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Administrative data

Description of key information

NOEL = 50 mg/kg in female rats

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:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Comparable to OECD guideline 407.
28-day repeated-dose oral toxicity and 2-week recovery test based on test guidelines of the Act on the Evaluation of Chemical Substances and Regulation of Their Manufacture, etc. [Partial Revisions of “Test Methods Pertaining to New Chemical Substances”] (5 December 1986 Environmental Conservation Project Notice No. 700 of the Environment Agency Planning and Coordination Bureau Head, Pharmaceutical and Food Safety Bureau Notice No. 1039 of the Ministry of Health and Welfare Pharmaceutical Affairs Bureau Head, and 1986 Basic Industries Bureau Notice No. 1014 of the Ministry of International Trade and Industry Basic Industries Bureau Head.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International Inc.
- Age at study initiation: 4 weeks
- Weight at study initiation: M: 137 - 161 g, F 120 - 132 g
- Housing: aluminium cages [380mm (L) × 300mm (W) × 180mm (H)] with inserted bedding that housed 2 or 3 rats each. The room interior was cleaned each day with a concentrated dust collection apparatus, and the cages, bedding, and water bottles were replaced with sterilised ones twice each week.
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY:
Feed given was a cobalt-60 radiation-sterilised solid feed (CE-2, Lot No. E-2010, E-2030, CLEA Japan Inc.) and inspected service water conforming to the Water Works Law water quality criteria was inserted into the water bottles as drinking water and given freely.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 10 %
- Air changes: 15 per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: test substance was dissolved by distilled water up to a dosage of 1 ml/kg


Duration of treatment / exposure:
28 days + 14 days recovery
Frequency of treatment:
once daily
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 sex/group (control, control + recovery, low dose, medium dose, high dose, high dose + recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a preliminary 7-day repeated-dose oral toxicity test on rats at doses of 10, 50, 100, 500, 1000 mg/kg. In particular, in the preliminary test, death was seen in the 1000 mg/kg group, and high GOT and GPT values were seen in the blood chemical analysis of 500 mg/kg and above groups; blackish feces was observed in general conditions, but no effects were observed resulting from the administration of test substances in the 100 mg/kg and under groups. Based on these factors, the high dose was set to 250 mg/kg, an amount by which the expression of toxic changes can be expected; the medium dose was set to 50 mg/kg with the following common ratio of 5, and the low dose was set to 10 mg/kg.
- Rationale for animal assignment: randomisation of weight
- Post-exposure recovery period in satellite groups: 14-days
Observations and examinations performed and frequency:
General conditions
The life and death of the animals and their general conditions were observed and recorded once daily during the quarantine and acclimation period, and then 3 times daily during the administration period (approximately 2 hours before administration, then approximately 2 and 5 hours after administration).

Food intake
The food intake and remaining amounts were measured for each cage utilising an electronic balance (FX-3000, A&D Company, Limited) once each week, and the daily food intake was calculated.

Body weight
Calculation was done utilising an electronic balance once each week.

Ophthalmological examination
An examination with the naked eye was carried out during the daily observations of general conditions.
Furthermore, as for machine-based examinations, a funduscopy was performed once before the start of administration, and then on the fourth week of administration and second week of recovery using a retinal camera on 5 cases of males and females each starting from the smaller animal number designations in each group. In addition, a mydriatic was applied to the eyes as a preliminary treatment during the funduscopy.

Clinical examination
a) urine analysis
The colour tone of the fresh urine, which was collected once before the start of administration and on the fourth week of administration and second week of recovery, was examined with the naked eye for pH, protein, sugar, ketone bodies, bilirubin, uric blood, and urobilinogen.
b) hematologic test
Blood was collected from the abdominal portion of the vena cava under ether anesthesia, utilising a syringe during dissection the day after completion of the final administration and completion of the recovery period. The EDTA-2K anticoagulant-treated whole blood was measured for the red blood cell count, leukocyte count, blood platelet count (electric resistance detection system), hematocrit value (pulse detection method), hemoglobin concentration (hemoglobin complex method), MCV (mean cell volume) and MCHC (mean corpuscular hemoglobin concentration), in each case utilising a multi-item automatic blood cell counting device; the same whole blood was measured for the reticulocyte count (New methylene blue / Wright double-staining technique) and differential leukocyte count (Wright staining technique), in each case utilising an automatic blood cell analyser. In addition, blood was collected in the same manner, and after performing anticoagulant treatment using a 3.8 % sodium citrate solution, the blood plasma obtained through centrifugal separation (3000 r.p.m., 15 minutes) was measured for the prothrombin time and activated partial thromboplastin time utilising an automatic blood coagulation time measuring device.
c) blood chemical analysis
Blood was collected from the ventral aorta utilising a syringe upon completion of the final administration and during dissection on the day after completion of the recovery period. After leaving this to stand for 40-60 minutes at room temperature, the serum obtained by centrifugal separation (3000 r.p.m., 15 minutes) was measured for GOT, GPT (JSCC-compliant formula), ALP (p-nitrophenylphosphate substrate method, LAP (L-leucyl-p-nitroanilide substrate method), γ-GTP (clathrate L-γ-glutamyl-p-nitroanilide substrate method), total bilirubin (alkaliazobilirubin method), total protein (biuret method), albumin (BCG method), A/G (calculation), total cholesterol (COD-DAOS method), triglyceride (GOP-DAOS method), cholinesterase (BTC-DTNB method), sugar (Glck / G-6-PDH method), BUN (urease-GLDH method), Na, K (electrode method), and Cl (coulometric titration method) utilising an automated analyser.



Sacrifice and pathology:
Organ weight
The absolute weight of the adrenal glands (left and right), testes (left and right), ovaries (left and right), brain (including the cerebellum), heart, lungs, liver, kidneys (left and right), and spleen were measured using an electronic balance and the weight relative to the body weight of each of these organs was simultaneously calculated.

Anatomicopathological examination
After collecting blood under ether anesthesia for a clinical examination the day after completion of the final administration and completion of the recovery period (after 16-22 hours of no eating), euthanisation was carried out by exsanguination, and the organs and tissue were observed with the naked eye.

Histopathological examination (optical microscopy)
The heart, spleen, marrow and bone (thigh bone), lungs, stomach (gastric corpus and pyloric region), liver, kidneys (left and right), urinary bladder, testes (left and right), ovaries (left and right), pituitary gland, thyroid gland, parathyroid gland, adrenal gland, cerebrum, cerebellum, brainstem, and eyeballs (left and right, including the Harderian gland and optic nerve) were suspended in 20 % neutral formalin (formaldehyde/glutaraldehyde liquid mixture in the case of the eyeballs, optical nerve and Harderian gland) and stored. Among these stored organs, thinly sliced specimens were produced in accordance with routine procedure for all cases of the heart, spleen, liver, kidneys (left and right), testes (left and right), ovaries (left and right), and adrenal gland. Such specimens were also made for the following cases in which an abnormality was observed with the naked eye: one male case (No. 32) of a kidney in the 50 mg/kg group, and two female cases (No. 38, 40) in the 50 mg/kg group and one female case (No. 58) in the 250 mg/kg group of a lung. In addition, such specimens were made for the following recovery test cases in which an abnormality was found: one male case (No.1) in the control group and two male cases (No. 41, 45) in the 250 mg/kg group of a kidney, and one female case (no. 54) in the 250 mg/kg group of a lung. The specimens were H/E stained and microscopically examined.
Statistics:
To start with, a homoscedasticity test was performed based on the Bartlett method regarding the data of each test group for body weight, hematologic test values, blood chemical analysis values and organ weight (absolute and relative weight).
In the case of equal variances, the variances were analysed using one-way classification. As a result, when a significance was observed, a paired comparison test of the mean values of each group was performed in relation to the control group based on the Dunnett method if the data quantity of each group was constant, and said test was performed based on the Scheffé method if the data quantity was indeterminate. If no homoscedasticity was observed based on the Bartlett method, the order was converted and a Kruskal-Wallis H test was performed.
As a result, when a significance was observed, a paired comparison test of the Dunnett model of the average of the ranks was performed if the data quantity of each group was constant, and said test of the Scheffé model of the average of the ranks was performed if the data quanitty was indeterminate.
A Fisher test was performed for the urine colour and general conditions with no evaluation ranking, and the percentage was calculated in relation to the control group for the food intake.
Using a COMPUTER (Micro VAX3600, DEC ), the less than 5 % risk ratio in these tests and calculations was deemed significant.
In this text it is indicated that “high values” or “low values” were found in relation to the toxic changes associated with statistical significance. These changes were compared with the background data (Shin Nippon Biomedical Laboratories Background Data Collection, Vol. 24-2, 1990) with regards to the rats of this test facility and while they are not significant changes, when judged to be a toxicological significance, they are expressed as “such trends were observed”.

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Only blackish feces was observed in all male and female cases from the 250mg/kg group. The blackish feces was observed daily from the 23rd day of administration in the earliest case and from the 27th day in the latest case until the day after the final administration in both cases, which corresponds to Day 0 of recovery. However, no blackish feces was observed in any cases following Day 1 of recovery.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
High values in the quantity and ratio of segmented neutrophils were observed in females from the 50 and 250 mg/kg groups during testing at the completion of the animation period, and high values in the quantity of segmented neutrophils were observed in males from the 250 mg/kg group during testing at the completion of the recovery period. However, no abnormalities were observed in the leukocyte count in either case, and it was determined that because there was no commonality between males and females, the values were not the result of the administration of the test substance.
In addition, while low values in the red cell blood count were observed in males from the 10 mg/kg group during testing at the completion of the administration period, there was no correlation between doses, and the values were determined to be incidental changes.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
High GOT values were observed in females from the 250 mg/kg group during testing at the completion of the administration period. In addition, low Cl values were observed in males from the 10 and 250 mg/kg groups, and high Na values were observed in females from the 250 mg/kg group during testing at the completion of the administration period. There was no commonality between males and females, and furthermore since no abnormal values were observed in other electrolytes, it was determined that these values were not the result of the administration of the test substance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The results of the statistical processing were that low pH values were observed at the completion of the administration period and low protein values were observed at the completion of the recovery period in females from the 250 mg/kg group. However, there was no commonality between males and females, and moreover, when taking the background data into account, it was determined that either change was incidental and caused by the fluctuations of the control group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
High values for the absolute weight of the liver were observed in females from the 250 mg/kg group in pathologic autopsy cases at the completion of the administration period. No other abnormalities were observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No notable changes were observed that could be attributable to the administration of the test substance.
In addition, while the following changes were observed, none of them were correlated with the volume. It was determined that since these changes were observed in a few cases, they were not toxic changes. At the completion of the administration period, slight pyelectasis was observed in one male case (No. 32) from the 50 mg/kg group, and rice grain-sized, adsuki bean-sized, or soybean-sized dark-red foci were found localised or scattered in the lungs in one female case (No.58) from the 250 mg/kg group. At the completion of the recovery period, slight pyelectasis was observed in one male case (No. 1) from the control group and in 2 male cases (No. 41, 44) from the 250 mg/kg group, and millet grain-sized dark brown foci were observed scattered in the lungs in one female case (no. 54) from the 250 mg/kg group.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No notable changes were observed that could be attributable to the administration of the test substance.
Furthermore, the following changes were observed as incidental or naturally occurring lesions. At the completion of the administration period, very minor to moderate extramedullary hematopoiesis was observed in the spleen in all male and female cases from the control group and 250 mg/kg group. Moreover, very minor pyelectasis was observed in the kidneys in one male case (No. 32) from the 50 mg/kg group, in which abnormalities were found in the anatomicopathological examination, and very minor peribronchiolar hyperplasia of the lymph follicles was observed in two female cases (No. 38, 40) from the 50 mg/kg group and in one female case (No. 58) from the 250 mg/kg group. In addition, minor to moderate red crystalline material was observed within the alveoli in one female case each (No. 40, 58) from the 50 and 250mg/kg groups. At the completion of the recovery period, very minor pyelectasis was observed in one male case (No. 1) from the control group and two male cases (No. 41, 44) from the 250 mg/kg group, and very minor hemosiderosis was observed within the alveoli and in the alveolar wall in one female case (No. 54) from the 250 mg/kg group.


Details on results:
Blackish feces was found in the 250 mg/kg group during the second half of the administration period during the observation of general conditions, but this disappeared following the first day of recovery, and no abnormality was observed afterward.
When taking into account the facts that the test substance is a black powder, no abnormalities were observed in the digestive tract in the results of the anatomicopathological examination, and that the blackish feces disappeared simultaneously together with the cessation of medication, and although it is inferred simply that the substance was mixed into the feces, the possibility is conceivable that some sort of change had taken place in the enzyme systems in living objects or the intestinal bacterial flora, which are considered to be involved in the decomposition or metabolism of the test substance in relation to the facts that the fastest expressed case was on the 23rd day of administration and the excretion was confirmed toward the completion of the administration period.
During testing at the completion of the administration period, high GOT values and high liver weight values in the organ weights were observed in females from the the 250 mg/kg group. The high GOT and GPT values of the 500 and 1000 mg/kg groups in the previously implemented preliminary 7-day repeated-dose oral toxicity test using rats were ascertained together with the high values of total cholesterol and triglyceride, and it was suggested that there was an effect on the liver. When also taking into consideration these preliminary test results, the results of this test were considered to be supportive of an effect upon the liver resulting from the administration of the test substance. However, since normality was restored during testing at the completion of the recovery period, such an effect was considered to be reversible.
Without any abnormalities observed in the food intake, body weight or ophthalmological tests, no changes attributable to the administration of the test substance were observed in either the urine analysis, hematologic test, pathologic autopsy or histologic examination. Moreover, given the facts that red crystalline material was confirmed with a light microscope and that peribronchiolar hyperplasia of the lymph follicles had been observed, it was conceivable that the few cases of dark red and dark brown folics observed in the lungs during the autopsy could also be a response profile based on the xenopneumonia resulting from the accidental ingestion of the test substance, and it appeared natural to consider that the test substance containing Fe, while only slight, had entered into the trachea due to some cause.
Nonetheless, since no abnormality of any kind had been expressed in general conditions during the administration, it was not necessarily determined to be a change resulting from an erroneous administration. '
From the above results, and given the suggestion of an effect, while negligible, upon the liver in females from the 250 mg/kg group under the conditions of this test, the NOEL was determined to be 50 mg/kg.
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
other: liver and related blood parameters
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
NOEL = 50 mg/kg
Based on high GOT and high liver weights female rats in the 250 mg/kg group. Normality was restored during testing at the completion of the recovery period, thus such an effect was considered to be reversible.


Executive summary:

Repeated dose toxicity of Basic Brown 022 was tested in rats at doses of 0 (control), 10, 50 and 250 mg/kg; rats were dosed for 28 days; a 14 days recovery period followed exposure for satellite groups of 5 rat/sex at 0 mg/kg and at 250 mg/kg.

Test doses were chosen based on a 7 -day preliminary test at 10, 50, 100, 500 and 1000 mg/kg, where mortality occurred at 1000 mg/kg and changes in blood parameters were seen at 500 mg/kg.

In the main test, no deaths occurred during the administration or recovery periods. Blackish feces was found in the 250 mg/kg group, but this disappeared following the first day of recovery, and no abnormality was observed afterward; no abnormalities were observed in the digestive tract in the results of the anatomicopathological examination; high GOT values and high liver weight values in the organ weights were observed in females from the the 250 mg/kg group.

When also taking into consideration the preliminary test results, the results of the main test were considered to be supportive of an effect upon the liver resulting from the administration of the test substance. However, since normality was restored during testing at the completion of the recovery period, such an effect was considered to be reversible.

Without any abnormalities observed in the food intake, body weight or ophthalmological tests, no changes attributable to the administration of the test substance were observed in either the urine analysis, hematologic test, pathologic autopsy or histologic examination.

From the above results, and given the suggestion of an effect, while negligible, upon the liver in females from the 250 mg/kg group under the conditions of this test, the NOEL was determined to be 50 mg/kg.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
System:
hepatobiliary
Organ:
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

28 -day repeated dose toxicity study in rats at doses of 0, 10, 50 and 250 mg/kg. A 14 -day recovery period followed dosing for in the control and high dose group.

High GOT values and high liver weight values were recorded in females from the 250 mg/kg group. However, as normality was restored during testing at the completion of the recovery period, such an effect was considered to be reversible.

Based on such effects, even if negligible, the dose of 50 mg/kg was taken as NOEL.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), classification for repeated dose toxicity is as follows:

- category 1: 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: (i) reliable and good quality evidence from human cases or epidemiological studies; or (ii) 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.

- category 2: 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.

Moreover, as reported in the CLP Regulation (EC 1272/2008), effects considered not to support classification for specific target organ toxicity following repeated exposures are: small changes in clinical biochemistry, haematology or urinalysis parameters and/or transient effects, when such changes or effects are of doubtful or minimal toxicological importance; changes in organ weights with no evidence of organ dysfunction.

The effects observed on GOT values and liver weight of female rats were reported to be light and reversible. Overall, effects reported in female rats at the dose of 250 mg/kg were considered as not sufficient to prompt a classification for repeated dose toxicity for Basic Brown 022.