Chromate(5-), bis[4-(hydroxy-κO)-7-[2-(2-hydroxy-1-naphthalenyl)diazenyl]-3-[2-[2-(hydroxy-κO)- 3-nitro-5-sulfophenyl]diazenyl]-2-naphthalenesulfonato(4-)]-, sodium (1:5)

Endpoint summary

Description of key information

NOEL (oral, 28 d) = 50 mg/kg/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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

SPF- Quality

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source: BRL Ltd., Basel, Switzerland
-Females nulliparous and non-pregnant: yes
-Age at study initiation: 6 weeks.
-Weight at study initiation: 165-168 g
-Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors.
-Diet: Free access to standard pelleted laboratory animal diet (Kliba 343 from Klingentalmühle AG, Kaiseraugst, Switzerland).
-Water: Free access to tap-water
-Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
-Temperature (°C): 22 ± 3 °C
-Humidity (%): 30-70 %
-Air changes (per hr): 15 air changes per hour
-Photoperiod (hrs dark / hrs light): 12 hours cycle dark/light

Route of administration:

oral: gavage

Details on route of administration:

A stainless steel stomach tube was used.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose volume was 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLE PROCEDURE
The formulations were analysed for test substance concentration, stability and homogeneity after preparation. Samples for the determination of the concentratlons and stability 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 using an analytical balance in a volumetrlc flask. All samples were further dlluted using Mllli-Q water to obtain suitable concentrations for analysis.

QUANTITATIVE ANALYSIS
Two independently prepared calibration Solutions of tghe substance in Milli-Q water were used each day of analysis to calíbrate the analytical method.

SPECTROPHOTOMETER CONDITIONS
-Band pass: 2 nm (during the final study)
-Detection wavelength: 609 nm
-Temperature: room temperature
-Cuvettes: plastic (path length 1.00 cm)
-Reference solvent: Milli-Q water
The instrument were calibrated

DATA HANDLING
Absorption: R = absorption test substance [units]
-Response factor:
RF = R/C [units*l/mg]
R= absorption calibration solution [units]
C= concentration of test substance in calibration solution [mg/l]
-Concentration analysed:
C= (R * d * V)/(RF * w) = [mg/l]
R = absorption sample [units]
d = dilution factor
V = volume volumetric flask [ml]
RF = mean response factor calibration solution [units*l/mg]
w = weight sample [g]
-Relative to nominal concentration:
(Concentration analysed/Nominal concentration) * 100 [%]
-Relative to mean concentration:
(Concentration analysed)/(Mean of concentrations analysed of 6 samples) * 100 [%]
-Relative Difference (Rel. Diff.)
(Concentration analysed t=4 - Concentration analysed t=0)/(Concentration analysed t=0) * 100 %
Where:
t = time of sampling [hours] and Concentration were mean concentration of duplicate samples

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily for 28 days, approximately the same time each day, 7 days per week.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females per dose of 50 and 200 mg/kg b.w./day; 10 males and 10 females per dose of 0 and 1000 mg/kg b.w./day

Control animals:

yes, concurrent vehicle

Details on study design:

-The dose level selection was based on the results observed in a 5-day dose range finding test. The study was performed with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day.
-A post-exposure recovery period of 14 days was observed for the dose levels of 0 and 1000 mg/kg b.w./day (5 males and 5 females per dose).

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
-Time schedule: once daily. The time of onset, degree and duration were recorded

MORTALITY / VIABILITY: Yes
-Time schedule: twice daily.

BODY WEIGHT: Yes
-Time schedule: Weekly and on the day preceding termination, prior to overnight fasting.

FOOD CONSUMPTION: Yes
-Time schedule: Weekly.

WATER CONSUMPTION: Yes
-Time schedule: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
-Time schedule: Both eyes were examined following instillation of tropicamide solution (5 mg/ml) during weeks 4 (all groups) and 6 (recovery groups).

CLINICAL LABORATORY INVESTIGATIONS
Blood samples were collected under light ether anaesthesia. 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 clottlng tests (1.0 ml) and untreated tubes for clinical biochemistry parameters (>2.0 ml).

1) HAEMATOLOGY: Yes
The following haematology parameters were determined from blood containing EDTA as an anti-coagulant: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelet count , Red cell distribution width, Total leucocyte count, Differential leucocyte count.
The following haematology parameters were determined from blood containing citrate as an anti-coagulant: Prothrombin time, Partial thromboplastin time.
For all parameters was used Sysmex K-1000, except for Differential leucocyte count (manual, microscope)

2) CLINICAL BIOCHEMISTRY: Yes
-Time schedule for collection of blood: daily between 7.30 and 9.30 a.m.
-Animals fasted: Yes
-The following clinical biochemistry parameters were determined from untreated blood (serum) samples after clotting and centrifugation: Alanine aminotransferase, Aspartate aminotransferase , Bilirubin (total), Cholesterol (total), Triglycerides, Creatinine, Glucose, Urea, Protein (total), Protein (albumin and globulin), Albumin Globulin ratio, Alkaline phosphatase, Sodium, Potassium, Chloride, Calcium, Phosphorus

Sacrifice and pathology:

NECROSCOPY
All animals surviving to the end of the observation period (day 29 for the Main Groups and day 43 for the Recovery Groups) were deeply anaesthetized using ether vapour and subsequently 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, Aorta, Brain, Cecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes (with optic nerve and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lung (infused with formalin), Lymph nodes (mandibular, mesenteric), Nasopharynx, Oesophagus, Ovaries, Pancreas, Pituitary gland, Preputial gland, Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum (with bone marrow), Stomach, Testes, Thymus, Thyrold( Including parathyroid), Tongue, Trachea, Urinary bladder, Uterus, Vagina. All gross lesions.

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded on the day of necropsy: Adrenal glands, Brain, Heart, Kidneys, Liver, Ovaries, Spleen and Testes.

HISTOPATHOLOGY
All organ and tissue samples, as defined under Histopathology were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
Slides of adrenals, heart, kidneys, liver, spleen, stomach and testes, collected at the scheduled sacrifice from all animals of the control and the highest surviving dose group and all gross lesions of all animals were examined by a pathologist. All abnormalities were described.

Statistics:

The following statistical methods were used to analyse the body weight, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance 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. The exact Fisher-test was applied to the ophthalmoscopic examination data. All 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 for discrete data (scores) in the summary tables.
Individual values, mean, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One female receiving 1000 mg/kg/day was noted with hunched posture and piloerection at the end of the treatment period.
No further signs of toxicity or behavioural changes were noted among treated animals.
Blue appearance of the skin (among nearly all treated animals) and dark faeces (among animals treated at 200 or 1000 mg/kg/day) were ascribed to the staining properties of the test substance and considered not to be a change of toxicological significance.
Excessive salivation was noted among treated animals, mainly in the 1000 mg/kg dose group. Salivation is often seen in rats after treatment with a xenobiotic agent via oral gavage and may be the result of a bad taste or irritant propertles of the substance. No toxicological significance was attached to this finding.
Other incidental findings included alopecia, scabs, rales, wound and chromodacryorrhoea. As these alterations were within the range of normal biological variation for rats of this age and strain or was a result of blood sampling procedures (chromodacryorrhoea), they were considered not to represent a sign of toxicity.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the 4 weeks of treatment and 2 weeks of recovery.

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.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmoscopic findings at the week 4 and week 6 examinations.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

-Red blood cell parameters
Toxicological changes were detected among males and females treated with test item at a dose level of 200 and 1000 mg/kg/day.
Red blood cell count (RBC), haemoglobin concentration (HB), haematocrit (HCT) and mean corpuscular haemoglobin concentration (MCHC) were noted to be decreased after 4 weeks of treatment when compared to controls. Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and red cell distribution width (RDW) were increased at week 4. A relationship with the dose was established in the majority of these differences. After 2 weeks of recovery, adaptive changes were clearly seen among males, previously treated with 1000 mg/kg/day. RBC and MCHC had returned to normal, while HB, HCT, MCV, MCH and RDW were found to be increased at the week 6 examination.
In females previously treated with 1000 mg/kg/day, physiological adaptation was less pronounced. Although HB, HCT and MCHC had returned to normal values, RBC was still decreased and MCV, MCH and RDW were still Increased at week 6 when compared to female controls.
Small changes in RBC, HB and HCT detected in males treated at 50 mg/kg/day did not reach statistical significance when compared to the (slightly high) control values and were still within the range of historical data established for rats of this age and strain. Therefore, these changes were considered to be of no toxicological significance.
-White blood cells and platelets
After 4 weeks absolute white blood cell counts were increased in males and females of the 1000 mg/kg dose group and differential counts of segmented neutrophils were increased in males of this dose group, thus causing a relative decrease in lymphocyte count.
At week 4, platelets were found to be increased in males treated at 200 or 1000 mg/kg/day and the same effect, although very slight, was observed in females receiving 1000 mg/kg/day.
After 2 weeks of recovery, white blood cell and platelet numbers were considered to be similar for control and treated animals.
Statistical differences arising in prothrombin time and differential monocyte counts in treated males or females were considered to have occurred by chance as no relationship with dose was detected nor any corresponding changes in the opposite sex.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Oral treatment for 28 days with test substance resulted in increased bilirubin concentrations in males and females receiving 1000 mg/kg/day and Increased triglyceride levels in females of this dose group when compared to controls.
Further differences occurring at week 4 between treated and control rats, were considered not to represent a clear sign of toxicity. Statistically significant changes resulted from slightly high control values or did not show any dose-relationship and the majority of values were still within the normal background range established for rats of this age and strain.
At the week 6 examination, no toxicologically significant changes were detected as all parameters in the serum of animals previously treated with 1000 mg/kg were considered to be within the normal range.

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Spleen weights of both males and females receiving 200 or 1000 mg/kg/day were increased after 4 weeks of treatment with test item.
Organ weights at week 4 of animals receiving 50 mg/kg or at week 6 of animals previously treated with 1000 mg/kg, were considered not to be clearly affected by the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The only toxicologically significant finding observed at necropsy was an enlarged spleen in all animals of the 1000 mg/kg dose group after 4 weeks of treatment. After recovery for 2 weeks no alterations were ascribed to toxicity of the test substance.
Blue discolouration of (a selection of) organs and tissues was seen among animals of all treated groups at week 4. In the 200 mg/kg dose group, bluish discoloration was observed in the mesenteric lymph node of all rats and in the mandibular lymph nodes, the mediastinal lymph nodes, the iliac lymph nodes, the kidneys, the lungs, and the liver of one or several rats. In the 50 mg/kg dose group one male rat showed bluish discolouration of the lungs. In animals of the 1000 mg/kg dose group all internal tissues showed blue discolouration and this was still present at the end of the recovery period. Findings were considered to be directly related to the staining properties of the test substance and not to have arisen as a result of toxicity.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the spleen of males and females receiving 200 or 1000 mg/kg for 28 days, increased haemopoiesis, increased haemosiderin deposits (as confirmed with Per's staining method for iron) and congestion was noted. In the liver of one female rat receiving 1000 mg/kg/day slight extramedullary haemopoiesis was seen in the liver as well. The incidence and degree of the histopathological changes in the spleen was more pronounced in the high dose group than in the mid dose group. In the 50 mg/kg dose group and in animals of the 1000 mg/kg dose group after recovery, type and incidence of changes in the spleen were comparable to those of controls. The small number of other findings recorded was within the normal range of background alteration which may be seen in untreated controls of age and strain; the spleen or other organs did not show any changes that were considered to represent a toxicological effect.

Details on results:

There were no difficulties in analysis of test substance preparations.
Toxicological effects of the test substance were seen in animals treated at 200 or 1000 mg/kg/day at week 4. The main organ affected was considered to be spleen. An increased breakdown of red blood cells was observed and this was reflected in haematological parameters (decreased RBC and HCT) and in clinical biochemistry (increased bilirubin). Confirmation of this anaemic state was found at necropsy examinations (spleen enlarged and increased in weight) and on microscopic evaluation (congestion, increased haemosiderin accumulation and increased haemopoiesis).
As a result of new formation of blood, more immature red blood cells were present in the circulation, reflected in the increased values for MCV and RDW and a decrease in MCHC.
The changes seen in white blood cell counts, neutrophils and serum triglyceride levels may be related to treatment, although their toxicological significance remains unclear.
After 2 weeks of recovery, the only changes seen were those in haematology parameters, indicative of an adaptive response to the breakdown of erythrocytes. The spleen appeared to have returned to normal function and activity.
There were no changes in body weights, food consumption and ophthalmoscopic examination that were considered to be an effect of treatment.

Key result

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

ANALYSIS OF DOSE PREPARATIONS

Test substance formulations in water were noted as stable for at least 4 hours at all concentrations tested. In addition, these test substance preparations formed a homogeneous suspension. Analysis of the accuracy of dose preparations revealed values within the range of 96 % to 106 % of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

DOSE LEVEL SELECTION

From the 5 -day dose range finding study these results were obtained:

-Mortality: No mortality.

-Clinical signs: No toxicological significant findings.

-Body weight: Normal.

-Food consumption: Normal.

-Macroscopic examination: No toxicological significant findings.

-Liver/Kidney weight: Normal.

Conclusions:

NOEL (oral, 28 d) = 50 mg/kg/day

Executive summary:

The repeated dose toxicity of the substance was evaluated in a subacute 28-day toxicity study, according to the OECD Guideline 407 (1981) and the method B.7 of the EEC-Directive 67/548 EEC. The substance was administered daily by gavage to Wistar rats for a period of 28 days, followed by a 14-day recovery period. The number of rats assigned to toxicity testing per group as well as the dose levels administered was as follows: 10 animals (5 females and 5 males) for doses of 50 and 200 mg/kg b.w.; 20 animals (10 males and 10 females) for doses of 0 and 1000 mg/kg b.w.. The dose levels selection was based on a 5-day dose-range finding. The suitability of the chosen vehicle (water) for the test item and its sufficient stability in the vehicle was analytically verified. No mortality occurred during the study period. No clinical signs considered of toxicological significance were found. There were no changes in body weights, food consumption and ophthalmoscopic examination that were considered to be an effect of treatment. Toxicological effects of the test substance were seen in animals treated at 200 or 1000 mg/kg/day at week 4. The main organ affected was considered to be spleen accompanied by various changes in haematological parameters. In the 50 mg/kg dose group and in animals of 1000 mg/kg dose group after recovery group, the spleen or other organs did not show any changes that were considered to represent a toxicological effect.

The NOEL was found to be 50 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

haematopoietic

Organ:

spleen

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 repeated dose toxicity of the substance was evaluated in a subacute 28-day toxicity study, according to the OECD Guideline 407 (1981) and method B.7 of the EEC-Directive 92/69 EEC. The substance was administered daily by gavage to Wistar rats for a period of 28 days, followed by a 14-day recovery period. The number of rats assigned to toxicity testing per group as well as the dose levels administered was as follows: 10 animals (5 females and 5 males) for doses of 50 and 200 mg/kg b.w.; 20 animals (10 males and 10 females) for doses of 0 and 1000 mg/kg b.w.. The dose levels selection was based on a 5-day dose-range finding. The suitability of the chosen vehicle (water) for the test item and its sufficient stability in the vehicle was analytically verified.

Based on the study findings, a NOAEL (No Observed Adverse Effect Level) of 50 mg/kg/d was found. Toxicological changes were detected among males and females treated with the test substance at dose levels of 200 and 1000 mg/kg.

Concerning the haematological parameters, an increased breakdown of red blood cells was observed as reflected by a decrease of RBC and HCT. This increased breakdown of red blood cells was also reflected by clinical biochemistry results that showed an increase of bilirubin. As a result of new formation of blood, more immature red blood cells were present in the circulation, resultingin increased values of MCV and RDW and a decrease in MCHC. Changes in white blood cell counts, neutrophils and serum triglyceride levels were noted and they may be related to treatment, but, their toxicological significance is unclear. After a 14-day recovery period, haematology parameters indicated an adaptive response to the breakdown of erythrocytes.

Microscopically, treatment-related findings were recorded in the spleen: congestion, increased haemopoiesis and increased haemosiderin deposits. The incidence and degree of the histopathological changes in the spleen were more pronounced in the high dose group than in the mid dose group. In the 1000 mg/kg dose group after the recovery period, the type and incidence of changes in the spleen were comparable to those of controls.

Macroscopically, an increase in the spleen weight was noted while at the highest dose the spleen was also enlarged. After a 14-day recovery period, the spleen was noted to have been returned to a normal weight, appearance, function and activity.

These findings are likely to be an indication of a dose-dependent anaemic state induced by test substance in both sexes. Considering that:

(i) the effects at 200 mg/kg are of similar nature but of a lower severity than those found at 1000 mg/kg;

(ii) the reversibility of the effects at the high dose level was proved by the complete/partial recovery observed in the two weeks after the exposure period,

it is likely that at the mid dose a full recovery could be have been observed. Even if a recovery period was not studied for this dose-group during the test, the reversibility of the effects is expected. Moreover, it could be noted that recovery at the high dose was observed in a 28-day study period and so it could be expected in a 90-day study period.

Justification for classification or non-classification

According to the CLP Regulation (EC n. 1272/2008), classification in Category 1 for repeated dose toxicity applies to substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure. Substances are classified in Category 1 for target organ toxicity (repeat exposure) on the basis of:

— reliable and good quality evidence from human cases or epidemiological studies; or

— observations from appropriate studies in experimental animals in which significant and/or severe toxic effects, of relevance to human health, were produced at generally low exposure concentrations.

Classification in Category 2 applies to substances that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to be harmful to human health following repeated exposure. Substances are classified in Category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations. If classification is based on results obtained from studies conducted in experimental animals, the dose/concentration guidance values that classify refer to effects seen in a standard 90-day toxicity study conducted in rats. These guidance values are presented in the CLP Regulation (EC n. 1272/2008) in Annex 1: 3.9.2.9.6., Table 3.9.2 for classification in Category 1 and in Annex 3.9.2.9.7., Table 3.9.3 for classification in Category 2. For a 28-day study the guidance values is increased by a factor of three.

Based on the results of Repeated Dose 28-Day Oral Toxicity Study OECD 407, no classification for repeated dose toxicity is warranted under the CLP Regulation (EC n. 1272/2008).