Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-Apr-16 to 1998-May-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
see below

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Gartenstr. 27, D-33178 Borchen
- Age at study initiation: approximately 6 weeks
- Housing: fully air-conditioned rooms in macrolon cages (type IV) on soft wood granulate in groups of 5 animals
- Diet (e.g. ad libitum): ssniff R(M-H (V15354), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: ad least 5 d

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration (in % (w/v)) in vehicle: 0, 1.25, 5, 20 (according to doses of 0, 62.5, 250, 1000 mg/kg bodyweight)
- Amount of vehicle (if gavage): 5 ml/kg bodyweight
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
stability is guaranteed for 15 days according to report of Analytical Toxicology dated Mar. 05, 1998
Duration of treatment / exposure:
29 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
62.5 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5/sex for 62.5 and 250 mg/kg bw
10/sex for 0 and 1000 mg/kg bw
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: acute oral toxicity testing of Reactive Red FC 73270 in rat yielded a LD50 above 2000 mg/kg bw in male and female animales
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
Behavior and state of health
- mortality: twice daily (a.m. and p.m.) during the week; once daily (a.m.) on weekends and public holidays
- behavior and general health: once daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 1/day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before first treatment, 1/week during exposure, at study termination

BODY WEIGHT: Yes
- Time schedule for examinations: before start of exposure, 2/weeks during the 28 d of exposure

FOOD CONSUMPTION:
- Food consumption was determined 2/week and food consumption per 100 g body weight/ d was calculated

WATER CONSUMPTION: Yes
- Time schedule for examinations: water consumption was determined 1/week over a period of 16 h and is given as water consumption/animal/16 h

HAEMATOLOGY: Yes
- Time schedule for collection of blood: termination of the study and for satellite groups after the additional 14 days follow up period
- Anaesthetic used for blood collection: Yes (narcosis with intraperitoneal injection of 67 + 6.7 mg/kg bw ketamine-hydrochloride + xylazine)
- Animals fasted: No
- How many animals: all animals
- Parameters checked: erythrocytes count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHM), leucocyte count, thrombocyte count, differential leucocyte count and red cell morphology, coagulation time
additional in control and 100 mg/kg bw/day groups only: reticulocyte count, Heinz bodies

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: termination of the study and for satellite groups after the additional 14 days follow up period
- How many animals: all animals
- Parameters checked: 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: overnight from day 26 to day 27 as well as from day 35 to day 36
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: appearance, color, volume, specific weight, pH-Value, hemoglobin, protein, glucose, bilirubin, urobilinogen, ketone bodies
additionally in control and 1000 mg/kg bw/day groups only: sediment

-OTHER: neurotoxicological examination, including "open field" observation, assessment of sensory function, motor activity, forelimb and hindlimb strength
at study termination additionally: sensory stimuli (auditory, visual and proprioceptive) including startle reflex (click response), response to approach with the finger to the nose of the animal and righting reflex; pupillary constriction
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After exsanguination, all animals were necropsied. Skin, orificed, eyes, teeth, oral mucosa and internal organs were macroscopically examined.
The lungs, including part of the trachea were removed and then fixed endotracheally with formalin solution.
Organ weights and organ to body weight ratios were measured/calculated for: heart, liver, kidneys, adrenal glands, spleen, testes, epididymides, thymus and brain

HISTOPATHOLOGY: Yes
following tissues or organs (or pieces of them) were preserved in a suitable fixative: heart, lungs, liver, spleen, kidneys, stomach, jejunum, colon, brain, thymus, trachea, thyroid glands with parathyroid glands, testes, epididymides, ovaries, uterus, urinary bladder, lymph nodes iliac, lymph nodes mandibular, adrenal glands, prostate gland, bone marrow (sternum), N. ischiadicus, spinal cord (cervical)
Statistics:
The following parameters were compared statistically with the control group values at the level of significance p=0.05:
body weight at the designated measurement times, hematological data, clinical chemistry parameters, urine analysis (volume, pH-value and specific weight), absolute organ weights and organ to body weight ratios, neurotoxicological measurements (motor actitvity, forelimb and hindlimb grip strength)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight development of the males was not influenced by the administration of the test compound. In the females of the intermediate and high dose groups, body weights were slightly higher from day 12 of the study through to the end of the treatment period when compared with the concurrent control group. The difference between body weights of high dose and control group female recovery animals at the end of the treatment period did not change during the 14 day recovery period.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological examinations at the end of dosing revealed a slight increase in thrombocyte counts of the high dose group males which is due to an incidentially low value in control males and was not seen in recovery animals
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the dosing period: The high dose group males showed a statistically significant decrease in calcium, and in the intermediate and high dose group males,
statistical evaluation revealed increased UREA and ASAT values when compared with the control group values. In the females, statistical evaluation showed a significant decrease in the calcium value for the high dose group and in the intermediate and high dose group, increases in inorganic phosphorus and albumin values were noted. Additionally, total bilirubin and cholesterol values were slightly increased in the high dose group females.
At the end of the recovery period, the males of the high dose group showed a statistically significant increase in cholesterol and the females in this group a
statistically significant increases in the albumin value when compared with the corresponding control group value.
All the deviations from control of clinicochemical parameters in the dosed groups at the end of the treatment and at the end of the recovery period as described above were only slight and the values still within the physiological range of rats. In addition, histological examination did not reveal any correlating organ damage. Therefore, these findings are considered not to be compound-related.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Reversible discoloration of the urine was due to the staining properties of the dye but without any toxicological relevance.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Neurotoxicological measurements including „open field" observations, assessment, of sensory function, motor activity and forelimb and hindlimb grip strength were not influenced by the administration of the test compound in all groups.
The result of the statistical evaluation showing a significant increase in motor activity of high dose males and a significant decrease in motor activity of intermediate dose females is considered a coincidental finding without any toxicological relevance. The values varied considerably, were contradictive in males and females and in addition, the result in females was not dose-dependent.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Discoloration of the kidneys due to the staining properties of the dye but without any toxicological relevance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologically, acute inflammatory cell infiltration with slight secretory disorder of the stomach mucous membrane (fundus) was found in high dose group males and females. This change was not accompanied by destructive processes and, as the recovery test shows, disappeared within an appropriate time after discontinuation of treatment. This finding is a local reaction diue to the mode of administration of s high salt/substance load and not toxicological relevant.
There were no other histological organ changes which could be related to the administration of the test compound and in particular, there was no histopatholoical
finding which could be related to the red discoloration of the kidneys.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths were encountered. Behavior and state of health remained unaffected by the administration of the test compound.

NEUROTOXICOLOGICAL EXAMINATIONS
Neurotoxicological measurements including ,open field" observations, assessment of sensory function, motor activity, forelimb and hindlimb grip strength were not influenced by the administration of the test compound in all groups.


BODY WEIGHT AND WEIGHT GAIN
Body weight development in the intermediate and high dose group females appeared to be dose-dependently accelerated, but were of no toxicological relevance. Males in all treated groups did not deviate from that of the control group.

HAEMATOLOGY
Hematological examinations at the end of dosing revealed a slight statistically significant increase in thrombocyte counts of the high dose group males which is due to an incidentially low value in control males and was not seen in recovery animals


CLINICAL CHEMISTRY
Observed changes were considered to be of no toxicological relevance, since:
- significantly decreased calcium, only observed in the male and female higher dosage group missed hematological or histological correlate findings.
- increased urea and ASAT values in the males in the intermediate and high dosage
- other changes in blood parameters (inorganic phosphorus and albumin) were increases,only observed in the female intermediate and higher dosage group
- changes were only marginal.

URINALYSIS
Urine showed a treatment-related reversible salmon pink discoloration in one intermediate dose group female and in all high dose female. Reversible discoloration of the urine was considered to be treatment-related but without any toxicological relevance.

ORGAN WEIGHTS
showed no compound-related changes in any of the dose groups.

GROSS PATHOLOGY
red discoloration of the kidneys was found in in all high dose group males and 2 intermediate and all high groups females at the end of the treatment periode and in all high dose group males and females at the end of the recovery period. There was no histopathological finding which could be related to the red discoloration of the kidneys.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological, acute inflammatory cell infiltration with slight secretory disorder of the stomach mucous membrane (fundus) was found in high dose group males and females. This change was not associated with destructive processes and reversible. It is caused by the method of administration of a high substance-/sal-load and is not considered toxicologically relevant.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: substance-related discolouration of plasma, urine and tissues/organs in mid and high dose animals due to the staining effect of the dye
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were seen. The only substance related effects were staining of organs/tissues which is due to the intended use as a textile dye and not toxicologically relevant.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Repeated doses of Reactive Red FC 73270 caused salmon pink discoloration of the urine and red discoloured kidneys. This discoloration was not associated with toxicity and reversible, though not completely within the period of 14 d recovery. 1000 mg/kg bw/d is therefore considered to be the NOAEL.
Executive summary:

Groups of male and female rats received Reactive Red FC73270 by oral gavage at dose levels of 0, 62.5, 250 and 1000 mg/kg body weight per day for 28 days. On day 29 five males and five females from each group were killed and necropsied. The remaining five animals in each of the control and high dose group were killed and necropsied after a recovery period of 14 days.

Behaviour and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly, and water consumption once weekly. Once before the first treatment and thereafter once a week detailed clinical observations were performed in all animals outside the home cage in a standard arena („open field"). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa, and impairment of dental growth. Neurotoxicological measurements including assessment of sensory function, motor activity, forelimb and hindlimb grip strength were conducted at the end of the treatment period. Haematological examinations, clinical chemistry and urine analysis were carried out at the end of the treatment period and after the recovery period. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs weighed and the organ to body weight ratios calculated. Many organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, haematological and clinical chemistry data, urine data (pH-value, volume, specific weight), absolute and relative organ weights, neurotoxicological measurements (motor activity, forelimb and hindlimb grip strength) were analysed with the aid of a statistical program to show differences compared to the controls.

No deaths occurred throughout the study. Behaviour, state of health, neurotoxicological parameters, food and water consumption remained unaffected by the administration of the test compound in all dose groups.

Body weight development of the males in all treated groups did not deviate from that of the control group. Body weight development in the intermediate and high dose group females appeared to be dose-dependently accelerated, which however was considered to be of no toxicological relevance. Haematological examinations showed a slight statistically significant increase in thrombocyte counts in males, which was considered to be due to the low values in control males. Clinical chemistry examinations revealed no compound-related changes in any dose group, but the urine showed a treatment-related reversible salmon pink discoloration in one intermediate dose group female and in all high dose group males and females, which is due to excretion of the test substance and its metabolites, which is a water soluble dye. This, however, was considered to be of no toxicological relevance. Organ weights showed no compound-related changes in any of the dose groups

At necropsy, compound-related red discoloured kidneys were observed in all high dose group males and in two intermediate and all high dose group females at the end of the treatment period and in all high dose group males and females at the end of the recovery period. There was no histopathological finding which could be related to the red discoloration of the kidneys, which is again due to the staining effect of the test substance.

Histopathologically, the test compound caused an acute inflammatory cell infiltration with slight secretory disorder of the stomach mucous membrane (fundus) in high dose group males and females. This change was due to the mode of test substance administration by means of a stomach tube and therefore not considered toxicologically relevant. Furthermore, it was not associated with destructive processes and reversible. There were no other histological organ changes which could be related to the administration of the test compound.

In conclusion, repeated oral administration of Reactive Red FC73270 (28 times during 29 days) at the dose level of 1000 mg/kg body weight/day did not cause any toxicological relevant adverse effects. The only test substance-related effects seen at the two highest dose level were a salmon pink discoloration of the urine and a red discoloration of the kidneys which was due to the dye nature of the test substance and not associated with organ toxicity and reversed after cessation of treatment.

No test substance-related effect was observed at the dose level of 62.5 mg/kg body weight/day, which is the 'No Observed Effect Level' (NOEL). With regard to the present study the 'No Observed Adverse Effect Level' (NOAEL) is 1000 mg/kg body weight/day.