A mixture of: sodium/potassium 7-[[[3-[[4-((2-hydroxy-naphthyl)azo)phenyl]azo]phenyl]sulfonyl]amino]-naphthalene-1,3-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:

05 June 1992 to 02 March 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

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:

yes

Remarks:

See overall remarks

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC Directive, Official Journal of the European Communities, 19.9.84, 84/449/EEC B.7., Sub-acute Toxicity (oral).

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Requirements of the Japanese Government under the revised Chemical Substance Law (1987) according to the notification of Dec. 9, 1986 by EA (No. 700), MHW (No. 1039) and MITI (No. 1014).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

code number: FAT 45155/B
batch No.: HT 3728/ TV 1
physical appearance: red powder
storage: room temperature, keep dark
expiration date: May. 15, 1997

Specific details on test material used for the study:

Test article: FAT 45155/B
Batch No.: HT 3728/TV 1
Purity: 60 - 70 %
Physical properties: powder; dark red
Storage conditions: room temperature; keep dark

Test animals

Species:

rat

Strain:

other: albino rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production CIBA-GEIGY Limited 4332 Stein / Switzerland
- Age at study initiation: 5 weeks
- Weight at study initiation: 142.3 - 171.0 g in males; 125.3 - 154.6 g in females
- Housing: housed in groups of 5 in macrolon cages type 4 with wire mesh tops and standardized granulated soft wood bedding (Société Parisienne des Sciures Pantin)
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%):55 ± 10
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light): 12 hours light per day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on oral exposure:

Preparation of solution: Solutions of the test article in the selected vehicle at the appropriate concentrations were prepared weekly and stored in the refrigerator.
Vehicle: Distilled water was used as a vehicle.
Volume of solution applied: 20 ml/kg bodyweight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Control analyses of the test article concentration in the vehicle were carried out at all dose levels on samples collected once per experimental week. The samples were collected after the weekly preparation of the test solutions, immediately deep frozen and sent to the analytical laboratories.

Rationale for dose selection:
The dose levels were based on the results of the previously conducted studies (Project no. 925014), 5-days range finding study in rats.
FAT 45155/B was administered to 3 male rats per dose at levels of 0, 10, 100 and 1000 mg/kg body weight. No clinical signs except for reddish discolouration of feces from treatment day 2 onwards were observed in animals treated at 1000 mg/kg. No death occurred during the study. Compared to the controls, slightly lower diet intake was recorded at day 1 of treatment, however values comparable or above the control level were attained from study day 2 onwards. Body weight development was not influenced by the treatment. At necropsy, no macroscopical changes were detected.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily for 28 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Middle dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

10/sex in the control and high dose group, and 5 /sex in the low and medium dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

The test article FAT 45155/B was administered by gavage for 4 weeks at daily doses of 0, 50, 200, and 1000 mg/kg bodyweight to a total of 60 albino rats, 10 males and 10 females in the control and high dose group, and 5 males and 5 females in the low and medium dose group. 5 animals per sex and group were sacrificed at the end of the treatment period, 5 animals per sex of the control and the high dose group were kept for a 4-week recovery period before sacrifice.

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

Mortality: All animals were checked daily
In-life observations: examination was carried out daily, and observations were recorded at least weekly
Bodyweight: The weight of all animals was recorded individually at weekly (midweek) weighing sessions. The first weights were recorded during the acclimatation period. Daily bodyweights for accurate dosing were taken but not recorded.
Food consumption: The food consumption was recorded weekly (cagewise).

Sacrifice and pathology:

Pathology
Macroscopical examination: At the end of the treatment period (experimental group I) and the treatment-free recovery period (experimental group II) all controls and treated animals were bled under ether anesthesia and subjected to detailed autopsy.
Microscopical examination: After the fixation, organ samples were taken from all animals of experimental group I, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin, and subjected to a microscopical examination.

Other examinations:

Laboratory investigations (haematology, blood chemistry and urine analysis) were carried out on all surviving animals of each dose group at the end of the treatment period (September 15/16, 1992), and additionally at the end of the recovery period (October 14, 1992) on animals of the control and high dose group kept for reversibility evaluation.

Statistics:

For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods were applied, to allow for non normal as well as normal data distribution.
Each treated group was compared to the control group either by Lepage's or by Wilcoxon's two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives. The Lepage test is a combination of Wilcoxon and Ansari-Bradley statistics, i . e . a combined test for location and dispersion. The Lepage test has a good power against the more general alternative that the distributions differ not only in location but also in dispersion. The Jonckheere's test is sensitive to monotone dose-related effects.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

During the treatment period, reddish discoloration of feces was noted in animals treated at 200 and 1000 mg/kg body weight.
Hair loss was found in one high dose male while skin lesion, neck was fond in few animals including control.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

From study start onwards and therefore considered unrelated to treatment, male control animals scheduled for sacrifice 1 (experimental group I) had lower mean bodyweights than control animals scheduled for sacrifice 2 (experimental group II) and animals of all treated groups (see table below). Consequently, during the treatment period the mean bodyweight of the control group was minimally lower than that of the treated groups, and higher than that of group 4 during the recovery period. At all tested dose levels, female animals gained less weight due to diminished food consumption compared to the control group. Since, however, there was no dose-dependency and mean bodyweights of treated groups were already lower during the acclimatisation period, this was considered rather to reflect biological variability than a treatment-related effect.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Overall food consumption (week 1-4) of treated male groups was considered comparable to the control value. Slightly decreased food consumption noted in all treated female groups was considered to reflect biological variability, since there was no dose-dependency, and decreased food consumption existed already before treatment start.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Both sexes revealed signs of a normochromic anemia, consisting in reduced values for red blood cells , hemoglobin and hematocrit in groups 3 and 4 (200, 1000 mg/kg), associated with an increased number of reticulocytes in group 4 (1000 mg/kg) as a sign of effective erythropoietic activity . Additionally, females of groups 3 and 4 (200, 1000 mg/kg) revealed a macrocytosis, substantiated by a higher value for MCV. An increased platelets count was observed in animals of the high dose group (1000 mg/kg). Other values reaching a level of statistical significance represent the physiological variation of the respective parameters and were therefore not considered treatment-related. In the absence of Heinz bodies in erythrocytes, the higher methaemoglobin levels recorded for group 4 animals are considered an analytical artifact due to discolouration of plasma samples by the test article. The effects on hematological parameters were reversible within the recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females revealed a marked increase of bilirubin in group 4 (1000 mg/kg), females additionally a moderate increase in group 3 (200 mg/kg). In the high dose group (1000 mg/kg), glucose and cholesterol concentrations were elevated in males, and potassium concentrations were minimally elevated in both sexes. These findings are considered treatment-related. The reddish discolouration of plasma samples caused by reabsorption of the test article did not interfere with the analysis methods used for these parameters. All the other statistically significant values obtained for blood chemistry parameters were considered to represent the normal physiological fluctuation and not to have been influenced by treatment. Reversibility within the recovery period of 4 weeks was demonstrated for all parameters.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of groups 3 and 4 (200, 1000 mg/kg) excreted discoloured urine during the treatment period. This effect was reversible within the recovery period.

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In male group 4 (1000 mg/kg), absolute and relative mean weights of liver and kidneys were significantly increased. Partial reversibility occurred during the 4-week recovery period. In the absence of corroborative findings, the toxicological meaning of the increased kidney weights is equivocal.

Gross pathological findings:

no effects observed

Description (incidence and severity):

However, the sporadic incidental findings included scab formation on the back of occasional control and treated animais. An enlargement of the thyroid (with parathyroid) gland was observed in one treated animal (male no. 21, group 4). This finding was due to the presence of accessory thymic tissue in the thyroid gland, which occurs naturally in some rats of our colony. Therefore, this lesion was devoid of toxicological relevance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of treatment period, the treatment-related findings were restricted to increased incidence and severity of congestion as well as increased severity of haemosiderosis in the spleen of male and female groups 3 and 4. Following the treatment -free recovery period spleen of animals from groups 1 and 4 (experiment group II) was examined. There was no difference with respect to the incidence and severity of congestion, but splenic haemosiderosis was still more prominent in male and female group 4 than in controls. All other microscopical changes found in this study were considered incidental and unrelated to treatment. They commonly occur in our colony of rats, and, neither their incidences nor their distribution and morphologic appearance gave any indication of a treatment-related association. Such findings included ulceration of skin, extramedullary haematopoiesis of spleen, lymphohistiocytic infiltration of myocardium and liver, inflammation with fibrosis of renal cortex, renal tubular atrophy, and, in female animals nephrocalcinosis. Despite the increased mean relative weight of the liver in male group 4, there were no microscopical findings indicating a treatment-related effect.

Effect levels

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analytical results: The results of these analyses showed that the contents of FAT 45155/B in the vehicle were in agreement with the nominal concentrations and that the samples were homogeneous and stable for both 4 hours and 7 days at room temperature.

Applicant's summary and conclusion

Conclusions:

The "no observable effect level" for FAT 45155/B when administered to rats by daily gavage over aperiod of 4 weeks is 50 mg/kg body weight.

Executive summary:

In a GLP-compliant study, FAT 45155/B was administered by gavage for 4 weeks at daily doses of 0, 50, 200, and 1000 mg/kg body weight to albino rats, 10/sex in the control and high dose group, and 5/sex in the low and medium dose group. 5 animals per sex and group were sacrificed at the end of the treatment period, 5 animals per sex of the control and the high dose group were kept for a 4-week recovery period before sacrifice. The study was carried out according to OECD guideline 407 and EU method B.7. Administered quantities of the test article solution were adjusted daily to individual bodyweight. On treatment day 1, animals received only half of the intended doses. Under the conditions of this test , treatment with FAT 45155/B did neither induce mortalities nor disturbances of food consumption and body weight development. Animals of groups 3 and 4 (200 and 1000 mg/kg) revealed a normochromic anemia, associated in group 4 with an increased number of reticulocytes as a sign of effective erythropoietic activity and an increased number of platelets. These effects were reversible within the recovery period. Males and females revealed marked increase of bilirubin in group 4 (1000 mg/kg), females additionally a moderate increase in group 3 (200 mg/kg). In the high dose group (1000 mg/kg), glucose and cholesterol concentrations were elevated in males, and potassium concentrations were minimally elevated in both sexes. Reversibility within the recovery period of 4 weeks was demonstrated for all changes. Animals of groups 3 and 4 (200, 1000 mg/kg) excreted discolored urine during the treatment period This effect was reversible within the recovery period. In male group 4 (1000 mg/kg), absolute and relative mean weights of liver and kidneys were significantly increased. Partial reversibility occurred during the 4-week recovery period. In the absence of corroborative findings, the toxicological meaning of the increased kidney weights is equivocal. There were no treatment related macroscopical findings. Microscopical examination indicated increased incidence and severity of congestion, as well as increased severity of hemosiderosis in the spleen of male and female groups 3 and 4 (200 and 1000 mg/kg) at the end of treatment period. Following a treatment-free recovery period, severity of hemosiderosis was still increased in the spleen of treated males and females of group 4 (1000 mg/kg). Based on the study results, the "no observable effect level" for FAT 45155/B when administered to rats by daily gavage over aperiod of 4 weeks is 50 mg/kg body weight.