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Description of key information

In repeated oral dose toxicity testing, no significant toxicity was observed.

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Guideline study under GLP
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
CiToxLab Hungary Ltd. H-8200 Veszprém, Szabadságpuszta, Hungary
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 360-392 g, Females: 228-261 g
- Fasting period before study: no, fasting only before sacrifice
- Housing: males housed in groups of 4, in polycarbonate cages. Females housed singly during and after mating. Lignocell (R) and Arbocel (R) natural crinklets
- Diet (e.g. ad libitum): at lib, ssniff (R)
- Water (e.g. ad libitum): ad lib, municipal water supply
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): appropriate vehicle for oily liquid test material
- Concentration in vehicle: 100, 300 and 1000 mg/kg bw/d
- Amount of vehicle (if gavage): stability measured at concentrations up to 250 mg/ml at room temperature.
- Lot/batch no. (if required): BCBQ0052V, Sigma-Aldrich
Analytical verification of doses or concentrations:
yes
Remarks:
See Analytical Report #16-178-316AN. Sarvari, Z., 22 March 2017. Analysis by LC-MS. Stable in PEG 400 for 16 days at room temperature (104% recovery). Stock solution stable for 7 days at 5 degrees C.
Details on analytical verification of doses or concentrations:
Analysed by LC-MS.
HPLC-MS: Thermo-Finnigan Surveyor HPLC with LCQ Duo MS detector
Balance: Sartorius BP221S
Water purification system: MILLIPORE, DIRECT Q 8 UV

Analytical Technique: LC-MS
Column: Luna CN, 50×4.6 mm, 3 µm
Column temperature: 25 °C
Mobile Phase: Methanol : water = 8:2 +0.1% acetic acid
Flow: 0.5 mL/min
Detector: SIM (409.1 m/z, negative ion)

Linearity test 1: Y = -50958.8+286873*X+8325.51*X^2 R^2 = 0.9993 W: Equal.
Linearity test 2:
Y = -689226+836889*X R^2 = 0.9955 W: 1/X

Recovery and Precision of Analytical test: 93-104%
Stabillity of Test Item in Vehicle: 104-109%



Duration of treatment / exposure:
28 days for males, 34-44 days for females
Frequency of treatment:
once daily, 7 days per week.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to first dose, and at least weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at randomisation, before treatment, at day 0, weekly and at necropsy. Females also at GD0, 3, 10, 17 and 20 and at PPD 0 and 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (pentobarital)
- Animals fasted: Yes, prior to sacrifice
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine sampling will be performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, on the control and high dose group animals.

Name: Euthanimal® (40% Pentobarbital sodium)
Batch No.: 1409236-06
Expiry Date: September 2017
Produced by: Alfasan Netherland BW, Kuipersweg 9, Woerden
Storage: Room temperature
Purpose of use: Euthanasia
Other examinations:
Functional Observation Battery on 5 animals/group.

Body and organ weight: At the time of termination, body weight and weight of the following organs of all adult animals will be determined:
uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus, adrenals, ovaries, thyroids with parathyroids. Testes and epididymides will be weighed individually. Individual and/or paired absolute organ weight will be reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable will be summarised. Relative organ weight (to body and brain weight) will be calculated and reported.

The number of implantation sites and of corpora lutea will be recorded in the females as applicable.
Statistics:
Data is collected using software PROVANTIS v.9. Statistical analysis is performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups will be checked by Bartlett's test. Where no significant heterogeneity is detected, a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant, then Duncan's Multiple Range test is used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data is examined by Kolmogorow-Smirnow test. In the case of non- normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance is applied. If a positive result is detected, the inter-group comparisons are performed using Mann-Whitney U-test. The Chi-squared test will be used for non-continuous data.
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree is applied automatically for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups will be checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log- transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test is carried out. If the obtained result is positive, Dunnett (Multiple Range) test is used to assess the significance of inter- group differences; identifying differences of <0.05 or <0.01 as appropriate.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control group male was found dead on day 17, due to technical error (perforation of the esophagus, confirmed at necropsy).
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Mean prothrombin time was significantly decreased (p<0.05) in mid and high dose males, but the treat ed group values were in the normal control range, and there was no clear dose response. A similar trend was noted in females, but didn't reach statistical significance. These effects are not considered biologically relevant or a test item related effect.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Glucose concentration was significantly increased (p<0.01) in mid and high dose males, but the treat ed group values were in the normal control range. These effects are not considered toxicologically significant or related to treatment.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weight (absolute and relative) was significantly increased (p,0.05) in both sexes at the high dose. For males (but not females) in the high dose group, this liver weight was just above the historical control range. Liver microscopic appearance and liver function tests remained unaffected. These effects are not considered a test item related adverse effect. There were significant decreases in the absolute weight of brain and epididymus for all test item related groups (males). Absolute weight of spleen was decreased significantly in high dose males, and that of the thumus in mid dose males. These were within the historical control range, with no evidence of histopathology. These were not considered a test item related adverse effect.

Neuropathological findings:
no effects observed
Description (incidence and severity):
.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
All high dose males showed a minimal increase in number of eosinophilic droplets in the kidney, compared with normal background presence in control group males. These cytoplasmic/luminal droplets had similar size to those noted in controls. Droplets were predominantly located in the proximal tubul es of the cortex. No accompanying degenerative/necrotic and inflammatory changes were recorded.
In the mid dose males, there was reduced sperm content and an occurrence of spermatocele in the epididymis (1/5) and dilation of renal tubule (1/5). In control males, there was inflammation of the prostate gland (3/5) this also occurred in 2/5 high dose males. In one high dose female, congestion/ haemorrhage of the thumus occurred.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant toxicity observed at the highest dose administered.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no

ANALYSIS OF DOSE FORMULATIONS:

The measured concentrations of ZWA 5496/100 evaluated for each dose group varied between 93% and 106% of the nominal value. No test item was detected in the control samples at any occasion. These results were within the acceptable range (90% - 110% of the nominal concentration) and were considered to be suitable for the study purposes.

 

All test item formulation samples were found to be homogeneous. Formulations were considered to be adequately stable under the study conditions.

Conclusions:
In an OECD 422 guideline study (28-day repeated dose toxicity and reproductive toxicity screening study) in Wistar rats at doses of the test material of 100, 300 and 1000 mg/kg bw/day, there were no adverse effects noted. There were minor changes in organ weights of liver and kidney of both sexes, and in males for epididymus, brain and spleen, but these were not associated with impaired function or histopathology findings. There was no reproductive toxicity observed. The conclusion is that the substance does not exert toxic effects at any dose tested under these conditions. The NOAEL for repeated dose toxicity effects is 1000 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
adequate
System:
other: no toxicity observed

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

Mode of Action Analysis / Human Relevance Framework

Not applicable

Additional information

In repeated oral (gavage) dose toxicity testing in the rat for 28 days according to the OECD 422 protocol, no significant toxicity was observed. The NOAEL is greater than 1000 mg/kg bw/d, the highest dose tested.

Justification for classification or non-classification

In repeated oral (gavage) dose toxicity testing in the rat for 28 days according to the OECD 422 protocol, no significant toxicity was observed. The NOAEL is greater than 1000 mg/kg bw/d. The criteria for Regulation EC No. 1272/2008 for classification for specific organ toxicity, repeated exposure, are not met. The substance is not classified.