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EC number: 220-562-2
CAS number: 2814-77-9
test item, Pigment Red 4, 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 (SPF quality) were used for testing. The test item was administered
in the form of a suspension in olive oil. 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/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 the study were
the requirement of the Study Monitor after evaluation of the results of
the DRFE (Annex 2).
first six males and six 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 twelve males
and females per group are a part of the reproduction study and examined
with respect to reproduction parameters.
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 – 49 days in total
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 in the 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 at the specified time intervals. Detailed clinical observation
was carried out weekly. Functional observations were performed at the
end of the application and observation period. 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 +
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
Dose Toxicity part of study:
The six 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 group (control: Nos. 101, 102, 103, 104,
107, 111, the lowest dose: Nos. 121, 122, 123, 124, 127, 130, the middle
dose: Nos. 141, 142, 143, 145, 147, 148 and the high dose: Nos. 161,
163, 164, 170, 171, 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.
During the Combined Repeated Dose
Toxicity Study with the Reproduction/Developmental Toxicity Screening
one female (at the dose level 1000 mg/kg/day) died during the study
probably for pregnancy and delivery complications.
Significantly decreased mean of body
weight was detected in females in all treated group during the pregnancy
period. The body weight increment in treated females was variable. In
males the body weight of males was relative balanced.
Decreased or variable the food
conversion and food consumption were recorded during the study, in
animals of both sexes.
No clinical findings revealed
influence of the test item on clinical status of treated animals and
satellite treated animals were recorded. Only coloured excrements was
recorded in all treated animals; this changes related to the colour of
the test item.
Haematological examination showed
statistically significantly decreased values of total erythrocyte count,
haemoglobin and hematocrite in males of all treated groups (with dose
dependence). Decrease of these parameters were recorded in females at
the end of recovery period. Statistically insignificantly increased
value of mean corpuscular volume was detected in males at the middle
(500 mg/kg/day) and the highest (1000 mg/kg/day) dose level.
In all treated females statistically
significantly and dose dependently increased percentual portion of
lymphocytes was recorded.
Biochemical examination in treated
animals showed following statistically significant differences:
reversible increased value of bilirubin total in animals of both sexes
(dose dependently), reversible decreased values of urea and creatinine
in all treated males, decreased value of chloride ions in males at the
lowest (250 mg/kg/day) and the highest dose level. Delayed statistically
significantly increased value of inorganic phosphorus in both sexes,
value of cholinesterase in males and activity of ALP in females were
During the urine examination of
males presence of bilirubin and leucocytes in males of treated groups
were recorded. Concentration of bilirubin in urine was higher than
measurable limit in two males at the lowest dose level and in all
examined males at the middle and the highest dose level. Change of
colour of urine from light yellow to dark yellow was recorded. The
change in urine colour was probably due to the presence of bilirubin in
decreased volume of urine in males at the middle dose level was
detected. At the highest dose level the volume of urine was decreased
Biometry of organs showed
statistically significantly increased absolute and/or relative weight of
liver and kidneys in treated males. Increasing of weight of kidneys in
treated males was irreversible. On the contrary significantly decreased
absolute weight of kidneys was detected in females at the end of
Absolute or relative weight of
spleen in males or females was reversible statistically significantly
These changes of weight of organs
related with changes detected during the hematological, biochemical
examination and examination of urine.
histopathological findings in gastrointestinal tract, kidneys, liver and
hemopoietic organs, which related with the test item treatment were
detected during the histopathological examination of organs in animals
at the highest dose level and satellite treated animals.
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