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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 24 April 2013 and 02 December 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Test Guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Di Lauroyl Peroxide
IUPAC Name:
Di Lauroyl Peroxide
Constituent 2
Chemical structure
Reference substance name:
Dilauroyl peroxide
EC Number:
203-326-3
EC Name:
Dilauroyl peroxide
Cas Number:
105-74-8
Molecular formula:
C24H46O4
IUPAC Name:
dodecanoyl dodecaneperoxoate
Test material form:
solid: flakes
Details on test material:
Identification: Di Lauroyl Peroxide (CAS# 105-74-8)
Description: White Solid Flakes
Chemical Name: Dilauroyl peroxide
Purity: 98.9%
Batch Number: 0905530079
Label: Laurox 0905530079 2KG
Date Received: 19 Febuary 2013
Storage Conditions: Stored in darkness and frozen at -20°C
Expiry Date: 01 June 2019

No correction for purity was made.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for six days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 177 to 210 g, the females weighed 134 to 163 g, and were approximately six to eight weeks old.

The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly temperatures and humidities are included in the study records. The temperature and relative humidity controls were set to achieve target values of 21 ± 2 °C and 55 ± 15 % respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.

The animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Test item preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Corn oil. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd. Analytical Services. Results from this study showed the formulations to be stable for at least twenty five days. Formulations were therefore prepared fortnightly and stored at approximately 4 °C in the dark.

Procedure
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of Corn oil.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
MATERIALS AND METHODS
Introduction
The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique.

Test item
The test item described in the main part of the study was also used as the analytical standard.

Analytical procedure
Preparation of standard solutions
Stock solutions of test item in tetrahydrofuran were prepared for external standard calibration. An aliquot, 100 mg of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with tetrahydrofuran to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in tetrahydofuran with a concentration of 0.1 mg/L.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were diluted with tetrahydrofuran. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with tetrahydrofuran this was then shaken to dissolve. Where necessary, sample solutions were further diluted with tetrahydrofuran to achieve the working concentration.

Preparation of accuracy samples
Samples of corm oil were accurately fortified with known amounts of test item equivalent to lowest and highest anticipated dose concentrations. These samples were then prepared for analysis in the same manner as the test samples.

Preparation of linearity standards
A range of standard solutions were prepared in tetrahydrofuran from a stock solution of 1 mg/mL by serial dilution covering the concentration range 0 to 0.1584 mg/mL.

Instrument setup
GC system: Agilent Technologies 5890, incorporating autosampler and workstation.
Column: DB-1 (30 m x 0.25 mm id x 0.25 µm film).
Oven temperature program: Oven: 60 °C for 0 minutes, with 10 °C/minute to 140 °C, for 0 minutes then 50 °C/minute to 300 °C, for 5 minutes.
Injection temperature: 250 °C
Flame ionisation detector temperature: 250 °C
Injection volume: 1 µL
Retention time: ~ 9 mins

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.


Results
Validation of analytical method
Specificity
The control dose samples and a solvent blank did not significantly affect the chromatogram at the retention time of the test item whose area changed accordingly with known concentration.

Linearity
The linearity of the analytical system used for sample analysis was demonstrated with a good relationship between peak areas measured and working standard concentrations. The regression coefficients calculated were found to be 0.999.

Accuracy
The fortified samples of corn oil were found to have a recovery value of ± 11 % of the fortification.

Test item formulations dietary admixtures
The formulations investigated during the study were found to comprise test item in the range of 91 % to 113 % and thus, the required content limit of ± 15 % with reference to the nominal content was met.
In addition, the test item was found to be stable in the formulation when kept 25 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time-zero mean.
In conclusion, the results indicate the accurate use of the test item and corn oil as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
The test item peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of test item and, therefore, the absence of the test item in the vehicle control samples was confirmed.

Discussion
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven suitable for use.
Duration of treatment / exposure:
Ninety consecutive days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

BODY WEIGHT: Yes
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION: Yes
Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5 % Tropicamide solution (Mydriacyl® 0.5 %, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

HAEMATOLOGY: Yes
Haematological investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices:
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count:
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).


CLINICAL CHEMISTRY: Yes
Blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)


URINALYSIS: Yes
Urinalytical investigations were performed on all test and control group animals during the final week of the study. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.
The following parameters were measured on collected urine:
Volume, Ketones, Specific Gravity, Bilirubin, pH, Urobilinogen, Protein, Blood, Glucose.


NEUROBEHAVIOURAL EXAMINATION:Yes
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Behavioural Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.
This test was developed from the methods used by Irwin (1968) and Moser et al (1988).

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20 % of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).

The following parameters were observed:
Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

OTHER:
Estrous cycle
The stage of estrus were classified according to the following criteria:

Di-estrus (D): Predominantly leucocytes present although some epithelial and cornified cells can be seen.
Proestrus (P): Predominantly epithelial cells, usually in significant numbers.
Early Estrus (E1): Predominantly cornified cells, usually seen as small groups or isolated cells.
Late Estrus (E2): Predominantly cornified cells usually seen as clumps of cells.
Metestrus (M): Large numbers of leucocytes with discrete clumps of cornified cells.

The estrous cycles are classified according to the following criteria:

Normal estrus: The pattern of daily stages of estrus show a four to five day cycle, which is generally repeated over 21 days.
Extended estrus: The observation of a predominance of epithelial/cornified cells for more than two days for more than one estrous cycle.
Extended di-estrus: The predominant cell type is the leucocyte for more than three consecutive days over more than one estrous cycle.
Irregular cycle: An irregular length of estrous cycle is observed over the 21 day evaluation period.
Acyclic: No evidence of an estrous cycle is observed over the 21 day evaluation period.
Sacrifice and pathology:
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver.


Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10 % formalin, except where stated:

Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)•
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Rectum
Caecum
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides ♦
Skin (hind limb)
Eyes*
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including Peyer’s patches)
Stomach
Jejunum
Testes ♦
Kidneys
Thymus
Liver
Thyroid/Parathyroid
Lungs (with bronchi) #
Tongue•
Lymph nodes (mandibular and mesenteric)
Trachea
Mammary glands
Urinary bladder
Muscle (skeletal)•
Uterus
Oesophagus

• Retained only and not processed
♦ Preserved in Bouin’s fluid and then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later
* Eyes fixed in Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues from control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Other examinations:
Semen Analysis
At necropsy of adult males the following evaluations were performed:
i) The left testis and epididymis were removed, dissected from connective tissue and weighed separately.
ii) For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approximately -20 ºC.
iii) For the epididymis the distal region was incised and a sample of the luminal fluid collected and transferred to a buffer solution for analysis of sperm motility. Approximately 200 individual sperm were assessed using an automated semen analyser, to determine the number of motile, progressively motile and non-motile sperm. The characteristics of motile sperm were also identified using the computer assisted sperm analyser (Hamilton-Thorne TOX IVOS system).
iv) The cauda epididymis was separated from the body of the epididymis, and then weighed. The cauda epididymis was then frozen at approximately -20 ºC.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight Change, Haematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any 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 shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the study.

There were no adverse clinical signs of toxicity detected.
Animals of either sex treated with 1000 mg/kg bw/day showed increased salivation from Day 2 onwards. Two males and two females treated with 300 mg/kg bw/day and one male treated with 100 mg/kg bw/day also showed isolated incidents of increased salivation. Observations of this nature are commonly observed following the oral administration of an unpalatable test item formulation and in isolation are not indicative of systemic toxicity.


BODY WEIGHT AND WEIGHT GAIN
No adverse effects were detected in body weight gain in treated animals when compared to controls.

Females from all treatment groups showed a statistically significant increase in body weight gain during Week 3 and Week 7. An increase in body weight gain is not considered to represent an adverse effect of treatment therefore the intergroup differences were considered not to be of toxicological importance. Females treated with 1000 and 300 mg/kg bw/day showed a reduction in body weight gain during Week 12. Overall body weight gain for these females was higher than control females therefore this slight reduction was considered not to be of toxicological significance.


FOOD CONSUMPTION
No adverse effect in overall food consumption or food efficiency was detected in treated animals when compared to controls.

WATER CONSUMPTION
There were no treatment related effects detected in water consumption.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment related ocular effects detected.

HAEMATOLOGY
There were no toxicologically significant effects detected in the haematological parameters examined.

Males treated with 1000 mg/kg bw/day showed a statistically significant increase in neutrophil count. Females from this treatment group showed a statistically significant reduction in mean corpuscular haemoglobin and mean corpuscular heamoglobin concentration. The effect on mean corpuscular haemoglobin also extended to females treated with 300 mg/kg bw/day. The majority of individual values were within normal background ranges for these parameters and in the absence of any associated changes the intergroup differences were considered not to be of toxicological importance.


CLINICAL CHEMISTRY
There were no toxicologically significant effects detected in the blood chemical parameters examined.

Males treated with 1000 mg/kg bw/day showed a statistically significant increase in chloride concentration and a statistically significant reduction in phosphorus, cholesterol and bilirubin. Females from this treatment group showed a statistically significant reduction in calcium concentration. The majority of individual values were within normal background ranges for these parameters and in the absence of any associated histopathological changes the intergroup differences were considered not to be of toxicological importance. Females treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in alanine aminotransferase. Females treated with 100 mg/kg bw/day showed a statistically significant increase in alkaline phosphatase. In the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance.


URINALYSIS
No toxicologically significant effects were detected in the urinalytical parameters measured.

Males treated with 1000 mg/kg bw/day showed a statistically significant increase in urine volume and a subsequent reduction in urine specific gravity. In the absence of any associated renal histopathology changes the intergroup difference was considered not to represent an adverse effect of treatment.

Females treated with 300 mg/kg bw/day showed a statistically significant increase in urine volume and a statistically significant reduction in specific gravity. Females treated 100 mg/kg bw/day also showed a statistically significant reduction in specific gravity. In the absence of a true dose related response or any renal histopathology changes the intergroup differences were considered not to be of toxicological significance.


NEUROBEHAVIOUR
Behavioural Assessments
There were no treatment-related changes in behavioural parameters measured.

Functional Performance Tests
There were no treatment-related changes in functional performance.
Statistical analysis of the data did not reveal any significant intergroup differences.

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

ORGAN WEIGHTS
No toxicologically significant effects were detected in the organ weights examined.

Females treated with 300 mg/kg bw/day showed a statistically significant increase in uterus/cervix weight both absolute and relative to terminal body weight. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.


GROSS PATHOLOGY
There were no toxicologically significant macroscopic abnormalities detected.

One male treated with 1000 mg/kg bw/day had a small and flaccid left testis and a small left epididymis. In the absence of any associated treatment related histopathology correlates the intergroup difference was considered not to be of toxicological importance. One control male had a fluid filled and increased renal pelvic space in the right kidney. This observation is considered to be a congenital abnormality and in the absence of treatment is considered to be incidental.


HISTOPATHOLOGY:
No treatment related microscopic abnormalities were detected.

An increase in vacuolation in the payer’s patches was detected in animals of either sex treated with 1000 mg/kg bw/day. Observations of this nature are not considered to represent a true adverse effect of treatment and can be observed following the ingestation of diets high in fatty acids.


OTHER FINDINGS
Estrous Cycle Assessments
There were no treatment-related effects on female estrous cycles or on the type or proportion of females with anomalous estrous cycles.

Sperm Analysis
There were no treatment-related effects on the concentration or motility of samples of epididymal sperm.

Effect levels

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: No toxicologically significant effects were noted

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Key to scoring system and explanation for behavioural assessments and sensory reactivity tests

 

NEUROLOGIC

These parameters are the first to be evaluated when the animal is placed in the arena:

 

Transfer Arousal (Time to Movement) and Locomotion

-2 = animal stays in one place

-1 = slow, deliberate movement

0 = moderate, undisturbed investigation of environment

1 = active progression around arena

2 = darting movements

 

Tail Elevation

0 = flattened

1 = rigidly horizontally extended

2 = rigidly diagonally elevated

3 = rigidly vertical

4 = diagonally retrograde (past vertical)

 

GAIT AND CO-ORDINATION

Tiptoe

Hindlimbs raised on tiptoe during movement

High stepping

Forelimbs lifted high during movement

Spastic

Shuffling of limbs

Waddling

Lateral movements

Dysmetric

Unco-ordinated movement with tremors

Splayed

Thighs splayed outwards, animal appears crouched

Scissor

Forelimbs cross over when extended


Ataxic

Lack of co-ordinated movement of trunk, pelvis and limbs.

Observation graded:  Slight
                                  Moderate (does not fall)
                                  Extreme (falls repeatedly)

 

 

CNS EXCITATION

Tremors

These are general involuntary movements of the muscles

Exertion

only occurs during movement

Rest

only occurs when animal is resting

Whole body

 

Head only

 

Body only

 

Tail only

 

 

When present, any observation is also graded:

Slight
Moderate
Extreme

 

Twitches

Identified as brief, coarse jerks of body or limbs - observation graded:

Slight

Moderate

Extreme

 

Convulsions

Clonic

Convulsions with alternate contraction and relaxation of muscles

Running excitement

Often accompanied by clonic convulsions

Champing

Clonus of the paws only

Hopping

Animal repeatedly “hops” into the air

Asphyxeal

Terminal convulsion, results in death from respiratory failure

Tonic

Seizure in which the head, body and limbs are arched backwards

Emprosthotonus

Seizure where the head, body and limbs are arched forwards

 

 

Bizarre Behaviour

Any bizarre behaviour is recorded using a unique code.

 

AUTONOMIC

Salivation*

Excessive visible wetness around the mouth

Pilo-erection*

Fur stands up - marked cases are described as “puff ball” appearance

Exophthalmia

Bulging eyes

Lachrymation

Tear staining (clear fluid)
Chromodacryorrhea (red/pink fluid)

Hyperthermia

 

Hypothermia

 

Skin colour**

Blue (cyanotic)
Redness of extremities
Skin pallor

Respiration

Decreased respiratory rate

 

Gasping

 

Increased rate

 

Laboured

 

Noisy

Palpebral closure

Degree of eyelid closure e.g. ptosis - observation is graded:

Slight

Moderate

Severe

Urination and defecation

During the study this can only be observed as urogenital wetness and/or diarrhoea. In the open arena, the number of occasions that the animal defecates/urinates is recorded

  

MANIPULATIVE TESTS

Performed in the open arena:

 

Grasp Response

The animal is grasped around the body and its reaction is scored:

0 = no response

1 = animal struggles slightly but becomes passive

2 = animal repeatedly struggles with/without vocalisation

3 = animal struggles violently with/without vocalisation

4 = animal attempts to bite operator

 

Vocalisation

The number of vocalisations exhibited by the animal during handling.

 

Toe Pinch

On handling one toe of a hindfoot is lightly pressed with a pair of forceps:

-3 = no response

-2 = animal shows awareness of actions

-1 = withdrawal

0 = rapid withdrawal with single vocalisation

1 = rapid withdrawal with multiple vocalizations

 

Tail Pinch

Whilst animal is in the arena, the end of the tail is lightly pressed with forceps:

-2 = no response

-1 = animal freezes/shows awareness of grasping

0 = animal attempts to escape/struggles with none or single vocalisation

1 = animal struggles violently with multiple vocalisations

 

Finger Approach

Whilst animal is in the arena the operator moves one finger towards the animal:

-1 = animal is oblivious to approach

0 = animal is aware of finger but is unmoved

1 = head sways from side to side

2 = moves towards finger

3 = investigates finger by moving onto it

 

Touch Escape

Whilst animal is in the arena the operator runs his finger down the body of the animal:

-2 = no response

-1 = animal twitches ears, investigates finger

0 = animal crouches

1 = animal moves away but is unhurried

2 = animal moves away as if startled

 

Pupil Reflex

The animal is placed in a darkened area to allow the pupils to dilate. A light beam is shone into each eye separately and the pupil is observed for immediate constriction.

The response recorded:

-2 = no response

-1 = sluggish

0 = normal

 

Blink Reflex (Palpebral Closure)

The operator attempts to touch the eye of the rat, the response is recorded:

-2 = no response

-1 = sluggish

0 = normal blink reflex

1 = rapid or repeated eye blink

 

Startle Reflex

A sharp sudden noise is produced by a noise generator or finger snap or similar. Each animal is held lightly but firmly facing the origin of the noise. The response is recorded:

-1 = no response

0 = normal

1 = hyper-responsive

*   When present, observation is graded slight or extreme.

**  When present, observation is graded slight, moderate, or extreme.

Applicant's summary and conclusion

Conclusions:
The oral administration of Di Lauroyl Peroxide (CAS# 105-74-8) to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

 

i)       The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

ii)     The EU Annex B Method B26 - “Subchronic Oral Toxicity Test - Repeated Dose 90-Day Oral Toxicity Study in Rodents” - updated 21 August 2001.

iii)   The United States Environmental Protection Agency (EPA), Health Effects Test Guidelines, OPPTS 870.3100 - 90 Day Oral Toxicity in Rodents.

 

This study was also designed to be compatible with 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™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Corn oil).

 

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals.

 

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

 

Results…….

Mortality

There were no unscheduled deaths during the study.

 

Clinical Observations

There were no clinical signs of toxicity detected.

 

Behavioural Assessment

There were no treatment-related changes in 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 effects were detected in body weight gain in treated animals when compared to controls.

 

Food Consumption

No adverse effect in overall food consumption or food efficiency was detected in treated animals when compared to controls.

 

Water Consumption

There were no treatment related effects detected in water consumption.

 

Ophthalmoscopy

There were no treatment related ocular effects detected.

 

Estrous Cycle Assessment

There were no treatment-related effects on female estrous cycles or on the type or proportion of females with anomalous estrous cycles.

 

Haematology

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

 

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.

 

Urinalysis

No treatment-related effects were detected in the urinalytical parameters measured.

 

Necropsy

There were no toxicologically significant macroscopic abnormalities detected.

Organ Weights

No toxicologically significant effects were detected in the organ weights examined.

 

Histopathology

No treatment related microscopic abnormalities were detected.

Sperm Analysis

There were no treatment-related effects on the concentration or motility of samples of epididymal sperm.

 

Conclusion

The oral administration of Di Lauroyl Peroxide (CAS# 105-74-8) to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore considered to be 1000 mg/kg bw/day.

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