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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Required, study is in develpment

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
purity: 99.5%

Test animals

Details on test animals or test system and environmental conditions:
The animals will be housed in a limited access rodent facility. Animal room controls will be set to maintain temperature and relative humidity at 22°C ± 2°C and 55% ± 15% respectively; actual conditions will be monitored, recorded and the records retained. There will be approximately 15 to 20 air changes per hour and the rooms will be lit by artificial light for 12 hours each day.
From arrival to mating, animals will be housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5x38x20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material will be provided inside suitable bedding bags and changed at least twice a week.
During mating, animals will be housed one male to one female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be inspected and changed daily.
After mating, the males will be re-caged as they were before mating. The females will be transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5x26.6x18.5 cm). Nesting material will be provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) will be provided as necessary. Nesting material will be changed at least 2 times a week

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item will be administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals will receive the vehicle alone at the same dose volume
corn oil
Analytical verification of doses or concentrations:
Duration of treatment / exposure:
Animals will be dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy, for a minimum of 28 days.
Dose volumes will be adjusted once per week for each animal according to the last recorded body weight.
Animals will be dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice.
Dose volumes will be adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes will be calculated according to the last recorded body weight.
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
37.5 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle


Observations and examinations performed and frequency:
Throughout the study, all animals will be checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays, a similar procedure will be followed except that the final check will be carried out at approximately mid-day. This will allow post mortem examinations to be carried out during the working period of that day.
Severely debilitated animals will be observed carefully. Animals judged to be in extremis will be killed. A complete necropsy will be performed in all cases as detailed in section 4.4.3 below.
4.2.2 Clinical signs
Once before commencement of treatment and at least once daily during the study, each animal will be observed and any clinical signs will be recorded. Observations will be performed at the same time interval each day; the interval will be selected taking into consideration the presence of post-dose reactions.
All observations will be recorded for individual animals.
Clinical observations (Functional Observation Battery Tests)
Once before commencement of treatment and at least once a week thereafter, each animal will be given a detailed clinical examination. Each animal will be removed from the home cage and observed in an open arena. The tests will include observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
All observations will be recorded for individual animals.
4.2.4 Grip strength and sensory reactivity to stimuli
Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters, where possible), 5 males and 5 females will be randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements will be performed using a computer generated random order.
4.2.5 Motor activity assessment
Once during the study, towards the end of treatment (on Day 12 post partum for females with viable litters where possible), 5 males and 5 females will be randomly selected from each group and the motor activity will be measured (for approximately 5 minutes) by an automated activity recording device.
4.2.6 Food consumption
The weight of food consumed by each cage of males and females will be recorded weekly (whenever possible) during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females will be measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.
4.2.7 Body weight
Males will be weighed weekly from allocation to termination.
Females will be weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams and pups will also be weighed on Days 1, 4, 7 and 13 post partum. Dams will also be weighed just prior to necropsy.
4.2.8 Vaginal smears and oestrus cycle
Stock females
Oestrus cycle will be monitored by vaginal smears in all stock females for at least 2 weeks before dosing in order to exclude from the study females with irregular cycle.
The vaginal smear data will be examined to determine the following:
a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears will also be taken from all females, before despatch to necropsy. No vaginal smears will be taken from females sacrificed for humane reasons.
4.2.9 Mating
Mating will be monogamous (one male to one female). Exceptions can arise in the case of occasional deaths of males. A vaginal smear will be taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female will be paired with the same male until positive identification occurs or 14 days have elapsed.
4.2.10 Parturition and gestation length
A parturition check will be performed from Day 20 to Day 25 post coitum.
Females which do not give birth after 25 days of post coitum period will be sacrificed shortly after.
Gestation length will be calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth. The day of birth (when parturition is complete) is defined as Day 0 post partum.
4.2.11 Pups identification, weight and observations
As soon as possible after parturition is considered complete (Day 0 post partum), all pups (live and dead) will be counted, sexed and live pups will be identified.
Live pups will be individually weighed on Days 1, 4, 7 and 13 post partum.
Observation will be performed once daily for all litters.
Pups killed or dying during the lactation period will be weighed before the despatch to necropsy.
After culling all pups will be sacrificed with the dams on day 14 post partum.
4.2.12 Culling and pups selection for blood collection (serum hormone) at necropsy
On day 4 post partum, the size of each litter will be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter depending. Partial adjustment (for example, 5 males and 3 females) is acceptable.
At least one culled male and one culled female will be selected for hormone determination.
If litters do not have extra pups to be culled (i.e.: litters with 8 pups or less), at least 1 female pup will be sacrificed for hormone determination in order to retain more male pups for nipple retention on Day 14 post partum. However, retained female pups in each litter should not be below 2. This means that for litters with 6 males and 2 females, no pups will be selected for hormone determination.
Anogenital distance (AGD)
The AGD of each pup will be measured on Day 1 post partum. The AGD will be normalized for the cube root of body weight collected on Day 1 post partum.
Nipple count
On Day 13 post partum, nipple areolas will be counted and recorded, if present, for all live male pups.
Sacrifice and pathology:
Clinical pathology investigations
Blood collection for hormone determination will be performed from all animals at termination under condition of food deprivation.
Blood samples for haematology, clinical chemistry and coagulation will be collected by random selection from 5 males and 5 females (females with viable litters if possible) under condition of food deprivation.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Microscopic observations
Treatment-related findings were seen in the kidneys, stomach, liver and thyroid of treated
animals of both sexes and in the spleen, thymus and adrenals in the treated females as
Kidneys: Increased hyaline droplet accumulation of marked degree in the kidneys of the
five randomly selected males of the high dose group, when compared to the controls.
The presence of hyaline droplets is generally considered a normal occurrence in younger
male rats; in this study an increased hyaline droplet accumulation was observed in the renal
tubules of high dose treated males. The pathogenesis, as reported in literature (1), could
be ascribed to low molecular weight protein accumulation within lysosomes due to the
disturbance of the normal balance of tubular reabsorption and hydrolysis, as a result of
either increased filtered protein loads or decreased catabolism and for this reason it could be
considered treatment-related.
Yellow/brown pigmentation in five randomly selected females of the high dose group was
also noted.
Stomach (non-glandular): The treatment-related changes, seen in the non-glandular stomach
of some high dosed animals of both sexes, generally consisted of minimal to mild diffused
hyperplasia of the squamous epithelium.
Epithelial hyperplasia was characterized by increased thickness of the nonkeratinized layers
of the epidermis, due to an increased number (layers) of epithelial cells, compared with the
same site in a control animal.
Liver: Treatment-related findings were seen in high dose treated animals of both sexes. The
treatment-related liver changes consisted of minimal to moderate centrilobular hepatocytic
hypertrophy with cytoplasmic eosinophilia, associated with minimal to mild degree of inflammatory
cell foci, morphologically represented by an enlarged size of the hepatocytes,
possibly correlated to the hepatic enzyme induction.
Thyroid: Pigmentation (yellow/brown) of the thyroid was noted in high dose treated males
and females. In males, the finding was characterized by yellow/brown pigmentation of follicular cells, whereas in females the pigmentation was characterized by exfoliative cells,
yellow/brown pigmentation of follicular cells with reactive hyperthrophy (cuboidal cells),
colloid concentration and depletion with yellow brown deposit.
Spleen: Treatment-related findings were seen in, high dose treated females. The treatmentrelated
spleen changes consisted of mild to moderate increased pigmentation (yellow/brown),
mainly in the red pulp of the spleen.
Thymus: Minimal to mild atrophy of thymus was observed in high dose treated females.
This lesion correlated with reduced thymus size and weight.
Adrenals: Cortical hypertrophy of the adrenal glands was of marked degree in high dose
females. The lesion was characterised by an increased cell size without an appreciable
increase in cell numbers in the zona fasciculata.
The remaining sporadic lesions reported in control and treated animals were considered
to be an expression of spontaneous and/or incidental pathology, commonly seen in this
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle
and to the integrity of the various cell types within the different stages; regular layering in the
germinal epithelium was noted in all control and treated males.
mortality observed, non-treatment-related
Description (incidence):
one miss-dose
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistical decrease in terminal body weight was observed in treated females, when compared to the controls.

Effect levels

Key result
Dose descriptor:
Effect level:
37.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs

Target system / organ toxicity

Key result
Critical effects observed:
Lowest effective dose / conc.:
75 mg/kg bw/day (nominal)
gastrointestinal tract
adrenal glands
thyroid gland
Treatment related:

Applicant's summary and conclusion

Detailed macroscopic observations were reported for individual animals in the study1. Microscopic
evaluation was performed on five randomly selected animals from the control
and high dose groups, including one male of the high dose group which was found dead. In
addition, all gross abnormalities from all animals were also examined. As treatment-related
findings were found in the thyroid, kidneys, liver and stomach of both sexes and spleen,
thymus and adrenals of high dose treated females, it is suggested to extend the histopathological
evaluation of the above mentioned organs and tissues in the intermediate groups and
in the remaining 5 males and 5 females of the control and the high dose groups