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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 February 1995 to 31 March 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recent EU & OECD test guidance in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: Testing Methods for New Chemical Substances (enacted July 13th, 1974; amended December 5th, 1986) of the Japanese Ministry of Health and Welfare.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Reactive Blue FC 75311

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winklemann GmbH
- Strain: SPF Bred, Hsd Cpb:WU
- Age at study initiation: 4 - 5 weeks old
- Weight at study initiation: Initial Mean weight - Males 122g (112-133g), Females 106g (100-114g)
- Fasting period before study: No
- Housing: Conventional conditions, Makrolon Type II cages, individually
- Diet (e.g. ad libitum):Altromin 1324 pellets, ad libitum (except during urine collection period)
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 5%
- Air changes (per hr): 10-15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark - light from 6am to 6pm

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Demineralised
Details on oral exposure:
For treatment of the animals the test compound was formulated once to twice weekly in the application vehicle {demineralized water) according to the dosage scheme. Calculation was based on a test compound content of 65%, the weighed portion being adjusted accordingly.

The test compound was orally administered by gavage once a day, 7 days per week, 28 times in total, to the animals of the treatment groups from the first day of treatment until spontaneous death or one day prior to scheduled death. Animals of the main groups were sacrificed one day after the last treatment, those of the recovery groups were observed for another 14 days. The application volume was 10 ml/kg body weight. Control animals received only the application vehicle (demineralized water). The individual application volume was based on the body weights determined before each treatment
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For treatment of the animals the test compound was formulated once to twice weekly in the application vehicle {demineralized water) according to the dosage scheme. Calculation was based on a test compound content of 65%, the weighed portion being adjusted accordingly.

All application formulations were prepared and stored at room temperature. The test compound content in the application formulations was checked once during the study period. For this purpose samples of the formulations used were withdrawn and analysed. Samples were also analysed to ensure stability and homogeneity of the test compound in the application vehicle for the duration of use.

High-performance liquid chromatography (HPLC) to an in-house SOP was used to determine the stability and homogeneity of the test samples. The two measurement concentrations were prepared by adding demineralized water to the test sample. The mixtures were homogenised by stirring on-a magnetic stirrer and analysed immediately and after 8 days, (storage at room temperature at about 23 °C) The low concentration was given as a homogeneous solution. The high concentration was given as a suspension, which was homogenised by stirring on a magnetic stirrer or, after 8 days, by stirring with a glass rod. The samples were taken during stirring in each case.

The formulations were found to be stable and homogeneous at, room temperature for the duration under test.
Duration of treatment / exposure:
Exposure 28 days
Frequency of treatment:
Once a day, 7 days a week for 28 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg body weight/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
40 mg/kg body weight/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg body weight/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg body weight/day
Basis:
actual ingested
No. of animals per sex per dose:
Male: 10 animals at 0 mg/kg bw/day (5 main study; 5 recovery satellite group)
Male: 5 animals at 40 mg/kg bw/day
Male: 5 animals at 200 mg/kg bw/day
Male: 10 animals at 1000 mg/kg bw/day (5 main study; 5 recovery satellite group)

Female: 5 animals at 0 mg/kg bw/day (5 main study; 5 recovery satellite group)
Female: 5 animals at 40 mg/kg bw/day
Female: 5 animals at 200 mg/kg bw/day
Female: 10 animals at 1000 mg/kg bw/day (5 main study; 5 recovery satellite group)

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages were selected taking into account the results from an acute oral toxicity study in male and female Wistar rats. A single dose of 2000 mg/kg was administered in this study and tolerated without mortality
- Rationale for animal assignment (if not random): The study was carried out in rats - a species recommended in test guidelines for toxicity studies.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
The animals were inspected at least twice daily {once on weekends and bank holidays) and any clinical signs or unusual features were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
A detailed weekly health inspection of individual animals assessed the following: body surfaces and orifices, posture, general behaviour, respiration and excretory products. Findings and unusual features were recorded.

BODY WEIGHT: Yes
Body weights were determined daily before application for determining the application volume. Online recording was done at the start of study prior to the first application and from then on once a week until the end of study. The final body weights determined immediately prior to necropsy were also used for the calculation of relative organ weights.

FOOD CONSUMPTION:
Feed and water intake were determined for each individual animal once a week from the difference between feed/water offered and not consumed. From these primary intake data, the following were calculated:
a) intake per animal and day
b) mean intake per animal and day
c) cumulative intake per animal
d) cumulative intake per kg body weight
e) mean cumulative intake per animal and day
f) mean cumulative intake per kg body weight and day

The calculation of cumulative data was based on a study period of 28 days. Intake data for the recovery groups were cumulated over an additional period of 14 days.
The algorithms for calculating feed and water intake are described in Part 2 of this report.

WATER CONSUMPTION: Yes
See above "FOOD CONSUMPTION".

HAEMATOLOGY: Yes
Blood samples for the determination of the glucose concentration in deproteinized whole blood were taken from one of the caudal veins of non fasted, non-anesthetised animals. Blood samples for the determination of the other parameters in peripheral blood were taken from the retro-orbital venous plexus of non-fasted animals under ether anesthesia. The following hematological parameters in peripheral blood were determined:

Differential blood count from blood smears: staining and differentiation using the Beckman Hematrak System

Erythrocyte morphology from smears in the course of differentiating the leucocyte population with the Hematrak System

Erythrocyte count using the TOA-Medical Sysmex Hematology System
Hemoglobin concentration in blood measured with the TOA-Medical Sysmex Hematology System

Hematocrit measured with the TOA-Medical Sysmex Hematology System

Leucocyte count using the TOA-Medical Sysmex Hematology System

Mean corpuscular hemoglobin(MCH) – calculation of the mean cell haemoglobin content from haemoglobin concentration in blood and erythrocyte count using the TOA-Medical Sysmex Hematology System

Mean corpuscular hemoglobin concentration (MCHC)- calculation of the mean cell-Hemoglobin concentration from hemoglobin concentration in blood and hematocrit using the Sysmex

Mean corpuscular volume (MCV)- calculation of the mean corpuscular volume of individual erythrocytes using the Sysmex

Reticulocyte count - using reticulocyte analyzer Sysmex

Thrombocyte count - measurement of resistance changes using the Sysmex Thromboplastin time - using the Hepato-Quick test as described by HEENE, D.L., Med. Welt, 25, 1529 (1974)


CLINICAL CHEMISTRY: Yes
The following parameters (enzymes, substrates, metabolic products) were investigated:

Enzyme Activities in plasma:
Alkaline phosphatase (EC-No. 3.1.3.1.),
Aspartate aminotransferase (*= glutamic-oxalacetic transaminase,EC-No. 2.6.1.1.) optimized,
Alanine aminotransferase (= glutamic-pyruvic transaminase EC-No. 2.6.1.2.) optimized all in accordance with recommendations by the German Society of Clinical Chemistry, Z. Klin. Chem. Klin. Biochem 10, 182 (1972)
Gamma-glutamyltransferase (EC-No. 2.3.2.2.) colorimetric as described by SZASZ, G., Z. Klin. Chem. Klin. Biochem. 12, 228 (1974)

Substrates in Deproteinised Whole Blood:
Glucose (hexokinase method) as, described by SCHMIDT,F.H., Klin. Wschr. 39, 1244 (1961)

Substrates and Metabolic Products in Plasma:
Albumin - according to DOUMAS, B.T. et al., Clin. Chim. Acta 31, 87 (1971)
Albumin/globulin ratio - Calculation was done according to the formula:

A/G ratio = albumin / (protein – albumin)

Bilirubin (total, DPD method) as described by WAHLEFELD, A.W., HERZ, G. and BERNT, E., Scand. J. Clin. Lab. Invest. 29, Suppl. 126, Abstract 11.12 (1972)
Cholesterol (enzymatic) as described by SIEDEL, J.; HAGELE, E.O.; ZIEGENHORN, J. and WAHLEFELD, A.W., Clin. Chem. 2_9_, 1075 (1983)
Creatinine (Jaffe kinetic} modified as described by BARTELS, H. et al., Clin. Chim. Acta 37, 193 (1972)
Total protein (biuret reaction) as described by WEICHSELBAUM, T.E., Amer. J. Clin. Path. 10, 40 (1946)
Urea (enzymatic UV test) as described by GUTMANN,I. and BERG-MEYER,H.U. in Bergmeyer: Methoden der enzymatischen (1974)
Triglycerides (full enzymatic) modified as described by WAHLEFELD, A.W. in: Bergmeyer, Methoden der enzymatischen Analyse, Bd. II, 3.Auflage,
Verlag Chemie, Weinheim (1974), and TRINDER, P. Ann. Clin. Biochem 6_, 24 (1969)

Electrolytes in Serum:
Calcium, potassium, sodium - Flame photometry (Li reference line)
Inorganic phosphate as described by SUGIURA, M. et al., Chem. Pharm. Bull. 29, 1451 (1981)
Chloride (coulometric) as described by LANGE, W. and BORNER, K., Z. Klin. Chem. Klin. Biochem. 10, 33 (1972)

URINALYSIS: Yes
- Time schedule for collection of urine: 16 hours (overnight)
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes

The following parameters were assessed:
Semiquantitative:
Blood, glucose, ketone bodies, pH value, bilirubin, urobilinogen and protein
using Ames Multistix or Multistix 10 SG (Bayer Diagnostics)
Sediment - microscopic examination following centrifugation

Quantitative:
Density measured using Urometer or Heraeus-Paar Digital Densimeter DMA 45
Volume using a measuring cylinder or graduated centrifuge tube

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On day 29 all animals of the main groups, and on day 43 all animals of the recovery groups, were anesthetized with diethyl ether and exsanguinated. The organs/tissues of the subsequently necropsied animals were subjected to thorough gross pathological evaluation. Changes were described in terms of site, size, colour and consistency. The following organs/tissues or parts thereof were fixed in 10% formaldehyde solution:

adrenal glands
aorta
auricles (tattooed)
bone marrow (in femur and sternum)
brain
epididymis
esophagus
extraorbital lacrimal glands
eyelids
eyes
femur (with knee-joint)
Harderian glands
head (remainder)
heart
intestine (cecum, colon, duodenum, i1eum, jejunum, rectum, remainder)
kidneys
larynx
liver
lymph nodes (mesenteric and mandibular)
mammary glands
musculature (thigh)
optic nerves
ovaries
oviducts
pancreas
pituitary
prostate gland
salivary glands
sciatic nerve
seminal vesicles
skin
spinal column with spinal cord (cervical,thoracal, lumbal)
spleen
sternum
stomach
testes
thymus (if present)
thyroid gland with parathyroids
tongue
tooth
trachea
ureters
urethra
urinary bladder
uterus
vagina
Zymbal glands

as well as all tissues showing macroscopic lesions. The lungs were instilled with 5% formaldehyde solution and then fixed in 10% formaldehyde solution.

HISTOPATHOLOGY: Yes
The following slides/organs were available for histopathological diagnostics:

Control and high dose groups (including recovery groups):
Liver, heart, testes, spleen, adrenal glands

All dose groups (including recovery groups):
kidneys, stomach

Dose groups(without recovery groups):
intestine (6x), tail

All dose groups:
macroscopic findings
Other examinations:
Organ Weights

The following organs of the animals sacrificed according to schedule on day 29 or 43 were weighed:
brain, heart, testes (in pairs), liver, spleen, kidneys (in pairs), adrenals (in pairs), ovaries (in pairs).
Statistics:
The statistical evaluation of data related to clinical chemistry, hematology, survival, body and organ weights as well as feed and water intake is performed using SAS® routines.

All variables that are not dichotomous are described by sex, dose group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum).

For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical tests are used, the choice of the test being a function of prior knowledge obtained in former studies. Provided that the variates in question can be considered approximately normally distributed with equal variances across treatments, the Dunnett test is used, if heteroscedasticity appeared more likely a p value adjusted Welch test is applied. If the evidence based on experience with historical data indicates that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA are carried out, i.e. the Kruskal-Wallis test followed1by adjusted Mann-Whitney-Wilcoxon tests (U tests) where appropriate.
Global tests including more than two groups are performed by sex and date, i.e. each sex x date level defines a family of tests in the context of multiple comparison procedures (MILLER, R.G., simultaneous statistical inference, 2nd edition. Springer, Berlin, Heidelberg, New York, Tokyo, 1981). Within such a family, the experiment wise error is controlled. If not otherwise noted, all pairwise tests are two-sided comparisons. Significant differences from the control group are indicated with "+" for p ≤ 0.05 and "++' for p ≤ 0.01.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
see below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
see below
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
No unusual features were observed in animals of all dose groups with respect to general behaviour, posture and respiration. At 200 and 1000 mg/kg a bluish discoloration of the feces became evident, which was no longer observed during the recovery period. No animal died during the study.

BODY WEIGHT AND WEIGHT GAIN:
Treatment with susbastance had no significant effect on body weights, of female rats of all dose groups and of male rats up to and including 200 mg/kg. Growth was retarded in male rats at 1000 mg/kg. At the end of the recovery period the body weight of treated males was still significantly lower compared to controls but the difference has become smaller.

FOOD CONSUMPTION:
Food intake was not affected in all female groups as well as in males up to and including 200 mg/kg. The 1000 mg/kg males consumed about 10% less food on average.

WATER CONSUMPTION:
The water intake in male and female rats at 1000 mg/kg was increased by about 17% to 34% (absolute) and 31% to 36% in relation of the body weight.
In the remaining groups water intake was roughly the same as in controls.

HAEMATOLOGY:
There was no effect on erythrocyte morphology in male and female rats up to and including 200 mg/kg. Poikilocytosis, microcytosis, polychromasia, anisocytosis and hypochromasia were to be seen in some males and females at 1000 mg/kg essentially at the end of the treatment period.

CLINICAL CHEMISTRY:
There were increased urea and potassium as well as decreased total protein and sodium concentrations in males at 1000 mg/kg at the end of the treatment period. In 1000 mg/kg females a significant increase in alkaline phosphatase activity was to be seen. At the end of the recovery period there was a still a significant increase in inorganic phosphate concentration in 1000 mg/kg females.
Otherwise the test substance had no noticeable effect on the plasma enzymes, substrates, metabolic products and electrolytes investigated.

As a general observation, there was a bluish discoloration of the plasma in all males and some females at 1000 mg/kg by the end of the treatment period.

URINALYSIS:
Toxicologically relevant effects caused by treatment with the substnace were found neither in the excreted volume nor in the density of the urine.
A bluish or green-yellowish discoloration of the urine was described in all rats at 1000 mg/kg by the end of the treatment period.

Semiquantitative findings gave no indication of test substance-related, toxicologically relevant effects up to and including 200 mg/kg. At week 4 there was an increased incidence of males and females with bilirubin-positive urine and of females with epithelia in the sediment at 1000 mg/kg. As there is a correlation to the degree of discoloration of the urine the "bilirubin"-positive findings are considered as a consequence of discoloration and not indicative of toxicity.

Investigation of the sediment produced no other unusual features.

ORGAN WEIGHTS:
According to these results, the test substance had no relevant effect on the absolute and relative organ weigths of males and females up to and including 1000 mg/kg. The absolute liver weight of males of the 1000 mg/kg group as well as the relative heart weight in the 40 and 200 mg/kg females marked as deviating in a statistically significant manner, are within the physiological range of historical controls and the difference from the control group is only slight.
The relative liver weight of 1000 mg/kg females was significantly increased over the controls at week 4. Relative kidney weights were conspicuously higher in 1000 mg/kg males and females at week 4 (p > 0.05). The remaining differences are slight and can essentially be explained by the differing body weights.


GROSS PATHOLOGY:
Blue discolorations were seen in the kidneys of all male and females at 200 and 1000 mg/kg. In addition, stomach, intestine, and tail skin were bluish discoloured at 1000 mg/kg in male and female rats. In all the recovery animals dosed with 1000 mg/kg blue discoloration of the kidneys was visible.

HISTOPATHOLOGY: NON-NEOPLASTIC:
In the kidneys of two males dosed with 1000 mg/kg degeneration of cortical tubules was observed which was characterized by necrosis, desquamation of tubular epithelia and focal tubular degeneration. There was also evidence for regeneration of tubular epithelium which was slightly basophilic and showed prominent nuclei. In the other males from this group there was evidence of minimal degenerative changes of the tubules of the inner cortex. In the stomach of males and females at 200 mg/kg and above, there was evidence of gastritis with increased mucus production between forestomach and glandular stomach.
In the tail skin traces of the dye were visible in the keratin layer of the proximal specimen.
At the end of the recovery period there was grossly visible dye deposition in the kidneys (blue discoloration) without histological correlate. In the stomach of one male hyperkeratosis was seen at the limiting ridge of the forestomach. The changes in the glandular stomach were completely reversible within the recovery period.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Five male and female rats each, received Reactive Blue FC 75311 in daily dosages of 0 (vehicle control), 40, 200 and 1000 mg/kg body weight by gavage for 28 days. In addition, 5 male and 5 female animals per group were treated with 1000 mg/kg or the vehicle (demineralized water) for 28 days and observed for another 14 days without treatment.

The animals were regularly observed, weighed, and their feed and water intakes were determined. In week 4 (main groups) and 6 (recovery groups) urine and blood samples were collected and various parameters determined. At necropsies organs were weighed and gross pathological investigations performed. A number of organs and tissues were processed histopathologically.

No substance-related clinical signs were found during the treatment period and the recovery period in animals of the dose groups of up to and including 1000 mg/kg. None of the animals died.
The blue discoloration of the feces (200 and 1000 mg/kg) and urine (1000 mg/kg) is most obviously due to excretion of the dye. As the discolorations of the gastro-intestinal tract observed in animals of the main groups could no longer be identified in animals of the recovery groups, these findings are most likely due to a transitory contact of the dye with the mucosa.

Feed intake and body weight gain were not affected in males up to and including 200 mg/kg as well as females of all groups. Males at 1000 mg/kg had lower body weights (up to about 15%) and slightly reduced feed intake. At the end of the recovery period the difference in body weights was slightly diminished and the feed intake comparable to controls. Water intake was increased in males and females at 1000 mg/kg.

The results of the hematological and histological investigations produced no evidence of test substance-related damage to blood or hematopoietic organs in the dose groups of up to and including 200 mg/kg. At 1000 mg/kg significantly reduced erythrocyte counts (with some changes in erythrocyte morphology), hemoglobin, hematocrit, MCV and MCH values in males point to a test substance induced hypochromic anemia. The increased percentage of reticulocytes is indicative of the increased hematopoiesis. Similar but less pronounced effects were to be seen in 1000 mg/kg females. In addition thrombocyte counts were significantly increased in both sexes. These effects were much less pronounced in males at the end of the recovery period and had disappeared in females, thus indicating reversibility, with regard to the leucocyte population there was a conspicuous shift from lymphocytes to segmented neutrophils in males at 1000 mg/kg. This could possibly be related to the effects on e.g. kidneys. But in view of the fact that the individual values were within the physiological ranges this is not considered to be of biological relevance.

Clinical laboratory investigations, necropsies and histological findings gave no indication of a change of the functional state or the morphology of the liver up to and including 1000 mg/kg. The slightly reduced total plasma protein concentration in 1000 mg/kg males in week 4 is therefore not considered to be a consequence of liver impairment. It may be related to the renal effects discussed below. Also the slightly increased activity of plasma alkaline phosphatase in 1000 mg/kg females in week 4 is not considered to be of indicating relevant toxicity in the absence of any correlate.

Urea and creatinine concentrations in plasma as well as the results of the urinalyses, necropsies and histopathological investigations gave no indication of treatment-related damage to the kidneys at 40 mg/kg. At 200 mg/kg the only noteworthy effect was a blue discolor¬ation of the kidneys at necropsy. In the absence of any findings indicative of functional or morphological changes this is not inter-preted as a toxic effect. It indicates absorption and renal excretion of the dye. At 1000 mg/kg degenerative changes of cortical tubules were found in males. Correlating effects could be the increased plasma urea and serum potassium concentration's as well as the decreased serum sodium concentration. As water intake and kidney weight were also increased in 1000 mg/kg females these changes are interpreted as an indication of slight functional changes. There were no morphological signs of nephrotoxicity after the recovery period, thus demonstrating reversibility.
In the absence of histological correlates the slightly increased number of 1000 mg/kg females with epithelia in the urinary sediment at week 4 might be incidental.

At 40 mg/kg there were no effects on the stomach. Males and females at 200 and 1000 mg/kg had a gastritis with increased mucus produc¬tion in the glandular stomach and hyperkeratosis at the limiting ridge between forestomach and glandular stomach. The latter finding was still present in one male animal at the end of the recovery period. Nevertheless reversibility can be assumed in principal but takes more than 2 weeks at least in some animals.

Hematological, clinico-chemical, organ-gravimetric, gross pathologi¬cal and histopathological investigations produced no evidence of damage to the other organs or tissues.

Thus, 40 mg/kg Reactive Blue FC 75311 were tolerated without adverse effects by males and females.
Executive summary:

Study conducted to EU test guidance 92/69/EEC B7 and OCED guideline 407 in compliance with GLP.

At 40 mg/kg there were no effect on the stomach. Males and females at 200 and 1000 mg/kg had a gastritis with increased mucus production in the glandular stomach and hyperkeratosis at the limiting ridge between forestomach and glandular stomach. The latter finding was still present in one male animal at the end of the recovery period. Nevertheless reversibility can be assumed in principal but takes more than 2 weeks at least in some animals.

Hematological, clinico-chemical, organ-gravimetric, gross pathological and histopathological investigations produced no evidence of damage to the other organs or tissues.

Thus, 40 mg/kg of the substance were tolerated without adverse effects by males and females.