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EC number: 309-269-1
CAS number: 100208-66-0
OECD 422 (read across substance
Leuco Sulphur Yellow 22, CAS 90268 -98 -7)
NOAEL for systemic toxicity of male/female
rats: 1000 mg/kg bw/day
NOAEL for reproductive performance of male/
female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 mg/kg bw/day
OECD 422 study
The objective of this study was to obtain
initial information on the toxic potential of the test item (Leuco
Sulphur Yellow 22, CAS 90268 -98 -7, source substance for read across to
Leuco Sulphur Brown 3) and on the possible effects of the test item on
reproduction and development when repeatedly administered orally (by
gavage) to rats at doses of 100, 300 and 1000 mg/kg bw/day (corrected
doses; corresponding to uncorrected doses of 110.46, 331.38 and 1104.61
mg/kg bw/day, respectively) compared to control animals according to
As a screening test, it was intended to
provide initial information on the possible health hazards likely to
arise from repeated exposure over a relatively limited period of time
and on the possible effects on male and female reproductive performance
such as gonadal function, mating behavior, conception, pregnancy,
parturition as well as on development of the F1 offspring from
conception to day 13 post-partum associated with administration of
repeated maternal doses.
The test item was administered orally (by
gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body
weight (mg/kg bw/day) doses to four groups of Han:WIST rats consisting
of 12 animals per sex per group in concentrations of 20, 60 and 200
mg/mL calculated by the active ingredient content and corresponding to a
5 mL/kg bw dosing volume. A group of vehicle (1 % methylcellulose)
treated animals (n= 12/sex) served as a control.
The suitability of the chosen vehicle for
the test item at the intended concentrations was analytically verified
The test item was stable in in the vehicle
in concentrations of 1 mg/mL and 200 mg/mL at room temperature for 1 day
and in a refrigerator (at 5 ± 3 °C) for 3 days.
The concentration of the test item in the
dosing formulations administered to the animals was checked two times
during the study. The test item concentrations in the dosing
formulations varied within the range of 94 % and 105 % in comparison to
the nominal values) and confirming the proper preparation of the dosing
All animals of the parent (P) generation
were dosed prior to mating (14 days) and throughout mating. In addition,
males received the test item or vehicle after mating up to the day
before the necropsy (altogether for 49 days). Females were additionally
exposed through the gestation period and up to lactation days 13-18,
i.e. up to the day before necropsy (altogether for 51-56 days).
Observations included mortality, clinical signs, body weight, food
consumption, mating, pregnancy and delivery process, as well as
development of offspring. Estrous cycle was monitored by examining
vaginal smears before the treatment for two weeks and for two weeks from
the beginning of the treatment period (two weeks pre-mating period) and
during the mating period until evidence of copulation.
The dams were allowed to litter and rear
their offspring up to day 13 post-partum. Litters were weighed and
offspring were observed for possible abnormalities and were euthanized
on post-natal day 13 or shortly thereafter.
Blood samples were collected for possible
determination of serum levels of thyroid hormones (T4 – Thyroxine, free
tetra-iodothyronine – and TSH – thyroid-stimulating hormone) from at
least two pups per litter (where it was feasible) on post-natal day 4,
from all dams and at least two pups per litter at termination on
post-partum/ post-natal day 13 and from all parent male animals at
All parental animals were subjected to gross
pathology one day after the last treatment. The body weight, brain
weight and weight of the testes and epididymides of adult male animals
were determined. Thyroid gland was preserved from all adult males and
females and one male and one female pup per litter for the intended
subsequent histopathological examination. Histopathology examination was
performed on ovaries, uterus, vagina, testes, epididymides, prostate and
seminal vesicles with coagulating gland in the control and high dose
groups (male or female). In addition, these organs were processed
histologically in not delivered female and male cohabited with in the
mid dose group. Five dams and their male mating partners were randomly
selected from each group to examine further signs of toxicity such as
functional observations, hematology, clinical chemistry, gross necropsy,
organ weighing and full histopathology examination.
In addition, organs showing macroscopic
findings were processed and examined histologically in animals of the
low and mid dose groups based on the macroscopic findings at the
There was no mortality at 100, 300 or 1000
mg/kg bw/day groups during the course of study (male and female).
Clinical signs of systemic toxicity were not detected at any dose level,
neither at the daily nor at the detailed weekly clinical observations or
at the functional observations. The behavior and physical condition of
the animals was not impaired at each dose level (100, 300 or 1000 mg/kg
bw/day) during the entire treatment period. Test item related changes in
the body weight or body weight gain were not detected. The body weight
development was not affected and it was comparable in the control and
test item treated groups. The mean daily food consumption was similar in
male or female animals in control and at 100, 300 and 1000 mg/kg bw/day
during the entire study (pre-mating and post-mating period for male
animals; pre-mating, gestation and lactation periods for female
animals). A test item influence on the estrous cycle was not found at
any dose level (100, 300 and 1000 mg/kg bw/day). There were no
significant differences between the control and test item treated male
or female animals in the examined parameters of reproductive performance
or in the delivery parameters of dams (100, 300 and 1000 mg/kg bw/day).
Hematological evaluation did not reveal adverse test item related
changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day.
There were no test item related adverse effects on the examined clinical
chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).
There were no test item related changes in the serum thyroid hormone (T4
and TSH) levels at any dose (parental male or 13-day offspring).
Macroscopic findings related to the effect of the test item were not
found in male and female animals at 100, 300 or 1000 mg/kg bw/day. There
were no test item related changes in the weights (absolute and relative
to body or brain weights) of brain, testes and epididymides of male
animals at any dose level. The examined organ weights of animals
selected for toxicity examinations were comparable in the control and
100, 300 and 1000 mg/kg bw/day groups at the end of the treatment
period. Histopathological examinations of the selected organs (ovaries,
uterus, vagina, testes, epididymides, prostate and seminal vesicles with
coagulating gland) did not reveal any test item related changes at 1000
mg/kg bw/day. There were no pathologic changes in the examined organs or
tissues of randomly selected male or female animals in the control or
1000 mg/kg bw/day groups.
No adverse effect on the mortality, clinical
signs, body weight development or necropsy findings were detected in the
offspring terminated as scheduled. The anogenital distance (male and
female) or nipple retention (male) were not affected.
Under the conditions of the present study,
the test item administered at 100, 300 or 1000 mg/kg bw/day (corrected
doses; respectively to uncorrected doses of 110.46, 331.38 and 1104.61
mg/kg bw/day) by oral gavage did not cause signs of systemic toxicity
and did not adversely influence the reproductive performance (gonad
function, mating behavior, conception, parturition) in parental male and
female Han:WIST rats. The development of the F1 offspring was not
impaired from birth to post-natal day 13 at any dose level after
repeated oral administration of dams.
Based on these observations the No Observed
Adverse Effect Levels (NOAEL) were determined as follows:
Labelling, and Packaging Regulation (EC) No 1272/2008
available experimental test data are reliable and suitable for
classification purposes under Regulation (EC) No 1272/2008. Based on
available data, the test item does not require classification for
reproduction toxicity according to Regulation (EC) No 1272/2008 (CLP),
as amended for the
twelfth time in Regulation (EU) No 2019/521.
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