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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2016 to June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BYK Chemie HJ-15-097
- Expiration date of the lot/batch: 1-Dec-2016
- Purity test date: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: grinding

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK)
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 44 - 50 days
- Weight at study initiation: males 213-276 g, females 158-201 g
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: Teklad 2014C diet; potable water from public supply

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were weighed. Small amounts of the vehicle were added and mixed with the test material in a mortar using a pestle. Any agglomerates were broken down to produce a smooth paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was poured into a measuring cylinder which had been wetted with the vehicle. The mortar was also rinsed with the vehicle and added to the cylinder and the suspension was made up to the required volume with vehicle. The suspension was transferred into a mixing container and mixed, using a Silverson mixer, carefully until a slight bitty suspension was achieved (not a paste) and then stirred magnetically for at least 30 minutes.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 20, 60, 120 mg/ml
- Amount of vehicle (if gavage): 5 ml / kg bw
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of dose formulations were analysed by HPLC with evaporative light scattering detector. Mean concentrations were within 10% of the nominal concentrations.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
The highest dose should have been at 1000 mg/kg bw/day but the dose formulation at this concentration turned out to be unable to be administered because the formulation could not be drawn up the catheter.
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: In an OECD422 study in the CD rat, oral administration of WS400517 (BYK-LP R 6075 WS) at dose levels of 100, 300 and 1000 mg/kg/day was well tolerated with no adverse effect of treatment on clinical signs, body weight gain, macroscopic finding or organ weight. It was concluded from these studies that the high dose of 1000 mg/kg/day was the no-observed-effect-level (NOEL).

Based on this information, it was considered appropriate to investigate a high dose level of 1000 mg/kg/day (equivalent to the limit dose for this study type) on the current study. The intermediate and low dose levels of 300 and 100 mg/kg/day were selected to assess any dose-responsiveness of any test substance-related finding following a longer (i.e. 13 week) treatment period.

On Day 1 of study, however, the 1000 mg/kg/day dose level was unable to be administered because the formulation could not be drawn up the catheter (8 or 9 choke). Reducing the concentration by increasing the dose volume was discounted because of the toxicity of larger amounts of the vehicle, propylene glycol. The highest practical dose, considered to be 600 mg/kg/day (120 mg/ml administered at 5 ml/kg) was, therefore, administered for the high dose group from Day 2.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once per week starting 1 week before first adminstration

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and in week 12
- Dose groups that were examined: all animals (pre-treatment), animals of high dose group and control group (week 12)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly arena observations
- Dose groups that were examined: all animals of all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: week 12, all animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Estrous cycles by vaginal smear: for 14 days during weeks 10 and 11 of treatment, all groups

Sperm analysis: after scheduled sacrifice of each male, sperm motility (all groups), morphology and homogenisation-resistant spermatid count (groups 1 and 4)
Statistics:
The following sequence of statistical tests was used for grip strength, motor activity, body weight, sperm analysis, organ weight and clinical pathology data: A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. Where there were only two groups, comparisons were made using t-tests.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test (Shirley 1977).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Figure body weight
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological investigations during Week 13 of treatment revealed slightly high reticulocyte counts, compared with controls (1.4X control), for males receiving 600 mg/kg/day. Activated partial thromboplastin time was slightly shorter than that of the controls for females receiving 300 or 600 mg/kg/day and the mean prothrombin time was shorter than that of the controls for females receiving 600 mg/kg/day.
A small decrease of mean cell haemoglobin concentration at all doses in males attained statistical significance, however, the differences were minor, confined to one sex and lacked dose-relationship and were therefore attributed to normal biological variation.
see "overall remarks, attachments" : Table Haematology group mean values
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Blood chemistry investigations during Week 13 of treatment revealed slightly low (0.88X control) urea concentration for females receiving 600 mg/kg/day.
A small number of other differences from controls attained statistical significance, but these were minor, confined to one sex and lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included a reduction in bile acids for males at 300 or 600 mg/kg/day and a slight increase in total protein in males at 600 mg/kg/day.
see "overall remarks, attachments" : Table Clinical biochemistry group mean values
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Analysis of organ weights for animals killed after 13 weeks of treatment revealed, when compared with the controls, slightly high absolute and body weight adjusted spleen weights for males receiving 600 mg/kg/day. Following microscopic examination of the spleen, no findings were seen to account for the reported slightly increased spleen weights for males that received 600 mg/kg/day.

Absolute and body weight-adjusted kidney weights at all doses in females were slightly lower than those of the controls. The differences from controls were, however, minor, there was no dose relationship and no similar trend in the kidney weights of males and, consequently, these differences from controls were considered of no toxicological significance.

All other inter-group differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.
see "overall remarks, attachments" : Table Organ weights
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with BYK-LP R 6075 WS were seen in the mandibular salivary gland of females. A minimal to moderate decrease in the granules in the striated ducts was seen in treated females and exhibited a dose-relationship. The toxicological significance of this finding in one of the three salivary glands was unknown.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
All other findings were considered incidental and unrelated to treatment, attributed to normal biological variation.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: NOAEL refers to the highest dose level that practically could be administered to the animals.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion