Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March 2010 to 19 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March 2010 to 19 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: unspecified EC No. 440/2008 guidelines
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 12 weeks old
- Weight at study initiation: males weighed 304-375 g, females weighed 189-234 g
- Fasting period before study: not reported
- Housing: initially housed in groups of 5 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper with one male and one female. Following successful mating, males were returned to their original cages while mated females were housed individually during gestation and lactation in solid floor cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum access to a pelleted diet of Rodent 2018C Teklad Global Certified Diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2 degrees C
- Humidity (%): 55 +/- 15 %
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 14 April 2010 To: 29 May 2010
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
(400)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: formulations were prepared twice monthly and stored at approximately 4 degrees C in the dark

DIET PREPARATION: Not applicable, dosing was via oral gavage

VEHICLE
- Justification for use and choice of vehicle (if other than water): This vehicle was considered to be the most suitable vehicle to provide a homogeneous and stable formulation following a vehicle trial performed in the preliminary study. The formulations were stable for at least 22 days.
- Concentration in vehicle: 0, 37.5, 87.5, 250 mg/ml
- Amount of vehicle (if gavage): up to 4 ml/kg
- Lot/batch no. (if required): not reported
- Purity: not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of FLL Sample 4 in the test item formulations was determined by gel permeation chromatography (GPC) using an external standard technique. The standard and sample solutions were analysed by GPC using the following conditions:
HPLC System : Agilent Technologies 1050, incorporating autosampler and workstation
Column: PL Gel HTS-D (150 x 7.5 mm id)
Column temperature: 40 degrees C
Mobile phase : Tetrahydrofuran
Flow-rate : 1 ml/min
Detector : UV at 232 nm
Injection volume : 100 μl
Retention time : ~ 3.9 mins
The concentration of the test item measured (as % of nominal) ranged from 87 to 100 % of nominal concentrations of 37.5 to 250 mg/ml. The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
Duration of treatment / exposure:
up to 8 weeks
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
0 mg/mL
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
37.5 mg/mL
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Remarks:
87.5 mg/mL
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
250 mg/mL
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: a preliminary study was performed with three groups of 6 Wistar rats (3 male and 3 female), with the test item administered by oral gavage for fourteen consecutive days at dose levels of 250, 500, and 1000 mg/kg/day. In all of the dose levels, there was no mortality, no adverse effects on body weight change, no macroscopic abnormalities detected upon necropsy, and no adverse effects on dietary or water intake.
- Rationale for animal assignment (if not random): used a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable
Positive control:
Not used in this study
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week
- Cage side observations included: overt signs of toxicity, ill-health and behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of treatment and at weekly intervals thereafter

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 mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, although the test item was not dosed in the water
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data, The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, although the test item was not dosed in the water
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 42 for males and day 4 post partum for females
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 5 males and 5 females from each test and control group (total of 40 animals)
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 42 for males and day 4 post partum for females
- Animals fasted: No
- How many animals: 5 males and 5 females from each test and control group (total of 40 animals)
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the 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.
- Dose groups that were examined: all dose groups
- Battery of functions tested: Table 3

OTHER: None
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, a full external and internal examination, and any macroscopic abnormalities were recorded. Also, organ weights were obtained for: adrenals, brain, epididymides, prostate, seminal vesicles, spleen, testes, heart, kidneys, liver, ovaries, pituitary, thymus, thyroid, uterus
HISTOPATHOLOGY: Yes (see table 4)
Other examinations:
Not applicable
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
Data for males and females prior to pairing, and functional performance test data, and where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The transformed data were analysed 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 showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data for females during gestation and lactation, and offspring 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 of variance. Where variances were shown to be homogenous, 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.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations included post-dose increased salivation, noisy respiration, and were considered unrelated to test item toxicity.

Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male treated with 350 mg/kg/day was found dead on Day 25, but this death was confirmed to be attributed to inappropriate dosing technique and not related to test material toxicity.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences in body weight change were evident for females during the pre-mating, gestation or lactation phases of the study. No adverse effects on body weight change were detected for treated males when compared to controls during the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
(even though not dosed via food)
Food efficiency:
no effects observed
Description (incidence and severity):
No treatment-related intergroup differences.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No treatment-related intergroup differences (even though not dosed via water).
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment related effects detected in the haematological parameters measured



Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters measured. Females treated with 1000 and 350 mg/kg/day showed a statistically significant reduction in lymphocyte counts, when compared to control values. The significance achieved was minimal (P<0.05), and all individual values for these animals were within the normally expected ranges. These minor reductions were therefore considered to be attributable to one higher than expected control value, and were unrelated to treatment with the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in sensory reactivity or functional performance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected in the organ weights measured. Males treated at all dose levels showed statistically significant increases in absolute and body weight-relative pituitary weights when compared to controls. In the absence of a convincing dose-related response or any histopathological changes to suggest an effect of treatment, these increases were considered not to represent test item toxicity.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant macroscopic abnormalities detected. The interim death male treated at 350 mg/kg/day showed reddened lungs and a fluidfilled thoracic cavity. These findings are associated with inappropriate administration of the test item resulting in death, and are unrelated to test item toxicity.
The remaining macroscopic findings consisted of reddened lungs for one female treated with 350 mg/kg/day and an irregular surface edge of the liver was observed for another female treated with 350 mg/kg/day. Hydronephrosis of the right kidney was evident for one male treated with 150 mg/kg/day. These findings were isolated to one animal on each occasion and in the absence of any histopathological correlations, were considered to have arisen incidentally, with no relation to treatment with the test item. Epithelial sloughing of the glandular region of the stomach, hydronephrosis of the left kidney and ovaries encased in a fluid-filled sac were observed in the female control group. These findings are occasionally observed in repeated dose studies of this type, and in the absence of treatment in this group, the findings were purely incidental in nature.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment related microscopic abnormalities detected in terminal kill animals. Marked necrosis of the trachea was evident for the interim death male. The cause of death for this animal was therefore confirmed to be attributable to inappropriate dosing technique and was unrelated to treatment. All other findings noted in this study were considered to be incidental findings commonly noted in rats of this strain and age or findings associated with the status post partum.
Histopathological findings: neoplastic:
no effects observed
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:
other: overall effects including: clinical signs; mortality; body weight; food consumption; food efficiency; water consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology
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:
other: reproduction
Key result
Critical effects observed:
no

Reproductive Performance: there were no treatment-related effects on any observed reproductive endpoint.

Conclusions:
No effects were seen at the highest concentration tested, therefore the 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity and reproductive toxicity was considered to be 1000 mg/kg/day.
Executive summary:

Study Summary:

Introduction. The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and meets the requirements 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 meet the requirements of Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods 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).

 

Methods. The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:HsdHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 150, 350 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study.

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 dose group on Day 4post partum. Haematology and blood chemistry were evaluated prior to termination on five males and five females from each dose group (due to a technical error, only four males were assessed from the 350 mg/kg/day dose group).

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

 

Results.

Adult Responses:

Mortality. There were no unscheduled deaths attributable to test item toxicity.

 Clinical Observations. No clinically observable signs of toxicity were detected.

 Functional Performance Tests. No treatment-related effects were detected.

 Body Weight. No adverse effects on body weight change were detected.

 Food Consumption. No adverse effects on dietary intake were detected.

 Water Consumption. Daily visual inspection of water bottles did not reveal any significant intergroup differences.

 Reproductive Performance:

 Mating. No treatment-related effects were detected in mating performance.

 Fertility. No treatment-related effects on fertility were detected.

 Gestation Lengths. No treatment-related effects were detected in gestation lengths.

 Litter Responses:

Offspring Litter Size and Viability. No treatment-related effects were detected.

 Offspring Growth and Development. No treatment-related effects were detected.

 Laboratory Investigations:

Haematology. No treatment-related effects were detected.

 Blood Chemistry. No toxicologically significant intergroup differences were detected.

Pathology:

Organ Weights. No toxicologically significant effects of treatment were detected.

 Necropsy. No treatment-related macroscopic abnormalities were detected.

 Histopathology. No treatment-related microscopic abnormalities were detected.

 

Conclusion. The oral administration of FLL Sample 4 to rats by gavage at a maximum dose level of 1000 mg/kg/day did not result in any toxicologically significant effects of treatment. The minor effects detected in this study were not considered to represent an adverse effect of treatment; therefore a ‘No Observed Adverse Effect Level’ (NOAEL) was established at 1000 mg/kg/day for systemic toxicity.

No treatment-related effects were detected for reproductive performance, hence a ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: other guideline: unspecified EC No. 440/2008 guidelines
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): FLL sample 4
- Physical state: viscous liquid
- Lot/batch No.: 240210
- Expiration date of the lot/batch: not reported
- Storage condition of test material: room temperature in the dark
- Other: yellow/brown color
- All other template details: Not reported

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 12 weeks old
- Weight at study initiation: males weighed 304-375 g, females weighed 189-234 g
- Fasting period before study: not reported
- Housing: initially housed in groups of 5 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper with one male and one female. Following successful mating, males were returned to their original cages while mated females were housed individually during gestation and lactation in solid floor cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum access to a pelleted diet of Rodent 2018C Teklad Global Certified Diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITION
S- Temperature (°C): 21 +/- 2 degrees C
- Humidity (%): 55 +/- 15 %- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 14 April 2010 To: 29 May 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
(400)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: formulations were prepared twice monthly and stored at approximately 4 degrees C in the dark

DIET PREPARATION: Not applicable, dosing was via oral gavage

VEHICLE
- Justification for use and choice of vehicle (if other than water): This vehicle was considered to be the most suitable vehicle to provide a homogeneous and stable formulation following a vehicle trial performed in the preliminary study. The formulations were stable for at least 21 days.
- Concentration in vehicle: 0, 37.5, 87.5, 250 mg/ml
- Amount of vehicle (if gavage): up to 4 ml/kg
- Lot/batch no. (if required): not reported
- Purity: not reported :
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of successful mating
- Proof of pregnancy: not reported
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not applicable
- Further matings after two unsuccessful attempts: not applicable, only one male:female mating attempt
- After successful mating each pregnant female was caged (how): individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Any other deviations from standard protocol: not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of FLL Sample 4 in the test item formulations was determined by gel permeation chromatography (GPC) using an external standard technique. The standard and sample solutions were analysed by GPC using the following conditions: HPLC System : Agilent Technologies 1050, incorporating autosampler and workstation; Column: PL Gel HTS-D (150 x 7.5 mm id); Column temperature: 40 degrees C; Mobile phase : Tetrahydrofuran; Flow-rate : 1 ml/minDetector : UV at 230 nm; Injection volume : 100 μl; Retention time : ~ 3.9 mins.
The concentration of the test item measured (as % of nominal) ranged from 88 to 104 % of nominal concentrations of 37.5 to 250 mg/ml. The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
Duration of treatment / exposure:
males: 42 days
females: 42-47 days
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: a preliminary study was performed with three groups of 6 Wistar rats (3 male and 3 female), with the test item administered by oral gavage for fourteen consecutive days at dose levels of 250, 500, and 1000 mg/kg/day. In all of the dose levels, there was no mortality, no adverse effects on body weight change, no macroscopic abnormalities detected upon necropsy, and no adverse effects on dietary or water intake.
- Rationale for animal assignment (if not random): used a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups
Positive control:
Not reported

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week
- Cage side observations included: overt signs of toxicity, ill-health and behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of treatment and at weekly intervals thereafter

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 mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, although the test item was not dosed in the water
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data, The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, although the test item was not dosed in the water
- Time schedule for examinations: daily
Oestrous cyclicity (parental animals):
Not reported
Sperm parameters (parental animals):
Parameters examined in all males: testis weight, epididymides weight, seminal vesicles and prostate weights
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, haematological and blood chemicals measured

GROSS EXAMINATION OF DEAD PUPS: not applicable, no pups died before the end of the study
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed following completion of female gestation and lactation phases.
- Maternal animals: All surviving animals were sacrificed on Day 5 post partum.

GROSS NECROPSY
- Gross necropsy consisted of full external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
See Table 4 for tissues removed and examined. Organ weights were obtained for adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary (post-fixation), protate, seminal vesicles, spleen, testes, thymus, thyroid, uterus.
Postmortem examinations (offspring):
SACRIFICE
- All offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of full external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
See Table 4 for tissues removed and examined. Organ weights were obtained for adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary (post-fixation), protate, seminal vesicles, spleen, testes, thymus, thyroid, uterus.
Statistics:
Data for females during gestation and lactation, and offspring 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 of variance. Where variances were shown to be homogenous, 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.
Reproductive indices:
Fertility indices (%): mating index and pregnancy index
Parturition index (%), implantation loss (%), live birth indes (%), viability index (%), sex ratio (% males)
Offspring viability indices:
= number of offspring alive on Day 4 / number of offspring alive on Day 1 x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations included post-dose increased salivation, noisy respiration, and were considered unrelated to test item toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male treated with 350 mg/kg/day was found dead on Day 25, but this death was confirmed to be attributed to inappropriate dosing technique and not related to test material toxicity.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
o differences in body weight change were evident for females during the pre-mating, gestation or lactation phases of the study. No adverse effects on body weight change were detected for treated males when compared to controls during the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related intergroup differences
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No treatment-related intergroup differences
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment related effects detected in the haematological parameters measured.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters measured. Females treated with 1000 and 350 mg/kg/day showed a statistically significant reduction in lymphocyte counts, when compared to control values. The significance achieved was minimal (P<0.05), and all individual values for these animals were within the normally expected ranges. These minor reductions were therefore considered to be attributable to one higher than expected control value, and were unrelated to treatment with the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
NEUROBEHAVIOUR: There were no treatment-related changes in sensory reactivity or functional performance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment related microscopic abnormalities detected in terminal kill animals. Marked necrosis of the trachea was evident for the interim death male. The cause of death for this animal was therefore confirmed to be attributable to inappropriate dosing technique and was unrelated to treatment. All other findings noted in this study were considered to be incidental findings commonly noted in rats of this strain and age or findings associated with the status post partum.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): no significant treatment-related differences in mating performance, fertility, gestation length, litter size or viability. There was one non-pregnant female from the control group.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects, no treatment-related effects were observed for any of the reproductive endpoints measured

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
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):
Necropsy
No treatment-related macroscopic abnormalities were detected for the one interim death offspring from the control group, or for remaining offspring at termination on Day 5 post partum. The incidental findings observed in the terminal kill offspring (small size, hydronephrosis of the kidneys, wound to the head) were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.
Histopathological findings:
no effects observed
Description (incidence and severity):
Histopathology
There were no treatment related microscopic abnormalities detected in terminal kill animals.
All findings noted in this study were considered to be incidental findings commonly noted in rats of this strain and age and were unrelated to treatment.
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Offspring Litter Size and Viability
No treatment-related differences in corpora lutea, implantation sites, or pre- and postimplantation losses were detected for females from treated groups when compared to controls. No differences in litter size or viability were evident for offspring from treated litters when compared to those from the controls. Statistical analysis of these data did not result in any significant intergroup differences.

Offspring Growth and Development
There were no differences in litter weights or mean offspring body weights between control and treated animals.
No treatment-related effects in litter weights, offspring weights or surface righting were detected for litters from treated groups when compared to control litters. Statistical analysis of these data did not result in any significant intergroup differences.
The number of interim offspring found dead or missing (cannabalised by the mother) was essentially similar throughout the control and treatment groups. No treatment-related clinical signs were observed. The clinical signs observed were low incidence findings commonly observed in reproductive studies of this type and unrelated to test item toxicity.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at the highest tested dose.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

Conclusions:
Based on a read-across from ana anlogue substance the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.
Executive summary:

Study Summary:

Introduction. The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and meets the requirements 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 meet the requirements of Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods 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).

 

Methods. The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:HsdHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 150, 350 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study.

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 dose group on Day 4post partum. Haematology and blood chemistry were evaluated prior to termination on five males and five females from each dose group (due to a technical error, only four males were assessed from the 350 mg/kg/day dose group).

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

 

Results.

Adult Responses:

Mortality. There were no unscheduled deaths attributable to test item toxicity.

 

Clinical Observations. No clinically observable signs of toxicity were detected.

 

Functional Performance Tests. No treatment-related effects were detected.

 

Body Weight. No adverse effects on body weight change were detected.

 

Food Consumption. No adverse effects on dietary intake were detected.

 

Water Consumption. Daily visual inspection of water bottles did not reveal any significant intergroup differences.

 

Reproductive Performance:

Mating. No treatment-related effects were detected in mating performance.

 

Fertility. No treatment-related effects on fertility were detected.

 

Gestation Lengths. No treatment-related effects were detected in gestation lengths.

 

Litter Responses:

 

Offspring Litter Size and Viability. No treatment-related effects were detected.

 

Offspring Growth and Development. No treatment-related effects were detected.

 

Laboratory Investigations:

Haematology. No treatment-related effects were detected.

 

Blood Chemistry. No toxicologically significant intergroup differences were detected.

 

Pathology:

Organ Weights. No toxicologically significant effects of treatment were detected.

 

Necropsy. No treatment-related macroscopic abnormalities were detected.

 

Histopathology. No treatment-related microscopic abnormalities were detected.

 

Conclusion. The oral administration of FLL Sample 4 to rats by gavage at a maximum dose level of 1000 mg/kg/day did not result in any toxicologically significant effects of treatment. The minor effects detected in this study were not considered to represent an adverse effect of treatment; therefore a ‘No Observed Adverse Effect Level’ (NOAEL) was established at 1000 mg/kg/day for systemic toxicity.

 

No treatment-related effects were detected for reproductive performance, hence a ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.