tetrasodium 3-amino-4-{[4-({4-fluoro-6-[phenyl(2-{[2-(sulfonatooxy)ethyl]sulfonyl}ethyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-5-hydroxynaphthalene-2,7-disulfonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Oct. 19, 2004 to Nov. 30, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

457-630-8

EC Name:

-

Cas Number:

2138836-26-5

Molecular formula:

Hill formula: C29H23FN8Na4O16S5

IUPAC Name:

tetrasodium 3-amino-4-[(1E)-2-[4-({4-fluoro-6-[phenyl({2-[2-(sulfonatooxy)ethanesulfonyl]ethyl})amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazen-1-yl]-5-hydroxynaphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat has proved to be a suitable species for subacute oral toxicity testing with many different substances and is the species of choice according to the international guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, D-33178 Borchen
- Age at study initiation: approximately 6 weeks
- Housing: In transparent macrolon® cages (type IV) on soft wood granulate in an air-conditioned rooms, 5 animals per cage, separated according to sex
- Diet: ssniff® R/M-H (V 1534) ad libitum, except for the period in which the animals were kept in diuresis cages
- Water: Tap water in plastic bottles ad libitum, except for the period in which the animals were kept in diuresis cages
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 50 ± 20%
- Photoperiod: 12 h light / 12 h dark cycle

IN-LIFE DATES: From: Oct. 19, 2004 To: Nov. 30, 2004

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route is considered to be a potential exposure route in man and provide the most reliable results for systemic toxicity.

Vehicle:

water

Remarks:

deionized

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 d (test groups) and 42 d (recovery groups)

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Acute oral toxicity testing of the test substance at a dose of 2,000 mg/kg in the rat (limit test) showed that the median lethal dose (LD50) is above 2,000 mg/kg bw in female animals. The dose of 2,000 mg/kg was tolerated by all the animals without signs of intoxication. Only temporary feces and urine discolorations were observed. Based on these results, dose levels of 0, 62.5, 250 and 1,000 mg/kg bw/day were
selected for the present study.
- Rationale for animal assignment (if not random): Computer generated algorithm
- Post-exposure recovery period in satellite groups: 14 d

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (on weekends and public holidays once daily) for survival, health condition and behaviour and once daily for recording individual clinical observations.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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'). 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, piloerection, 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 other abnormal motor movements (such as excessive grooming, repetitive circling or other stereotypes) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded. In addition, defecation and urination were evaluated.

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: Yes
- Time schedule: two times per week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes (intraperitoneal injection of 67 + 6.7 mg/kg bw Ketamine-Hydrochloride + Xylazine)
- Animals fasted: No data
- How many animals: 5/sex/dose
- Parameters examined: erythrocyte counts (RBC), Heinz body counts, hematocrit (packed cell volume), hemoglobin, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), reticulocyte counts, differential leukocyte counts (DLC), leukocyte counts (WBC), coagulation time (clotting time), thrombocyte counts (platelets).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the termination of the study and after the recovery period, after blood sampling for hematological investigations
- Animals fasted: No data
- How many animals: 5/sex/dose
- Parameters examined: γ-glutamyltranspeptidase (GGT), alanine aminotransferase (ALT), albumin, albumin / globulin ratio (calculated), alkaline phosphatase (ALP), aspartate aminotransferase (AST), bilirubin total, calcium, chloride, cholesterol, creatinine, globulin (calculated), glucose, inorganic phosphorous, potassium (K+), sodium (Na+), total protein, triglycerides, urea and uric acid.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight from Day 23 to Day 24 and from Day 37 to Day 38
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food and water was withdrawn during the period of sampling)
- Parameters examined: appearance, specific weight, volume, bilirubin, blood, glucose, ketone bodies, protein, urobilinogen, microscopic examination, (Sediment), color and pH.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the termination of the study,
- Dose groups that were examined: all,
- Battery of functions tested: Sensory reactivity to stimuli of different types (auditory, visual, and proprioceptive) were 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 a 16-station automated motor activity monitoring device. Fore- and hindlimb grip strength were measured by a strain gauge device (FMI, Föhr Medical Instruments GmbH).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, the autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.

HISTOPATHOLOGY: Yes, for the control and high dose animals and all animals which died early as well as gross lesions in all other groups.
Additionally, stomach (forestomach and glandular stomach) from all animals in the low and mid dose group as well as from all recovery group animals were histopathologically examined because of findings in the high dose group at the end of the treatment period.
- organs and tissues for histopathological examination were: adrenals, bone marrow / sternum, brain with medulla oblongata, epididymides, heart, small intestine (ileum), large intestine (colon), kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (iliac), sciatic nerve, ovaries, prostate, seminal vesicle, spinal cord (cervical), spleen, stomach, testes, thymus, thyroid gland with parathyroids, trachea, urinary bladder, uterus and all other gross lesions.

Other examinations:

Organ weights: The following organs were weighed and the organ to body weight ratios calculated: adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, and thymus

Statistics:

Dosing and recovery phase data of body weight (from Day 1 of dosing, twice weekly) was analyzed for statistical significance (p ≤ 0.05) within each sex by a 1-way ANOVA with a two sided ordinal step-down trend test (Tukey et al., 1985). Hematological parameters, serum chemistry parameters, urinalysis and organ weights were analyzed either as described above, if normally distributed, or by the Jonckheere trend test with corrections for ties (Jonckheere AR,1954; Lin, F.O. and Haseman, J.K., 1975), if not normally distributed.

Results and discussion

Results of examinations

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

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

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):

only local irritant effects in the stomach were seen. These are without relevance for human hazard assessment

Details on results:

CLINICAL SIGNS AND MORTALITY: No death occurred throughout the study. General health condition and behavior showed no test substance related alterations. One male animal from the mid dose group had irregular respiration (Day 4 – 6) and reddish discolored body surface from Day 4 up to the end of the study on Day 29. It was considered to be the outcome of faulty intubation on Day 4. This might also be the reason that the animal was the only one in this group showing test item related discoloration of serum as well as of various internal organs at necropsy. All animals of the high dose group showed test item related discolorations of feces (reversible on Day 31) and urine (still present after recovery period). One male and one female from the intermediate dose group showed discolorations of urine. One female animal of the low dose group also showed red discolored urine.

BODY WEIGHT AND WEIGHT GAIN: Body weight gains were not influenced by the administration of the test compound in all groups.

FOOD CONSUMPTION: Food consumption remained unaffected by the administration of the test compound throughout the study in all groups.

HAEMATOLOGY: No relevant test substance related alterations were seen for test groups. Some statistically significant alterations were observed in male animals from low and mid dose groups for erythrocyte count, hematocrit, reticulocyte count, leucocyte count, lymphocytes and large unclassified cells. However, none of these values showed distinct dose-dependency and moreover, no mean values outside the available laboratory historical control data ranges were detected in all dose groups treated with the test item. The findings were therefore not considered to be relevant. Female animals showed no corresponding alterations in final values.
No statistics could be performed with males of the high dose group, because samples of two animals coagulated and consequently only 3 values were available which was not sufficient for statistics. Coagulation occurred obviously incidentally and was no indication of a substance related effect, because measured blood coagulation of all 5 animals of this group showed no relevant alteration in male animals compared to the control group.
In the high dose recovery groups eosinophil levels showed slight alterations in contradictory directions in both sexes compared with the control. These were considered as incidental findings. Coagulation time was slightly decreased in male recovery animals of the high dose group but not outside the historical control range. Moreover, the control value was relatively high. The finding was therefore not considered to be toxicologically relevant.

CLINICAL CHEMISTRY: Serum chemistry showed increased total bilirubin levels in both sexes in the high dose group. Male animals in the low and intermediate dose group also had slight but significant increases in bilirubin levels. However, the mean values in the intermediate and low dose groups corresponded to the historical control range and therefore not considered as adverse finding. Male animals showed additionally dose-dependent increases in aspartate aminotransferase (statistically significant in the intermediate and high dose group), but values were within the historical control range and below the final recovery value and therefore not considered to represent toxicologically relevant findings.

URINALYSIS: Urinalysis showed no biologically relevant alterations after the administration of the test compound in all groups. A slight dose-dependent decrease in pH (high dose group) and an increase in specific urine weight reaching statistical significance in all dose groups was observed in female animals at the end of the treatment period. However, all values were within the historical control range and no histopathological findings in kidneys were observed. Hence these findings were toxicologically irrelevant. At the end of the recovery period female animals of the high dose group showed also slightly lowered pH, and increased specific weight correlated with reduced urine volume. However, values were also within the historical normal range.

NEUROBEHAVIOUR: Neurotoxicological parameters including 'open field' observations, assessment of sensory function, and forelimb and hindlimb grip strength were not influenced by the administration of the test compound in all groups. Male animals in the low dose group showed a statistically significant increase in the number of movements. As no dose-dependency was observed, this finding was considered to be incidental and not substance related.

ORGAN WEIGHTS: Organ weight analysis showed no biologically relevant alterations in all dose groups, both after treatment and recovery period. Absolute weights of adrenal glands in male animals of the high dose group (Day 29) were statistically significant reduced at the end of the treatment period, but not outside the historical control range and therefore not toxicologically relevant. Moreover, no histological correlate was observed.

GROSS PATHOLOGY: Organ discolorations were observed at necropsy at the end of the treatment period (Day 29): One female from the low dose group showed light red discolored skin as well as red discolored kidneys and one male and two females of the intermediate group showed red discolored kidneys. Furthermore, all animals of the high dose group showed red or light red discolorations in organs such as kidneys (4 male and 4 female animals); skin (all male and all female animals); testes and epididymides (all male animals) and rectum (one female animal). After the end of the recovery period organ discolorations were observed in all males and 4 females of the high dose group. Red discolored kidneys (4 males and 3 females), light red discolored skin (4 males, 2 females), light red discolored testes (2 males), and light red discolored epididymides (one male) were observed. These test substance related discolorations were considered to have no correlated physiological or histopathological findings and therefore did not represent adverse effects. Less number of animals and organs being affected at the end of the recovery period compared with the end of the treatment period indicated that organ discolorations were reversible. This was supported by the observation that urine discoloration was still observed in the recovery period indicating ongoing elimination of the test substance.

HISTOPATHOLOGY: NON-NEOPLASTIC: Histopathological assessment revealed irritations in stomach, characterized by mixed cellular infiltrates in the submucosal area of the glandular stomach which occurred in all male and female animals of the high dose group at the end of the treatment time as well as in 4 males and 4 females in the mid dose group. These alterations were reversible and no longer detected at the end of the recovery period.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Dose-dependent reversible discolouration of diverse organs and tissues

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

3

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the 'No Observed Effect Level' (NOEL) for the test substance was determined to be 62.5 mg/kg bw/day, as no toxicologically relevant adverse effects were seen during the entire study period, the 'No Observed Adverse Effect Level' (NOAEL) for the test substance was determined to be >= 1000 mg/kg bw/day

Executive summary:

OBSERVATIONS AND MEASUREMENTS

Groups of male and female rats received Reactive Red F00-0314 by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight per day for a period of 28 days. On day 29 five males and five females from each group were killed and necropsied. Five males and five females from 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.Once before the first treatment and weekly thereafter 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 and damage to the oral mucosa.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, serum chemistry as well as urine analysis were carried out at the end of the treatment period and alter the recovery period. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, haematological and serum chemistry data, urinalysis, absolute and relative organ weights and 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.

RESULTS

No deaths occurred throughout the study. Behaviour, state of health, organ weights, neurotoxicological parameters, body weight development and food consumption remained unaffected by the administration of the test compound in all dose groups. Haematology and organ weight analysis showed no biologically relevant alterations, both after treatment and recovery period. All animals of the high dose group showed test item related discolorations of faeces (reversible on day 31), serum (also reversible in the recovery period) and urine (still present alter recovery period) as well as of several organs (kidneys, skin, testes, epididymides, and/or rectum; largely reversible after the recovery period). Individual animals of the intermediate dose group showed discolorations of urine (one male, one female) and kidneys (one male, two females). One female animal of the low dose group showed also red discoloured urine, kidneys and skin at the end of the treatment period.

Serum chemistry showed increased total bilirubin levels in both sexes in the high dose group. Male animals in the low and intermediate dose group showed also slight but significant increases in bilirubin levels. This finding is proof of internal exposure to the test substance, as the tinctorial effect of the test substance interferes with the photometrical assay for bilirubin. Male animals showed additionally dose-dependent increases in aspartate aminotransferase (statistically significant in the intermediate and high dose group), but values were within the historical control range and below the final recovery value and therefore not considered to represent toxicologically relevant findings. Urinalysis showed no toxicologically relevant alterations after the administration of the test compound in all groups.

Histopathological assessment showed irritations in stomach, characterized by mixed cellular infiltrates in the submucosal area of the glandular stomach which occurred in all male and female animals of the high dose group at the end of the treatment time as well as in 4 males and 4 females in the mid dose group. These alterations were reversible and no longer detected at the end of the recovery period. This finding is not of toxicological relevance for human hazard assessment, at it is owed to the mode of test substance administration.

CONCLUSION

In conclusion, repeated administration of Reactive Red F00-0314 at dose levels of 62.5, 250 and 1000 mg/kg body weight over a period of 28 days caused no systemic toxic effects. The only effects seen, were reversible staining effects due to the tinctorial properties of the test substance.

Dose-dependent reversible acute topical effects were noted after administration of 250 and 1000 mg/kg in form of mixed-cellular infiltration in the stomach. This local irritation is of no toxicological relevance as it is the result of the mode of test substance administration combined with a high salt concentration in the test material.

With regard to local effects the 'No Observed Adverse Effect Level' (NOAEL) is 62.5 mg/kg mg/kg body weight per day. For systemic toxicity, the NOAEL is considered to be 1000 mg/kg body weight per day.