Registration Dossier

Administrative data

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:
2009-01-02 to 2009-06-24
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): LCE08081
- Physical state: Liquid
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Wistar Han HsdRccHan: WIST strain rats were obtanied from Harlan Laboratories UK Ltd. The animals were acclimatised for twelve days during which time their health status was assessed.
At the start of treatment, the males weighed 310 to 383 g; the females weighed 184 to 217g and were approximately twelve weeks old.
Housing:
- Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake
bedding.
- Mating phase; animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one
female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were
housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
Food & water: free access (pelleted diet (Rodent 2018C Teklad Global Certified Diet Harlan Laboratories U.K.)
Air exchange: At leat 15/h
Light: 12 hours continuous light/day
temperature: 21 +/- 2°C
Hunidity: 55 +/- 15%

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
For the purpose of the study, the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test material formulations were determined and show the formulations to be stable for at least twenty days. Formulations were therefore prepared four times during the study and stored at approximately 4°C in the dark.
The test material was administered daily by gavage using a stainless cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day arachis oil BP.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of LCE08081 in the test material formulations was determined by gas chromatography using an external standard technique.
The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/ml.
Duration of treatment / exposure:
Groups of ten male and ten female animals were treated daily at the appropriate dose level for 46 consecutive days.
Frequency of treatment:
the test material was administered daily by gavage using stainless steel cannula attached to a disposal plastic syringe.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
no post-exposure recovery period in satellite groups was performed
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
Clinical observations:
All animals were examined for overt signs of toxicity, ill-health and gehavioral change immediately before dosing, up to thirty minutes after dosing and one and five hours after docing, during the working week. Immediatly before dosing, soon after dosing, and one hour after dosing at weeends and public holidays.

Functionnal observations:
Prior to start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioural assessments:
detailled individual clinical observations were performed for each animal using a purpose-built arena.
This test was developped from the methods used by Irwin (1968) and Moser at al (1988).

Functional performance tests:
motor activity/Forelimb-Hindlimb Grip Strength/sensory activity

Bodyweight:
recorded on day 1 (prior to dosing) ant then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on day 0, 7, 14 and 20 post coitum, and on days 1 and 4 post partum. Body weights were also recorded for all animals on the day of termination.

Food consumption:
During the mating period, weekly food consumption was recorded for each cage of adults. This was continued for males after mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. for females with live litters, food consumption was recorded on days 1 and 4 post partum.
Food efficiency was calculated retrospectively for males throughout the study period and for females during maturation and the first two weeks of gestation.

water consumption:
Daily visual inspection.

Laboratory investigations:
Haematological and blood chemical investigations were performed on five males and five females selected from each test group and control group on day 14.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Reproduction and Revelopmental observations (data not added in this file)
Statistics:
Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating
Levene's test for homogeneity and variance.Where variances were shown to be homogeneous, pairwise comparisons were conducted using
Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and
Mann-Whitney 'U' test.
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the
individual sexes:
-Chi-squared analysis for differences in the incidence of lesions with an overall frequency of 1 or greater.
-Kruskal-Wallis one-way non-parametric analysis of evidence for the comparison of severity grades for the more frequently observed graded
conditions.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no unscheduled deaths during the study Instances of increasing salivation either sex treated with 1000 mg/kg/day
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no unscheduled deaths during the study Instances of increasing salivation either sex treated with 1000 mg/kg/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slight reduction in bodyweight gains was observed for males treated with 1000 mg/kg/day when compared to controls throughout the treatment period, with statistically significant reductions in cumulative bodyweight change observed during week 5.
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
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Weekly open fiel arena assessment confirmed the signs of noisy respiration observed for one male treated with 1000 mg/kg/day during week 5. There were no other behavioural changes considered attributed to treatment
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Stomach: A greater incidence of agglomeration of secretion in the mucosa adjacent to the limiting ridge and acanthosis/hyperkeratosis of the limiting ridge were seen in relation to treatment for animals of either sex treatment with 1000 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Isololated instances of acanthosis and hyperkeratosis of the forestomach were evident at 1000 mg/kg/day
Histopathological findings: neoplastic:
no effects observed
Details on results:
The oral administration of LCE08081 to rats, for a period of forty-two days for males and a two week maturation phase, pairing, gestation and early
lactation for females, at dose levels of 100, 300 and 1000 mg/kg/day, resulted in treatment-related systemic effects at 1000 mg/kg/day.
clinical signs were mainly confined to occasionnal excessive salivation detected soon after administration of the test material formulations for both
females and males treated with 1000 mg/kg/day and incidentals of noisy respiration. Noisy respiration was confirmed following the weekly
behavioural assessments. These observations are occasionally observed following the oral administration of an unpalatable or tlightly irritant test
material formulation, and irritancy was confirmed with greater incidence of agglomeration of secretion in the mucosa adjacent to the limiting ridge
and acanthosis/hyperkeratosis of the forestomach were also evident at 1000 mg/kg/day. there were no associated degenerative changes in the
stomach. Slighly reduced bodyweight gains were evident for males treated with 1000 mg/kg/day although there were no adverse effects on dietary
intake, water intake or food conversion efficiency. The bove changes were therefore not considered to represent serious damage to health, as
defined by the criterion given in EC labelling guideline of commission directive 2001/59/EC.

Effect levels

Key result
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:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
haematology
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of LCE08081 to rats, for a period of up to forty-six consecutive days at dose levels of up to 100, 300 and 1000 mg/kg/day, resulted in treatment-related systemic effects at 1000 mg/kg/day, although, there findings were considered not to represent an adverse health effect, therefore, the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 1000 mg/kg/day for systemic toxicity.
Executive summary:

The study was designed to investigate the systemic toxicity and potential effects of the test material on reproduction (including offspring development) and complies with the recommendations of the OECD Guidelines for testing of Chemicals No.422 "Combined repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity screening Test" (adopted 22 March 1996).

This study was also designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test method pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

The test material was administrered by gavage to three groups each of ten male and female Wistar Han:HsdRccHan:WIST strain rats, for up to forty-six consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose level of 100, 300 and 1000 mg/kg/day. a control group of ten males and ten females was dosed with the vehicle alone ( Arachis oil BP).

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected males and females from each dose group.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each group on Day 4 post partum.

Males were terminated on day 43, followed by the termination of all females and offspring on day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results:

Motality. There were no unscheduled deaths.

Clinical signs. Isolated instances of increased salivation were detected soon after dosing for animals of either sex treated with 1000 mgkg/day during the treatment period.

No clinical observable signs of toxicity were evident for animals of either sex treated with 300 or 100 mg/kg/day.

Behavioural Assessment. No treatment-related changes were detected for treted animals when compare to controls.

Functional Performance Tests. There were no treatment-related differences in functional performance assessment fo treated animals when compared to controls.

Sensory Reactivity Assessments. No treatment-related effects were detected.

Bodyweights. A silght reduction in bodyweight gains was observed for males treated with 1000 mg/kg.day when compared to controls.

No adverse effect on bodyweight change was detected for treated females throughout the pre-mating, gestation or lactation phases of the study when compare to controls.

Food Consumption and food Efficiency. No adverse effect on food consumption or food efficiency was detected.

Water Consumptions. No treatment-related intergroup differences in water intake were detected for treated animals when compared to controls.

Haematology. No toxicologically significant differences were detected in the haematological parameters for treatment animals when comprared to control.

Blood chemistry. no treatment-related effects were detected in the blood chemical parameters for treated animals when compared to controls.

Organ weights. There were no treatment-related differences in organ weights from treated animals when compared to controls.

Histopathology. Histophatological examinations revealed the following treatment-related effects:

- Stomach: A greater incidence of agglomeration of secretion in the mucosa adjacent to the limiting ridge and acanthosis/hyperkeratosis of the limiting ridge were seen in relation to treatment for animals of either sex treatment with 1000 mg/kg/day. Isolated instances of acanthosis and hyperkeratosis of the forestomach were also seen at this treatment level. A similar effect was not seen at any other treatment level for either sex.

Conclusion.

The oral administration of LCE08081 to rats, for a period of up to forty-six consecutive days at dose levels of up to 100, 300 and 1000 mg/kg/day, resulted in treatment-related systemic effects at 1000 mg/kg/day, although, there findings were considered not to represent an adverse health effect, therefore, the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 1000 mg/kg/day for systemic toxicity.