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Description of key information

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:
other: read across from similar substance
Adequacy of study:
key study
Study period:
04-1994 to 08-1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, test procedure in accordance with OECD 407 methods, meets generally accepted scientific principles, acceptable for assessment. Not GLP compliant. Justification for Read Across will be provided in section 13.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding Velaz Black Ox
- Housing: animals were housed in plastic cages T3 (supplier Velaz, Prague); in each cage there were 2 or 3 animals separated by sex. Were used as bedding dry and clean shavings of soft wood. During the study the animals were separated in experimental group; in each group there were 10 animals (5 males and 5 females). Animals were individually identified with colored patches on their coat with number from 1 to 10.
- Diet: animals were fed with mixture ST-1, supplied by Bergman, Jesenice u Prahy.
- Water: drinking water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 40-60%
- Photoperiod: 12 hours of light and 12 hours of darkness
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 100, 500 and 1000 mg/kg in addition of 0.5 % of methylcellulose (MC)
The substance in the form of an aqueous suspension was administered by gavage daily into the stomach.

MAXIMUM DOSE VOLUME APPLIED: 1000 mg/kg
Duration of treatment / exposure:
28 days
Frequency of treatment:
The administrations were made at the same time of day.
Remarks:
Doses / Concentrations:
100, 500 1000 mg/kg
Basis:
nominal in diet
No. of animals per sex per dose:
5 males and 5 females.
Control animals:
other: yes; animals were administered 0.5% methylcellulose solution
Details on study design:
- Duration of observation period following administration: 42 days.

Observations and examinations performed and frequency:
The animal's health was assessed by food consumption, weight of all animals included in the trial, by clinical examination, haematological, biochemical examinations, performed before the start of the study, 28th and 42nd day of the study. The urinalysis was performed on 28th and 42nd day of the study.
Pathological examination was performed on 28th and 42nd day of the study

EXAMINATIONS:
-Clinical observations: all animals were monitored daily. The parameters observed were: animal behaviour, anomalous physical activity, skin elasticity, color of the saliva and of the coat, urine discoloration.

- weight monitoring

-Haematological examination: Blood intended for haematological tests were collected in heparinized glass capillary orbital plexus into PVC tubes covered by anti-coagulant solution potassium (K2EDTA). The parameters that are assessed were: total number of leukocytes and erythrocytes, hematocrit, mean corpuscular volume, hemoglobin content using the hematology analyzer Coulter Counter CBC 5 and the total number of platelets using the Coulter Counter ZM computer; the shape, size of the blood cells .

- Biochemical examination: Blood samples were taken from the orbital plexus under general anesthesia . Animals were starved approximately 18 hours prior to sampling, but received drinking water ad libitum. Serum samples were prepared by spontaneous coagulation, and then spun for 10 minutes in a centrifuge MPW 360th. Automatic biochemical analyzer to Vitalab 210 were set the following parameters: total protein, glucose, transaminases (AST, ALT), bilirubin and creatinine. Urea, albumin and alkaline phosphatase (ALP) were measured on the 11th .The sodium and potassium was assessed potentiometrically using an ion-selective electrodes sodium and potassium (Crytur tour). Measurements were performed after diluting the blood serum of the appropriate buffer for the pH meter MS-22 (Laboratory Instruments Prague). Chloride and calcium ions were determined using kits BIO-LA-TEST Diagnostica spectrophotometer at 195 D

- Urine analysis: Tests were carried out using diagnostic strips Heptaphan (Lachema Brno). The parameters observed were: the number of erythrocytes, hemoglobin, ketones, bilirubin, urobilinogen, glucose, protein in urine and its pH.

Sacrifice and pathology:
- Pathological and histophatological examination: After the experiment the animals were killed. Following parameters were investigated: organ weight ( liver, adrenal , kidney, brain, testes)
Statistics:
Data were elaborated by Mann-Whitney test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the entire study there was no death of any animal. Post-treatement angry behaviour
Mortality:
mortality observed, treatment-related
Description (incidence):
During the entire study there was no death of any animal. Post-treatement angry behaviour
Body weight and weight changes:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
some very light effects on ions concentrations and cholesterol only at the highest dose
Urinalysis findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
The clinical observations reported changes in animal behavior applied in any group. Before the applications the animals were quiet, and after they reacted angrily to the treatment. The skin was elastic, the visible mucous membranes physiologically colored, shaped droppings. Only in males treated with the highest dose was revealed brown discoloration of the coat around the anus and vulva. For females, coat color was weak, observed in only three cases. During the study there was no death of any animal.

BODY WEIGHT AND WEIGHT GAIN
Average body weight was monitored at weekly intervals. By comparing the control animals with the treated animals, it was found that the substance did not affect significantly the weight at all doses. Only in male and female at the highest dose (1000 mg / kg) weight gains compared to the control were higher, although differences were not noted in feed consumption. In the 5th week of the study, the average weight of males at the highest administered dose increased by 46 g, for females the difference was 22 g. In the 6th and 7 week of the study, the difference in weight persisted only in males.

HAEMATOLOGY
On examination of the satellite surviving animals administered at the highest dose, a reduced level of hemoglobin was measured, which was in the physiological range for both sexes. The total number of leukocytes was moved at the upper limit of normal range.
During examination of the group of animals administered with the lowest dose (100 mg / kg) for each sex, no changes from the control group were observed. All parameters were in the physiological ranges.
In females group in which was administered the medium dose (500 mg / kg) it was found a statistically significant decrease of hemoglobin and increase hematocrit and MCV (mean corpuscular voIume). None of these values deviated from physiological range.
On investigation of this application in male group an increased incidence of leukocytes was found, whose number was at the upper limit of normal range.
In the groups with the highest dose applied, an increased hematocrit value was observed, although varied in the physiological range. In particular for males, the leukocyte count was at the upper limit. In differential leukocyte count young evolutionary forms appeared (especially leukocyte and myelocytes metamyelocyty).

BIOCHEMICAL
During the biochemical examination it was found that the males' group in which it was adminstered the lowest dose (100 mg/kg) it was found a statistical significant increased of cholesterol concentration and K + ions and a decreased concentrations of albumin. The females with the same dose showed a decreased of serum creatinine, total bilirubin and cholesterol and an increased concentrations of K + ions. All measured values fell within the physiological range.
During the examination of the group of animals administered with the medium dose of 500 mg / kg, it was measured in males a significant increased concentrations of K + ions and a decreased concentration of the Na ions. The females showed a reduced AST, creatinine, and Ca ions and a elevated cholesterol levels and K + ions.
In the groups of animals in which was applied the highest dose, decreased concentrations of Na 'ions and increased concentration of Cl' ions were measured, whose value were above the upper limit of normal range. During the examination of females were found a reduced concentrations of Na and Ca2+ ions and a increased cholesterol concentration and K ions. The measured concentrations of Cl 'ions are similar to the males, this value was above the upper limit of normal range. The parameters varied in the physiological range.

PATOLOGICAL EXAMINATION
The rats, that were killed at the end of the study, were registered under the histological numbers 260 - 269 / 94, 280 - 289/94, 300 - and 320 309/94 - 329/94, the rats killed at the end of the observation period, were registered with the following numbers 424 - 428/94 and 439 to 453 /94.

OTHER FINDINGS
Test results on the 28th day study:
At the end of application period, in 5 males and in one female from the group in which was applied the medium dose (500 mg / kg), it was observed a blue-black pigment on to the gastric mucosa . In one male with the highest dose administered (1000 mg / kg) it was observed in lung tissue a blue-black coloration , in particular in the hilus and in other males from this group were stained on blue-black color the skin of the tail.

With the histological examination in rats in which was applied only the 0.5% of methylcellulose solution, it was found an hematoxylin staining eozine (HE) histopathological changes. The staining for the presence of neutral lipids (oil red) have been detected small fat droplets in the plasma of hepatocytes localized exclusively in the periphery of liver lobes (peripheral steatosis). One male was so affected about 10% of hepatocytes in various wattles, isolated hepatocytes with fatty droplets localized in irregular wattles (irregulární steatosis) were observed in the liver, additional male and one female.
In the group with the lowest dose applied (100 mg / kg) it was demonstrated with HE staining the presence of grains in the gastric mucosa and in the liver of one male and in one female it was found the presence of small nodules in the immediate vicinity of the central vein. In the mid dose group administered (500 mg / kg) it was seen with the HE staining fine grains of pigment in the gastric mucosa of all males and in one female without a noticeable reaction of the mucosa. In the liver of one male and one female it was found small nodules. In two males administered with the highest dose (1000 mg / kg), it was detected pulmonary affections. In the vicinity of blood vessels there was perivascular infiltration. In the alveoli and in the septum, there were numerous macrophages with a blue-black granular pigment. In one case it was evident signs of inflammation.

- The result of examination 42nd day study: The animals in which was applied the solution of 0.5% methylcellulose did not show any histopathological changes. The animals, in which the highest dose was administered , during the histological examination of organs showed with a macroscopically coloration of the lung tissue, alveolar and interstitial macrophages coloured with blue-black pigment and insignificant reaction of lung tissue.



Dose descriptor:
NOEL
Effect level:
ca. 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
gross pathology
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
Analogue substance 1 was administered to rats Wistar by gavage into the stomach in the form of aqueous suspension at doses of 100, 500 and 1 000 mg / kg of animal weight daily for 28 days. This substance had no significant toxic effects on the organism of experimental animals. Only the highest dose administered 1000 mg / kg caused a reversible changes in haematological and biochemical parameters.
Based on the results of this study can be considered a zero effect level (NOEL) dose of 500 mg / kg.
Executive summary:
Saturonova modr LBRR 200 was tested in 28-day study according to OECD guidelines 407. In this study, Wistar rats were administered the test substance into the stomach tube in the form of aqueous suspension at a dose of 100, 500 and 1000 mg/kg weight of the animal. All experimental and control rats were monitored daily for their health, and detailed clinical examination was carried put weekly, measured by feed consumption and weight of the animals, which was corrected by application of a substance volume. B
lood samples for determination of hematological and biochemical parameters and urinalysis were performed at 28th and 42nd days of the study. All animals at the end of application or observation periods were investigated with macroscopic dissection of chest, abdomen and skull and organs were removed or further histological processing.
The test substance in the lowest (100 mg/kg) and in the mid dose (500 mg/kg) did not affect behaviour and growth of animal weight, or cause changes in haematological and biochemical parameters that deviate from physiological norms. Also the results of histopathological examinations did not show any change, that might be due to tested substance. Consequently, the dose of 500 mg/kg can be regarded as the zero level effect (NOEL).
The test substance in the highest dose (1000 mg/kg) has made the hematology changes in differential leukocyte count, in which young evolutionary forms of leucocytes, especially myelocytes and metamyelocyt were detected.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 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

In the framework of REACH Regulation animal testing has to be carefully evaluated and a sub-chronic toxicity study (90 days) (Annex IX, section 8.6.2) shall be proposed by the registrant only if the frequency and duration of human exposure indicates that a longer term study is appropriate. Relevant human exposure can be excluded in accordance with Annex XI section 3 where testing in accordance with sections 8.6 and 8.7 of Annex VIII, Annex IX and Annex X (Repeated dose toxicity and reproductive toxicity) may be omitted, based on the exposure scenario(s) developed in the Chemical Safety Report.

In the attachment a full Chemical Safety Report completed with exposure scenarios has been presented, where only very basic Risk management measures have been recommended to ensure Risk control for the described exposure and where the exposure has been conservatively overestimated taking into account the fact that the use is always fractioned in hundreds of sites with very diluted solutions.

Some general effects have been reported on similar substance 1 at the highest tested doses in the 28 days subacute test (500 mg/Kgbw), a dose that reported to the human body means about 30 g per day actually ingested.

This value was used in the Chemical safety report based on the fact that the oral exposure is the most conservative way of absorption with respect to the dermal and inhalative routes. As a consequence no further study is proposed on the substance, neither for repeated dose, nor for teratogenicity.

Based on read across consideration the same value can be used to assess the endpoint repeated dose toxicity of target substance.

Justification for classification or non-classification

No classification for repeated dose toxicity is warranted under Regulation 1272/2008.