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Description of key information

A study was performed according to OECD guideline 407 and in accordance with GLP. Based on the results of this study, 50 mg/kg body weight/day of FAT 40827/A was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of read across substance FAT 40827/A 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For a detailed read across justification please refer to chapter 13.
Reason / purpose:
read-across source
No. of animals per sex per dose:
Groups 1 and 4: 10 males; 10 females

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
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:
CLINICAL SIGNS AND MORTALITY
No animal died during the course of study.
During the treatment period blue coloring of the feces was observed in animals of all treated groups.
There were no other treatment-related findings.

BODY WEIGHT AND WEIGHT GAIN
There were no effects on the mean body weight or body weight development in any dose group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Absolute and/or relative food consumption were increased in males and females of group 4 (1000 mg/kg bw/day) during the third and fourth week of treatment.
No test article related changes were observed in any other group.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ophthalmoscopic findings.

HAEMATOLOGY
The assessment of hematological data indicated the following effects in rats of groups 3 (200 mg/kg bw/day) and/or 4 (1000 mg/kg bw/day) at termination of the treatment:
- Slightly increased reticulocyte count (relative/absolute) in both sexes of group 4 with a tendency towards higher values in group 3, slightly increased HFR and slightly decreased LFR reticulocyte fluorescence ratio in males of group 4, and markedly increased methemoglobin (MET-HB) concentration in both sexes of group 4 with a slight increase observed in group 3. In addition, an increased incidence of polychromatophilia was noted in both sexes of group 4.
The findings were considered to be treatment-related and suggest methemoglobinemia with an increase in hemopoietic activity as indicated by the increase
in reticulocytes. At termination of the treatment-free recovery period these findings were found to be reversed and comparable to those of the controls.

CLINICAL CHEMISTRY
In the assessment of clinical biochemistry data the following effects were recorded in rats of groups 3 (200 mg/kg bw/day) and/or 4 at termination of the
treatment:
- Slightly increased urea level in females (group 4), slightly increased creatinine level in both males and females (group 4), markedly increased uric acid and total bilirubin level in both males and females (group 4) and slightly increased in both males and females (group 3), slightly increased total cholesterol level in both males and females (group 4), slightly decreased triglyceride level in both males and females (group 4), slightly increased phosphorus level in females (group 4), marginally increased chloride level in females (group 4), slightly increased albumin and total protein level in both males and females (group 4), and slightly increased albumin to globulin ratio in males (group 4). In addition, a dark blue discoloration of the plasma was observed in all animals
of group 4 and a light blue discoloration in all animals of group 3. These findings were considered to be test article related. At termination of the treatment-free recovery period most of these findings were found to be reversed and comparable to those of the controls, except for a slightly increased uric acid and total bilirubin level in both sexes and a slightly increased phosphorus level in females of group 4. Moreover, a light blue discoloration of the plasma was yet to be noted in all animals of this group.

URINALYSIS
Urinalysis data indicated no changes which could be considered of toxicological significance at termination of the treatment. The only alterations noted were a deep brown to brown urine discoloration in both sexes of group 4 (1000 mg/kg bw/day) and a deep yellow urine pigmentation in three males and five females of group 3 (200 mg/kg bw/day). Also noted was a slight increase in the urine pH in females of group 4, slight increase in urine protein in both males and females of group 4 and marked increase in urine bilirubin (scores) in both males and females of group 4 with a slight increase in group 3. The discoloration was an expected effect of the test article, whereas the increase in urine bilirubin suggests a false positive reaction due to interference of the test article or metabolites thereof with the reagent test-strip reaction. This was also supported by the fact that "Ictotest" used to confirm positive bilirubin reaction was found to be negative in all cases nor was this latter observation supported by any underlying morphological findings which would suggest disturbed bilirubin metabolism, liver dysfunction or biliary obstruction. At termination of the treatment-free recovery period these findings were generally reversed and comparable to those of the controls, except for a slight increase in urine bilirubin and a yellow grey urine discoloration in both sexes of group 4.

ORGAN WEIGHTS
In comparison with the control group, no treatment-related effects on the organ weights or organ weight ratios were noted.

GROSS PATHOLOGY
At necropsy general and kidney discoloration (bluish) was observed in animals of group 3 and 4 (200, 1000 mg/kg bw/day). This was reversible after cessation of treatment. At histopathological examination slightly increased extramedullar haematopoesis was observed in 2 males and 1 female of group 4 (1000 mg/kg bw/day) at the end of the treatment period and also in 1 male each of group 1 and 4 (0, 1000 mg/kg bw/day) after recovery.
No other treatment-related macroscopic or microscopic findings were noted.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
The "no-observed-adverse-effect level" of FAT 45168/A is 50 mg/kg bw/day for male and female rats when administered orally by gavage for a period of 28 days.
Executive summary:

In this subacute toxicity study, FAT 45168/A was administered daily by gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg bw/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.

The only toxicologically significant observation during the in-life phase was an increase in food intake noted for both sexes of group 4 (1000 mg/kg bw/day) during the third and fourth week of treatment. At clinical laboratory investigations a number of slight to marked effects on biochemical, hematological and urinary parameters were noted for rats of group 4 (1000 mg/kg bw/day) and - in some cases - also of group 3 (200 mg/kg bw/day). The findings reflected methemoglobinemia with an increase in hemopoietic activity and with the corresponding histopathological findings of slightly increased splenic extramedullar hematopoiesis.

Based upon the results obtained in this study, the "no-observed-adverse-effect level" of FAT 45168/A is 50 mg/kg bw/day for male and female rats when administered orally by gavage for a period of 28 days.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A high quality GLP compliant guideline study
System:
haematopoietic
Organ:
spleen

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

Currently no 28-days or 90-days repeated dose toxicity study is available to assess the toxicity potential of Reactive Orange 035. However, a sub-acute oral toxicity study conducted with the lithium sodium salt of Reactive Black 039 has been used to fulfill this data gap. The lithium sodium salt of the Reactive Black 039 was evaluated in a GLP-compliant repeated dose toxicity study, performed according to OECD guideline 407. In this study, Wistar rats of both sexes were treated with test substance at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days by oral gavage. Dose groups 0 and 1000 mg/kg bw/day containing 10 males and 10 females; and dose groups 50 and 200 mg/kg bw/day containing 5 males and 5 females. 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. The only toxicologically significant observation during the in-life phase was an increase in food intake noted for both sexes of group 4 (1000 mg/kg bw/day) during the third and fourth week of treatment. At clinical laboratory investigations, a number of slight to marked effects on biochemical, haematological and urinary parameters were noted for rats of group 4 (1000 mg/kg bw/day) and - in some cases - also of group 3 (200 mg/kg bw/day). In conclusion, these findings reflect methaemoglobinemia with an increase in haematopoietic activity. However, confirmatory histopathology, i.e. increased splenic extramedullary haematopoiesis, was confined to the high dose group (1000 mg/kg bw/day) and was only minimal to slight in degree in the affected animals. At termination of the treatment-free recovery period, most of these haematological, biochemical and urinary findings were generally reversed and comparable to those of the controls, except for a slight increase in urine bilirubin and a yellow grey urine discoloration in both sexes of group 4; a slightly increased uric acid and total bilirubin level in both sexes and a slightly increased phosphorus level in females of group 4. Moreover, a light blue discoloration of the plasma was yet to be noted in all animals of this group. During the treatment period blue colouring of the faeces was observed in animals of all treated groups. At termination of treatment, a dark blue discoloration of the plasma was observed in all animals of group 4 and a light blue discoloration in all animals of group 3. At necropsy general and kidney discoloration (bluish) was observed in animals of group 3 and 4 (200 and 1000 mg/kg bw/day), this was reversible after cessation of treatment. Based upon the results obtained in this study, the "no-observed-adverse-effect level" of the test substance is 50 mg/kg bw/day for male and female rats when administered orally by gavage for a period of 28 days.

Repeated dose toxicity: inhalation

Currently no study to assess the repeated dose inhalation toxicity potential of Reactive Orange 035 is available. However, the vapour pressure for the substance can be considered low owing to the high melting point (>270 °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 334 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. Further, results of exposure to test animals from the subacute repeated dose toxicity study via oral route with the read-across chemical, Reactive Black 039 lithium sodium salt are available. Taking above arguments into account, no elevated toxicity other than already seen in repeated dose oral toxicity study with the source chemical, is expected via the 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 with Reactive Orange 035 is scientifically not necessary.

Repeated dose dermal toxicity: dermal

Currently no study to assess the repeated dose dermal toxicity of Reactive Orange 035 is available. However, the molecular weight of the chemical is 814.11 g/mol, indicating it being too large for dermal absorption. It has water solubility of 334 g/L and n-octanol/water partition coefficient (log P) of -3.46, 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. Absence of systemic toxicity in acute dermal, skin irritation and sensitisation studies with Reactive Orange 035, further supports the conclusion that low toxicity is expected for the chemical via the dermal route. Further, results of exposure to test animals from the subacute repeated dose toxicity study via oral route with the read-across chemical, Reactive Black 039 lithium sodium salt are available. Taking above arguments into account, no elevated toxicity other than already seen in repeated dose oral toxicity study with the source chemical, is expected via the 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 Orange 035 is considered to be scientifically not necessary.

Justification for classification or non-classification

In a 28-days repeated dose oral toxicity study with a read across substance in the rat statistical changes were evident in clinical pathology data indicating methaemoglobinemia in the 200 and 1000 mg/kg bw/day dose group. Confirmatory histopathological splenic changes were confined to the high dose group at the end of the 28-days treatment period. A clear trend to reversibility of the clinical pathology findings was evident in recovery animals after a 14 -days treatment free recovery period. By then the incidence and severity of splenic microscopic findings did no longer distinguish recovery animals of the high dose group from concurrent controls. Based on these findings the test substance is classified STOT Re. Exp. 2: H373 according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.