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EC number: 237-600-9
CAS number: 13863-31-5
Dose range finding
Before the start of study with laboratory
animals, the stability and homogeneity of application form were
determined at the test facility. A dose-range finding experiment (DRFE)
was performed to determine the dose levels for the main study.
The doses of 80, 250, 750 mg/kg/day were
determined on the basis of results of DRFE and approved by Sponsor.
Wistar rats (SPF quality) were used for
testing. The test item was administered in the form of a solution in
aqua pro iniectione. Oral application by stomach tube was performed
daily. The animals were without feed two hours before application and
two hours after application of the test item. The main study includes 3
treated groups (doses 80, 250, 750 mg/kg/day) and one control group
(vehicle only) and two satellite groups of animals - one control group
(vehicle only) and one treated group (750 mg/kg/day).
Each main group consisted of 12 males and
12 females; each satellite group consisted of 6 males and 6 females.
The first 6 males and 6 mothers who
delivered pups per group (as per internal SOP) and a satellite groups of
animals (control and treated) are part of the repeated dose toxicity
study and examined with respect to toxicity of the test item. Satellite
animals were used for observation of reversibility, persistence or
delayed occurrence of systemic toxicity effects up to 14 days post
treatment. All 12 males and females per group are a part of the
reproduction study and examined with respect to reproduction parameters.
The treated groups were administered daily
for the following periods: males and females – 2 weeks prior to the
mating period and during the mating period; pregnant females – during
pregnancy and up to the 12th day of lactation; males – after mating
period – 49 days in total; non-pregnant females (mated females without
parturition) – for 25 days after confirmed mating; non-mated females –
for 25 days after the end of the mating period. After the end of the
administration period, the animals in the main groups were sacrificed
and satellite animals were observed for the next 14 days without
During the study, clinical observation and
health status controls were performed daily. The body weight and food
consumption were measured weekly or at the specified time intervals.
Detailed clinical observation was carried out weekly. Functional
observations were performed at the end of the application and
observation periods. Vaginal smears were prepared daily, 2 weeks before
start of the administration period (oestrous cycle monitoring), during
the mating period (until the presence of spermatozoa) and at necropsy.
Reproduction parameters relevant to pups (number of pups, weight of
litters and weight of pups, sex and vitality of pups, measurement of
anogenital distance, nipple retention, serum levels of thyroid hormones
(T4 and TSH in pups) were also recorded. The study was completed with
urinalysis, haematological and biochemical analysis and gross necropsy
of animals. In all males of the main groups, the sperm parameters, sperm
motility and sperm morphology were examined. Selected organs from adult
animals and pups were removed for weighing and histopathological
Results - repeated dose toxicity
part of the study
Repeated oral administration of the test
item did not cause any mortality (except two females from the dose level
80 mg/kg/day – female No. 126 died due to intubation error and female
No. 127 during the complicated delivery). These deaths were accidental
and not treatment-related.
The 6 males per group (Nos. 1-6, 21-26,
41-46, 61-66) and first six mothers per group that delivered pups were
examined from the main groups (control: Nos. 101,103,106,109,110,112;
the lowest dose: Nos. 121,123,124,125,128,131; the middle dose: Nos.
142,143,144,146,148,150 and the highest dose: Nos.
161,162,165,166,170,172). Also, satellite animals (control: males - Nos.
81-86, females – Nos. 181-186 and high dosed: males - Nos. 91-96 and
females Nos. 191-196) were part of the examination of the repeated dose
toxicity of the test item.
Test item treatment did not produce
clinical changes in health status of animals, did not affect the normal
growth of treated parental males and females.
The haematological examination did not
reveal toxic effect of the test item on administered animals.
Haematological examination of treated males and females showed only
sporadic significant differences in haematological parameters in treated
groups compared to control group. These isolated findings in
haematological examination did not reveal toxic effect of the test item
on administered animals.
During biochemical examination of animals,
several statistically significantly changed values were detected.
In males, significantly increased value of
calcium ions (Ca) and decreased value of creatinine (Crea) were detected
at all dose levels, without dose dependence. The decreased values of
creatinine were out of the historical control range at the dose levels
80 and 750 mg/kg/day. The values of alanine aminotransferase (ALT) were
dose-dependently increased in males at all dose levels, at the dose
level 250 and 750 mg/kg/day with statistical significance, but
reversible (not observed at the satellite treated males). The value of
ALT was out of the historical control range at the highest dose level.
This exceed of the historical control range was due to a marked increase
of ALT value in male No. 61 at the highest dose level. The value altered
at all dose levels (statistically significantly) compared to control
animals was cholinesterase (CHE) – decreased at the dose level 80
mg/kg/day and increased at the dose levels 250 and 750 mg/kg/day (in
range of the historical control range). Significantly decreased values
of chlorides ions were reported in males at the dose levels 250 and 750
mg/kg/day, but still in range of historical control range. The
statistically significantly increased values of total bilirubin (in
historical control range) were recorded in males at the dose levels 80
and 750 mg/kg/day. In males at the dose level 250 mg/kg/day was detected
statistically significantly increased value of phosphorus (in the
historical control range). All these changes in biochemical parameters
in males were reversible, not observed in satellite treated males.
A statistically significantly decreased
values of total protein, triglycerides and albumin were recorded in
satellite treated males in comparison with the satellite control males,
but in range of the historical satellite control. Values of other
biochemical parameters of satellite treated males were similar to the
satellite control group.
During the biochemical examination of
females, significantly changed biochemical values were recorded only
sporadically in treated females. Significantly increased value of total
cholesterol was reported in females at the dose level 250 mg/kg/day (in
historical control range). Significantly increased values of calcium
ions were recorded in females at the dose levels 250 and 750 mg/kg/day,
but also in range of the historical control. Values of ALT and AST were
insignificantly increased and out of the historical control range at the
dose level 750 mg/kg/day. These exceeds of the historical control ranges
were due to a markedly increased values of ALT and AST in one female at
this dose level (female No. 170). The value of triglycerides was out of
the historical control range for the same reason at the dose level 250
mg/kg/day, the value of triglycerides was markedly increased in female
No. 142 at this dose level. Other biochemical parameters of treated
females were comparable to the control group.
Statistically significantly decreased
value of cholinesterase and increased value of total cholesterol were
noted in satellite treated females in comparison with the satellite
control females (in range of the historical satellite control). Values
of other biochemical parameters of treated satellite females were
comparable to the control group.
The biochemical examination of treated
males revealed some dose-dependently changed values and some values out
of historical control range associated with the test item
administration. Specifically, these parameters are: ALT, creatinine and
cholinesterase. But this changes were not observed in satellite treated
Biochemical examination of treated females
did not show a toxicologically significant effect of the test item.
Urinalysis did not reveal any
statistically significant changes of pH. The volume of urine was
statistically significantly decreased in males at the dose level 80
mg/kg/day and statistically significantly increased in the satellite
treated males. The presence of proteins, blood and leucocytes were
recorded in treated males as well as in the control males and were not
associated with the application of the test item.
During biometry of organs in males and
females, significant changes of absolute and relative weights of organs
related to the administration of the test item were not recorded.
During the macroscopic examination, no
findings related to the test item treatment were found out.
Histological examination showed changes
related to the test item treatment only in the males. The focal
polymorphonuclear inflammation (hepatitis) was detected in liver of 5
males at the dose level 750 mg/kg/day. This finding was recorded only in
1 female at the highest dose level. This finding was not fully
reversible, because of occurrence of the focal polymorphonuclear
necrotic inflammation in 2 satellite males.
The histopathological examination of the
genital tract of organs revealed the tubular degeneration of testicles
in 1 control male and in 3 males at the dose level 750 mg/kg/day.
Because the relative weight of testicles and spermiogenesis of the males
were not affected with the test item treatment, this change was
considered as non-specific, unrelated to the effect of the test item
treatment. The incidence of other microscopical findings were in most
cases spontaneous or agonal changes, or sporadic, not associated with
the test item treatment.
Accordingly, the NOAEL for repeated dose
toxicity in males was established as 250 mg/kg body weight/day and in
females was established as 750 mg/kg body weight/day.
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