Dilauroyl peroxide

Endpoint summary

Description of key information

Based on the results of an apparently well conducted GLP 90-day oral gavage study, the NOAEL of dilauroyl peroxide is 1000 mg/kg/day, the highest dose tested.  There were no mortalities and no effects on any parameters evaluated.    

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

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

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.

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

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

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)

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.

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

Critical effects observed:

not specified

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.

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Rat, RccHanTM: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals

- Source:
RccHanTM: WIST(SPF) strain rats from Harlan Laboratories B.V. Kreuzelweg 53 5961 NM Horst / Netherlands

- Age at study initiation:
Approximately six to eight weeks old

- Weight at study initiation:
224 to 369g (male), 166 to 226g (female)

- Fasting period before study:
Not applicable

- Housing:
The animals were housed in groups of five in clear plastic cages with wire mesh tops and standard softwood bedding.

- Diet:
A pelleted diet Harlan Teklad 2914 C batch no. 82/09) rodent maintenance diet was available (ad libitum). The diet was considered not to have any level of contaminant.

- Water (e.g. ad libitum):
ad libitum mains water was supplied from polycarbonate bottles attached to the cage. The drinking water was considered not to have any level of contaminant.

- Acclimation period:
Seven days

ENVIRONMENTAL CONDITIONS

- Temperature:
: (°C): 22 ± 3

- Humidity (%):
: 30 - 70%

- Air changes (per hr):
: 10 – 15 air changes per hour

- Photoperiod (hr dark / hrs light):
: 12 hours continuous light and 12 hours darkness with background music played during the light period

IN-LIFE DATES:
The in-life phase of the study was conducted between 25 February 2010 (Day 1 of treatment) and 25 March 2010 (Day 29, day of necropsy).

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Method of administration:
Oral Gavage

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations in corn oil.

DIET PREPARATION
- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable.

- Storage temperature of food:
Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable

- Concentration in vehicle:
Group 1: 0 mg/mL/day
Group 2: 20 mg/mL/day
Group 3: 60 mg/mL/day
Group 4: 200 mg/mL/day

- Amount of vehicle (if gavage):
5 ml/kg bodyweight

- Vehicle Lot/Batch no.:
409110276

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories study C69855), the stability of the test item formulations was considered to require daily preparation. The test item formulations in this study (C69866) were thereforeprepared daily at the appropriate concentrations in corn oil. The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

Dilauroyl peroxide (CAS# 105-74-8) was weighed into a glass beaker on a tared balance. A small amount of vehicle was added and the remaining vehicle was added. The mixtures were stirred using a magnetic stirrer and used at room temperature (20 ± 5 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

After experimental start, duplicate samples of the control group as well as three samples (top,
middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hours).

The samples were delivered to the analytical department (Harlan Laboratories Ltd., Analytics, Itingen / Switzerland) at ambient temperatures and stored there at -20 ± 5 °C until analysis.

The test item was used as analytical standard.

Dose formulation samples (primary samples or duplicates) were discarded upon written confirmation by the study director after acceptance of the draft report.

The results of analysis are summarized in the attached Appendix 4.

Duration of treatment / exposure:

The test item was administered daily, for twenty-eight consecutive days, by oral gavage to all rats. Control animals were treated in an identical manner with 5 ml/kg/day of corn oil.

Frequency of treatment:

Dosing regime: 7 days/week

Remarks:

Doses / Concentrations:
dose levels of 100, 300 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Group 1: 5 males and 10 females (Control)
Group 2: 5 males and 5 females
Group 3: 5 males and 5 females
Group 4: 5 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on available toxicity information and compliance with regulatory hazard classification.

- Rationale for animal assignment (if not random):
Random.

- Rationale for selecting satellite groups:
Not applicable

- Section schedule rationale (if not random):
Not applicable

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Daily Observations
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change once daily before commencement of administration as well as daily on days 1 - 28 (twice daily during days 1 - 4) during the treatment period.

- please see attached group summary and individual clinical signs data.

NEUROBEHAVIOURAL EXAMINATIONS: Yes see attached summary and individual data.

Behavioral Observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.

Functional Observational Battery (Screen) were performed in all animals during week 4 using a modified Irwin screen test.

Grip Strength were performed in all animals during week 4. Assessment was based on Fore and hind limb grip strength measurements.

Locomotor Activity were performed in all animals during week 4. Assessment was based on decreased or increased locomotor activity measured.

BODY WEIGHT: Yes
- Time schedule for examinations:
Body weights were recorded weekly during acclimatization and treatment periods and before necropsy.

FOOD CONSUMPTION: Yes
Food consumption was recorded once during the acclimatization period and weekly thereafter.

FOOD EFFICIENCY: Yes
- Weekly food efficiency (bodyweight gain/food intake) was calculated.

WATER CONSUMPTION: No
- Time schedule for examinations:
Water intake was visually assessed throughout the study for overt inter group differences.

OPHTHALMOSCOPIC EXAMINATION:
Not applicable

HAEMATOLOGY: Yes- please see attached group summary and individual data.

- Time schedule for collection of blood:
At the end of the treatment period.

- Anaesthetic used for blood collection:
Isoflurane

- Animals fasted:
18 hours fasting period

- How many animals:
5 per sex and group

Routine haematological parameters were examined.

CLINICAL CHEMISTRY: Yes- please see attached group summary and individual data.
- Time schedule for collection of blood:
At the end of the treatment period.

- Animals fasted:
18 hours fasting period

- How many animals:
5 per sex and group

Routine biochemical parameters were examined.

URINALYSIS: No.
- Time schedule for collection of urine:
Not applicable.

- Metabolism cages used for collection of urine:
Not applicable.

- Animals fasted:
Not applicable.

- Routine urinalysis parameters were examined.
Not applicable.
- Vaginal Smear for Estrus Stage
Vaginal smears were taken over four days from all females, and the stage of estrus was evaluated.

Sacrifice and pathology:

- GROSS PATHOLOGY: Yes- please see attached group summary and individual data.

- NECROPY: Yes- please see attached group summary and individual data.
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed
by exsanguination.

- ORGAN WEIGHTS: Yes- please see attached group summary and individual data.
The organs from animals were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.

- HISTOPATHOLOGY: Yes- please see attached group summary and individual data.
All control and high dose animals were subjected to a full histological examination and low and intermediate group animals were routinely subjected to examination of liver and spleen.

Other examinations:

MORTALITY DATA
Observations for viability / mortality were recorded twice daily.

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

• The Dunnett-test [see References (1)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test [see References (2)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test [see References (3)].

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not specified

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

Details on results:

See attached tabulated summary and individual animal data.

Viability / Mortality
All animals survived until the end of the treatment period.

Daily Observations
No clinical signs during daily observations were evident at any dose level throughout the treatment period.

Weekly Behavioral Observations
No clinical signs during weekly behavioral observations (performed during week 1 - 3) were evident at any dose level during the treatment period.

Functional Observational Battery (Screen)

No clinical signs during the functional observational battery (performed during week 4) were evident at any dose level during the treatment period.

Grip Strength
The were no test item-related differences in the mean fore- or hindlimb grip strength values performed at week 4, when compared with the control values. In males treated with 300 mg/kg/day, the hindlimb grip strength was significantly elevated (p<0.05) when compared with controls, but in the absence of a dose response relationship at the highest dose level, lack of similar changes in the forelimb grip strength values or similar differences in the females, this difference was considered to be incidental.

Locomotor Activity
There were no test item-related differences in the mean locomotor activity (measured as the number of light-beam interruptions) performed at week 4, when compared with the control. In females treated with 300 mg/kg/day, significantly greater locomotor activity was recorded during 0 - 10 minutes (p<0.05) and during 20 - 30 minutes (p<0.05) when compared with the controls. The total locomotor activity (0 - 60 minutes) of these females was also elevated, but did not attain statistical significance when compared with controls. As the value recorded in the females at 1000 mg/kg/day was similar to that of the controls, the difference seen in the females treated with 300 mg/kg/day was considered to be incidental.

Body Weights
The mean daily body weight gain values of the males and females treated with 1000 mg/kg/day were marginally lower than those of the respective controls during the treatment period, but were not statistically significant. This difference was considered to be related to the treatment with the test item. The mean body weights of the males and females of the remaining groups were unaffected.

Vaginal Smear for Estrus Stage
Although the frequency of diestrus was marginally higher than that of the control females, the pattern of the estrus over the four days of measurement was considered to be normal and therefore the difference was considered to be incidental.

Clinical Laboratory Investigations

Hematology
No test item-related differences in the hematology values were noted. At 1000 mg/kg/day, the mean red cell count in females was significantly elevated (p<0.01) when compared with the control females, but remained within the ranges of the historical control data. The reticulocyte maturity indices for the middle-fluorescence reticulocytes were elevated with statistical significance (p<0.05) in males when compared with controls. The low-fluorescence reticulocytes, however, were significantly higher (p<0.05) than that of the control males. In females, the red cell distribution width was lower than the controls and marginally exceeded the lower limit of the historical control values. The mean relative number of ‘large unstained cells’ was significantly higher than seen in the control females. This difference marginally exceeded the upper limit of the historical control values. At 300 mg/kg/day, the mean red cell count in males was significantly elevated (p<0.05) when compared with the control males, but did not exceed the upper limit of the historical control data. In females, the red cell distribution width was lower than the controls but remained within the lower limit of the historical control values. At 100 mg/kg/day, there were no differences of toxicological relevance seen in males or females when compared with the respective controls.

In general, although the mean absolute and relative reticulocyte counts remained within the ranges of the historical control data, the values of the reticulocyte maturity indices of the control and test item-treated groups were out of range of the historical control data.

Clinical Biochemistry
No test item-related differences in the clinical biochemistry values were noted. At 1000 mg/kg/day, the mean sodium levels in males were significantly elevated (p<0.05) when compared with the controls, but remained within the upper limit of the historical control values. Females at this dose level showed significantly elevated creatine kinase activity (p<0.05), but this value also remained within the upper range of the historical control data. At 300 mg/kg/day, the mean sodium levels in males were significantly elevated (p<0.01) when compared with the controls, but remained within the upper limit of the historical control values, and exceeded the values seen in male rats treated with 1000 mg/kg/day. Therefore this difference was considered to be incidental. Males also showed statistically significant but (p<0.05), but dose unrelated increases in protein levels (p<0.05) which were within the ranges of the historical control values. No differences were ascertained at 100 mg/kg/day.

Thyroid Hormone Analysis
There were neither macro- nor microscopic changes which would be commonly associated with possible effects upon thyroid hormones and therefore these optional investigations were considered to be superfluous.

Pathology

Organ Weights
The mean absolute and relative organ weights of the test item-treated rats were unaffected. No statistically significant differences were noted when compared with the controls

Macroscopic Findings
There were no gross lesions that could be attributed to treatment with the test item.
All gross lesions recorded were considered to be within the range of normal background alterations. At 1000 mg/kg/day, male no. 17 showed reddish foci on the lungs and thymus, as well as dilated renal pelvis. No macroscopical findings were noted in the remaining males or in any female. At 300 mg/kg/day, male no. 14 showed reddish foci on the stomach mucosa. Female no. 33 had reddish foci on the lungs. No macroscopical findings were noted in the remaining males or females. At 100 mg/kg/day, male no. 8 had dilated renal pelvis. No macroscopical findings were noted in the remaining males or in any female.

Microscopic Findings
All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Based on the results of this study, 300 mg/kg body weight/day of Dilauroyl peroxide (CAS# 105-74-8) was established as the no-observed-effect-level (NOEL).

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Based on the results of this study, 1000 mg/kg bodyweight/day of Dilauroyl peroxide (CAS# 105-74-8) was established as the no-observed-adverse-effect-level (NOAEL).

Critical effects observed:

not specified

See Attached Study Tables and Appendices

Conclusions:

Based on the results of this study, 300 mg/kg body weight/day of Dilauroyl peroxide (CAS#105-74-8) was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of Dilauroyl peroxide (CAS# 105-74-8) as the no-observed-adverse-effect-level (NOAEL).

Executive summary:

- Mortality / Viability

All animals survived the treatment period.

 

- Clinical Signs (Daily and Weekly)

No clinical signs during daily observations or weekly observations (weeks 1 - 3) were evident at any dose level throughout the treatment period.

 

- Functional Observational Battery

No clinical signs during weekly behavioral observations (performed during week 1 - 3) were evident at any dose level during the treatment period.

 

- Grip Strength

The were no test item-related differences in the mean fore- or hindlimb grip strength values performed at week 4, when compared with the control values.

 

- Locomotor Activity

There were no test item-related differences in the mean locomotor activity (measured as the number of light-beam interruptions) performed at week 4, when compared with the control.

 

- Food Consumption

The mean daily food consumption values of the test item-treated rats of all dose groups were similar to that of the control rats. The mean relative food consumption of the test item-treated rats was also unaffected.

 

- Body Weights

The mean daily body weight gain values of the males and females treated with 1000 mg/kg/day were marginally lower than those of the respective controls during the treatment period, but were not statistically significant. There were no test item-related differences at any other dose level.

 

- Estrus Stage

Although the frequency of diestrus was marginally higher than that of the control females, the pattern of the estrus over the four days of measurement was considered to be normal and therefore the difference was considered to be incidental.

 

- Hematology

No test item-related differences in the hematology values were noted.

 

- Clinical Biochemistry

No test item-related differences in the clinical biochemistry values were noted.

 

- Organ Weights

The mean absolute and relative organ weights of the test item-treated rats were unaffected. No statistically significant differences were noted when compared with the controls.

 

- Macroscopic / Microscopic Findings

There were no gross lesions that could be attributed to treatment with the test item. All gross lesions and all microscopic changes recorded were considered to be within the range of normal background alterations.

 

- Conclusion

Based on the results of this study, 300 mg/kg body weight/day of Dilauroyl peroxide (CAS# 105-74-8) was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of Dilauroyl peroxide (CAS# 105-74-8) as the no-observed-adverse-effect-level (NOAEL).

 

Reference 3

Endpoint:

chronic toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential effect of dilauroyl peroxide following repeated oral gavage was evaluated in rats in an OECD 408 study conducted in accordance with Good Laboratory Practice regulations. Wistar rats were administered 100, 300 and 1000 mg/kg/day of the test substance for 90 -days. Based on the results of this study the NOAEL was 1000 mg/kg/day.

In an English translation of a poorly documented 1971 literature article, male rats were administered the test article via the diet for 4 months at dose levels of 1/50 LD50, 1/250 LD50, 1/50 LD33 and 1/250 LD33.

There were no effects reported in the 1/250 LD50 or 1/250 LD33 dose groups. Effects on some hematology parameters, methemoglobin and peroxidase activity were reported in the 1/50 LD50 and 1/50 LD33 dose groups when compared to controls. However an assessment of the results is not possible due to the insufficient details of the study design and results.

Therefore the OECD 408 90 -day repeated dose report is considered the key study.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Apparently well conducted GLP repeat dose study.

Justification for classification or non-classification

No classification is required. There were no adverse effects following the repeated oral gavage administration of 1000 mg/kg/day to rats for 90 -days.