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Description of key information

Oral (OECD 422), rat: NOAEL (systemic), males/females ≥ 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Oct - 06 Dec 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Comission Regulation (EC) No. 440/2008, L142, Appendix Part B
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl: WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: males / females: 10-11 weeks
- Weight at study initiation: males mean: 271 g, females mean: 194 g
- Fasting period before study: no data
- Housing: the animals were kept individually in IVC cages (except during the mating period), type III H, polysulphone cages on Altromin saw fibre bedding; females were separated and housed individually after confirmation of the mating
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 0856), ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulation was prepared freshly on each day of administration. After cooling the test item in a refrigerator for 10 minutes at 2-8 ºC, the test item was grinded to a fine powder using a mortar and pestle or laboratory electric mill. When mortar and pestle was used for grinding, it was ensured that not too much force was applied during the grinding and grinded with little force in circulating movements to prepare a very fine powder. The powdered test item was weighed into a tared plastic vial or container on a suitable precision balance and then the vehicle (corn oil) was added to give the appropriate final concentration of the test item. The formulation vials/container was shaken by hand for short period to ensure proper homogenistation of the formulation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle has been selected on the basis of the test item’s characteristics and the results of the dose range finding study.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Batch no. (if required): MKBH 4894 V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, stability and homogeneity of the formulated samples were analytically verified using HPLC with UV detection.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) (Total 16 samples) for all dose groups. The mean recoveries observed in the low mid and high dose groups were 98.7 %, 103.8 % and 98.0 % of the nominal concentration, respectively.
Stability of formulation samples was investigated in week 1 for low and high dose groups. Two aliquots per dose were analyzed immediately after preparation, another two per dose were stored for 6 hours at room temperature and analyzed thereafter. After 6 hours storage at room temperature recovery compared to starting value was 105.0 % and 134.9 % for low and high dose samples. Since the initial consistency of freshly prepared formulation samples is a suspension transforming within approx. an hour into a jellylike lump it is assumed that paralelly the density of the formulation, and therefore its concentration increases. This effect is observed at the higher concentration only. Because animals in the main study were administred within 1 hour, the 6 hour stability measurement was not relevant for the study and therefore the stability of samples at the concentration of high dose group was investigated again for the storage period of 1 hour. Investigation of 1 hour stability of high dose samples confirmed that the samples are stable and revealed 99.3 % recovery.
Samples for homogeneity analysis were taken in week 1 and week 5 of the study from top, middle and bottom of low, mid and high dose preparations. The mean recovery observed for low dose group was 97.2 and 94.1 % of the nominal value, for mid dose group was 101.0 and 106.3 % and 100.4 and 100.1 % for high dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 6.4 and 5.7 % in low dose group, 8.8 and 0.6 % in mid dose group and 2.0 and 0.7 % in high dose group.
Duration of treatment / exposure:
females: 54 days (14 days of pre-mating and maximum 14 days of mating, during the gestation period and up to post-natal day 3)
males: 28 days
Frequency of treatment:
daily, 7 days/week
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The choice of test substance concentrations was based on a dose range finding study (BSL project no. 123581). The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: no data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly, on the corresponding days of the body weight measurements, except during the mating period in female animals and the mating and post-mating period in male animals.
- Mean weekly diet consumption per sex calculated as g food/kg body weight/week: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: no data
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminal sacrifice
- Anaesthetic used for blood collection: No (Animals were sacrificed using an anaesthesia (ketamine/xylazin, 2:1). Blood was taken from the abdominal aorta of the animals.)
- Animals fasted: No data
- How many animals: five males and five randomly selected females from each group
- Parameters checked: activated partial thromboplastin time (aPTT), basophils (Baso), eosinophils (Eos), haematocrit value (Hct), haemoglobin content (Hb), lymphocytes (Lym), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), monocytes (Mono), neutrophils (Neu), platelet count (PLT), prothrombin time (PT) , red blood cell count (RBC), reticulocytes (Re), white blood cells (WBC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at terminal sacrifice
- Animals fasted: No data
- How many animals: five males and five randomly selected females from each group
- Parameters checked: alanine aminotransferase (ALAT), alkaline phosphatase (AP), aspartate-aminotransferase (ASAT), albumin (Alb), creatinine (Crea), glucose (Gluc), potassium (K), sodium (Na), total bile acids (TBA), total cholesterol (Chol), total protein (TP), urea

URINALYSIS: Yes
- Time schedule for collection of urine: at terminal sacrifice
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- How many animals: from five randomly selected males from each group
- Parameters checked: appearance, bilirubin, blood, glucose, ketone bodies (ketones), leukocytes, nitrite, ph-value (pH), protein, specific gravity, urine colour, urobilinogen (ubg)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the week before the treatment and at the end of the study
- Dose groups that were examined: 5 randomly selected males and on day 3 of the lactation period in 5 randomly selected females
- Battery of functions tested: sensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (All surviving male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4. All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.)

HISTOPATHOLOGY: Yes (on the following preserved organs and tissues of 5 randomly selected male and female animals of the control and high dose groups: adrenal glands, brain (cerebrum, cerebellum and pons), bone with bone marrow (sternum), epididymides, eye, gross lesions, heart, kidneys, liver, lungs and trachea, lymphnodes, mammary glands, oesophagus, ovaries, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, pituitary gland, prostate and seminal vesicles with coagulating glands as a whole, skin, small and large intestines (including Peyer´s patches), spinal cord, spleen, stomach, testes, thymus, thyroid, urinary bladder, uterus with cervix, vagina.
The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for eyes, testes and epididymides which were preserved in modified Davidson’s Solution.)
The wet weight of the following organs of 5 sacrificed males and 5 females randomly selected from each group was recorded as soon as possible (Paired organs were weighed separately. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded): adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary gland, prostate (seminal vesicles and coagulating glands), spleen, testes, thymus, thyroid/parathyroid glands, uterus with cervix
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 and V.6.01 software (p<0.05 was considered as statistically significant).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred in the control or any of the dose groups during the treatment period of this study.
No test item related clinical signs were observed in male and female animals during entire study period. Few predominant spontaneous clinical signs observed occasionally in male and female animals were alopecia on various body parts, red nasal discharge, slight to moderate salivation, abnormal breathing, slight piloerection and moving the bedding.

BODY WEIGHT AND WEIGHT GAIN
In both males and females, no statistically significant effect was observed on body weight and body weight gain in treatment groups during the most study phases. The only exception was a statistical significant decrease in male body weight gain in mid dose males during mating and postmating days 7-14. Due to the lack of dose dependency, this statistically significant decrease in male body weight gain was considered to be biologically irrelevant.

FOOD CONSUMPTION
In both males and females, no statistically significant effect was observed on food consumption during the whole study period.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopic findings in any of the animals of this study.

HAEMATOLOGY
In males, a statistically significant increase in platelets in the mid dose group and decrease in reticulocytes in the low dose and mid dose group was observed when compared with controls. Since all haematological parameters were within the normal range of variation and due to lack of dose dependency, statistically significant differences in male hematology parameters are not assumed to be biologically relevant. All remaining haematological parameters in males were remained unaffected.
In females, no statistically significant difference was observed in any of the haematology parameters in treated groups when compared with the controls.

CLINICAL CHEMISTRY
In males and females, no statistically significant difference was observed in any of the clinical biochemistry parameters in treated groups when compared with the controls.

URINALYSIS
The urinalysis performed in randomly selected male animals revealed no test item related effect in any of the treatment groups compared to the control group except high protein and erythrocyte levels in few animals. As these effects were not dose-related and also found in the urine of control animals animals, they were considered to be by chance findings.

NEUROBEHAVIOUR
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.

ORGAN WEIGHTS
In males and females, no statistically significant difference in the absolute and relative organ weights was observed in the treatment groups except a statistically significant increase in relative liver weight in mid and high dose males and increase in relative adrenal weights in low and high dose males when compared with the control group. As the histopathological evaluation did not reveal any effect, the effects on liver and adrenal weights were considered to be spontaneous in nature and not treatment-related.

GROSS PATHOLOGY
At necropsy of males and females, macroscopic examination of the animals revealed few findings in males and females like small thymus (1/10 in low dose female), fluid filled uterus (1/10 in high dose female) and yellow spots on epididymis (2/10 in low dose and 2/10 in mid dose males). The yellow spots on epididymis were histologically confirmed to represent spermatic granuloma, a finding occasionally seen as spontaneous change in untreated rats of this strain and age.
Taken together, the gross pathological findings were spontaneous in nature and were assumed to be common background findings in this strain and as such not due to a systemic effect due to the test item administration.

HISTOPATHOLOGY: NON-NEOPLASTIC
The pathological evaluation did not reveal any test item-related effect on reproductive organs or on the other organs and tissues evaluated.
Spermatic granulomas were noted in 2/10 low dose and 2/10 mid dose males. Although this lesion was not noted in control males of this study, it was considered as a spontaneous change, as there was no dose relationship, and this finding is occasionally seen in untreated rats of this strain and age used at the test facility.
In the kidney, minimal numbers of debris-filled tubules were observed at the inner cortex of one single high dose male, but were not considered sufficient evidence of a test item related effect.
Minor inter-group differences were observed in the incidences of some findings. Minimal interstitial mononuclear cell infiltrates in the epididymis (2/10 in the control, 1/10 in the low dose, 5/10 in the mid dose and 7/10 in the high dose group), minimal cortical hyaline droplets in the kidney (2/10 in the control and 4/10 in the high dose group), minimally dilated glands in the trachea (1/10 in the control and 3/10 in the high dose group) and minimal or mild presence of germinal centers in the mesenteric lymph nodes (minimal: 1/10 in the control and 3/10 in the high dose group; mild: 2/10 in the control and 2/10 in the high dose group) were noted in males. In females, minimal basophilic tubules in the kidney (1/10 in the control and 3/10 in the high dose group) and minimal or mild sinusoidal erythrocytes in the mesenteric lymph nodes (minimal: 0/10 in the control and 3/10 in the high dose group; mild: 1/10 in the control and 0/10 in the high dose group) were observed. However these changes had a background incidence in the control group, the inter-group difference was not noted in both sexes and there was no tendency towards higher severity levels in the treated animals. Therefore, all of these inter-group differences were considered to be fortuitous in nature and not treatment-related.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL corresponding to the highest dose tested.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A combined repeated dose/developmental toxicity screening study was conducted with UMA121 under GLP conditions according to OECD 422 in male and female Crl: WI(Han) rats (Takawale, 2013). Ten male and female rats each were once daily orally treated with test substance at concentrations of 100, 300, 1000 mg/kg bw/day dissolved in corn oil. Females were treated 2 weeks before mating until day 3 of lactation (about 50 days) and the males for 28 days. Control animals were treated with the vehicle. No mortality and no test item-related clinical signs were observed. No statistically significant effects were observed on food consumption, ophthalmoscopic examination, clinical chemistry, urinalysis and neurobehaviour. Statistically significant effects were noted on body weight gain as it was decreased in mid dose males during mating and postmating days 7-14, on organ weights (increased relative liver weight in mid and high dose males and increased relative adrenal weights in low and high dose males) as well as on haematology (increase in platelets in the mid dose males and decrease in reticulocytes in the low dose and mid dose males). Those effects were considered to be not biologically relevant due to the lack of dose dependency and as the histopathological evaluation did not reveal any effect on the respective organs. Gross pathology revealed few findings like small thymus in one low dose female and fluid filled uterus in one high dose female as well as yellow spots on epididymis in low and mid dose males. The yellow spots on epididymis were histologically confirmed to represent spermatic granuloma, a finding occasionally seen in untreated rats of this strain and age. As there was no dose-response relationship, spermatic granuloma were considered to be an incidental finding. Further histopathological observations showed minimal numbers of debris-filled tubules at the inner kidney cortex of one high dose male and few minor inter-group differences in males and females that had a background incidence in the control group. As the respective inter-group difference was not noted in both sexes and there was no tendency towards higher severity levels in the treated animals the inter-group differences were considered to be fortuitous in nature and not treatment-related. Taken together, due to the lack of dose-response relationship, the gross pathological and histopathological findings were considered to be spontaneous in nature and occurrence as they were assumed to be common background findings in this rat strain and as such not related to a systemic effect due to the test item administration. Therefore, the systemic NOAEL was determined to be 1000 mg/kg bw/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
There is only one study available.

Justification for classification or non-classification

The available data on repeated dose toxicity of the test substance does not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification.