Registration Dossier

Administrative data

Description of key information

As only adverse effect, a local irritation in the stomach of rats was observed due to the high local concentration of the dye as tetrasodium salt. This effect is not deemed relevant for human risk assessment, as this kind of test substance administration will not occur in humans.

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:
05 February 1998 to 05 March 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recent EU & OECD test guidance in compliance with GLP.
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
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:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HARLAN WINKELMANN
- Age at study initiation: approximately 5-6 weeks
- Weight at study initiation: No data
- Fasting period before study: No
- Housing: in fully air-conditioned rooms in macrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet (e.g. ad libitum): ssniff R/M-H (V 1534) ad libitum, except for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water in plastic bottles ad libitum, except for the period in which the animals were kept in diuresis cages
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (± 3°C)
- Humidity (%): 50 (± 20)
- Photoperiod (hrs dark / hrs light): 12 hours daily

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionised
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

The test compound was dissolved homogeneously in the vehicle at the following dates:

1. February 05, 1998
2. February 19, 1998
After each measurement of the body weight, the calculation of the application volume was repeated.

Dose Concentration Volume applied Vehicle
(mg/kg b.w.) in % (w/v) ((mg/kg b.w.)

0.0 0.00 5 deionized water
62.5 1.25 5 deionized water
250.0 5.00 5 deionized water
1000.0 20.00 5 deionized water

Route of application: Orally by gavage
Vehicle: Deionized water
Schedule: 28 applications within 29 days, 7 days per week
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not measured. Based on the stability and homogenity of the test substance in water for 14 days reactions with water seems to be unlikely. The test substance in water can be assumed as stable for 15 days.
Duration of treatment / exposure:
28 applications within 29 days
Frequency of treatment:
7 days per week
Remarks:
Doses / Concentrations:
62.5 mg/kg body weight/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250.0 mg/kg body weight/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000.0 mg/kg body weight/day
Basis:
actual ingested
No. of animals per sex per dose:
Group Dose (mg/kg b.w.) Number of animals Animal number
male female male female
1 0.0 5 5 01-05 21-25
2 62.5 5 5 06-10 26-30
3 250.0 5 5 11-15 31-35
4 1000.0 5 5 16-20 36-40
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): Random. At the beginning of the acclimatization period, the test animals were randomized and assigned to the following groups according the randomization tables
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable.
- Section schedule rationale (if not random): Random
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
The behavior and general health condition of the animals were observed once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first treatment and thereafter once a week

BODY WEIGHT: Yes
- Time schedule for examinations: Before the start of the study and then twice weekly throughout the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was determined continuously (2 times per week). The values on the printouts refer to the intervals between one measurement and the next. They are converted to the food consumption per 100 g body weight over a 24 hour period.

WATER CONSUMPTION : Yes
- Time schedule for examinations: once weekly over a period of 16 hours and is given in the results as water consumption / animal /16 h (from approx. 3.00 p.m. to 7.00 a.m.)

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study
- Anaesthetic used for blood collection: No.
- Animals fasted: No

At the termination of the study, hematological examinations were performed on all animals without previous withdrawal of food. Blood samples were taken from the retrobulbar venous plexus in narcosis ((intraperitoneal injection of approx. 67 + 6.7 mg/kg body weight Ketamin-Hydrochloride + Xylazin). In order to prevent systematic errors, blood sampling was conducted in a randomized order.

The following hematological parameters were determined:

Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Leucocyte count
Thrombocyte count
Differential leucocyte count and red cell morphology
Reticulocyte count
Heinz bodies
Coagulation time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After blood sampling for hematological testing, the animals were killed by section of the vena cava cranialis in deep narcosis and exsanguinated.
- Animals fasted: No

After blood sampling for hematological testing, the animals were killed by section of the vena cava cranialis in deep narcosis and exsanguinated. In order to prevent sys¬tematic errors, exsanguination was conducted in a randomized order.

The following serum values were determined:

Sodium
Potassium
Inorganic phosphorus
Uric acid
Bilirubin total
Bilirubin direct
Creatinine
Glucose
Urea
Calcium
Chloride
Aspartate aminotransferase (ASAT/GOT)
Alanine aminotransferase (ALAT/GPT)
Alkaline phosphatase (AP)
Gamma-glutamyltranspeptidase (GGT)
Cholesterol
Triglycerides
Total protein
Albumin


URINALYSIS: Yes
- Time schedule for collection of urine: Urine analysis was performed on all animals a few days before termination of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

Urine analysis was performed on all animals a few days before termination of the study. For this purpose, the urine was collected by using metabolism cages (overnight from day 27 to day 28). Food and water were withdrawn during this period.

The following parameters were checked:

Appearance
Color
Volume
Specific weight
pH-Value
Hemoglobin
Protein
Glucose
Bilirubin
Urobilinogen
Ketone bodies
Sediment (high dose and control groups only)

NEUROBEHAVIOURAL EXAMINATION: Yes
Once before the first treatment and thereafter once a week detailed clinical observa¬tions were performed in all animals outside the home cage in a standard arena („open field"). Each animal was assessed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, salivation, nasal discharge, pile-erection, pupil size, and unusual respiratory pattern. Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, tremor, and any any other abnormal motor movements (such as excessive grooming, repetitive circling or other stereotypies) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. In addition, defecation and urination was also evaluated.
At the termination of the study sensory reactivity to stimuli of different types (auditory, visual, and proprioceptive) was evaluated including startle reflex (click response), response to approach with the finger to the nose of the animal, and righting reflex. The presence and absence of pupillary constriction was assessed using a pen flashlight directed into the eye. Assessments of motor function were performed including measurement of motor activity, and forelimb and hindlimb grip strength. The animals were evaluated for motor activity during a 60-minute period in an 16-station automated motor activity monitoring device (FMI, Fohr Medical Instruments GmbH). Activity counts were recorded by the interruption of photocells in 3-minute-intervals to give a total of 20 intervals. Fore- and hindlimb grip strength were measured by a strain gauge device (FMI, Fohr Medical Instruments GmbH).



Sacrifice and pathology:
NECROPSY AND MICROSCOPIC EXAMINATIONS:

After exsanguination, all animals were necropsied and checked for macroscopically visible abnormalities.

The lungs were fixed endotracheal with a formalin solution using a needle inserted into the trachea. The instillation pressure was between 20 and 30 cm water column. Following completion of the endotracheal fixation the lungs were fixed, together with the other organs, in formalin solution.
The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.

All abnormal findings were recorded.

HISTOPATHOLOGY:

The following tissues or organs (or pieces of them) were preserved in a suitable fixa¬tive and processed for histopathological investigations:
Heart
Lungs
Liver
Spleen
Kidneys
Stomach
Jejunum
Colon
Brain
Thymus
Trachea
Thyroid glands
Testes
Epididymides
Ovaries
Uterus
Urinary bladder
Lymph nodes iliac
Lymph nodes mandibular
Adrenal glands
Prostate gland
Bone marrow
N. ischiadicus
Spinal cord (cervical)
Other examinations:
None
Statistics:
The following parameters were compared statistically with the control group values at the level of significance p = 0.05:

Body weights at the designated measurement times
Neurotoxicological measurements
Hematological data
Clinical chemistry parameters
Urine analysis (Volume, pH-value and specific weight)
Absolute organ weights and organ to body weight ratios
Evaluation was performed by IS Research, HMR Deutschland GmbH with the aid of a program package for the evaluation of toxicological studies. The calculation methods used are referred to on the computer printouts.

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):
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 examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Inflammatory reaction in the submucosal and muscular mucosal layer of the glandular stomach. Considered as a reversible adaptive response in connection with the route of excretion, not a toxicological effect of the test substance.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
No compound-related deaths occurred throughout the study. Behavior and state of health remained unaffected by the administration of the test compound in all dose groups.

Opacity of the refracting media of the eyes and changes of the oral mucosa were not observed.

BODY WEIGHT AND WEIGHT GAIN:
Body weight development of the animals was not impaired by the administration of the test compound and was comparable in all groups with the exception of a statistically significant increase on study days 8 and 12 in males of the high dose group. This effect is not considered to be of toxicological relevance since the other values of the high dose group, especially at the termination of the study, were comparable to the control values.

FOOD CONSUMPTION:
Absolute and relative food consumption of the animals remained unaffected by the administration of the test compound throughout the study.

WATER CONSUMPTION:
The administration of the test compound did not alter the water consumption

HAEMATOLOGY:
Hematological examinations revealed a statistically significant increase in coagulation time in females of the high dose group. Additionally, males of the high dose group showed decreases in thrombocytes count.

The changes in thrombocytes count were within the physiological range for rats and thus, are considered not to be compound-related. The increase in coagulation time is also not considered to be compound-related since no other red blood cell parameters were adversely influenced.

CLINICAL CHEMISTRY:
Statistical evaluation revealed increases in inorganic phosphorus levels in males of the high dose group and decreases in albumin, calcium and alkaline phosphatase levels in females of the high dose group.

In all cases the values were within the physiological range of rats. Therefore, a com-pound-related effect is not evident.

URINALYSIS:
The specific weight of the urine was significantly increased in males of the high dose group.

The sediments were inconspicuous.

The increase in specific weight is not considered to be of toxicological relevance since no other parameters were adversely affected, and especially no changes in kidney function were found. This finding is presumably a consequence of the reduced urine volume.

ORGAN WEIGHTS:
Statistical evaluation revealed increased absolute spleen weights in males of the high dose group. In females of the high dose group increased relative thymus weights were observed.

The values of the spleen weights were within the normal range of rats. Thus, the changes are not considered to be compound-related. The decreases in relative thymus weights in females of the high dose group were slightly beyond the normal range. Since the effect was not dose-dependent and the control values were also at the lower bound of the historical range, this finding is not considered to be compound-related.

GROSS PATHOLOGY:
At necropsy, no organ alterations attributable to the administration of the compound were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Histopathological examinations revealed an inflammatory reaction in the submucosal layer and beyond the muscular mucosal layer of the glandular stomach in animals of all dose groups with dose-dependent increase in severity and incidence in the intermediate and high dose group. Thus, a compound-related effect cannot be excluded.
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
Conclusions:
In conclusion, the substance caused an increase in severity and incidence of an inflammatory reaction under the glandular layer of the stomach in male and female rats when administered 28 times during 29 days at the dose level of 1000 mg/kg body weight per day.

At the dose level of 250 mg/kg body weight per day there was still an increase in severity and incidence of an inflammatory reaction under the glandular layer of the stomach in male and female rats compared to the control animals.

At the dose level of 62.5 mg/kg body weight incidence and severity of the inflammatory reaction in the glandular stomach was comparable to the control group.

In study reference „8.6 98-0696 Amendment Patho deutsch zu Bericht 98-0437 28-Day Study“ it is proposed that inflammatory reaction in the submucosal layer and beyond the muscular mucosal layer of the glandular stomach in animals of all dose groups could be considered as reversible, following further evaluation of the pathological data provided. This is based on the fact that a number of parameters associated with being a permanent toxicological effect are not noted within the study groups, given that independent effects such as suspect hyperplastic changes and cell proliferation do not occur on a dose dependant basis, and as such there is no evidence that such changes could evolve on the basis of the data available. It is proposed that the pathological findings in a possible follow-up study are likely to be reversible if this were to be conducted (although on animal welfare grounds this would not be conducted).

Therefore it is considered that the effects may be considered an adaptive response in connection with the route of excretion rather than a toxicological effect of the test substance. However, without further evidence, the NOAEL of 62.5 mg/kg/day will be applied to the substance.
Executive summary:

Study conducted to EU test guidance 92/69/EEC B7 and OECD test guideline 407 in compliance with GLP.

The substance caused an increase in severity and incidence of an inflammatory reaction under the glandular layer of the stomach in male & female rats at the highest dose level (1000 mg/kg body/day).

At the dose level of 250 mg/kg body weight/day there was still an increase in severity and incidence of an inflammatory reaction under the glandular layer of the stomach in male & female rats compared to the control animals.

At the dose level of 62.5 mg/kg body weight/day incidence and severity of the inflammatory reaction in the glandular stomach was comparable to the control group.

It is considered that the effects may be considered an adaptive response in connection with the route of excretion rather than a toxicological effect of the test substance. However, without further evidence, the NOAEL of 62.5 mg/kg/day will be applied to the substance. No classification and labelling is applicable however.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
62.5 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 only effects attributed to the substance in a 28-day subacute oral toxicity caused an increase in severity and incidence of an inflammatory reaction under the glandular layer of the stomach in male and female rats when administered 28 times during 29 days at dose levels of 1000 and 250 mg/kg body weight per day. At the dose level of 62.5 mg/kg body weight incidence and severity of the inflammatory reaction in the glandular stomach was comparable to the control group.

However it is proposed that inflammatory reaction in the submucosal layer and beyond the muscular mucosal layer of the glandular stomach in animals of all dose groups could be considered as reversible, following further evaluation of the pathological data associated with the substance. This is based on the fact that a number of parameters associated with being a permanent toxicological effect are not noted within the study groups, given that independent effects such as suspect hyperplastic changes and cell proliferation do not occur on a dose dependant basis, and as such there is no evidence that such changes could evolve on the basis of the data available. It is proposed that the pathological findings in a possible follow-up study are likely to be reversible if this were to be conducted (although on animal welfare grounds this would not be conducted).  

Therefore it is considered that the effects may be considered an adaptive response in connection with the route of excretion rather than a toxicological effect of the test substance. However, without further evidence, the NOAEL of 62.5 mg/kg/day will be applied to the substance.

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. Furthermore the results of laboratory animal studies show negligible acute dermal toxicity. In the 28 - days repeated dose study via oral gavage, administration does not exacerbate systemic toxicity effects which suggest bioavailability is low, thereby there is low toxicity potential. This intrinsic property/toxicity potential can therefore be extrapolated to repeated dermal and inhalation routes of administration. Further studies for these endpoints are therefore not appropriate both on predictive toxicology and animal welfare grounds.


Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach

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.