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Repeated dose toxicity: oral

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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:
09 April 2010 to 21 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study (OECD 422)
Justification for data waiving:
other:
Cross-reference
Reason / purpose for cross-reference:
reference to same study

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

Test material

Constituent 1
Reference substance name:
Oils, fish, oxidized, bisulfited, sodium salts
EC Number:
307-037-4
EC Name:
Oils, fish, oxidized, bisulfited, sodium salts
Cas Number:
97488-98-7
Molecular formula:
not aplicable
IUPAC Name:
Oils, fish, oxidized, bisulfited, sodium salts
Details on test material:
- Name of test material (as cited in study report): FLL sample 3
- Chemical name: Oils, fish, oxidized, bisulfited, sodium salts (triglycerides, C12 to C24, even, saturated and unsaturated, bisulfited, sodium salt)
- Physical state: viscous liquid
- Purity: 90%
- Impurities: 10% water content by Karl Fischer
- Lot/batch No.: LS06921
- Expiration date of the lot/batch: 13 October 2010
- Storage condition of test material: room temperature in the dark
- Other: amber color
- All other template details: Not reported

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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 310-371 g, females weighed 190-237 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: seven 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: 13 April 2010 To: 28 May 2010

Administration / exposure

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 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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of FLL Sample 3 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 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:
up to 8 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 150, 350, 500 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 37.5, 87.5, 250 mg/ml
Basis:
actual ingested
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

Examinations

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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
(even though not dosed via water)
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
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
Details on results:
CLINICAL SIGNS AND MORTALITY: Episodes of increased salivation and noisy respiration were detected in animals of either sex treated with 1000 and 350 mg/kg/day and in males treated with 150 mg/kg/day throughout the treatment period. One female treated with 1000 mg/kg/day also had an increased respiratory rate between Days 37 and 42. Generalised fur staining was evident in animals of either sex treated with 1000 and 350 mg/kg/day and in females treated with 150 mg/kg/day. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are not considered to be of toxicological importance.
Incidences of generalised fur loss were evident in one female treated with 1000 mg/kg/day and two females treated with 150 mg/kg/day. Two control females also had fur loss between Day 27 and Day 45. Observations of this nature are commonly observed in animals during the lactation phase and are not considered to be related to treatment.

BODY WEIGHT AND WEIGHT GAIN: no significant intergroup differences

FOOD EFFICIENCY, FOOD AND WATER CONSUMPTION: no treatment-related intergroup differences

HAEMATOLOGY: no treatment related effects detected in the haematological parameters measured

CLINICAL CHEMISTRY: There were no toxicologically significant effects detected in the blood chemical parameters measured. Males treated with 1000 mg/kg/day showed a statistically significant increase (P<0.05) in alanine aminotransferase. In the absence of any associated histology correlates the
intergroup difference was considered not to be of toxicological importance. Males treated with 1000 mg/kg/day showed a statistically significant increase (P<0.01) in bile acids. Males treated with 350 mg/kg/day also showed a statistically significant reduction (P<0.05) in sodium concentration. In the absence of any associated changes or a true dose related response the intergroup difference was considered not to be of toxicological significance.

NEUROBEHAVIOUR: There were no treatment-related changes in sensory reactivity. There were no treatment related changes in functional performance. Males treated with 1000 mg/kg/day showed a statistically significant increase in mean hind limb grip strength and a statistically significant reduction in overall activity. The intergroup difference in hind limb grip strength was confined to one out of the three tests and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, these findings were considered to be of no toxicological significance.

ORGAN WEIGHTS: No toxicologically significant effects were detected in the organ weights measured. Males from all treatment groups showed a statistically significant reduction in thyroid weight both absolute and relative to terminal body weight. In the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance.

GROSS PATHOLOGY: There were no toxicologically significant macroscopic abnormalities detected. One control male, one male treated with 1000 mg/kg/day and two males treated with 150 mg/kg/day had red lungs at necropsy. One male treated with 350 mg/kg/day had a mass present at necropsy. One control female and one female treated with 350 mg/kg/day showed epithelial sloughing of the glandular region of the stomach whilst a further female treated with 350 mg/kg/day showed epithelial sloughing of both the non glandular and glandular region of the stomach. In the absence of a true dose related response, any associated histology correlates and in view of the fact the observations were also seen in control animals the intergroup differences were considered not to be of toxicological importance. One female treated with 1000 mg/kg/day, one female treated with 150 mg/kg/day and two control females showed fur loss at necropsy. Observations of this nature are commonly observed following lactation and due to the observation also being present in control females the intergroup differences were considered unrelated to treatment.
The female treated with 1000 mg/kg/day that had a total litter loss showed pale adrenals, a misshapen diaphragm protruding into the thoracic cavity, the gastro intestinal tract spreading into the thoracic cavity, a misshapen right kidney, a small liver with a pale median lobe and missing caudate lobe and small, malformed and asymmetric lungs. Histopathological examination of this animal did not reveal any microscopic abnormalities.

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 or findings associated with the status post partum.

Effect levels

open allclose all
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
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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 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 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 selected males and females from each dose group.

 

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.

 

Clinical Observations. There were no clinical signs of toxicity detected.

 

Behavioural Assessment. There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests. There were no treatment-related changes in functional performance.

 

Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

 

Body Weight. No adverse effect on body weight change was detected for treated animals when compared with controls.

 

Food Consumption. No adverse effects on food consumption or food efficiency were detected for treated animals when compared with controls.

 

Water Consumption. No intergroup differences were detected.

 

Reproductive Performance:

Mating. There were no treatment-related effects on mating.

 

One female treated with 350 mg/kg/day and one female treated with 150 mg/kg/day failed to show any positive evidence of mating but subsequently gave birth to live young.

 

Fertility. There were no treatment-related effects in conception rates for animals of either sex treated with 1000, 350 or 150 mg/kg/day.

 

Gestation Lengths. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

 

Litter Responses:

Offspring Litter Size and Viability. Of the litters born, litter size at birth and subsequently on Day 1 and Day 4post partumwas comparable to controls.

 

Offspring Growth and Development. Offspring body weight gain and litter weights at birth and subsequently on Day 1 and Day 4post partumwas comparable to controls.

 

No adverse effects were detected in surface righting reflex on Day 1.

 

Laboratory Investigations:

Haematology. There were no toxicologically significant effects detected in the haematological parameters measured.

 

Blood Chemistry. There were no toxicologically significant effects detected in the blood chemical parameters measured.

 

Pathology:

Organ Weights. No toxicologically significant effects were detected in the organ weights measured.

 

Necropsy. No toxicologically significant macroscopic abnormalities were detected.

 

Histopathology. There were no treatment-related microscopic abnormalities detected.

 

Conclusion. The oral administration of FLL Sample 3 to rats by gavage, at dose levels of 150, 350 and 1000 mg/kg/day, did not result in any toxicologically significant effects.

 

The 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg/day.

 

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