Registration Dossier

Administrative data

Description of key information

The no-observed-adverse-effect level (NOAEL) of Reactive Orange 136 by oral route of administration was 1000 mg/kg.

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:
04-DEC-1996 to 01-APR-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
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. certificate)
Remarks:
Swiss GLP
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WP2/96
- Expiration date of the lot/batch: November 30, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature at about 20 °C; away from direct sunlight
- Stability of the test substance in the solvent/vehicle: Stable for at least 48 h at 20 °C.
Species:
rat
Strain:
Wistar
Details on species / strain selection:

Rationale: Recognized by the international guidelines as the recommended test system
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4
4414 Fuellinsdorf/Switzerland - Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 126 - 172 g (mean 152 g); Females: 99 - 145 g (mean 131 g)
- Housing: Groups of five in Makrol on 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) was available ad libitum (Batch no. 81/96)
- Water: Tap water was available ad libitum in water bottles
- Acclimation period: 7 days under test conditions following a health
examination. Only animals without any visible signs of illness were used for the study

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h fluorescent light/ 12 h dark (light period between 6.00 a.m. and 6.00 p.m.), music during the light period

IN-LIFE DATES: From 11-DEC-1996 to 22-JAN-1997
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:
Dose volume: 10 ml/kg body weight per treatment day,
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean concentrations of the homogeneity samples were found to be 101.0 %, 96.4 %, and 100.0 % of the nominal concentrations of dose group 2 (5 mg/ml), dose group 3 (20 mg/ml), and dose group 4 (100 mg/ml), respectively. The individual concentrations varied in the range from -4 % to +6 % of the mean concentrations.
FAT 45'176/A was found to be stable in bi-distilled water at room temperature for at least two hours.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week for a total of 28 days.
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 / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Medium
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High
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:
TEST ARTICLE PREPARATION
FAT 45'176/A 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. The study comprised 5 animals per group and sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.
Clinical signs, food consumption and body weights were recorded periodically during the treatment and recovery periods. Ophthalmoscopic examinations were performed at pretest, the end of the treatment and recovery periods.
At the end of the dosing period and the treatment-free period, blood samples were withdrawn for hematology and plasma chemistry analyses, and urine samples were collected for urinalysis. All animals were killed, necropsied and examined post mortem. Samples of major organs from all group 1 (0 mg/kg) and group 4 (1000 mg/kg) animals, as well as gross lesions from all animals were processed as hematoxylin and eosin stained slides and examined by light microscopy.


FAT 45'176/A was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using an magnetic stirrer. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer.
Frequency of preparation: Daily, prior to each application
Positive control:
no data
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 using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.
BODY WEIGHTS: Body weight was recorded once weekly during pretest, treatment and recovery, and twice before necropsies; using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.
OPHTHALMOSCOPIC EXAMINATIONS: At 4 weeks and at 6 weeks
Sacrifice and pathology:
NECROPSY: At 4 weeks and at 6 weeks
All animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. At the end of the treatment or recovery period the animals designated according to the necropsy schedule were anesthetized by intraperitoneal injection of sodium pentobarbitone and sacrificed by exsanguination.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS: The following organ weights were recorded on the scheduled dates of necropsy: Adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroid including parathyroid gland. The organ to terminal body weight ratios as well as the organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOTECHNIQUE: All organ and tissue samples, as defined under Histopathology were processed, embedded, cut at an approximate thickness of 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY: 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 all gross lesions from rats of all groups were examined by a pathologist.
Other examinations:
Clinical Laboratory Investigations:
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia.The animals were fasted in metabolic cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected between the hours of 06.30 and 07.35 to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
Urine was collected during the 18-h fasting period into a specimen vial.
Statistics:
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:
effects observed, treatment-related
Description (incidence and severity):
All animals survived the scheduled treatment period. One group 4 (1000 mg/kg) female (no. 53) died during blood sampling on the day of the scheduled necropsy.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
All animals survived the scheduled treatment period. One group 4 (1000 mg/kg) female (no. 53) died during blood sampling on the day of the scheduled necropsy.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
However, females at 50 mg/kg showed generally slightly lower absolute food consumption throughout the whole treatment period. In view of the slightly lower food consumption already recorded during pretest for this group, the difference is considered to be incidental.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
However, ophthalmologic findings were noted in a small proportion of animals from all treated groups. They included persistent pupillary membranes, slight posterior lenticular opacity and corneal opacity. These findings occurred at similar incidences in the control and treated groups. No test article-related effect was noted comparing the ophthalmologic findings during pretest, the end of the treatment period and the recovery period. Therefore, they are considered to be unrelated to treatment with test article.
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period - slightly increased uric acid level in females (p<0.01), moderately increased total bilirubin level in both males and females (p<0.01), slightly increased total cholesterol and phospholipid levels in males (p<0.01), slightly increased triglyceride level in females (p<0.01), slightly lower potassium level and slightly increased total protein and globulin levels in males (p<0.05). In addition, orange plasma discoloration was observed in all animals of group 4.
Recovery period - with the exception of a slightly lower potassium level in males (p<0.05), all findings recorded during the treatment period indicated reversibility and/or were similar to those of the controls, and no longer statistically significant. Subsequently, no further discoloration of the plasma was observed.
All other statistical differences in the results of the clinical biochemistry during the treatment or recovery period were considered to be incidental and unrelated to the treatment, and of normal biological variation for rats of this strain and age.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment period - apart from a higher incidence in the score for urine bilirubin (statistically non-significant) and urine discoloration in both sexes of group 4, no remarkable findings were noted. Urine discoloration as observed in both sexes of group 4 ranged from a deep-yellow (2 males, 6 females) to light-orange (4 males, 2 females) to orange (2 males) to deep-orange (2 males). Moreover, a deep-yellow urine discoloration was also noted in 4 out of 5 males and 2 out of 5 females of group 3 (200 mg/kg) and in 3 out of 5 males and 1 out of 5 females of group 2 (50 mg/kg).
Recovery period - reversibility in the score for bilirubin and in the urine discoloration was observed in all animals of the recovery group. All other values were unremarkable and found to be similar between the control and treated rats.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Most of the macroscopic findings were considered to be spontaneous in nature and did not distinguish treated animals from the controls.
One rat, no. 48 (group 3, female) had a closed vagina which on macroscopic examination was diagnosed imperforation, a malformation abnormality. Another group 3 female, no. 47 had a dark red nodule in the pancreatic adipose tissue which was found to represent infarcted ectopic hepatic tissue. Neither of these findings were related to treatment with the test article.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All gross and microscopic lesions recorded in this study were considered to be spontaneous in origin. Their incidence and severity was similar in control and treated rats at both sacrifices.
Other effects:
no effects observed
Details on results:
Clinical Signs: There were no clinical signs noted in groups 2 (50 mg/kg) and 3 (200 mg/kg). Animals of group 4 showed dark feces from treatment day 13 throughout the treatment period and during the first day of the treatment-free recovery period.

Hematology / Clinical Biochemistry / Urinalysis
The results of the hematology, clinical biochemistry and urinalysis data indicated no changes of toxi cological significance at termination of the treatment nor at the end of the treatment-free recovery period. However, the following changes recorded between the controls and treated rats of group 4
(1000mg/kg) at termination of the treatment were considered to be test articlerelated:
Clinical Biochemistry: slightly increased uric acid level in females (p<0.01), moderately increased total bilirubin level in both males and females (p<0.01), slightly increased total cholesterol and phospholipid levels in males (p<0.01), slightly increased triglyceride level in females (p<0.01), slightly lower potassium level and slightly increased total protein and globulin levels in males (p<0.05). In addition, orange plasma discoloration was observed in all animals of group 4.
Urinalysis: apart from a higher incidence in the score for urine bilirubin (statistically non-significant) and urine discoloration in both sexes of group
4, no remarkable findings were noted. Urine discoloration as observed in both sexes of group 4 ranged from a deep-yellow (2 males, 6 females) to light-orange (4 males, 2 females) to orange (2 males) to deep-orange (2 males). Moreover, a deep-yellow urine discoloration was also noted in 4 out of 5 males and 2 out of 5 females of group 3 (200 mg/kg), and in 3 out of 5 males and 1 out of 5 females of group 2 (50 mg/kg).
Recovery period: with the exception of a slightly lower potassium level in males (p<0.05), all of the findings recorded during the treatment period indicated reversibility and/or were similar to those of the controls, and no longer statistically significant. Subsequently, no further plasma or urine discoloration was observed.
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
urinalysis
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Overall study findings
Critical effects observed:
not specified
Conclusions:
The no-observed-adverse-effect level (NOAEL) of FAT 45'176/A was established 1000 mg/kg.
Executive summary:

FAT 45'176/A 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. The study was carried out according to OECD 407 and EU B.7 guideline.

The study comprised 5 animals per group and sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, food consumption and body weights were recorded periodically during the treatment and recovery periods. Ophthalmoscopic examinations were performed at pretest, the end of the treatment and recovery periods.

The oral administration of FAT 45'176/A to Wistar rats at doses of 50, 200 and 1000 mg/kg body weight/day, for 28 days, resulted in no effects on mortality, food consumption, body weight, ophthalmoscopical findings, organ weights, macroscopic and microscopic findings.

Animals of group 4 (1000 mg/kg) showed dark feces from treatment day 13 throughout the treatment period and during the first day of the treatment-free recovery period.

In group 4 (1000 mg/kg) slightly increased uric acid level in females, moderately increased total bilirubin level in both males and females, slightly increased total cholesterol and phospholipid levels in males, slightly increased triglyceride level in females, slightly lower potassium level and slightly increased total protein and globulin levels in males, higher incidence in the score for urine bilirubin were considered to be related to treatment with test article. Plasma discoloration was observed in all animals of group 4 (1000 mg/kg) and urine discoloration in most of the animals of group 4 (1000 mg/kg) and in some animals of groups 3 (200 mg/Kg) and 2 (50 mg/kg). Urine discoloration was reversible after the treatment-free recovery period.

Based on the results of this study, 200 mg/kg FAT 45'176/A was established as the no-observed-effect level (NOEL) - except for urine discoloration - and 1000 mg/kg 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:
GLP compliance and according to OECD guideline

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
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

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
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

FAT 45'176/A 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. The study was carried out according to OECD 407 and EU B.7 guideline.

The study comprised 5 animals per group and sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, food consumption and body weights were recorded periodically during the treatment and recovery periods. Ophthalmoscopic examinations were performed at pretest, the end of the treatment and recovery periods.

The oral administration of FAT 45'176/A to Wistar rats at doses of 50, 200 and 1000 mg/kg body weight/day, for 28 days, resulted in no effects on mortality, food consumption, body weight, ophthalmoscopical findings, organ weights, macroscopic and microscopic findings.

Animals of group 4 (1000 mg/kg) showed dark feces from treatment day 13 throughout the treatment period and during the first day of the treatment-free recovery period.

In group 4 (1000 mg/kg) slightly increased uric acid level in females, moderately increased total bilirubin level in both males and females, slightly increased total cholesterol and phospholipid levels in males, slightly increased triglyceride level in females, slightly lower potassium level and slightly increased total protein and globulin levels in males, higher incidence in the score for urine bilirubin were considered to be related to treatment with test article. Plasma discoloration was observed in all animals of group 4 (1000 mg/kg) and urine discoloration in most of the animals of group 4 (1000 mg/kg) and in some animals of groups 3 (200 mg/Kg) and 2 (50 mg/kg). Urine discoloration was reversible after the treatment-free recovery period.

Based on the results of this study, 200 mg/kg FAT 45'176/A was established as the no-observed-effect level (NOEL) - except for urine discoloration - and 1000 mg/kg as the no-observed-adverse-effect level (NOAEL).

Repeated dose toxicity: Inhalation

Currently no study to assess the repeated dose inhalation toxicity potential of Reactive Orange 136 is available. However, the vapour pressure for the substance can be considered low (5.75 x 10-20 Pa) along with the high melting point (>400 °C). Hence the substance is considered to have low volatility. Synthesis and formulation of this chemical is performed in a closed process; the final product consists of liquid formulations only. 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, based on vapour pressure and/or the likelihood of an exposure to aerosols, particles or droplets. Referring to the expected low volatility of the substance, the fact that the substance is imported into the EU in a formulated form as liquid formulation, the exposure via inhalation is considered to be unlikely. Further, in case the substance is entering the respiratory tract, it will be trapped in the mucus and cleared, thereby further limiting the absorption.No systemic toxicity was observed when Reactive Orange 136 was administered upto 1000 mg/kg bw/day via gavage in a 28 day repeated dose oral toxicity study. 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 upon inhalation exposure and not after systemic exposure. Taking into consideration the above arguments, no elevated toxicity is expected via the 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 Orange 136 is considered to be scientifically not necessary. 

Repeated dose toxicity: Dermal

Currently no study to assess the repeated dose dermal toxicity of Reactive Orange 136 is available. However, the molecular weight of the chemical is 838.7 g/mol, indicating it being too large for dermal absorption. It has water solubility of 330 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. Absence of systemic toxicity in acute dermal, skin irritation and sensitization studies with Reactive Orange 136, 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 sub-acute oral repeat dose toxicity study is available and no systemic toxicity was observed when Reactive Orange 136 was administered upto 1000 mg/kg bw/day via gavage in a 28 day repeated dose oral toxicity study. Taking into consideration the above arguments, no elevated toxicity is expected via the 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 dermal route for Reactive Orange 136 is considered to be scientifically not necessary.

Justification for classification or non-classification

Based on the findings of the repeated dose oral toxicity study, the Reactive Orange 136 does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.