[No public or meaningful name is available]

Endpoint summary

Description of key information

Reactive Blue 50 is considered to have a NOAEL of 1000 mg/kg bw/day on repeated exposure.

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

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study; RA from supporting substance

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System:
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 9-10 weeks old, females: 9-10 weeks old.
Body weight at the allocation of the animals to the experimental groups: males: 248 - 281 g (mean: 267.83 g, ± 20 % = 214.26 – 321.39 g) females: 171 - 202 g (mean: 184.33 g, ± 20 % = 147.46 – 221.19 g). The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.The animal study was authorized by local government under file no. 55.2-1-54-2532.2-11-11.
Housing and Feeding Conditions: Full barrier in an air-conditioned room
Temperature: 22 ± 3 °C
Relative humidity: 55± 10 %
Artificial light, sequence being 12 hours light, 12 hours dark
Air change: 10 x / hour
Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 131113)
Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
Animals were housed in groups of 2 animals/ sex/ cage in IVC cages (type III H, polysulphone cages) during the premating period in both males and females and during postmating period in males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to its original cage. Each cage was provided with Altromin saw fibre bedding (lot no. 1526)
Certificates of food, water and bedding are filed at BSL BIOSERVICE- Adequate acclimatisation period (at least 5 days) under laboratory conditions
Preparation of the Animals: Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animals showed any adverse clinical signs before the study initiation. Before the first administration, all animals to be used for the study were weighed. Mean body weight of the group housed animals was used to assign all animals to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively, while ensuring to keep each animal with its initial cage partner if possible. Each animal was marked with its identification number by individual ear tattoo.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test item was weighed into a tared plastic vial on a precision balance and suspended in aqua ad injectionem. Homogeneity of the test item in the vehicle was maintained by using an ultra turrax. The test item formulations were prepared once in every ten days and stored at 2-8 °C. Every day before the dose administration the formulation samples were allowed to reach the room temperature and the homogeneity was ensured by votexing the sample on vortex machine. The following doses were evaluated:
Control: 0 mg/kg bw/d
Low Dose:100mg/kg bw/d
Medium Dose: 300mg/kg bw/d
High Dose: 1000mg/kg bw/d
Since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg bw/d corresponding to a limit dose for this study. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad injectionem using the same volume as used for the dose groups.For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured. The administration volume was 5 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations.Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples). Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples). Samples for stability analysis were taken in the first week of the study [0 hours, 6 hours (at RT) and 10 days after the preparation (stored at 2-8 °C)] from high and low dose formulations (6 samples). All formulation samples were analysed on the day of sample collection and were stored at -20 °C. These samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140307. The results are reported in the annex of the final report.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

None

Dose / conc.:

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

Remarks:

Low Dose

Dose / conc.:

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

Remarks:

Medium Dose

Dose / conc.:

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

Remarks:

High Dose

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes - Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: daily Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on lactation days in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests.

BODY WEIGHT: Yes
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.

FOOD CONSUMPTION: Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: yes

HAEMATOLOGY: Yes
Time schedule for collection of blood: at the end of the treatment period
Anaesthetic used for blood collection: Yes (ketamine/xylazin, 2 :1)
Animals fasted: No
How many animals: five randomly selected males and females of each group
Parameters examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc), prothrombin time (PT) and activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: at the end of the treatment period
Animals fasted: No
How many animals: five randomly selected males and females of each group
Parameters examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na) and potassium (K)

URINALYSIS: Yes
Time schedule for collection of urine: 5 randomly selected males and females at necropsy.
Metabolism cages used for collection of urine: No
Animals fasted: No
Parameters were examined: urine colour/ appearance, specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
Time schedule for examinations: in the week before the first treatment and during the last week of the treatment
Dose groups that were examined: all- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for testes and epididymides which were fixed in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.


HISTOPATHOLOGY: Yes
A full histopathology was carried out on the preserved organs and tissues of 5 randomly selected male and female animals (only lactating females were evaluated) of the control and high dose groups which were sacrificed at the end of the treatment period. These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in any organ of the high dose group. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 μm and stained with hematoxylin and eosin and were examined in all animals. All gross lesion macroscopically identified was examined microscopically in all animals. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

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

Water consumption and compound intake (if drinking water study):

not examined

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:

Mortality: No mortality occurred in the control or any of the dose groups during the study period.

Clinical Observations: In males and females, there were no clinical signs of toxicological relevance in any of the dose groups when compared to the control. However, there were isolated incidences of nasal discharge, alopecia on various body parts, moving the bedding, regurgitation, slight to moderate piloerection, aggressiveness, chromodacryorrhea at the right eye, slightly reduced spontaneous activity and emisis (blue mouth and nose) in a few control and/or dose group animals. There were also discoloured blue faeces observed in male and female animals of high dose group which could be attributed to the colur of the test item. During the weekly detailed clinical observation, no significant changes or differences between the groups were observed.

Functional Observations: No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups. However, in males statistically significantly lower body temperature was observed in the last week of treatment in MD and HD group when compared with the controls. As this difference was marginal and overall there was no effect on the general health condition, body weight development and food consumption of these animals this effect on body temperature is not considered to be adverse.

Body Weight Development: In males, no adverse effects of toxicological relevance of FAT 20033/L were observed throughout the study period on body weight and body weight gain when compared to the controls. A statistical analysis of data revealed no statistically significant difference between the dose groups and corresponding control group. In females, no statistically significant difference between the dose groups and corresponding control group was observed on body weight and body weight gain. However, marginally lower (2-6 %) group mean body weight during gestation and lactation period and body weight gain during gestation period was observed in high dose group when compared to the controls. As this difference was marginal, values were within the range of biological variation and this effect on body weight development was attributed to low body weight values from single high dose female (no. 80) in which no live pups were born, no toxicological relevance could be attributed to this effect on female body weight development.

Food Consumption:Throughout the study period, there were no statistically significant effect of FAT 20033/L on food consumption of males and females of the dose groups when compared to the control group. However, marginally lower group mean food consumption from gestation day 7 to post-natal day 4 was observed in high dose group when compared to the controls. As this difference was correlated with body weight development it is not considered to be no toxicologically relevant.Haematology and Coagulation: In males and females, there were no adverse effects of toxicological relevance on parameters of haematology and blood coagulation parameters in dose groups when compared with corresponding control. However, in males there was marginally but statistically significantly higher reticulocyte count in LD and HD group when compared with the controls. As the mean value was in the range of historical control data and in the absence of dose dependency, the finding was not considered to be toxicological relevant.In females, statistical analysis of haematology and blood coagulation data revealed statistically significantly higher eosinophils in LD group (0.62 %) when compared with the controls (0.16 %). As the mean value was in the range of historical control data and due to lack of dose dependency, this effect was not considered to be toxicologically relevant.

Clinical Biochemistry:In males and females, there were no adverse changes of toxicological relevance for clinical biochemistry in dose groups when compared to the controls. However, statistically significantly higher creatinine in HD males (26.80) and statistically significantly lower potassium in HD females (3.45) was observed when compared with the corresponding controls. Since all mean and most individual values of clinical biochemistry of males and females were within the historical control range, the effect on creatinine in male HD group and potassium in female HD group was not considered to be an adverse effect due to the treatment.Urinalysis: There were no signs of toxicological relevance in urine parameters of males and females dose groups when compared to the controls.

Pathology: No gross lesions were observed in this study that could be attributed to treatment with the test item. All gross lesions recorded in male animals (yellow spots and soft nodule on epididymides, dilated renal pelvis of kidneys, increase in size of thymus, reduced size of thyroid and parathyroid, lobus cadatus (liver) herniation, increase in size of seminal vesicles with coagulating glands, prostate gland and heart, slightly reduced size of spleen, testes and pituitary gland in control and/ or dose group animals) and in female animals (kidneys doscoloured pale and enlarged adrenals in LD group ) were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered incidental macroscopic findings that did not correlate with microscopic changes.Bluish or greenish doscolouration of various organs (esophagus, stomach, small and large intestines, Peyer’s patches, mesenteric lymph node, and/or trachea) of the high-dose was observed in males and was considered to reflect the colour of the test item or the color being formed by the test item mixing with the intestinal contents. However, there was no histologic alteration correlated with macroscopic appearances.Greenish/green discoloration was also recorded in the kidney of some animals including the control animals. The expression of the colour was the same as the above mentioned alteration. However, the coloration of this case is unlikely to be derived from the test item, because this was observed in the control animals too, and probably this is due to the status of blood removal at the necropsy. For example, it is empirically known that the kidney appears greenish/green after the whole body or renal blood perfusion was performed with the use of saline or fixative. In any event, there were no histologic abnormalities which might be related to macroscopic coloration. All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered to be incidental macroscopic appearances that did not correlate with microscopic changes. Organ Weight:In males and females, there were no statistically significant differences on absolute and relative organ weights of the dose groups when compared with the corresponding controls and all group mean and individual values were comparable with the controls.

Histopathology: Macroscopic alteration of various organs due to bluish /greenish discolouration was considered to be the coloration derived from the unique colour of the test item and/or the colour being formed by the test item mixing with the intestinal contents. However, there was no histologic alteration correlated with macroscopic appearances.Discoloured kidney of some animals including the control animals was probably due to the status of blood removal at the necropsy. There were no histologic abnormalities which might be related to macroscopic coloration. All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered to be incidental macroscopic appearances that did not correlate with microscopic changes. Micropscopically, no histomorphologic evidence of toxicological properties observed in any organs and tissues including the male reproductive organs (testes, epididymides, prostate and seminal vesicles) and the female reproductive organs (ovaries, uterus with cervix and vagina). In addition, as a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. All findings recorded in the organs and tissues examined in this study were within the range of normal background lesions which may be recorded in animals of this strain and age.In conclusion, no histomorphological evidence of toxicity in any organs and tissues of male and females was established up to 1000 mg/kg bw/d.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L TE is considered to be 1000 mg/kg/d for systemic toxicity and also for reproduction/ developmental toxicity in males and females.

Critical effects observed:

not specified

Conclusions:

Based on this combined repeated dose oral toxicity and reproduction/developmental toxicity screening test with FAT 20033/L in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d the following conclusions can be made: There were no adverse toxicological effects in the adult males and females or significant reproductive and developmental effects in pups at any administered dose. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg/d for systemic toxicity and also for reproduction/ developmental toxicity in males and females.

Executive summary:

The study was conducted to assess the possible effects of FAT 20033/L at the dose level of 100, 300 and 1000 mg/kg repeated dose administration to male and female Wistar rats according to OECD 422 guideline. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 or 29 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad injectionem, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

 

The following doses were evaluated:

Control (C): 0 mg/kg/d

Low Dose (LD): 100 mg/kg/d

Medium Dose (MD): 300 mg/kg/d

High Dose (HD): 1000 mg/kg/d

 

The test item formulation was once in every ten days and stored at 2-8 °C. The administration volume was 5 mL/kg body weight. During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment and at the end of the study. Haematological, clinical biochemistry and urine analysis evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected lactating females from each group. A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups. Gross lesions from all groups were examined by light microscopy. No treatment related adverse effects were found in any of the dose group.No gross lesions were observed in this study those could be attributed to treatment with the test item. All gross lesions were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered to be incidental macroscopic findings that did not correlate with microscopic changes. There were no adverse toxicological effects in the adult males and females at any administered dose. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg/d for systemic toxicity in males and females.

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 guideline study. For justification of the read-across please refer the justification document attached to chapter 13.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

Data on repeated dose toxicity are not available for the target substance i.e FAT 40069. To fill the data gaps, read across approach is adapted using similar substance FAT 20033/L. Read-across is claimed basis of structural relationship of the target and the source chemicals. Read-across substance is FAT 20033/L and have been investigated for repeat dose toxicity.

Based on this toxicity screening test with FAT 20033/L in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d conducted according to OECD 422 guideline (GLP compliant), there were no adverse toxicological effects in the males and females observed at any administered dose level. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg/d for systemic toxicity in males and females.

Inhalation:

Currently no study to assess the repeated dose inhalation toxicity potential of Reactive Blue 050 is available. However, the vapour pressure for the substance can be considered low owing to the high melting point (>320 °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 257 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 when the source substance, Acid Blue 277, was administered up to 1000 mg/kg bw/day via gavage in a combined repeated dose toxicity study with reproductive/developmental toxicity screening. 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 inhalation exposure and not after systemic application. Taking into consideration the above arguments, low toxicity potential is expected on repeated exposure of Reactive Blue 050 via inhalation route and safety for human health can be estimated using the principles of read across and route to route extrapolation. Hence, the conduct of repeated dose toxicity study via inhalation route for Reactive Blue 050 is considered to be scientifically not necessary.

Dermal:

Currently no study to assess the repeated dose dermal toxicity of Reactive Blue 050 is available. However, the molecular weight of the chemical is 773.93 g/mol , indicating it being too large for dermal absorption. It has water solubility of 257 g/L and n-octanol/water partition coefficient (log P) of -3.51, 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 when the source substance Acid Blue 277 was administered upto 1000 mg/kg bw/day via gavage in a combined repeated dose toxicity study with reproductive/developmental toxicity screening. Similarly, absence of systemic toxicity in skin irritation and sensitisation 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 above arguments into account, low toxicity potential is expected on repeated exposure via dermal route and safety for human health can be estimated using the principles of read across and route to route extrapolation. Hence, the conduct of repeated dose toxicity study via dermal route for Reactive Blue 050 is considered to be scientifically not necessary.

Justification for classification or non-classification

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