mortality; body weight; food consumption; ophthalmoscopic examination; haematology; clinical chemistry; urinalysis; organ weights; macroscopic and microscopic observations

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, CD rats of Sprague Dawley origin were treated with the test substance (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. For the post-exposure recovery period two satellite groups exposed to 0 and 1000 mg/kg bw for 28 days consisting of five male and five female rats each were monitored for an additional 14 days after the end of the 28 days treatment period. No treatment related effects were observed on mortality, clinical signs, body weight, food consumption, ophthalmic examinations, clinical pathology investigations, and organ weight. An abnormal blue color was observed at the macroscopic examination in several organs and tissues mainly of animals receiving 1000 mg/kg/day. Blue pigmentation was observed microscopically in the kidneys and testes of treated animals receiving 1000 mg/kg/day, sacrificed after 4 weeks of treatment. This finding was still evident after two week of recovery. The toxicological significance of this finding is not known in detail, but is considered to be a sign of test substance absorption, distribution and excretion. This data indicates that the test substance is well tolerated at all dose levels, suggesting a NOAEL of 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

In a GLP compliant repeated toxicity study, performed according to OECD guideline 407, CD rats of Sprague Dawley origin were treated with the test substance (50, 200, and 1000 mg/kg bw) by repeated oral gavage, for a period of 28 days (RTC 1995). The study was comprised of four groups, each containing five male and five female rats. For the post-exposure recovery period two satellite groups exposed to 0 and 1000 mg/kg bw for 28 days consisting of five male and five female rats each were monitored for an additional 14 days after the end of the 28 days treatment period. No treatment related effects were observed on mortality, clinical signs, body weight, food consumption, ophthalmic examinations, clinical pathology investigations, and organ weight. An abnormal blue color was observed at the macroscopic examination in several organs and tissues mainly of animals receiving 1000 mg/kg/day. Blue pigmentation was observed microscopically in the kidneys and testes of treated animals receiving 1000 mg/kg/day, sacrificed after 4 weeks of treatment. This finding was still evident after two week of recovery. The toxicological significance of this finding is not known in detail, but is considered to be a sign of test substance absorption, distribution and excretion. This data indicates that the test substance is well tolerated at all dose levels, suggesting a NOAEL of 1000 mg/kg bw.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only 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.