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EC number: 304-149-5
CAS number: 94246-75-0
test substance, Reactive Blue 234, was tested for reproduction and
subacute toxicity using the OECD Test Guideline No. 422: Combined
Repeated Dose Toxicity Study with the Reproduction/Developmental
Toxicity Screening Test, Adopted by the Council on 29th July 2016.
rats of SPF quality were used for testing. The test substance was
administered in the form of solution in water for injection. Oral
application by stomach tube was performed daily. The study includes four
main groups and two satellite groups of animals. Each main group
consisted of 12 males and 12 females; each satellite group consisted of
6 males and 6 females. Main groups contained 3 treated groups (doses
250, 500, 1000 mg/kg of body weight /day) and one control group (vehicle
only). The satellite groups contained one control group (vehicle only)
and one treated group (1000 mg/kg/day). The dose levels for study were
determined on the basis of results of a dose-range finding experiment
(see the Annex 2) and approved by Sponsor.
treated groups were administered daily for the following periods:
and females – 2 weeks prior to the mating period and during the mating
during pregnancy and till the 12th day of lactation,
males – after
mating period – totally for 49 days,
females (mated females without parturition) – for 25 days after the
females – for 25 days after the end of mating period
the end of administration period the animals of main groups were
sacrificed and satellite animals were observed for the next 14 days
the study clinical observation and health status controls were performed
daily. The body weight and food consumption were measured weekly or in
the specified time intervals. Detailed clinical observation was carried
out weekly. The functional observation was performed at the end of
application and observation period. Vaginal smears were prepared daily,
2 weeks before start of administration period (oestrous cycle
monitoring), during the mating period (until the presence of
spermatozoa) and at necropsy day. Reproduction parameters relevant to
pups (number of pups, weight of litters, weight, sex and vitality of
pups, measurement of anogenital distance, nipple retention) were also
study was finished by urinalysis, haematological and biochemical
analysis and gross necropsy of animals. In all males of main groups the
sperm parameters, sperm motility and sperm morphology were examined. The
selected organs from parental animals and pups were removed for weighing
and histopathological examination.
oral administration of Reactive Blue 234to rats by gavage at the dose
levels of 250, 500 and 1000 mg/kg/day did not cause any mortality.
Dose Toxicity part of study:
test substance treatment did not produce changes detected in health
condition control, daily clinical observation after application (except
coloured faeces) and functional observation of animals.
test substance did not interfere with normal growth of treated parental
animals. Body weight and body weight increments of treated animals were
not significantly affected by the test substance administration.
examination of males did not show negative effect of the test substance
on blood and coagulation parameters. Only the value of reticulocytes was
statistically significantly increased in males at the dose level 1000
mg/kg/day (out of historical range). This change was reversible, so it
can be considered as adaptive response of organism to chemical stress.
examination of females showed significant changes only in females at the
dose level 500 mg/kg (increased concentration of haemoglobin and
increased APTT – still in range of historical control). Haematological
parameters of high-dosed females in comparison with the control females
were not influenced by the test substance administration.
findings observed in both sexes (but in absence of a treatment-related
clinical signs of toxicity) were considered to be of no toxicological
significant changes in biochemical examination were observed in males at
all treated groups. Decreased values of total protein and albumin
concentration in comparison with the control group were recorded in
males at the dose level 1000 mg/kg. Increased values of concentration of
triglycerides, cholinesterase were recorded in males at the dose level
500 mg/kg. Increased values of concentration of cholinesterase was
recorded in males at the dose level 250 mg/kg. Decreased concentration
of chloride ions was recorded in males at all dose levels -
statistically significant and dose-dependent. Decreased concentration of
natrium ions was recorded in males at all dose levels - statistically
significant. Increased concentration of kalium ions was recorded in
males at all dose levels – dose-dependent.
changes were reversible.
significant differences were also found in satellite treated males –
increased concentration of calcium, decreased concentration of
phosphorus and cholinesterase. Other parameters of treated animals were
well-balanced with control animals.
statistically significant changes in biochemical examination were
observed in females at all treated groups. Slightly and insignificantly
decreased concentration of chloride ions was recorded in females at all
dose levels. Decreased concentration of natrium ions was recorded only
in females at dose level 1000 mg/kg. Other parameters of treated females
were similar to the control females. Statistically significant
difference was found only in satellite treated females – concentration
of natrium was decreased. Other parameters were well-balanced.
manifested the influence of the test substance administration on value
of pH in males at the dose level 1000 mg/kg. The pH was decreased and
this change persisted into the end of recovery period. The volume of
urine was significantly changed in males at the dose level 1000 mg/kg –
decreased at the end of application period and increased at the end of
biometry of organs, statistically significant changes in absolute
weights of prostate gland with seminal vesicles and thyroid gland in
males were recorded. Decreased weight of prostate gland with seminal
vesicles was noted in males at the dose level 250 and 1000 mg/kg.
Decreased weight of thyroid gland was recorded in males at the highest
dose level only. Vice versa absolute weight of thyroid gland in treated
satellite males was statistically significantly increased. Absolute
weight of kidneys was increased in males at all treated groups (without
statistical significance but dose-dependently). Statistically
significantly increased relative weight of kidneys was recorded in males
at all dose levels (without dose dependency). In satellite treated males
no significantly changed the weight of organs was reported.
biometry of organs, significant changes in absolute weights of kidneys
and thyroid gland in females were recorded. Increased weight of kidneys
was noted in females at the dose level 1000 mg/kg. Increased weight of
thyroid gland was recorded in females at the dose level 250 and 500
mg/kg and simultaneously decreased in females at the highest dose level.
Statistically significantly increased relative weight of thyroid gland
was recorded in females at the dose levels 250 and 500 mg/kg. Relative
weight of kidneys in females at the dose level 1000 mg/kg. In satellite
treated females significantly decreased absolute and relative weight of
thymus and decreased absolute weight of heart was reported.
changes related to test substance color were recorded in males and
females of the dose level 500 and 1000 mg/kg. Colored content of small,
large intestine, caecum and colored kidneys were observed. Other
findings were probably of spontaneous character.
evaluation showed that the test substance orally administered at the
dose of 1000 mg/kg/day (the highest dose level) did not cause
histopathological changes indicative of a toxic effect in any examined
organs. Mild hydronephrosis of kidneys was reported in dosed animals as
well as in a control animals.
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