Sodium 2,4-diamino-5-(4-(2-sulfoxyethanesulfonyl)phenylazo)benzenesulfonate

Endpoint summary

Description of key information

Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg bw/day produced no toxicologically significant changes in the parameters measured.The "No Observed Effect Level" (NOEL) was, therefore, considered to be 1000 mg/kg bw/day.

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

29 May 1996 to 09 September 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to recent EU test guidance in compliance with GLP and reported with a GLP certificate. Read across is applicable based on the content of sodium ions. The presence of these is determined by the pH of the isolation of the dyestuff itself. Therefore the substance to be registered is deemed to be a mixture of free acid, mono and di sodium salts. Upon comparison of the NMR-Spectra of the substance to be registered and the read across chemical it is evident that the chemical shifts as well as the integrations are the same, hence it is difficult to quantify free acid, mono and di variants. The CAS number proposed for the substance to be registered covers the sodium element. The associated free acid has a unique CAS number; this is referenced in the substance identity, as does the disodium variant, which is also referenced.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

67/548/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Sprague-Dawley Crl : CD ® BR strain rats were obtained from Charles River (UK) Limited, Manston, Kent. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for eight days during which time their health status was assessed.

A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 133 to 169g, and the females weighed 129 to 160g, and were approximately five to six weeks old.

The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food and water. A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK) was used. A certificate of analysis of the batch of diet is given in Appendix VIII. Mains water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Safepharm Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print¬outs of hourly mean temperatures and humidities were included in the study records. The temperature and relative humidity were controlled to remain within target ranges of 21 ± 2 C and 55 ± 15% respectively. Occasional deviations from the humidity targets were considered not to have affected the purpose or integrity of the study.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled water

Details on oral exposure:

Method of administration: Gavage

The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 10 ml/kg/day of distilled water.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the purpose of this study the test material was prepared at the appropriate concentrations as a solution/suspension in distilled water. The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory, Results are documented within the report and show the formulations to be stable for at least ten days. Formulations were therefore prepared weekly and stored at 4 deg C in the dark.

Samples were taken of each test material formulation and were analysed for concentration of Everzol Yellow GSP at Safepharm Analytical Laboratory. The method used for analysis of formulations and the results obtained are documented within the report. The results indicate that the prepared formulations were within ± 8% of the nominal concentration.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Remarks:

Doses / Concentrations:
0, 15, 150, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

DOSE LEVEL TREATMENT VOLUME CONCENTRATION ANIMAL NUMBERS
(mg/kg/day) (ml/kg) (mg/ml)
MALE FEMALE
0 10 0 5 (1 – 5 ) 5 (6 – 10)
15 10 1.5 5 (11 – 15) 5 (16 – 20)
150 10 15 5 (21 – 25) 5 (26 – 30)
1000 10 100 5 (31 – 35) 5 (36 – 40)

Control animals:

yes, concurrent vehicle

Details on study design:

For the purpose of this study the test material was prepared at the appropriate concentrations as a solution/suspension in distilled water. The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory and show the formulations to be stable for at least ten days. Formulations were therefore prepared weekly and stored at 4⁰C in the dark.

Samples were taken of each test material formulation and were analysed for concentration of Everzol Yellow GSP at Safepharm Analytical Laboratory. The results indicate that the prepared formulations were within ± 8% of the nominal concentration.

The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 10 ml/kg/day of distilled water.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals

Observations and examinations performed and frequency:

Clinical Observations:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends. All observations were recorded.

Bodyweight:
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

Food Consumption:
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Laboratory Investigations:
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

Haematology:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
mean corpuscular haemoglobin (MCH)
mean corpuscular volume (MCV)
mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count
neutrophils (Neut)
lymphocytes (Lymph)
monocytes (Mono)
eosinophils (Eos)
basophils (Bas)
Platelet count (PLT)
Methaemoglobin (Meth)
Reticulocyte count (Retic)

Clotting (prothrombin) time (CT) was assessed by 'Hepato Quick' using samples collected into sodium citrate solution (0.11 mol/l).

Blood Chemistry:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na + )
Potassium (K + )
Chloride (CI-)
Calcium (Ca + +)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total bilirubin (Bili)

Sacrifice and pathology:

Pathology:
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.

All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Gonads, Kidneys , Pituitary, Brain, Heart, Liver, Spleen

Histopathology:
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:

Adrenals Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain, Caecum, Colon, Duodenum, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Muscle (skeletal), Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skin (hind limb), Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Lungs, Lymph nodes (cervical and mesenteric), Trachea, Urinary bladder, Uterus

The following preserved tissues from all control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5μm and stained with haematoxylin and eosin for subsequent microscopic examination:
Adrenals, Heart, Kidneys, Liver, Spleen, Testes

Liver and spleen from all 15 and 150 mg/kg/day dose group animals were also processed.

Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE 84 pathology computerisation system for tabulation and report production.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate. Absolute and relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating 'F-max' test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal-Wallis non-parametric analysis of variance and Mann Whitney U-Test.

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:

not examined

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:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Mortality
There were no deaths during the study.

Clinical Observations
No clinically observable signs of toxicity were detected in test or control animals throughout the study period.
Animals of either sex treated with 1000 mg/kg/day showed dark coloured faeces on the cage tray liners from Day 2 onwards. By Day 4 orange staining was also noted on the tray liners. This was considered to be colouration of the urine by the test material. Orange/yellow fur staining was noted for three females treated with 1000 mg/kg/day on Day 17 persisting in these and the other two females from the group throughout the study period. These observations are consistent with oral administration of a coloured test material and merely represent normal excretion of the coloured compound. Such observations are therefore, of no toxicological significance.
Animals treated with 150 mg/kg/day showed dark faeces from Day 2 onwards followed by orange staining on the cage tray liners from Day 25.
No clinically observable signs were detected at 15 mg/kg/day.

Bodyweight
All animals showed normal gains in bodyweight throughout the study period. Bodyweight gain in test animals was comparable with that seen in controls

Food Consumption
There was no adverse effect on food consumption during the study. Food efficiency in test animals was comparable with that seen in controls.

Water Consumption
Visual inspection of water bottles revealed no overt intergroup differences.

Haematology
There were no treatment-related changes in the haematological parameters measured.
A statistically significant reduction in reticuolyte count was detected for females treated with 1000 mg/kg/day in comparison with controls. The reduction was attributable to unusually high control values and, in the absence of any other haematological changes, was considered to be without toxicological significance.

Blood Chemistry
There were no blood chemical changes which could be considered toxicologically significant.
Statistically significant increases in total plasma protein, albumin and bilirubin were detected for males treated with 1000 mg/kg/day when compared with controls. All individual values were within the respective normal ranges and, as minimal, isolated changes, all were regarded as fortuitous. An increase in plasma potassium concentration was detected for males from this group but, in the absence of any evidence to suggest renal dysfunction, the increase was considered not to be toxicologically significant. An increase in inorganic phosphorus for 15 and 1000 mg/kg/day males was detected but no convincing dose relationship was evident and, as such, the increase was considered to be unrelated to treatment with the test material. Plasma urea was reduced for 150 and 1000 mg/kg/day males. All values were within the normal range for rats of this strain and age and a reduction in this parameter cannot be regarded as toxicologically significant.
The remaining statistically significant difference detected between test and control groups was confined to 150 mg/kg/day females and, as such, showed no dose-related response.

Pathology
Organ Weights
There were no changes in the organ weights measured which could be considered attributable to treatment with the test material.
A statistically significant increase in heart weight, relative to bodyweight, was detected for females treated with 1000 mg/kg/day in comparison with controls but, in the absence of any histopathological correlates this was considered not to be toxicologically significant. The remaining statistically significant differences detected between test and control groups were confined to a reduced relative liver weight for 150 mg/kg/day females which showed no dose-related response and an increase in absolute heart weight for 1000 mg/kg/day males. No effect on relative heart weight was apparent and the increase was, therefore, considered to be fortuitous.

Necropsy
No treatment-related macroscopic abnormalities were detected. The incidental finding recorded at terminal kill for a 1000 mg/kg/day female, identified as hydronephrosis of the left kidney, was consistent with normally expected low incidence findings in laboratory maintained rats.

Histopathology
No treatment-related changes were observed.
All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

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 observed

Critical effects observed:

not specified

Conclusions:

Oral administration of the test material, Everzol Yellow GSP, to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day produced no toxicologically significant changes in the parameters measured.

The "No Observed Effect Level" (NOEL) was, therefore, considered to be 1000 mg/kg/day.

Executive summary:

The results are read across from the free acid form. Read across is applicable based on the content of sodium ions. The presence of these is determined by the pH of the isolation of the dyestuff itself. Therefore the substance to be registered is deemed to be a mixture of free acid, mono and di sodium salts. Upon comparison of the NMR-Spectra of the substance to be registered and the read across chemical it is evident that the chemical shifts as well as the integrations are the same, hence it is difficult to quantify free acid, mono and di variants. The CAS number proposed for the substance to be registered covers the sodium element. The associated free acid has a unique CAS number; this is referenced in the substance identity, as does the disodium variant, which is also referenced.

The study was designed to investigate the systemic toxicity of the test material and complies with the testing method described in Council Directive 67/548/EEC (Method B7).

 

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl : CD ® BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (distilled water).

 

Clinical signs, bodyweight, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

 

All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

 

The results are summarised as follows:

 

Mortality

There were no deaths during the study.

 

 

Clinical Observations

No clinically observable signs of toxicity were detected. Animals treated with 1000 mg/kg/day showed dark faeces from Day 2 followed by orange-coloured staining on the cage tray liners from Day 4. Orange/yellow staining of the fur was evident in the females from Day 17 onwards. These observations are consistent with oral administration of a coloured test material and merely represent normal excretion of the coloured compound. Such observations are, therefore, of no toxicological significance.

Animals treated with 150 mg/kg/day showed dark faeces from Day 2 onwards followed by orange staining on the cage tray liners from Day25.

No such effects were detected at 15 mg/kg/day.

 

Bodyweight

No adverse effect on bodyweight development was detected.

 

Food Consumption

No adverse effect on dietary intake was detected.

 

Water Consumption

No overt intergroup differences were detected.

 

Haematology

No treatment-related effects were detected.

 

Blood Chemistry

No treatment-related effects were detected.

 

Organ Weights

No treatment-related effects were detected.

Necropsy

No treatment-related macroscopic abnormalities were detected.

 

Histopathology

No treatment-related microscopic changes were observed.

 

Conclusion

Oral administration of the test material, Everzol YellowGSP, to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day produced no toxicologically significant changes in the parameters measured.

 

The "No Observed Effect Level" (NOEL) was, therefore, considered to be 1000 mg/kg/day.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 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 results are read across from the free acid form. Read across is applicable based on the content of sodium ions. The presence of these is determined by the pH of the isolation of the dyestuff itself. Therefore the substance to be registered is deemed to be a mixture of free acid, mono and di sodium salts. Upon comparison of the NMR-Spectra of the substance to be registered and the read across chemical it is evident that the chemical shifts as well as the integrations are the same, hence it is difficult to quantify free acid, mono and di variants. The CAS number proposed for the substance to be registered covers the sodium element. The associated free acid has a unique CAS number; this is referenced in the substance identity, as does the disodium variant, which is also referenced.

Oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day produced no toxicologically significant changes in the parameters measured.The "No Observed Effect Level" (NOEL) was, therefore, considered to be 1000 mg/kg/day.

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. In addition 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.

The test substance appears to be absorbed from the gastrointestinal tract however a significant bioaccumulation potential can most probably be excluded due to the marked hydrophilic properties, plus the lack of toxicity observed in the study. From the mutagenicity assays it appears that the test substance is not metabolised toward genotoxic structures. Review of the available data indicates that the substance does not exhibit conspicuous toxicokinetic behaviour. The results from all studies with dermal exposure indicate that the test substance has insignificant or no dermal absorptive potential. Bioaccumulation of the test substance can therefore most probably be excluded from all routes of exposure.

Justification for classification or non-classification

The above study has 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.