Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
combined repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD 422)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 05 June 2018 and 19 March 2019 (date of thyroid hormone phase report). The in-life phase of the study was conducted between 14 August 2018 (first day of treatment) and 07 October 2018 (final day of necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
purity >85 % (w/w)

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han(TM), sub-strain: RccHan(TM):WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, UK
- Age at study initiation: 11 weeks (males) and 14 weeks (females) at start of treatment
- Weight at study initiation: At the start of treatment the males weighed 275 to 333g. The females weighed 198 to 234g.
- Fasting period before study:
- Housing: Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet: the animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used.
- Water: Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: the animals were acclimatized for twenty days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.

DETAILS OF FOOD AND WATER QUALITY, ENVIRONMENTAL CONDITIONS: The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

- Temperature (°C): 22 ± 3 °C;
- Humidity (%): 50 ± 20%;
- Air changes (per hr): at least 15;
- Photoperiod (hrs dark / hrs light): the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: between 14 August 2018 (first day of treatment) and 07 October 2018 (final day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): the test item was prepared at the appropriate concentrations as a suspension in Distilled water. The homogeneity of the test item formulations was confirmed analytically. The test item was administered within two hours of it being formulated. It is assumed that the formulation was stable for this duration.

VEHICLE
- Concentration in vehicle: 23.2., 69.8, 233.0mg/mL
- Amount of vehicle: 5ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken on two occasions and analyzed for concentration of the test item.
The results indicate that the prepared formulations were within 102-110% of the nominal concentration.
Duration of treatment / exposure:
Males were treated for approximately five weeks and females up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
Once daily throughout the treatment period (with the exception of females during parturition, if applicable)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were chosen based on available toxicity data which included a 14-day range finder toxicity tudy in the rat;
- Fasting period before blood sampling for clinical biochemistry: animals were not fasted.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable). All observations were
recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase, females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND FOOD EFFICIENCY: Yes
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

OPHTHALMOSCOPIC EXAMINATION: No

HEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).
- Animals fasted: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity;
- Battery of functions tested: sensory activity, grip strength, motor activity;

IMMUNOLOGY: No

OTHER: YES (endocrine function: Thyroxin (T4) levels)
Statistics:
Where appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Data were analyzed using the decision tree from the Provantis. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression. Where the data were unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 100, 300 or 1000 mg/kg bw/day.
One female treated with 300 mg/kg bw/day had either scab formation or an open wound on the neck between Days 41 and 52. This was considered to be a physical injury and unrelated to treatment. One male and five females treated with 1000 mg/kg bw/day showed isolated incidences of increased salivation during the study. Observations of this nature are commonly observed following the oral administration of an unpalatable test item formulation and is generally considered of no toxicological importance.
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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry 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:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100, 300 or
1000 mg/kg bw/day.
Details on results:
The pathology evaluation was subject to peer review.

Effect levels

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:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Remarks on result:
other: One male and five females treated with 1000 mg/kg bw/day showed isolated incidences of increased salivation

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of Reaction mass of Cucurbit[6]uril, Cucurbit[7]uril and Cucurbit[8]uril to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any adverse treatment-related effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

Following a 14-day non-GLP range-finding study in rats with an oral (gavage) NOAEL of 1000 mg/kg bw/day, a dosage sequence of 0 (control), 100, 300 and 1000 mg/kg bw/day, was used in the GLP repeated dose toxicity study with reproduction/developmental toxicity screen in accordance with OECD testing guideline 422. The treatment did not result in any adverse treatment-related effects. The NOAEL for systemic toxicity was therefore considered to be 1000 mg/kg bw/day. The NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day