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Toxicological information

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:
2011-07-27 and 2012-10-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
It is a GLP compliant study conducted in compliance with agreed protocols, with no or minor deviations for the standard test.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Names of test material (as cited in study report): lauric aldehyde; dodecanal; n-dodecylic aldehyde; lauraldehyde
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test dates: 2011-06-23 & 2011-07-29
- Batch Nos: 0004053090 & 0004140189
- Expiration dates of the batches: 2012-07-01 / 2012-08-01
- Stability under test conditions:
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, United Kingdom
- Age at study initiation: 6-8 wks
- Weight at study initiation: Males 199 g - 253 g; females 153 g - 205g
- Fasting period before study:
- Housing: In groups of ≤ 4 by sex, in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet: Ground diet, ad libitum
- Water: Mains water, ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 ºC; short term deviations did not affect study integrity.
- Humidity: 55 ± 15 %; short term deviations did not affect study integrity.
- Air changes: ≥ 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 d
Frequency of treatment:
In diet
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200 ppm; mean achieved dosage level (MADL) 14.6 mg/kg bw/d
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2000 ppm; MADL 143.8 mg/kg bw/d
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
6000 ppm; MADL 430.8 mg/kg bw/d
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
20000 ppm; MADL 1409.7 mg/kg bw/d
Basis:
nominal in diet
No. of animals per sex per dose:
10 animals per sex per dose (including control group).
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary concentrations were chosen based on the results from previous toxicity work (Harlan Laboratories Ltd Project Number 41102081).
- Rationale for animal assignment: 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.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Examined for any toxicity, ill-health or behavioural changes
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: On day 1 and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION
- Time schedule for examinations: Daily, per group

OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: pre-treatment, before termination (during wk 12)
- Dose groups that were examined: Control, high dose
- 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
- Time schedule for collection of blood: Wk 7 and at end of study
- Animals fasted: No
- How many animals: All
- Parameters examined: 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).

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Wk 7 and at end of study
- Animals fasted: No
- How many animals:
- Parameters checked in table [No.?] were examined: 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 (Bile), calcium (Ca²⁺).
- Additionally, 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)

URINALYSIS
- Time schedule for collection of urine: Wk 7 and at end of study
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters examined: Volume, ketones, specific gravity, bilirubin, pH, urobilinogen

BEHAVIOURAL ASSESSMENT
- Time schedule for examinations: Weekly
- Dose groups that were examined: All
- 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.

FUNCTIONAL PERFORMANCE TESTS
- Time schedule for examinations: Weekly
- Dose groups that were examined: All
- Battery of functions tested: Sensory activity; grip strength; motor activity.

OESTROUS CYCLE
- Females only
- Time schedule for examinations: Daily during wks 6, 7, 12, 13
- Each sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain. The slides were examined microscopically and the stage of oestrus was recorded
Sacrifice and pathology:
GROSS PATHOLOGY
- On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
- All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

THYROID HORMONE ASSESSMENT
- At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately - 20 ºC. No treatment related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

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; Brain; Epididymides; Heart; Kidneys; Liver; Ovaries Pituitary; Prostate; Seminal Vesicles (with coagulating gland); Spleen; Testes; Thymus; Thyroid/Parathyroid (post fixation); Uterus (with cervix)

HISTOPATHOLOGY
- Samples of the following tissues were removed from all animals and preserved in buffered 10 % formalin, except where stated: Adrenals; Aorta (thoracic); Bone& bone marrow (femur including stifle joint); Bone & bone marrow (sternum); Brain (including cerebrum, cerebellum and pons); Caecum ; Colon ; Duodenum; Gross lesions; Heart ; Ileum (including Peyer’s patches); Jejunum; Kidneys; Liver; Lymph nodes (cervical and mesenteric) ; Mammary glands ; Muscle (skeletal); Oesophagus ; Ovaries; Pancreas; Pituitary; Prostate; Rectum; Salivary glands (submaxillary); Sciatic nerve; Seminal vesicles; Skin (hind limb); Spinal cord (cervical, mid-thoracic lesions and lumbar); Spleen; Stomach; Thymus; Thyroid/Parathyroid; Tongue; Trachea; Urinary bladder; Uterus; Vagina.
- Eyes fixed in Davidson’s fluid
- Epididymides and testes: Preserved in Bouin's fluid then transferred to Industrial Methylated Spirits (IMS) approximately 48 hrs later
- Lungs with bronchi were inflated to approximately normal inspiratory volume with buffered 10 % formalin before immersion in fixative
- All tissues were sent for processing. All tissues from control and 20000 ppm 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.
- Since there were indications of treatment related liver and stomach changes, examination was subsequently extended to include similarly prepared sections from animals in the low and intermediate treatment groups.

SEMEN ASSESSMENT
- At necropsy the following calcuations were performed on all males:
-- 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 -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in a suitable saline/detergent mixture. Samples of the homogenate were stained with a DNA specific fluorescent stain and a sub-sample was analysed for numbers of homogenisation resistant
spermatids.
-- 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 and sperm morphology. A minimum of 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) A sample of semen was preserved in formalin and then stained with eosin. A subsample was placed on a glass slide with a coverslip and a morphometric analysis of sampled semen was performed manually.
-- v) The cauda epididymis was separated from the body of the epididymis and then weighed. The cauda epididmis was then stored frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in an appropriate saline/detergent mixture. Samples of the homogenate were stained with eosin and a sub-sample was analysed for homogenisation resistant
spermatids.
-- vi) For both ii) and iv) above, samples from Groups 1 and 5 were evaluated only as no significant effects were seen.
Statistics:
- Data were processed to give group mean values and standard deviations where appropriate.
- All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using Fisher's ANOVA or ANCOVA and Bartlett’s test.
- The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for nonparametric data.
- If no dose-response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (nonparametric) test to determine significant differences from the control group.
- Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric)

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:
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- There were no unscheduled deaths during the study
- There were no toxicologically significant effects detected throughout the study period
- 2 females treated with 20000 ppm had generalised fur loss between Days 76 and 90. Observations of this nature are commonly observed following group housed animals and in isolation is considered not to be of toxicological importance.

BODY WEIGHT AND WEIGHT GAIN
- There were no toxicologically significant effects detected in body weight development.
- Females treated with 20000 and 6000 ppm showed a statistically significant (P<0.05) increase in body weight gain during Week 5. 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 20000 ppm also showed a statistically significant (P<0.01) reduction in body weight gain during the final week of treatment. In the absence of an effect on overall body weight development the isolated intergroup difference was considered not to be of toxicological significance.

FOOD CONSUMPTION AND COMPOUND INTAKE
- No adverse effect in overall food consumption was detected in treated animals when compared to controls.
- At 20000, 6000, 2000 and 200 ppm, mean achieved intake was fairly consistent for animals of either sex throughout the treatment period and generally maintained a 3, 10 or 100 fold interval (respectively) when compared to the high dose group.

FOOD EFFICIENCY
- No adverse effect in overall food efficieny was detected in treated animals when compared to controls.

WATER CONSUMPTION
- Daily visual inspections of the water consumption did not result in any treatment related effects throughout the study.

OPHTHALMOSCOPIC EXAMINATION
- There were no treatment related ocular effects detected.

HAEMATOLOGY
- No toxicologically significant effects were detected in haematological parameters examined.
- Animals of either sex treated with 20000 ppm and females treated with 6000 ppm showed statistically significant (P<0.05) increases in eosinophil count during Week 7 assessment. In the absence of a similar effect detected at the end of the treatment period the intergroup differences were considered not to be of toxicological importance. Males from all treatment groups showed a statistically significant (P<0.05) increase in prothrombin time during the Week 13 assessment. In the absence of a true dose related response the intergroup differences were considered not to be of toxicological significance. Females treated with 20000 ppm showed a statistically significant reduction in lymphocyte count during both Week 7 (P<0.05) assessment and at the end of the treatment period (P<0.01). In the absence of any supporting histology correlates to suggest an effect of treatment the intergroup differences were considered not to be of toxicological significance.

CLINICAL CHEMISTRY

URINALYSIS
- No treatment related effects were detected in the urinalytical values of all treated animals

NEUROBEHAVIOUR

ORGAN WEIGHTS
-There were no toxicologically significant effects detected in the organ weights measured.
- Males treated with 20000 ppm showed a statistically significant (P<0.05) reduction in adrenal weight both absolute and relative to terminal body weight whilst females treated with 20000 and 6000 ppm showed statistically significant (P<0.01) reductions in absolute and relative liver weight. In the absence of any associated histology correlates the intergroup differences were considered not to be of toxicological importance.

GROSS PATHOLOGY
- No toxicologically significant macroscopic abnormalities were detected.
- One male treated with 20000 ppm had small and flaccid testes at necropsy. Microscopic examinations of this animal revealed unilateral tubular atrophy in the right testis. In the context of this study these findings were considered within the range of normal background lesions which may be recorded in rats of this strain and age used and as such were considered of no toxicological significance.

SPERM ANALYSIS
- There were no toxicologically significant effects detected in sperm motility values, morphological assessments or in homogenisation-resistant spermatid counts.
- Males treated with 20000 ppm showed a statistically significant (P<0.01) increase in the number of abnormal sperm during morphological assessment. In the absence of any associated effects on motility or any histology correlates the intergroup difference was considered not to be of toxicological importance. One male treated with 20000 ppm had a very low sperm concentration and no sperm motility. Macroscopic and microscopic observations correlated with these findings (small and flaccid testes and tubular atrophy) therefore in the absence of any similar effects in the remaining treated males this was considered incidental and not related to test item toxicity.

HISTOPATHOLOGY:
- There were no treatment related microscopic abnormalities detected.
- The findings recorded were within the range of normal background lesions which may be recorded in rats of the strain and age used.

Effect levels

Dose descriptor:
NOAEL
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Overall effects.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test substance was assessed for repeat dose oral toxicity according to OECD guideline 408. The oral administration of lauric aldehyde to rats at dietary concentrations of 200, 2000, 6000 and 20000 ppm (equivalent to a mean achieved dose of 14.6, 143.8, 430.8 and 1409.7 mg/kg bw/day), did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore considered to be 20000 ppm (1409.7 mg/kg bw/day).