A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex

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:

Treatment: 29 November 1995 to 26 December 1995. Blood sampling: 27 December 1995. Necropsy: 27 December 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex;

EC Number:

423-940-7

EC Name:

A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex;

Cas Number:

85585-91-7

IUPAC Name:

4-[2-(7-{2-[4-(ethenesulfonyl)-2-hydroxy-5-methoxyphenyl]diazen-1-yl}-8-hydroxy-6-sulfonaphthalen-2-yl)diazen-1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid 4-[2-(8-hydroxy-7-{2-[2-hydroxy-4-(2-hydroxyethanesulfonyl)-5-methoxyphenyl]diazen-1-yl}-6-sulfonaphthalen-2-yl)diazen-1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid dicopper tetrasodium hydride

Test material form:

solid

Specific details on test material used for the study:

Identification: Pacified Reactive Black 31
Description: Dark blue crystals
Batch: 9T-55
Purity: 89%
Storage: At room temperature in the dark
Stability in vehicle: Stable in water for at least 96 hours

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognised by international guidelines as the recommended test system. Females were nulliparous and non-pregnant.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age at start of treatment
Approximately 6 weeks

Number of animals
20 females, 20 males

Conditions
Air-conditioned room with approximately 15 air changes per hour and the environment controlled with optimal conditions considered as being a temperature of 21°C and a relative humidity of 50%. Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity. Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

Accomodation
Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. Acclimitisation period was at least 5 days before start of treatment under laboratory conditions.

Diet
Free access to standard pelleted laboratory animal diet. Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.

Water
Free access to tap=water. Certificates of analysis (performed quarterly) were examined and archived.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a stainless steel stomach tube.

Vehicle:

water

Details on oral exposure:

Formulations (w/w) were prepared daily immediatelt prior to dosing. stored at ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for formulations, prepared after the in-life phase of the study, were analysed to check for homogeneity (highest and lowest concentrations) and accuracy of preparations (all concentrations).

Sampling procesdure
Samples for the determination of the concentrations were taken at 50% height from the formulation. For the determination of the homogeneity, three samples were taken: one at the top (at 90% height), one at the middle (at 50% height) and one at the bottom (at 10% height).

Sample pretreatment
All samples were weighed accurately in volumetric flasks using an analytical balance. All samples were further diluted using a 0.05% tetrabutyl ammonium bromide in Milli-Q water, to obtain suitable concentrations for analysis.

Quantitative analysis
Quantiative analyses were based on the largest peak in the HPLC chromatogram of Pacified Reactive Black 31.

Claibration solutions
Two independently prepared calibration solutions of Pacified Reactive Black in Milli-Q water were used each day of analysis to calibrate the analytical method.

HPLC conditions
Column: LiChrospher 100 RP-18, 125 x 4 (I.D.) mm; dp = 5 µm.

Mobile phase: 60/40/0.05 (v/v/w) Milli-Q water / Acetonitrile / Tetrabutyl ammonium bromide

Flow: 1 ml/min

Detection: Visible at wavelength pf 565 nm

Injection volume: 10 µl

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

5 females, 5 males

Control animals:

yes, concurrent vehicle

Details on study design:

Randomisation
At least 5 days before study start, by computer-generated algorithm according to bodyweight, with all animals within +/- 20% of the sex mean. A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Identification
Earmark and tattoo

Positive control:

None

Examinations

Observations and examinations performed and frequency:

Moratlity / Viability
Twice daily

Clinical signs
At least once daily from day 1 onwards. The time of onset, degree and duration were recorded.

Body weights
Weekly and on the day preceeding termination, prior to overnight fasting

Food consumption
Weekly

Water consumption
Subjective appraisal was maintained during the study, but no quantitative inhvestigation introduced as no effect was suspected.

Clinical laboratory investigations
Blood samples were collected under light anaesthesia immediately prior to post-mortem examination, between 7.30 and 9.30 am. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/ sex/ group and collected into tubes prepared with EDTA for haematological parameters (0.6 ml), with citrate for clotting tests (1.0 ml) and untreated tubes for clinical biochemistry parameters (>2.0ml).

Haematology
The following haematology parameters were determined from blood prepared with EDTA as an anti-coagulant.
Eryhtrocyte count
Haemoglobin
Haematocrit
Mean corpuscular volume
Mean corpuscular hameoglobin
Mean corpuscular haemoglobin concentration
Platelet count
Red cell distribution width
Total leucocyte count
Differential leucocyte count

The following haemotology parameters were determined from blood prepared with citrate as an anti-coagulant:
Prothrombin
Partial thrombroplasti time

Clinical biochemistry
The following clinical biochemistry parameters were determined from serum samples. Serum samples were stored at -80°C for 16 days prior to analysis of sodium, potassium and calcium.
Alanine aminotransferase
Aspartate aminotransferase
Bilirubin, total
Creatinine
Glucose
Urea
Protein, total
Protein, albumin
Alkaline phosphatse
Sodium
Potassium
Chloride
Calcium
Phosphorous

Sacrifice and pathology:

Necropsy
all animals surviving to the end of the observation period were deeply anaesthetised using ether vapour and subsequetly exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:
Adrenal glands
Heart
Kindneys
Liver
Spleen
Stomach
Testes
All gross lesions

Organ weights
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands
Heart
Kidneys
Liver
Spleen
Testes

Hostopathology
Slides of adrenals, heart, kidneys, liver, spleen, stomach and testes, collected at the schedules sacrifice from all animals of the control and the highest dose group and all gross lesions of all animals were examined by a pathologist. Based on the treatment related morphological chnages, kidneys were also examined from all rats of the intermediate dose groups. All abnormalities were described and included in the report.

Statistics:

Univariate one-way analysis of variance was used to assess the signficiance of intergroup differences.

If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.

The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.

all the tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated foe discrete data (scores) in the summary tables.

Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant changes in clinical signs were noted during the study.

Black staining of the faces and tail, noted in all animals treated at 100 mgkg bw/day, was ascribed to the phsyico-chemical properties of the test substance without any toxicological significance.

Excessive salivation was seen among animals of the 1000 mg/kg bw/day dose group. This is often noted in rats of this age and strain following oral gavage and considered to be related to multiple intra-oesophageal intubation and/or an irritant effect or bad taste of the test substance. Therefore it was considered not to be a sign of systenic toxicity,

Hunched posture and piloerection, noted in one female receiving 50 mg/kg bw/day, were considered to be incidental findings. In the absence of similar changes among animals of the higher dose groups, no toxicological significance was attached to these findings.

Other findings noted in control and treated animals were considered to be within the range of biiologica variation for these animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females receiving 50 or 1000 mg/kg bw/day died during blood sampling.

No mortality occurred in the other dose groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights nad body weight gain of treated animals remained in the same range as controls over the 4-week study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences in food consumption before or after allowance for body weight between treated and control animals.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes in haematology parameters were noted.

Mean corpuscualr haemoglobin concentration was decreased and red cell distribution width increased in females receiving 1000 mg/kg bw/day. All values were within the range of historical data for rats of this age and strain and no other red blood cell parameters were affected, these changes were considered to be of no toxicological relevance.

Other minor statisticallu sigi=nificant differences arising between controls and animals receiving 50 or 200 mg/kg bw/day were considered not to represent a sign of toxicity as no dose relationship was seen and no concurrent changes wre observed in the other sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences notes between control and treated rats that were considered to be related to treatment with Pacified Reactive Black 31.

Changes in clinical biochemistry parameters achieving a level of statistical significance when compared to controls, were considered to be of no biological relevance (ALAT) or had occureed by chance as values remained within the range of historical data for rats of this age and starin. In the absence of a dose-response relationship, increased ALP levels in the serum of males receiving 50 r 200 mg/kg bw/day were considerd to be of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney: body weight ratios were increased with statistical significance in males receiving 1000 mg/kg bw/day.

A statistically significant decrease in liver weights of 50 mg/kg bw/day trated males was considered to have arisen as a result of a slightly high control value and therefore not to be a sign of toxicity.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of systenic toxicity.

bluish or blackbrown discolouration of (parts of) the gastro-intestinal tract, mesenteric lymph nodes and/or kidneys were noted in male and female rats receiving 200 or 1000 mgkg bw /day.

These discolourations were considered to result from the staining properties of Pacified Reactive Black 31 and not to represent a toxicological effect.

Other findings noted among treated animals were considered to be within the range of biological variation of rats of thi =s age and strain in this type of study and not to represent a change of toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Kidneys of males and females of the high-dose group showed an increased incidence and severity of a degenerative change which was characterised by cortical tubular hyalin resorption bodies. the hyalin resorption bodies wre more pronounced in the kidneys of male rats than female rats.

The small number of chnages recorded in treated animals wer within the range commonly seen for rats of this age and strain.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

200

System:

urinary

Organ:

kidney

Applicant's summary and conclusion

Conclusions:

Test substance preparations in distilled water appeared to be homogenous and sufficently accurate concentrations were encountered for the purpose of this study.

An increase in kidney weights was seen in males receiving 1000 mg/kg bw/day. In addition, mciroscopic examination revelaed degnerative change sin the kidneys of the animals of this dose group. Males appeared to be more sensitivefor thse effects thab females. However, no indication of impairment of renal function was established on the clinical biochemistry parameters.

There were no changes in clinical appearance, body weights, food consumption, clinical laboratory investigatins and macroscopic examination that were considered to be a toxicological effect of treatment.

From the results preseneted from th =e study a definitive No Obsevred Efefct Level (NOEL) of 200 mg/kg bw/day was established.

Executive summary:

Summary

Subacute 28 -day oral toxicity with Pacified Reactive Black 31 by daily gavage in the rat.

Based on the results of a 5 -day range-finding study, the dose levels for the 28 -day toxocity study were selected to be 0, 50, 200 and 1000 mg/kg bw/day.

The study was based on the following guideline - EEC Directive 92/69/EEC, B.7 Sub-acute toxicity - Oral, 1992.

The test substance was administered aily for 28 -days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.

The following parameters were evaluated:

clinical signs daily; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Results

Formulations

Accuracy and homogeneity of test substance formulations wre demonstrated by analyses.

50 mg/kg bw/day

No treatment-related findings noted

200 mg/kg bw/day

No treatment-related finding noted.

1000 mg/kg bw/day

1) Increase in kidney weights

2) Increased incidence and severity of cortical tubular hyalin resorption bodies in kidneys

Conclusion

From the resulst presented in this report a efinitive No Obsevred Effect Level (NOEL) of 200 mg/kg bw/day was established.