main component 1 (isomer 1): 2-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonapht-7-ylamino]-1,3,5-triazin-2-ylamino}-3-{6-fluoro-4-[3-(1,5-disulfonaphth-2-ylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt;main component 1 (isomer 2): 2-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-3-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt;main component 2: 2,3-bis-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt;main component 3: 2,3-bis-{6-fluoro-4-[3-(1,5-disulfonaphth-2-ylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt

Endpoint summary

Description of key information

Based on the results obtained in the 28-day repeated toxicity study by gavage, the no observed adverse effect level (NOAEL) was determined to be 200 mg/kg bw for male and female rats.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

08 November 1996 to 14 February 1997

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identity: FAT 40557/A
Batch-no.: PV1
Purity: 65 %
Appearance: Solid powder, red-brown
Solubility (g/l): >100
Stability of Test Article in Vehicle: Stable in bi-distilled water for 48 hours.
Stability of Test Article: Stable under storage conditions; expiration date: 30-SEP-2002
Expiration date: 30 September, 2002
Storage conditions: At room temperature at about 20 °C, away from direct sunlight.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat, HanIbm:WIST (SPF)
Rationale: Recognized by the international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wölferstrasse 4, CH-4414 Füllinsdorf
- Age at delivery: 6 weeks
- Weight at acclimatization/pretest: Males: 127 - 172 g (mean 152 g), Females: 110 - 146 g (mean 134 g)
- Housing: In groups of five in Makrolon type-4 cages with standard softwood bedding ('Lignocel' Schill AG, 4132 Muttenz / Switzerland).
- Diet: Pelleted standard Kliba no. 343 rat maintenance diet (KLIBA Mühlen AG, 4303 Kaiseraugst/Switzerland) ad libitum (Batches nos. 78/96, 81/96).
- Water: Tap water was available ad libitum in water bottles.
- Acclimation period: Seven days.

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

In life phase: 08-N0V-1996 to 20-DEC-1996

Route of administration:

oral: gavage

Details on route of administration:

Rationale: Accidental oral ingestion is a possible route of human exposure.

Vehicle:

water

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 10 mL/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 determined in samples taken during acclimatization and during week 3 of the treatment.

Duration of treatment / exposure:

up to 28 days

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

Control

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Middle dose

Dose / conc.:

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

Remarks:

High dose

No. of animals per sex per dose:

The number of rats assigned to toxicity and recovery testing per group were:
- 5 per sex/dose for the toxicity test
- 5 per sex for 0 and 1000 mg/kg bw for the recovering test

Control animals:

yes, concurrent vehicle

Details on study design:

FAT 40557/A was administered daily to SPF-bred Wistar rats 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. The test article/vehicle mixture was administered on a mg/kg bw basis by oral gavage. The dose was based upon data received from acute studies and a 5-day oral (range-finding) gavage study. The animals of the control group were treated similarly with the vehicle alone. 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.

Observations and examinations performed and frequency:

- Mortality/viability and clinical signs: Observations for mortality/viability were recorded twice daily and clinical signs of toxicity were recorded at least once daily.
- Food consumption: The food consumption was recorded once during the pretest period and weekly thereafter.
- Body weights: Body weights were recorded weekly during pretest, treatment weeks 1 to 3 and recovery week 1, twice weekly in weeks 4 and 6.
- Ophthalmoscopic examination: The ophthalmoscopic examinations of both eyes of all animals were performed after the application of a mydriatic solution (Ciba Vision AG, CH-3172 Niederwangen) using a Heine BETA 200 Ophthalmoscope (Eisenhut Vet. AG, CH-4123 Allschwil).
- Haematology/clinical chemistry: Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia at termination of treatment. The animals were fasted in metabolic cages for approximately 18 hours before blood sampling but water was provided. Blood samples were collected from each animal between the hours of 6.35 and 7.35 a.m. to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube. Parameters being measured: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, reticulocyte fluorescence ratios, nucleated erythrocytes, heinz bodies, methemoglobin, total leukocyte count, differential leukocyte count, red cell morphology, thromboplastin time, activated partial thromboplastin time, glucose, urea, creatinine, uric acid, bilirubin, cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl-transferase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total, globulin, albumin/globulin ratio.
- Urinalysis: Urine was collected during the 18-hour fasting period into a specimen vial. Parameters being measured: volume (18 hour), colour, appearance, specific gravity, osmolality, pH, protein, glucose, ketone, bilirubin, blood, 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 taken from all animals necropsied: Adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroid including parathyroid gland.

HISTOPATHOLOGY: Yes
- Slides of adrenals, heart, kidneys, liver, lungs, spleen, stomach and testes collected at terminal sacrifice from the animals of group 1 (control) and group 4 (high-dose) as well as kidneys, stomach and all gross lesions from rats of all groups were examined by a pathologist.

Statistics:

The following statistical methods were used to analyse the body weights, organ weights all ratios as well as clinical laboratory data:
- When 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 for each sex.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The Fisher's exact test was applied to the ophthalmoscopy data and macroscopical findings.

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

Description (incidence and severity):

Food consumption values were similar for treated groups and controls, except females at 1000 mg/kg showed slightly higher absolute food consumption throughout the whole treatment period. In view of the slightly higher food consumption already recorded during pre-test for this group, the difference is considered to be incidental.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Ophthalmologic findings were noted in a small proportion of animals from all treated groups. They included haemorrhage in vitreous body, pupillary membranes and corneal opacity. These findings occurred at similar incidences in treated groups at pre-test, the end of the treatment period and the recovery period. Therefore, they are considered to be unrelated to treatment with test article.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

High Dose:
Treatment period -
slightly lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) incidences in males (p <0.05), slightly increased platelet count in males (p <0.01), slightly increased reticulocyte count (rel.& abs.) in males (p <0.05 - p <0.01), slightly increased total leukocyte count as well as a slight increase in lymphocytes of the differential leukocyte count (abs.) in females (p <0.05). In addition, slightly shorter thromboplastin times (PT) were recorded in both sexes (p <0.01).

Recovery period -
no remarkable findings were noted. All findings recorded during the treatment period were, with the exception of a slightly shorter thromboplastin time (PT) in females (p <0.05), no longer statistically significant and generally similar to those of the controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

High dose:
Treatment period -
slightly decreased glucose level in both males (p<0.05) and females (statistically non-significant), slightly increased uric acid level in males (p<0.01), markedly increased total bilirubin level in males (statistically non-significant) and females (p<0.01), slightly increased total cholesterol,
triglyceride and phospholipid level in both sexes (p<0.01), slightly decreased potassium level in females (p<0.05), slightly increased total protein and globulin level (p<0.01 - p<0.05), and decreased albumin to globulin ratio (p<0.05) in males.
In addition, orange plasma discoloration was observed in all animals of group 4.

Recovery period -
all findings recorded during the treatment period were found to be reversible and generally similar to those of the controls. However, a slightly but statistically significantly decreased glucose and increased total bilirubin level in males (p <0.05 - p <0.01), and increased triglyceride level in females (p <0.01) were still to be noted.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

High dose:
Treatment period - the urinalysis data was generally unremarkable, however, a slight to moderate increase in the score for blood was recorded in both males (statistically non-significant) and females (p <0.05).
In addition, urine discoloration was observed in both sexes, ranging from light orange (8 males, 10 females) to orange (2 males). Also noted, was a deep-yellow urine discoloration in 3 out of 5 males and females of group 3 (200 mg/kg).

Recovery period -
a moderate increase in the score for blood was recorded in both sexes (p <0.05). Moreover, a light-orange urine discoloration was still to be noted in 5 males and 4 females as well as a deep-orange in 1 female.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 4 weeks of treatment with test article absolute kidney weights were higher in animals at 1000 mg/kg (males:+8 %; females +17 %, p <0.01) and in females at 200 mg/kg (+11 %, p <0.05). After the treatment-free recovery period absolute kidney weights of animals at 1000 mg/kg were marginal higher (+6 %) but not statistically significant different from the controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic findings attributed to treatment with the test article consisted of reddish discolorations in several organs including: tongue, gastrointestinal tract, kidneys, urinary bladder, testes, skin, mammary gland and lymph nodes. These findings were recorded with a high incidence in group 4 animals of the main test and the recovery.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Main Test:
In the stomach, vacuolation of the limiting ridge epithelium increased in severity and incidence with increasing dose (from minimal to moderate degrees). In the glandular mucosa, hyaline droplets were recorded. Those increased also in incidence and severity with increasing dose (from minimal to moderate degrees). Furthermore, inflammatory cells increased in the submucosa of the glandular stomach (group 3: minimal degree, group 4: minimal to slight degree). In the kidneys of group 4 males, hyaline droplets in tubular cells increased in severity (from minimal to slight degrees in group 1 to slight and moderate degrees in group 4). In group 4 females, a brownish exogenous pigment (slight to moderate degree) was recorded in kidneys. All changes of group 4 animal kidneys were accompanied by multifocal tubular degeneration (minimal to slight degree).
The brownish, exogenous pigment was also recorded in intestinal segments and several lymph nodes mainly of group 4 animals. In addition, in one group 4 male, the pigment was also recorded in alveolar macrophages of the lung.

Recovery:
In the glandular stomach mucosa, the hyaline droplets recorded were still higher in incidence and mean severity in group 4 animals compared with the controls.
There were no other findings recorded in the stomach. In the kidneys of group 4 males, hyaline droplets in tubular cells were higher in mean severity compared with group 1 males. The exogenous pigment was still present in group 4 females. Multifocal tubular degeneration were recorded at higher grades of severity in group 4 recovery animals compared with the rats from the main test. Moreover, the brownish exogenous pigment was seen at higher degrees of incidence and severity in the group 4 recovery animals compared with the findings of the main test. In addition, in one recovery group 4 male the pigment was also recorded in alveolar macrophages of the lung. A basophilic focus in the liver was recorded in one group 4 female. Furthermore, multifocal hepatocellular vacuolization was recorded at higher grades of incidence and severity in group 4 females compared with those of group 1.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on haematology, clinical biochemistry, urinalysis, kidneys, gastrointestinal tract and lymph nodes were noted in animals treated at 1000 mg/kg body weight.

Critical effects observed:

no

Conclusions:

The no-observed-adverse-effect level (NOAEL) of FAT 40557/A in 28 day repeated dose oral toxicity study in Wistar rats was determined to be 200 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 (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 toxicological relevant effects were observed on mortality, clinical signs, food consumption, body weight and ophthalmoscopic examinations. Slightly altered clinical laboratory findings were observed in animals treated with 1000 mg/kg bw. These effects were predominantly reversible within the recovery period. Macroscopically it was observed that after 4 weeks of treatment, absolute kidney weights were higher in animals of the 1000 mg/kg bw dose group and in females of the 200 mg/kg bw dose group. After the treatment-free recovery period kidney weights of animals of the 1000 mg/kg bw dose group were marginally higher but not statistically significantly different from the controls. In animals treated with 1000 mg/kg body weight morphological alterations in the kidneys, gastrointestinal tract and lymph nodes were noted. Microscopically, an increased storage of hyaline bodies in the stomach of both sexes and kidneys of males was recorded with increasing dose down to the lowest dose group. In females of the 1000 mg/kg bw dose group, a brownish, exogenous pigment was recorded in the kidneys. In the kidneys, the finding was accompanied by a minimal to slight tubular degeneration. The finding in the stomach was accompanied by a slightly higher incidence of inflammatory cells in the gastric submucosa of animals of the 200 and 1000 mg/kg bw groups and by a dose dependent, slightly increased incidence of vacuolization of the limiting ridge epithelium. Except the storage of hyaline bodies, all other findings in the gastrointestinal tract resolved after the recovery period. However, the findings in the kidneys were still present and most often recorded at higher degrees of mean severities. Also, deposits of an exogenous pigment were recorded in intestinal segments, several lymph nodes and in one case in lung alveolar macrophages. The deposits in the lymph nodes were seen at higher degrees of severity in recovery animals. The brownish pigment is deemed to be the test article or a metabolite thereof. In recovery females, the above findings were accompanied by a higher mean severity and incidence of vacuolization and, in one case by a basophilic focus in the liver. Based on the results of this study, 200 mg/kg bw was established as the no-observed-adverse-effect level (NOAEL), a no-observed-effect-level (NOEL) could however not be established.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-compliant guideline study, Klimisch code 1

System:

urinary

Organ:

kidney

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

Repeated dose toxicity: Oral

In a GLP compliant repeated toxicity study, performed according to OECD guideline 407, Wistar rats 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 toxicological relevant effects were observed on mortality, clinical signs, food consumption, body weight and ophthalmoscopic examinations.

Slightly altered clinical laboratory findings were observed in animals treated with 1000 mg/kg bw. These effects were predominantly reversible within the recovery period. Macroscopically it was observed that after 4 weeks of treatment, absolute kidney weights were higher in animals of the 1000 mg/kg bw dose group and in females of the 200 mg/kg bw dose group. After the treatment-free recovery period kidney weights of animals of the 1000 mg/kg bw dose group were marginally higher but not statistically significantly different from the controls. In animals treated with 1000 mg/kg body weight morphological alterations in the kidneys, gastrointestinal tract and lymph nodes were noted.

Microscopically, an increased storage of hyaline bodies in the stomach of both sexes and kidneys of males was recorded with increasing dose down to the lowest dose group. In females of the 1000 mg/kg bw dose group, a brownish, exogenous pigment was recorded in the kidneys. In the kidneys, the finding was accompanied by a minimal to slight tubular degeneration. The finding in the stomach was accompanied by a slightly higher incidence of inflammatory cells in the gastric submucosa of animals of the 200 and 1000 mg/kg bw groups and by a dose dependent, slightly increased incidence of vacuolization of the limiting ridge epithelium. Except the storage of hyaline bodies, all other findings in the gastrointestinal tract resolved after the recovery period. However, the findings in the kidneys were still present and most often recorded at higher degrees of mean severities. Also, deposits of an exogenous pigment were recorded in intestinal segments, several lymph nodes and in one case in lung alveolar macrophages. The deposits in the lymph nodes were seen at higher degrees of severity in recovery animals. The brownish pigment is deemed to be the test article or a metabolite thereof. In recovery females, the above findings were accompanied by a higher mean severity and incidence of vacuolization and, in one case by a basophilic focus in the liver.

Based on the study results, 200 mg/kg bw was established as the no-observed-adverse-effect level (NOAEL), a no-observed-effect-level (NOEL) could however not be established.

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.