Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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 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.
Details on mating procedure:
Animals were paired on a 1male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each
morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. The
presence of sperm within the vaginal smear and/or vaginal plug in situ was taken positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original cages. Mated females were housed individualy during the period of gestation and lactation.
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.
Details on study schedule:
- Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturation where applicable). The first day of dosing was designed as Day 1 of the study.
- On day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
- Following evidence of mating (designed as day 0 post coitum) the males were returned to their original cages and females transferred to individual cages.
- Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum.
- Following completion of the female gestation and lactation phases, the male dose groups were killed.
_ At Day 5 post partum, all females and surviving offspring were killed
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:
not applicable

Examinations

Parental animals: Observations and examinations:
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.
Oestrous cyclicity (parental animals):
Each pregnant female was observed at approximately 830, 1230 and 1630 hours and around the period of expected parturation. Observations were carried out at approximately 830 and 1230 hours at weekands and public holidays. The following was recorded for each female:
Date of pairing
Date of mating
Date and time of observed start of parturation
Date and time of observed completion of parturation
Sperm parameters (parental animals):
not evaluated
Litter observations:
On completion of parturation (Day 0 of post partum), the number of live and dead offspring was recorded. Offsprings were individualy identified within each litter by tatoo on Day 1 post partum.
For each litter the following was recorded:
Number of offspring born
number of offspring alive recorded daily and reported on Day1 and Day4 post partum
Sex of offspring on Day 1 and 4 post partum
Clinical condition of offspring from birth to day 5 post partum
Individual offspring and litter weights on Day 1 and 4 post partum
All alive offspring were assessed for surface righting reflex on Day 1 post partum
Postmortem examinations (parental animals):
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded.
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- Organ weight
- hitopathology
Postmortem examinations (offspring):
All offspring animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
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.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
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.
Reproductive indices:
- Mating performance and fertility
- Pre-coital interval
- Fertility indices
- Gestation and partiration data
- Gestation length
- Parturation index
- Litter response
- Implantation losses
Offspring viability indices:
- Live birth and viability indices
- Sex ration

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Instances of increasing salivation either sex treated with 1000 mg/kg/day during the treatment period. One female also displayed noisy respiration on Day 31 and one male showed noisy respiration on Day 34.
There were no further clinical observation considered to be attributed to treatment with the test material.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
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 (P<0.05).
No adverse effects on bodyweight change were detected for treated females throughout the pre-mating, gestation or lactation phases of the study when compared to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on dietary intake was observed for treated males when compraed to controls of for treated females during the pre-mating, gestation and lactation phases of the study compared to control females.
Food efficiency:
no effects observed
Description (incidence and severity):
Food efficiency was unaffected.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No significant intergroup differences in water consumption for treated animals when compared to controls.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Exophalmia and opacity of the left eye was recorded for one female treated with 1000 mg/kg/day during Week 6. These signs were considered to be related to pregnancy rather than related to treatment with the test material.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant differences were detected prior to pairing on Day 14. Further haematological analysis was therefore not required.
Males treated with 1000 mg/kg/day showed a statistically significant increase in neutrophil counts when compared to controls. The significance achieved was minima and in absence of corresponding changes in any associated haematological parameters, this increase was not considered to represent an adverse effect of treatment.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related effects were detected in the blood chemical parameters investigated prior to pairing on Day 14.
Statistical analysis of the data did not reveal any significant intergroup differences between control and treated groups.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
All remaining inter and intra group differences in urination were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no behavioural changes considered attributed to treatment.
Tip-toe gait, hunched posture and pilo-erection were evident for one female treated with 300 mg/kg/day during the Week 5 assessments and hunched posture was still evident for this animal during Week 6. These signs were considered to be related to pregnancy rather than related to treatment with the test material.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
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
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating: No treatment-related changes in mating performance were observed for treated animals when compared to controls. All animals mated within the first four days of pairing.
Fertility: No treatment-related differences in fertility were evident for treated animals when compared to controls. All females treated with 100 and 300 mg/kg/day achieved pregnancy. There was one non-pregnant control female ans two females treated with 1000 mg/kg/day did not achieved pregnancy. The control male and one of the high dose males which failed to produce a pregnancy in the female partner showed small testes during the post-mortem procedure and histopathological examinations revealed supression of spermatogenesis of the testes for the control male and testicular atrophy for the hight dose male. This would explain the fealure to produce a pregnancy in the female partners. The remaining high dose male or female partner which failed to produce a pregnancy did not show any abnormalities which may have result in infertility. Non-pregnancies are occasionally observed in reproductive studies and these findings were not considered to represent a toxic effect of the test material in this study.

Details on results (P0)

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.
No significant reproduction effects were detected during the study. There were no differences in mating performance, or gestation lengths

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
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
ophthalmological examination
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive performance
Remarks on result:
other:

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Offspring viability was slightly lower at 1000 mg/kg/day when compared to control litters, although this was du to three offsping deaths at birth for one litter in this dose group, and statistical analysis of the data did not reveal any significant intergroup differences, hence this finding was without toxicological importance.
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treated-related macroscopic abnormalities were detected for offspring dying during lactation or at termination on day 5 post partum
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
A slight lower number of offspring from the 1000 mg/kg/day dose group successfully passed the surface righting reflex assessment on Day 1 post partum, however, the higher standard deviation and absence of statistical significance would suggest that this finding was attributed to unsuccessful result obtained from one litter from the 1000 mg/kg/day. This litter also revealed the three dead offspring at birth and the interim offspring deaths. these findings were confined to only one litter from this dose level, therefore were not considered to be representative of the dose group. As such, these findings were not considered to represent toxicity of the test material.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No significant reproductive effects were detected during the study. There were no differences in mating performance, or gestation lengths. There were two non-pregnant females treated at the highest dose level. One of these pregnancies was attributed to the presence of small testes and testicular atrophy of testes for the male partner. A similar effect was also evident for one control male which failed to produce a pregnancy. There were no associated findings which suggested a cause for the second non-pregnancy at the highest dose level, however, in the absence of any further effects which would suggest a reproductive effect of the test material, the non-pregnancies observed in this study were considere to be low incidence findings occasionally observed in reproductive studies of this type, and were not considered to be related to treatment.
One 1000 mg/kg/day female was considered not to be pregnant but was confirmed as pregnant following post-mortem procedures, which revealed the presence of a dead foetus in utero. Such findings are occasionally observed in reproductive studies, and in isolation, this event was not considered to be attributed to test material toxicity. Any discrepancies in liter response between control and 1000 mg/kg/day were attributable to one litter from the 1000 mg/kg/day dose group which showed deaths at birth, interim deatchs and lower reflexological responses. These findings were confined to one litter from this group, there was no indication that these findings were a consequence of test material toxicity.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Remarks:
developmental toxicity in progeny
Generation:
F1
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
gross pathology
developmental neurotoxicity

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

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 and a 'No Observed Effect Level' (NOEL) was established at 1000 mg/kg/day for reproductive toxicity including fertility and mating in adults for developmental toxicity in their subsequent progeny
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.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

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.

Reproductive Performances:

Mating. no treatment-related changes in mating performance were observed for treatment animals when compared to controls.

Fertility. No significant differences in fertility were evident for treated animals when compared to controls.

Gestation Length. No significant differences in gestation lengths were detected for treated females when compared to controls.

Litter Responses.

Offspring Litter Size and Viability. There were no differences in corpora lutea, implantations sites or implantation losses for control and treated females. Litter size at birth and during lactation were comparable between control and treated animals, and no differences in sex ratio were detected for treated litters when compared to controls.

Offspring Growth and Development. There were no differences in litter weights or mean offspring bodyweights between litters from treated animals when compared to controls. No clinically observable signs of toxicity were detected.

Pathology:

Necropsy. No treatment-related macroscopic abnormalities were detected for offspring dying during lactation or at termination on Day 5 post partum.

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 and a 'No Observed Effect Level' (NOEL) was established at 1000 mg/kg/day for reproductive toxicity including fertility and mating in adults for developmental toxicity in their subsequent progeny.