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Administrative data

Description of key information

NOEL (28d, oral, rat, male and female) = 50 mg/kg/day

NOAEL (28d, oral, rat, male and female) = 200 mg/kg/day

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
November 27 to January 08, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
Details for read across approach are included into the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted on May 12, 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
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd., Basel, switzerland.
- Age at study initiation: approximately 6 weeks old.
- Housing: animal were housed 5 to a cage (same sex) in stainless steel suspended cages with wire mesh floors.
- Diet: standard pelleted laboratory animal diet (Kilba, Klingentalmuehle AG, 1303), ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 7 days.

DETAILS OF FOOD AND WATER QUALITY
Each batch of food was analysed for contaminants, as well as tap water.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C
- Relative humidity: 55 %
- Air changes: 15 air changes per hour.
- Photoperiod: 12 hrs dark / hrs light.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- Dose volume: 5 ml/kg bw
- Actual dose volume: calculated weekly according to the latest body weight.

FORMULATION
- Formulation: test substance was weighed into a glass flask on an analytical balance and the vehicle added (w/w).
- Frequency of substance formulation: daily, immediately prior to dosing.
- Homogeneity of test article in vehicle: by the use of electric shaker. From chemical analysis, the test susbtance appeared to be a homogeneous suspension in water at all concentrations used in the test.
- Storage for test item fomulation: at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis: samples of formulations prepared during week 1 were analysed to check the accuracy of preparation.
Actual concentrations of preparations were in agreement with the treatment levels as per protocol.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, approximately the same time each day, 7 days per week
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
900 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males and 5 females per group
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
MORTALITY
Twice daily. Animals showing pain, distress or discomfort and which were considered not transient in nature or was likely to become more severe were sacrificed for human reasons.

DETAILED CLINICAL OBSERVATIONS
At least once daily. Severity of observations were graded.

BODY WEIGHT
Weekly and on the day preceding termination, prior to overnight fasting.

FOOD CONSUMPTION
Weekly.

OPHTHALMOSCOPIC EXAMINATION
Both eyes were examined following instillation examinations of tropicamide solution (5 mg/ml) before commencement of treatment and during the last week of treatment.

BLOOD SAMPLING
Blood samples were collected under light ether anaesthesia immediately prior to post mortem examination, between 8.00 and 10.00 a.m.. 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) and untreated tubes for clinical biochemistry parameters (>2 0 ml). Blood samples were analysed with the exception of serum samples for the determination of Chloride. These were kept deep frozen and sent to testing laboratory.

HAEMATOLOGY
The following haematology parameters were determined from blood containing EDTA as an anti-coagulant.
Parameter: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, red cell distribution, total leucocyte count, differential leucocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes).

CLINICAL CHEMISTRY
The following clinical biochemistry parameters were determined from serum samples after clotting and centrifugation.
Parameter: glucose, urea, creatinine, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin protein.
Sacrifice and pathology:
NECROPSY
All animals surviving to the end of the observation period (day 29) and all moribund animals were deeply anaesthetised by intraperitoneal injection of sodium pentobarbital 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, esophagus, 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, 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, thyroid inc1uding parathyroid, tongue, trachea, urinary biadder, uterus, vagina, all gross lesions.

ORGAN WEIGHTS
The following organ weights (and terminai body weight) were recorded at termination: adrenal glands, heart, kidneys, liver, spleen, testes.

HISTOTECHNOLOGY
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. Sections of liver were also stained with Gomori’s method for reticular fibres, Best's Carmine method for glycogen and van Gieson.

HISTOPATHOLOGY
Slides of adrenals, heart, kidneys, liver and spleen, collected at termination from all animals of the control and high dose group as well as from all animals of all dose groups, which died spontaneousiy or were terminated in extremis and all gross lesions of all animals were examined by a pathologist. Based on the treatment-related morphologic changes, liver and adrenals were also examined from all rats of group 2 (50 mg/kg/day) and 3 (200 mg/kg/day). All abnormalities were described and included in the report.
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.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. 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).
Individual values, means, 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):
Changes in clinical appearance or behaviour that were considered to be an effect of toxicity included lethargy, dark appearance of the eyes, rough coat, weight loss/emaciated appearance, hunched posture and pale appearance of the skin, all of which were noted in animals of the 900 mg/kg dose group.
Excessive salivation was noted intermittently in treated males and females receiving 200 or 900 mg/kg/day. Since this may be attributed to a possible irritant effect or bad taste of the test substance, it was considered not to represent a clear sign of toxicity. In addition, all treated animals showed dark or blue appearance of the faeces over the 4 weeks of observation. Considering the physico-chemical properties of test item, colour changes were considered to be due to test substance that had not been absorbed in the gastro-intestinal tract.
Regurgitation of test substance was noted intermittently in animals receiving 200 or 900 mg/kg/day and on a few occasions in males receiving 50 mg/kg/day. Since this phenomenon is very rare in rats and possibly may be associated with the above mentioned irritant effect of the test substance, it was considered not to be of toxicological significance.
Alopecia, which was noted incidentally among females receiving 200 or 900 mg/kg/day, is commonly noted in rats of this age and strain and, at the degree and incidence noted, considered not to represent a sign of toxicity.
Description (incidence):
One male of the 900 mg/kg dose group was found dead on day 8 of treatment. In addition, 1 other male of the 900 mg/kg dose group was killed for humane reasons on day 9.
No mortality occurred among animals receiving 200 or 50 mg/kg/day during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of males receiving 900 mg/kg/day were statistically significantly lower than controls over the 4 week study period. No changes were noted between body weights of control animals and animals receiving 200 or 50 mg/kg/day.
As a result of control weights tending towards the lower limits of that normally expected, multiple statistically significantly increased body weights were noted among treated females in comparison with controls during the study period. High body weights of treated females were therefore considered not to be an effect of toxicity.
Body weight gain was lower than controls among males receiving 900 mg/kg/day over the study period, although this decrease was not noted as statistically significant after 1 week of treatment. Body weight gain of females receiving 900 mg/kg/day showed an apparent decrease over the last 2 weeks of treatment when compared to body weight gain of other treated and control rats over the same weeks. Although values remained in the same range as controls, the clear change in body weight gain was considered to be of toxicological significance. Body weight gain of animals receiving 200 or 50 mg/kg/day remained similar to control animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was noted as decreased in comparison with controls in males receiving 900 mg/kg/day over the 4 weeks of treatment. Food consumption by treated females and males receiving 200 or 50 mg/kg/day were similar to those of control rats.
Relative food consumption by males receiving 900 mg/kg/day was considered to be lower than control males after 1 week of treatment and higher than controls after 2, 3 and 4 weeks of treatment, indicating an initial loss of appetite and later, associated, lack of body weight gain. Relative food consumption of males receiving 200 or 50 mg/kg/day or treated females were considered to be within the normal biological range for rats of this age and strain.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no differences between the pretest examination and the examination at week 4 that could be attributed to treatment with test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cell parameters of treated rats were considered not to have been affected by treatment. Minor statistically significant differences arising between red blood cell parameters of females receiving 900 mg/kg/day and control females were within normal biological variation for rats of this age and strain and were considered to have arisen by chance.
Statistically significantly increased total white blood cells were noted in males receiving 900 mg/kg/day when compared to control males. White blood cell numbers in females receiving 900 mg/kg/day or males and females treated at 200 or 50 mg/kg/day, remained in the range of controls.
Statistically significantly increased platelets in males receiving 900 mg/kg/day and decreased platelets in females receiving 200 mg/kg/day in comparison with corresponding controls were considered to be the result of an incidentally high or low individual platelet value and considered not to be of biological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Creatinine values of males receiving 900 mg/kg/day were statistically significantly decreased when compared to control males. Creatinine values of females receiving 900 mg/kg/day and animals receiving 200 or 50 mg/kg/day were comparable to control values.
Total protein was noted to be statistically significantly lower than controls in animals receiving 900 mglkg/day. Animals receiving 200 or 50 mg/kg/day were not thus affected.
Comparing to control values, markedly statistically significantly increased values were noted in total bilirubin (males and females receiving 900 mg/kg/day), aspartate aminotransferase (males receiving 900 mg/kg/day), alanine aminotransferase (males and females receiving 900 mg/kg/day) and alkaline phosphatase (males and females receiving 900 mg/kg/day). No such changes were noted in animals receiving 200 or 50 mg/kg/day.
Other values in treated animals achieving a level of statistical significance when compared to controls, were within the range normally expected for rats of this age and strain and considered to have arisen fortuitously.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no differences in organ weights of treated animals and control animals that were considered to have arisen as an effect of toxicity.
Statistically significant changes were noted to have arisen in male organ/body weight ratios. These were considered to be the result of extremely low body weights, beyond which organ/body weight ratios are meaningful, and therefore not to represent a sign of toxicity.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopically observed blue/black appearance of tissues was noted in animals treated at 900 mg/kg/day, both intercurrent deaths as well rats killed at scheduled sacrifice. In addition blue discolouration of the kidneys was observed in 1 male rat receiving 200 mg/kg/day.
Black areas in the lungs were noted in 1 female receiving 200 mg/kg/day and in 1 female receiving 900 mg/kg/day.
Other autopsy findings noted in 1 or 2 animals treated at 900 mg/kg/day comprised of: ascites, distended stomach with red areas, distended urinary bladder, small and pale liver with irregular surface and small seminal vesicles.
The small number of other pathological findings were those of spontaneous nature commonly seen in rats of this age and strain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic observation of the organs listed revealed distinctly treatment related changes in the liver and adrenals of animals receiving 900 mg/kg/day. There were no obvious treatment related changes noted in animals treated at 200 or 50 mg/kg/day.
In the liver the lesions consisted of severe hepatocellular changes and bileduct hyperplasia.
The hepatocytes were enlarged, the cytoplasm appeared homogeneous eosinophilic or showed fine vacuolisation. Sometimes there were large intracytoplasmic vacuoles. The nuclei were large and vesicular and they contained prominent nucleoli. An increased mitotic rate was observed.
Apoptotic bodies and necrotic liver cells were regularly encountered. The hepatocellular changes seemed to start in the periportal area, and were observed throughout the liver in more severe affected rats.
In all males receiving 900 mg/kg/day a striking bile duct hyperplasia was noted in the periportal zone. Sometimes this hyperplasia extended to interlobular bridges, and was accompanied by slight fibrosis. All males receiving 900 mg/kg/day showed hepatic changes as described above, and in most female rats of this group very slight liver changes in the hepatocytes were noted as well. Accumulations of blue stained (test) substance were noted in 1 male rat receiving 900 mg/kglday.
In the adrenals there was an increased incidence and degree of vacuolation in the cortical zone in 4/5 male rats receiving 900 mg/kg/day.
Depletion of the white pulp in the spleen was observed in the male rat of the 900 mg/kg group that died intercurrently. The male receiving 900 mg/kg/day that was killed in extremis showed necrosis in the white pulp.
In 1 female of the 900 mg/kg group, that showed black areas in the lungs, accumulations of blue test material accompanied by cellular reactions were observed. In 1 female receiving 200 mg/kg/day, also showing black areas in the lungs, a slight cellular reaction was noted only. Apart from the accumulations in the liver and the lung in the rats mentioned, there were no signs of blue stained organs upon microscopy.
Other histopathological changes that were probably treatment related consisted of haemorrhages in the stomach of 2 males, depletion of colloid in the seminal vesicles of 1 male and interstitial edema in the pancreas.
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall adverse effects
Critical effects observed:
no
Conclusions:
NOEL (28d) in males and females: 50 mg/kg/day
Executive summary:

A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 150 and 750 mg/kg/day) to provide a basis for selection of dose levels for a study of longer duration. No differences of biological significance were observed in clinical appearance, body weight, food consumption, macroscopic appearance or liver weights between the treated groups. However, mortality was noted in the oral LD50 study among animals treated at 1000 mg/kg body weight.

Based on these observations, a high treatment level of 900 mg/kg/day was selected for a study of 28 days duration.

In the subacute 28-day toxicity study, test item was administered daily by gavage to SPF-bred Wistar rats. The study included four groups. 5 male and 5 female rats were assigned to toxicity testing per group per dose level; doses administrated were 0, 50, 200 and 900 mg/kg.

At 50 mg/kg/day no treatment-related changes were noted. At 200 mg/kg/day macroscopically observed black areas were noted in the lungs of 1 female; cellular reaction was noted in the lungs of such female.

At 900 mg/kglday clinical signs of ill health were noted in males and females. One male was found dead on day 8 and 1 male was killed in extremis on day 9 of treatment. Markedly low body weights and body weight gain was noted in males over the 4 week study period. Females were noted with slightly low body weight gain after 3 and 4 weeks of treatment. Decreased food consumption was noted in males over the study period. Relative food consumption of males was noted as low after 1 week of treatment and high after weeks 2, 3 and 4 of treatment. Increased numbers of white blood cells were noted in males only.

Decreased serum creatinine was noted in males only and decreased total protein was noted in both males and females. Increased total bilirubin, alanine aminotransferase and alkaline phosphatase was noted in males and females and increased aspartate aminotransferase was noted in males only.

Macroscopically observed black areas were noted in the lungs of 1 female. Other findings included ascites, distended stomach with red areas, distended urinary bladder, small pale liver with irregular surface and small seminal vesicles. Microscopically observed lesions consisted of hepatocellular changes and bile duct hyperplasia noted in the liver; increased incidence and degree of vacuolation noted in the adrenal cortex; depletion of the splenic white pulp; cellular reaction and accumulation of test substance noted in the lungs; haemorrhage in the stomach (2/5 males); colloid depletion in the seminal vesicles (1/5 males) and interstitial edema in the pancreas (1/5 males).

Conclusion

From the results presented in the report a definitive No Observed Effect Level (NOEL) of 50 mg/kg/day was established. Toxicological changes were considered to be sex related and generally noted in male rats only. The principle target organ was established as being the liver with associated changes being observed in blood chemistry.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Organ:
kidney
liver

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

No studies on the repeated dose toxicity potential are available for Direct Blue 267. Therefore, an available study on Similar Substance 01 is used within a read across approach.

Read across approach can be considered reliable and appropriate to investigate the property (details for the approach are included into the IUCLID section 13).

In the subacute 28-day toxicity study, Similar Substance 01 was administered daily by gavage to SPF-bred Wistar rats. The study comprised of 4 groups. 5 male and 5 female rats were assigned to toxicity testing per group per dose level; doses administrated were 0, 50, 200 and 900 mg/kg. At 50 mg/kg/day no treatment-related changes were noted. At 200 mg/kg/day macroscopically observed black areas were noted in the lungs of 1 female; cellular reaction was noted in the lungs of such female. At 900 mg/kg/day clinical signs of ill health were noted in males and females. One male was found dead on day 8 and 1 male was killed in extremis on day 9 of treatment. Markedly low body weights and body weight gain was noted in males over the 4 week study period. Females were noted with slightly low body weight gain after 3 and 4 weeks of treatment. Decreased food consumption was noted in males over the study period. Relative food consumption of males was noted as low after 1 week of treatment and high after weeks 2, 3 and 4 of treatment. Increased numbers of white blood cells were noted in males only. Decreased serum creatinine was noted in males only and decreased total protein was noted in both males and females. Increased total bilirubin, alanine aminotransferase and alkaline phosphatase was noted in males and females and increased aspartate aminotransferase was noted in males only.

Macroscopically observed black areas were noted in the lungs of 1 female. Other findings included ascites, distended stomach with red areas, distended urinary bladder, small pale liver with irregular surface and small seminal vesicles. Microscopically observed lesions consisted of hepatocellular changes and bile duct hyperplasia noted in the liver; increased incidence and degree of vacuolation noted in the adrenal cortex; depletion of the splenic white pulp; cellular reaction and accumulation of test substance noted in the lungs; haemorrhage in the stomach (2/5 males); colloid depletion in the seminal vesicles (1/5 males) and interstitial edema in the pancreas (1/5 males).

From the results presented in the report, a definitive No Observed Effect Level (NOEL) of 50 mg/kg/day was established. Toxicological changes were considered to be sex related and generally noted in male rats only (Reijnders, 1991).

At 200 mg/kg/day, black areas and a slight cellular reaction in the lungs was noted in one female; blue discolouration of kidneys was noted in one male. Black areas in lungs was also reported for one female at 900 mg/kg/day.

Based on the type of effects and the number on involved animals, the dosage of 200 mg/kg bw/day can be considered as not causing significant health effects.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement, relying on all available evidences, including the use of recommended guidance values, which take into account the duration of exposure and the dose, which produced the effects, and are placed in one of two categories, depending upon the nature and severity of the effects observed.

To help reach a decision about classification of a substance, guidance values are provided to categorise a substance showing toxic effects. These values refer to effects seen in a standard 90-day toxicity study conducted in rats. However, they can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration (the assessment shall be done on a case-by- case basis). For example, for a 28-day study, guidance values are increased by a factor of three.

 

In a 28-day study in rats, the low dose of 50 mg/kg bw/day is identified as NOEL; the medium dose of 200 mg/kg bw/day is not associated to significant health effects and may be interpreted as NOAEL; the high dose of 900 mg/kg is associated to adverse effects on body weight, food consumption, clinical biochemistry, histopathological findings, thus it is considered as LOAEL.

By extrapolating between a dose causing no significant toxic effects, i.e. 200 mg/kg/day, and a dose causing toxic effects, i.e. 900 mg/kg/day, on the bases of the severity and type of effects seen at the highest dose compared to 200 mg/kg/day, it is likely that no significant health effects would occur at the threshold dose for classification of 300 mg/kg/day.

Consequently, the substance is not classified for repeated dose toxicity within the CLP Regulation (EC 1272/2008).