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Administrative data

Description of key information

The No Observed Effect Level (NOEL) is 1000 mg/kg body weight/day for male and 250 mg/kg body weight day for female animals, due to renal re-absorption of dye particles at 1000 mg/kg body weight/day. However, no adverse effects resulted from this re-absorption. Consequently, as there was no evidence of a toxic effect in male and female rats after administration of up to the highest dose administered, the NOAEL was considered to be 1000 mg/kg body weight/day.

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1. Feb to 1 Mar 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU test guidance in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG
- Strain: Hoe:WISKf (SPF71)
- Age at study initiation: approx. 6 weeks
- Housing: 5 rats/cage
- Diet (ad libitum): Altromin 1324
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 50±20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Preparation: daily
- Concentration in vehicle: 1.2.5, 5, 20%
- Amount of vehicle (if gavage): 5 ml/kg

Route of administration: oral by gavage
Vehicle: deonized water
Number of applications: 28 in 29 days
Frequency of application: a total of 28 applications, 7 days per week
Time of application: between 9.30 and 12.00 a.m.
Frequency of test compound preparation: daily, immediately prior to application
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Single exemplary analysis of formulation concentrations was conducted during the study after application.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
Remarks:
Doses / Concentrations:
62.5, 250, 1000 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Reaktiv-Rot F-66 813 FW was administered orally by gavage to SPF-Wistar rats over a period of 29 days (a total of 28 applications, 7 days a week) at dose levels of 0.62.5.250 or 1000 mg kg body weight. Behavior and general state of health were examined in all study groups. Body weights and food consumption were recorded twice a week, water consumption once weekly.
Hematology, clinical chemistry and urinalysis were performed at study end. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Relevant organs of the animals were processed for histopathological examination and checked for microscopically visible changes.
Body weights, hematological and clinical chemistry data, albumin and globulin values, urine data (pH-value and specific weight) as well as the absolute and relative organ weights were analyzed with the aid of a statistical program to show differences compared to control groups.
Positive control:
Not assessed.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

Behavior and general state of health

Behavior and general state of health of all animals were checked twice dailyI on weekends and public holidays once daily. All rats were weekly examined for neurological disorders, turbidity of the refracting media of the eyes, lesions of the oral mucosa and disturbances of the dental growth.

Bodyweight

Body weight of all animals was recorded once prior start of study and twice weekly thereafter

Food and Water consumption

Food consumption was calculated continuously (2 measurements / week). The printed measurements refer to the intervals between the body weight measurements. They are calculated as food consumption /100g body weight /day.

Water consumption was measured once weekly. It is expressed as consumption /100g body weight within 16 hours (15.15 p.m. -7.15 a.m.).

Hematology

At study end the hemogram was examined in all male and female animals without previous withdrawal of food. Blood was sampled from the retrobulbar venous plexus. In order to avoid systematic errors blood sampling was conducted according to a Randomization scheme
The following hematological parameters were determined:

Erythrocyte counts
Hemoglobin
Hematocrit
Leukocyte counts
Thrombocyte counts
Differential blood count
Reticulocyte counts*
Heinz Body counts*
Coagulation Time

* These parameters were only evaluated in the animals from the control and 1000 mg/kg/d group.

Furtheron the values for MCV, MCH, and MCHC were calculated.

Clinical chemistry

After blood sampling from the retrobulbar venous plexus for hematological investigations, the animals were killed in Nembutal narcosis (injection of approx. 50 mg/kg Lp.) by cutting of the vena cava cranialis and exsanguination. In order to avoid systematic killing and exsanguination was conducted randomly according to a Randomization scheme.

The following Clinical chemistry parameters were examined:
Sodium
Potassium
Inorganic phosphorous
Uric acid
Bilirubin total
Creatinine
Glucose in serum
Urea-N (Bun)
Calcium
Chloride
ASAT (GOT)
ALAT (GPT)
Alkaline phosphatise (AP)
Gamma-Glutamyltranspeptidase
Total Protein
Albumin and Globulin

Urinalysis
The urine was collected in metabolism cages overnight from study day 26/27 (approx. 16 h) from non-fed and non-watered animals. Urinalysis was performed in all male and female animals and consisted of the following parameters:

Color
pH-Value
Hemoglobin
Protein
Glucose
Keton bodies
Bilirubin
Uribilinogen
Specific weight
Sediment*

* These parameters were only evaluated in the animals from the contral and 1000 mg/kg/d group as weil as in one male animal of the 250 mg/kg group which showed Hemoglobin in urine.
Sacrifice and pathology:
Necropsy and macroscopic observations

After killing and exsanguination the animals were necropsied. The autopsy included the macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs. All abnormal findings were recorded.

Organ weights

The following organs were weighed and the organ to body weight (100g) ratio calculated:

Heart Spleen Lung Testes Liver Adrenals Kidneys

Histological examinations

The following organs or pieces of them were submitted into fixative according to Dissection

Heart
Jejunum
Lung
Colon
Liver
Thymus
Kidneys
Testes
Spleen
Adrenals
Stomach
Bone marrow (Femur)
Other examinations:
None
Statistics:
The following parameters were evaluated for statistically significant differences in comparison to the control (p= 0.05):

Body weight at each specific measurement point
Body weight course from study day -2 to 29
Hematological parameters (except Differential Blood Count)
Clinical Chemistry parameters (except GGT)
Albumin and Globulin values
Organ weights. absolute and relative
pH-Value and specific weight of urines

The evaluation was performed with the aid of a program package for the evaluation of toxicological studies, according to the Standard Operating Procedure by Dr. Passing, Department of Applied Mathematics, Department of Pharma Research Informatics.
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):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Behaviour and general state of health and lethality
Behaviour and general state of health of the animals remained unobtrusive during the whole course of the study. No neurological disorders, opacity of the refracting media of the eyes, disturbances of the dental growth or alterations in the oral mucosa were detected which are related to the administration of the test compound. Faeces of animals of the 1000 mg/kg test group were red discolored, beginning with day 6. There were no unscheduled deaths throughout the study.

Body weight development
Statistical evaluation of the body weight development revealed no deviations compared to the control.

Food and water consumption
Absolute and relative food consumption remained unaffected by the test compound in all dose groups throughout the study.
Relative water consumption also remained unimpaired by the administration of the test compound.

Hematology
Hematological examinations resulted in male animals of the 62.5 mg/kg group in statistically significant decreased coagulation times. Due to a lack of dose dependency a compound related effect can be excluded. Differential blood counts showed no abnormal deviations in both sexes.

Clinical chemistry
Statistical evaluation of clinical chemistry parameters resulted in male animals of the 250 mg/kg group in statistically significant increased sodium values. Furtheron, inorganic phosphorus, uric acid, serum glucose and alkaline phosphatase values were decreased in female rats of the 62.5 mg/kg group and calcium values in the 250 mg/kg group. In all cases, the values were within the physiological range of the used rat strain and did not show a dose dependency. Hence, they were considered to be not compound related.
Urea values were statistically significant increased in female rats of the 250 and 1000 mg/kg group. Again, the individual data were within the physiological range of the used rat strain and a compound related effect is supposed to be unlikely.
Serum electrophoresis resulted in increased alpha 2 globulin values in female rats of the 250 mg/kg group and slightly decreased beta 1 globulin values in the 250 and 1000 mg/kg group.
As a result of the minor changes and the missing dose dependency, a correlation with the administration of the test compound is not obvious.

Urinalysis
Urinalysis remained inconspicuous. Only in the high dose group a discoloration of the urine was observed.

Organ weights
Relative organ weights of the lungs were decreased in male rats of the 250 mg/kg group but, due to the absence of a dose dependency a compound related effect is considered to be unlikely.

Macroscopic and microscopic findings

Macroscopic examination showed isolated pyelectasis. The kidneys of the animals of the 1000 mg/kg group were red discolored, and additionally the testes of the male animals. One animal's testes were atrophied.

Microscopic examination revealed resorption vacuoles with a homogeneous content in the tubular epithelial cells of the convoluted segment of the proximal tubulus of the renal cortex in two female animals of the highest dose group. This finding indicates a of the dye from the primary urine and its intracellular Iysosomal deposition.

A deposition of substance in the kidneys of a kind as described was not detected in the remaining animals of this dose group. Likewise, a deposition of the dye in the tissue of the testes was not detected
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects observed at any dose-level
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: histopathology - renal re-absorption of dye at 1000 mg/kg bw/day- no toxicological adverse effect
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 1000 mg/kg: Discoloration of exctreta and organs. Renal re-absorption of dye in females - not considered to be toxicological relevant adverse effects.
Critical effects observed:
not specified
Conclusions:
The oral administration of Reaktiv-Rot F-66813 FW at a dose of 1000 mg/kg body weight/day for 4 weeks resulted in microsopically visible re-absorption of the dye in the kidneys of two female animals. There was no evidence in clinical chemistry and histopathology of an adverse effect on the renal function. The administration of the test compound in a dose of 1000 mg/kg body weight/day did not induce any evident relevant compound-related changes in male animals. The NOAEL was therefore considered to be 1000 mg/kg bw/day.
Executive summary:

Reaktiv-Rot F-66 813 FW was administered orally by gavage to SPF-Wistar rats over a period of 29 days (a total of 28 applications, 7 days a week) at dose levels of 0.62.5.250 or 1000 mg kg body weight. Behavior and general state of health were examined in all study groups. Body weights and food consumption were recorded twice a week, water consumption once weekly.

Hematology, clinical chemistry and urinalysis were performed at study end. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Relevant organs of the animals were processed for histopathological examination and checked for microscopically visible changes.

Body weights, hematological and clinical chemistry data, albumin and globulin values, urine data (pH-value and specific weight) as well as the absolute and relative organ weights were analyzed with the aid of a statistical program to show differences compared to control groups.

 

Behavior, general state of health, body weight development as well as food and water consumption remained unaffected by the administration of the test compound. Feces of animals of the 1000 mg/kg test group were red discolored, beginning with day 6.

There was no evidence for compound-related toxicity in hematology and clinical chemistry. Urine was red-brown discolored in all animals of the 1000 mg/kg test group.

Evaluation of absolute and relative organ weights showed no compound-related effects.

Necropsy revealed red discoloration of the kidneys in all animals of the 1000 mg/kg test group. Additionally, the testes of the male animals were red discolored. Vacuolization of the epithelial cells of the renal cortex was microscopically observed in two female animals of this test group which are considered to be a result of an increased re-absorption and subsequent deposition of the test compound.

Summarizing, the 29-day oral administration of Reaktiv-Rot F-66813 FW at a dose of 1000 mg/kg body weight/day resulted in two female animals in microsopic renal findings as described above. There was no evidence in clinical chemistry and histopathology of an adverse effect on the renal function. The administration of the test compound in a dose of 1000 mg/kg body weight/day did not induce any evident relevant compound-related changes in male animals. The NOAEL was therefore considered to be 1000 mg/kg bw/day.

With regard to the present study the No Observed Effect Level (NOEL) is 1000 mg/kg body weight/day for male and 250 mg/kg body weight day for female animals.There was no clear evidence of a toxic effect in female animals even after administration of 1000 mg/kg body weight / day.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

The subacute oral toxicity study (28 applications within 29 days) produced the following effects; faeces of animals of the 1000 mg/kg test group were red discoloured, beginning with day 6. Urine was red-brown discoloured in all animals of the 1000 mg/kg test group. Necropsy revealed red discoloration of the kidneys in all animals of the 1000 mg/kg test group. Additionally, the testes of the male animals were red discoloured. Vacuolization of the epithelial cells of the renal cortex was microscopically observed in two female animals of this test group which are considered to be a result of an increased re-absorption and subsequent deposition of the test compound. Summarising, the 29-day oral administration of the substance at a dose of 1000 mg/kg body weight/day resulted in two female animals in microscopic renal findings as described above. There was no evidence in clinical chemistry and histopathology of an adverse effect on the renal function. The administration of the test compound in a dose of 1000 mg/kg body weight/day did not induce any evident relevant compound-related changes in male animals. The NOAEL was therefore considered to be 1000 mg/kg bw/day. With regard to the present study the No Observed Effect Level (NOEL) is 1000 mg/kg body weight/day for male and 250 mg/kg body weight day for female animals. There was no clear evidence of a toxic effect in female animals even after administration of 1000 mg/kg body weight / day.

Furthermore, it is considered that the substance is unlikely to be inhaled and the physicochemical and toxicological properties suggest low potential for significant rate of absorption through the skin. In addition the results of laboratory animal studies demonstrated negligible no acute dermal toxicity. In the 28 - days repeated dose study via oral gavage, administration does not exacerbate systemic toxicity effects which suggest any toxicity associated with bioavailability to be low, thereby there is low toxicity potential. This intrinsic property/toxicity potential can therefore be extrapolated to repeat dermal and inhalation routes of administration. Further studies for these endpoints are therefore not appropriate both on predictive toxicology and animal welfare grounds.

The test substance appears to be absorbed from the gastrointestinal tract however a significant bioaccumulation potential can most probably be excluded due to the marked hydrophilic properties and lack of solubility in fat, plus the lack of toxicity observed in the study. From the mutagenicity assays it appears that the test substance is not metabolised toward genotoxic structures. Review of the available data indicates that the substance does not exhibit conspicuous toxicokinetic behaviour. The results from all studies with dermal exposure indicate that the test substance has insignificant or no dermal absorptive potential. Bioaccumulation of the test substance can therefore most probably be excluded from all routes of exposure.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. As the effects are considered adaptive rather than toxicological, no classification is proposed.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for prolonged effects is therefore required.