Tetra(sodium/potassium)-7-[(E)-{2-acetamido-4-[(E)-(4-{[4-chlor-6-({2-[(4-fluor-6-{[4-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonato-1-naphthyl)diazenyl]-5-methoxyphenyl}diazenyl]-1,3,6-naphthalintrisulfonate

Endpoint summary

Description of key information

Based on the test results, 50 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of the test item was established as the no-observed-adverse-effect-level (NOAEL).

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

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:

Experiment start date - 18 July 2006; Experiment end date - 17 January 2006; Study completion date - 08 September 2006.

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

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identity: FAT 40827/A
Batch: T2 5572 BOP 01/06
Purity: determined in this study
Appearance: black sticky powder
Expiration date: 28.02.2011
Storage: at room temperature

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animal: Rat, HanRcc:WIST(SPF)
Total number of animals per group: Groups 1 and 4: 10 males; 10 females; groups 2 and 3: 5 males and 5 females
Total number of animals: 30 males and 30 females
Age at delivery: 6 weeks;
Body weight range (acclimatization): males: 130.9-158.6 g; females: 113.0-130.8 g

Conditions
Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with a target range for temperature of 22 ± 3 °C and for relative humidity between 30-70 %. There was 12 hours fluorescent light/ 12 hours dark cycle with music during the light period.
Accommodation: Groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding.
Diet: Pelleted standard Kliba no. 343 rat maintenance diet was available ad libitum (Batches no. 23/06 and 36/06).
Water: Community tap-water was available ad libitum in water bottles.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Oral, by gavage, 10ml/kg body weight, daily for 28 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Several application formulations were prepared at the test facility and representative analytical samples were collected and dispatched to the test site internally. The test item concentrations were determined by HPLC coupled to an UV detector and quantified with the area under the peak.
The identity of test item was confirmed by its retention time which was similar to that measured in the working standards. The test item content in all samples was found to be within the accepted range of ±20 % of the nominal content. In addition, the homogenous distribution of test item in bidistilled water was demonstrated. The application formulations were considered to be stable for at least 2 hours and 7 days when kept under storage conditions. In conclusion, the results obtained within this phase confirm the correct preparation and storage of application formulations during the conduct of this study.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

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

Remarks:

Group 1 - Control group

Dose / conc.:

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

Remarks:

Group 2 - Low dose group

Dose / conc.:

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

Remarks:

Group 3 - Mid dose group

Dose / conc.:

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

Remarks:

Group 4 - High dose group

No. of animals per sex per dose:

Groups 1 and 4: 10 males; 10 females; groups 2 and 3: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

In this subacute toxicity study, test article was administered daily by gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. After termination of the treatment period half of the animals of the control group and of the high dose group were observed for a further 14-day treatment-free recovery period. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observation battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and stomach and gross lesions from all animals.

Observations and examinations performed and frequency:

Mortality/Viability: twice daily;
General cageside observation: once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).
Detailed clinical observations: in random sequence once before commencement of administration and once weekly (week 1-3) thereafter.
Food consumption: once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.
Body weight: weekly during pretest, treatment and recovery and before necropsy.
Functional observational battery: during week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.

Sacrifice and pathology:

All animals were weighted and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy were anesthetized by intraperitoneal injection of sodium pentobarbitone and sacrificed by exsanguination. Samples were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution.

Other examinations:

Hematology, clinical biochemistry, urinalysis, absolute and relative organ weights, histotechnology and histopathology.

Statistics:

The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of 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.
Student’s T-test was applied to grip strength and locomotor activity.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of adverse toxicological relevance were noted at any dose level.
Dark feces were found in animals treated with 1000 mg/kg/day. This finding disappeared during the recovery period in animals treated with 1000 mg/kg/day and was therefore not considered to be adverse. In animals treated with 1000 mg/kg/day, dark feces was noted from treatment day 3 until the end of the treatment.
During the recovery period dark feces was noted from day 1 to 4 of the recovery period in animals previously treated with 1000 mg/kg/day. Although test item-related, dark feces were considered to be a typical passive effect resulting from oral administration of a dyestuff.

Mortality:

no mortality observed

Description (incidence):

All animals survived until their scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related changes of toxicological relevance were noted in body weight and body weight gain of male and female rats at any dose level.
In males treated with 1000 mg/kg/day, the slightly reduced body weight gain during the treatment (day 8, 15, 22, and 28) and the recovery period (day 1, 8, and 14) resulted in slightly reduced body weight during the recovery period (day 1, 8, and 14). These findings were considered to be incidental and therefore of no toxicological relevance.
No further test item-related changes of toxicological relevance were noted in body weight and body weight gain of male and female rats at any dose level.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No findings of toxicological relevance were noted. Mean daily intake and relative food consumption in rats of both sexes at any dose level showed no relevant alterations to their respective controls.

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related effects were noted in hematological parameters after the treatment with FAT 40827/A (after week 4) or recovery period (after week 6). The following findings were considered to be of no toxicological relevance because they were incidental and within the biological variation:
After week 4: In males treated with 1000 mg/kg/day, the absolute reticulocyte count (Reti G/l) was significantly increased (p<0.05). In females treated with 1000 mg/kg/day, the hemoglobin level (Hb) (p<0.01) and the prothrombin time (PT) (p<0.05) were significantly reduced and the methemoglobin level (Met-Hb) was significantly elevated (p<0.05).
After week 6: In males previously treated with 1000 mg/kg/day, the erythrocyte count (RBC) (p<0.05) and the absolute eosinophil count (Eos G/l) (p<0.01) were significantly increased. In females previously treated with 1000 mg/kg/day, the mean corpuscular hemoglobin
concentration (MCHC) was significantly reduced (p<0.05).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related changes of toxicological relevance were noted in clinical biochemistry parameters after the treatment or recovery period. The following findings were within the range of typical biological variation and considered to be incidental. Therefore they were considered to be of no toxicological relevance.

After week 4
In males treated with 200 and 1000 mg/kg/day, the total bilirubin level was significantly decreased (each p<0.05). In males treated with 1000 mg/kg/day, the Cholesterin level was significantly elevated (p<0.05) and the glutamate dehydrogenase level (GLDH) was
significantly decreased (p<0.05). The sodium (p<0.01) and the phosphorus (p<0.05) blood level were significantly increased in males treated with 1000 mg/kg/day. In males treated with 200 mg/kg/day, the calcium blood level was significantly elevated (p<0.05).
After week 6
In males previously treated with 1000 mg/kg/day, the blood glucose level, the total protein level, and the globulin level were significantly reduced (each p<0.05). In females previously treated with 1000 mg/kg/day, the potassium blood level was significantly increased (p<0.01).

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urine color and urine pH were considered to be affected by the treatment with FAT 40827/A. These changes were considered to be either excreted test item or a possible metabolite.
Test item-related findings were noted in urine color after the treatment period in males treated with 1000 mg/kg/day and in females treated with 200 and 1000 mg/kg/day. Due to the reversibility of this finding after the recovery period, changes in urine color was not considered to be adverse. The test item-related increase of the urine pH was more clear in females treated with 1000 mg/kg/day than in males treated with 1000 mg/kg/day. After the recovery period, the increase of the urine pH was disappeared and therefore not considered to be adverse.
After week 4: The color of the urine was different in all animals (except one female) treated with 1000 mg/kg/day and in all females treated with 200 mg/kg/day compared to the respective control. In males treated with 1000 mg/kg/day, the urine color was light orange in 2/10, orange in 4/10, deep orange in 1/10. brown in 1/10, and deep brown in 2/10 animals. In females treated with 1000 mg/kg/day, the color of the urine appeared as light yellow in 1/10, as light orange in 1/10, as orange in 2/10, in deep orange in 1/10, as light brown in 2/10, as brown in 1/10, and as deep brown in 2/10. In all females treated with 200 mg/kg/day the urine color was light brown.
A significant increase (p <0.05) of the urine pH was noted in animals treated with 1000 mg/kg/day. These findings were considered to be test item-related. In males treated with 1000 mg/kg/day, the urine protein was significantly increased (p <0.05). This finding was considered to be a normal background lesion in males of this age and strain and therefore of no toxicological relevance.
After week 6: The urine color and pH of animals previously treated with 1000 mg/kg/day and the urine color of females previously treated with 200mg/kg/day were not different compared to the respective control. Therefore these changes were not considered to be adverse. No further test item-related findings were noted.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observational Battery
During functional observational battery (week 4) no test item-related adverse findings of toxicological relevance were noted. Dark feces were noted in single males and females at 1000mg/kg/day. In one female treated with 200 mg/kg/day miosis in right and left eye was noted. This isolated finding was considered to be incidental and therefore of no toxicological relevance. No further test item-related findings were recorded.

Grip Strength: No test item-related alterations in the mean fore- and hindlimb grip strength were recorded in rats at any dose level. In females treated with 1000 mg/kg/day, the mean hindlimb grip strength was significantly increased (p <0.05). This finding was considered to be incidental and therefore of no toxicological relevance.

Locomotor Activity: No test item-related effects in mean locomotor activity were noted in rats at any dose levels. In animals treated with 1000 mg/kg/day, the mean locomotor activity showed a significant increase (each p <0.05) during the 0-10 min measurement interval. In females treated with 50 and 200 mg/kg/day, the mean locomotor activity during the 30-40 min measurement interval was significantly reduced (each p <0.05). These findings were incidental and therefore of no toxicological relevance.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no differences in the absolute or relative organ weights indicating an effect of test item after the treatment or recovery period.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopical findings were considered to be test item-related.
All findings recorded were considered to be within the range of normal background lesions, which may be seen in rats of this strain and age. They consisted of pelvic dilation in kidneys, uterus dilation, seminal vesicles reduced in size, focus/foci reddish or dark red in stomach and thymus and discoloration in testes, ileum, rectum and ovaries.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The test item FAT 40827/A revealed alterations in the stomach consisting of increased inflammatory infiltrate. This finding in the stomach was recorded at higher incidences in females treated with 1000 mg/kg/day after the treatment period. As the male and female control group also showed increased inflammatory infiltrate in the stomach, this finding was considered to be incidental and therefore of no toxicological relevance.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Critical effects observed:

not specified

Conclusions:

Based on the results of this study, 50 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of the test item was established as the no-observed-adverse-effect-level (NOAEL).

Executive summary:

In a GLP-compliant study conducted according to OECD test guideline 408, the test article was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. After termination of the treatment period half of the animals of the control group and of the high dose group were observed for a further 14 -day treatment-free recovery period. No treatment-related effects were noted on any parameter observed during the study (mortality, changes of toxicological relevance in detailed clinical observations, in grip strength and locomotor activity, in mean absolute and relative food consumption, in body weights and body weight gains, in hematology and clinical biochemistry parameters, in organ weights and ratios as well as in macroscopical and microscopical findings. Test item-related findings were generally restricted to dark feces. Noted during general cage side observations and functional observational battery (week 4), in animals treated with 1000 mg/kg/day. In animals treated with 1000 mg/kg/day and in females treated with 200 mg/kg/day, the urine color was affected by the treatment, but was reversible after the recovery period. In animals treated with 1000 mg/kg/day, the urine pH was increased after the treatment, but was reversible after the recovery period. Based on the results of this study, 50 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of the test item was established as the no-observed-adverse-effect-level (NOAEL).

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:

This finding was confirmed by the results of the OECD 421 study performed on the same substance with equivalent NOAEL (1000 mg/kg bw/d)

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 oral toxicity

In the subacute toxicity study, test article was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. No treatment-related effects were noted on any parameter observed during the study (mortality, changes of toxicological relevance in detailed clinical observations, in grip strength and locomotor activity, in mean absolute and relative food consumption, in body weights and body weight gains, in hematology and clinical biochemistry parameters, in organ weights and ratios as well as in macroscopical and microscopical findings. Test item-related findings were generally restricted to dark feces. Noted during general cage side observations and functional observational battery (week 4), in animas treated with 1000 mg/kg/day. In animals treated with 1000 mg/kg/day and in females treated with 200 mg/kg/day, the urine color was affected by the treatment, but was reversible after the recovery period. In animals treated with 1000 mg/kg/day, the urine pH was increased after the treatment, but was reversible after the recovery period. Based on the test results, 50 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of the test item was established as the no-observed-adverse-effect-level (NOAEL). This finding was supported by the observations on parental animals in an OECD 421 reproductive screen study which equally resulted in a NOAEL for parental animals of 1000 mg/kg bw/d.

 

Repeated inhalation toxicity waiver

Currently no study to assess the repeated dose inhalation toxicity potential of Reactive Brown 051 is available. The calculated value for vapour pressure was found to be 3.6E-37 Pa at 25 °C. Hence, the substance is considered to have low volatility. Synthesis and spray drying of this chemical is performed in a closed process; the final product consists of non-dusty granules. Hence, the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Further, the chemical is found to have water solubility of >306 g/L, hence in the case of dust of the substance entering the respiratory tract, it will be trapped in the mucus and cleared, thereby further limiting the absorption. No systemic toxicity was observed upto 1000 mg/kg bw/day via gavage in a 28-day repeated dose toxicity study. Taking above arguments into consideration, low toxicity potential is expected on repeated exposure of Reactive Brown 051 via inhalation route and safety for human health can be estimated using the principles of route to route extrapolation. Hence, the conduct of repeated dose toxicity study via inhalation route for Reactive Brown 051 is considered to be scientifically not necessary.

 

Repeated dermal toxicity waiver

Currently no study to assess the repeated dose dermal toxicity of Reactive Brown 051 is available. However, the molecular weight of the chemical is 1272.65 g/mol, indicating it being too large for dermal absorption. It has water solubility of >306 g/L and n-octanol/water partition coefficient (log P) of <-5.4, indicating it being too hydrophilic to cross the lipid rich environment of the stratum corneum. Hence, the dermal uptake for the substance will be low. No systemic toxicity was observed upto 1000 mg/kg bw/day via gavage in a 28-day repeated dose toxicity study. Similarly, absence of systemic toxicity in skin irritation studies, further supports the conclusion that low toxicity is expected for the chemical via the dermal route. Further, experience with similar chemical substances has demonstrated that it is very unlikely that toxicity related to the intrinsic properties of the chemical only show up upon dermal exposure and not after systemic application. Taking the above arguments into consideration, low toxicity potential is expected on repeated exposure of Reactive Brown 051 via dermal route and safety for human health can be estimated using the principles of route to route extrapolation. Hence, the conduct of repeated dose toxicity study via dermal route for Reactive Brown 051 is considered to be scientifically not necessary.

Justification for classification or non-classification

The test substance is not classified under CLP (Regulation EC No.1272/2008) for repeated dose toxicity effects.