Cineole

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 14 February 2012 and

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: japanese Ministry of Economy Trade and Industry Guideline for 28 day repeat oral dose toxicity study.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cineole

EC Number:

207-431-5

EC Name:

Cineole

Cas Number:

470-82-6

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Constituent 2

Reference substance name:

Eucalyptol

IUPAC Name:

Eucalyptol

Details on test material:

- Name of test material (as cited in study report): Eucalyptol
- Physical state: Liquid
- Analytical purity: 99.8%
- Lot/batch No.: AX00012750
- Expiration date of the lot/batch: 10 October 2012
- Storage condition of test material: 4ºC in the dark (neat test item used under safety lighting)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: approximately six to eight weeks old
- Weight at study initiation: males weighed 200 to 235g, the females weighed 155 to 177g
- Fasting period before study: No data
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): Rodent 2014C Teklad Global Certified Diet, ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage, ad libitum
- Acclimation period: Seven days during which time their health status was assessed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined.
The concentration of Eucalyptol in the test item formulations was determined by gas chromatography (GC) using an external standard technique.The test item formulations were extracted with methanol to give a final, theoretical test item concentration of approximately 0.1 mg/ml.
Standard solutions of test item were prepared in methanol at a nominal concentration of 0.1 mg/ml.
The standard and sample solutions were analysed by GC using the following conditions:
GC system: Agilent Technologies 5890, incorporating autosampler and workstation
Column: DB-5 (30 m x 0.25 mm id x 0.25 µm film)
Oven temperature program: initial 50 ºC for 1 mins
rate 10 ºC/min
temp 120 ºC for 0 mins
rate 50 ºC/min
final 325 ºC for 5 mins
Injection temperature: 250 ºC
Flame ionisation detector temperature: 250 ºC
Injection volume: 1 µl
Retention time : ~ 5 mins
The test item formulations were sampled and analysed within two days of preparation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 300 and 600 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Five/sex/dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Observations
Clinical Observations
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. During the treatment-free period, animals were observed daily. All observations were recorded.

Functional Observations
Prior to the start of treatment (Day -1) and on Days 7, 14, 21 and 27, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait and co-ordination
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behaviour
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper/Hypothermia
Skin colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation

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 day, 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 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. The animal was pulled by the base of the tail until its grip was broken. 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 scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Blink reflex
Startle reflex

Body Weight
Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill and, in the case of recovery group animals, on Days 36 and 43 prior to terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

Water Consumption
Water intake was measured and recorded daily for each cage group.

Laboratory Investigations
Haematological and blood chemical investigations were performed on all non-recovery test and control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43. Animals were not fasted prior to sampling.
Urinalytical investigations were performed on all non-recovery test and control group animals during Week 4 and on all recovery group animals during Week 6. 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.

Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
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)
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).

The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Gamma glutamyltranspeptidase (γGT)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Triglycerides (Tri)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids

Urinalysis
The following parameters were measured on collected urine:
Volume
Specific Gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

Sacrifice and pathology:

Pathology
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free 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°C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were not analysed and were discarded.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals
Brain
Epididymides
Heart
Kidneys
Pituitary (post-fixation)
Prostate and Seminal Vesicles (with coagulating glands and fluids)
Liver
Ovaries
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
Epididymides ♦
Eyes *
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)#
Lymph nodes (mandibular and mesenteric)
Mammary gland
Muscle (skeletal)
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles (with coagulating
glands and fluids)
Skin (hind limb)
Spinal cord (cervical, mid thoracic
and lumbar)
Spleen
Stomach
Testes ♦
Thymus
Thyroid/Parathyroid
Trachea
Urinary bladder
Uterus & Cervix
Vagina

* Eyes fixed in Davidson’s fluid
♦ Preserved in Bouin’s fluid then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Sections of testes and epididymides were stained with Periodic Acid-Schiff (PAS) stain and examined. Any macroscopically observed lesions were also similarly processed together with the liver and spleen from all 30 and 300 mg/kg bw/day dose group animals.
Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the liver for all recovery group animals and the kidneys of all 30 and 300 mg/kg bw/day males and all recovery group males.

Statistics:

Data were processed to give summary incidence or group mean and standard deviation values were appropriate. All data were summarised in tabular form.
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 below:
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 covariates. 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:

effects observed, treatment-related

Description (incidence and severity):

See results

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See results

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:

effects observed, treatment-related

Description (incidence and severity):

See results

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See results

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality
There were no unscheduled deaths on the study.

Clinical Observations
Clinical signs were restricted to post-dosing salivation at 300 and 600 mg/kg bw/day, which may reflect distaste of the dosing formulations rather than any systemic toxicity of the test item. At 300 mg/kg bw/day, two males and all five females were affected and at 1000 mg/kg bw/day all males and females were affected.

Functional Observations
Behavioural Assessments: Assessment of the animals in a standard arena did not reveal any obvious adverse effects of treatment at 30, 300 or 600 mg/kg bw/day.
A higher incidence of urination was noted for females at 600 mg/kg bw/day during the last week of the study. No increased incidence of urination was observed for these females in previous arena assessments or during routine daily assessment of clinical signs.

Functional Performance Tests
At 30 mg/kg bw/day, higher overall mobile score for males during the assessment of motor activity attained statistical significance. No other statistically significant differences in motor activity from control were apparent for either sex at 30, 300 or 600 mg/kg bw/day.
Assessment of grip strength did not reveal any obvious adverse effect of treatment at 30, 300 or 600 mg/kg bw/day.

Sensory Reactivity Assessments
Sensory reactivity to different stimuli (auditory, visual and proprioceptive) did not reveal any obvious adverse effect of treatment at 30, 300 or 600 mg/kg bw/day.

Body Weight
At 600 mg/kg bw/day mean body weight gain of males were slightly lower than control during Week 1, with differences attaining statistical significance. Thereafter body weight during the remaining treatment period and the recovery period was similar to control.
For males at 30 and 300 there were no adverse effects of treatment on body weight gain.
Lower mean body weight gains was apparent for all treated groups compared to control during Week 3 but, there was no dosage relationship and no other statistically significant differences in body weight gain was apparent at 30, 300 or 600 mg/kg bw/day.

Food Consumption
There were no adverse effects of treatment on food consumption of either sex observed throughout the study at any of the dosages investigated.
Food conversion efficiency was also considered to be unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Water Consumption
Intergroup comparison of water consumption for males did not reveal any consistent differences in water intake that indicated any treatment related effect.
For females at 600 mg/kg bw/day water consumption was notably lower than control on the first day of treatment. Thereafter water intake was consistently higher than control throughout the remaining treatment period, although, at times, values were only slightly higher than that observed at 30 mg/kg bw/day. Following the cessation of treatment, water consumption for these females was generally similar to control.
At 30 and 300 mg/kg bw/day, intergroup comparison of water consumption for females did not reveal any consistent differences in water intake that indicated any treatment related effect although, on occasions, water intake at 30 mg/kg bw/day was noticeable higher than control.

Laboratory Investigations
Haematology:
For males at 300 and 600 mg/kg bw/day mean platelet count at the end of the treatment period was higher than control with differences attaining statistical significance. At 600 mg/kg bw/day, mean values and the majority of individual values for these animals exceeded the historical control range. At 300 mg/kg bw/day, the mean value still exceeded this historical control range but only two individual values were outside this range.
For females at all dosages, lower mean neutrophil counts at the end of the treatment period attained statistical significance when compared with control, however there was no dosage relationship or any statistically significant differences for total leucocyte count. All individual values for treated animals were within the historical control, while values for three of control animals exceeded this historical control range.
For females at 600 mg/kg bw/day, higher mean reticulocyte count also attained statistical significance when compared with control, however only one individual value exceeded the historical control range.
Following the two week recovery period, there were no statistically significant differences observed for haematology parameters for either sex at 600 mg/kg bw/day when compared with control.

Blood Chemistry
Males
For males receiving 300 and 600 mg/kg bw/day, mean values for creatinine at the end of the treatment period were statistically significantly higher than control; however only one individual value at 600 mg/kg bw/day exceeded the historical control range. Lower bilirubin levels at these dosages also attained statistical significance when compared with control; but no dosage relationship was apparent. All individual values were within the historical control range, while one control value exceeded this historical range.
At 600 mg/kg bw/day, mean albumin/globulin ratio at the end of the treatment period was statistically significantly lower than control; all individual values were within the historical control range. There were no corresponding statistically significant differences from control observed for mean total protein or for albumin.
Additionally for males at 600 mg/kg bw/day higher mean calcium and potassium levels attained statistically significance when compared with control. For calcium, all individual values were within the historical control range but, for potassium two values exceeded this historical range.
No statistically significant differences from control were apparent for biochemistry parameters at the end of the treatment period for males receiving 30 mg/kg bw/day.
At 600 mg/kg bw/day, following the two week recovery period, higher total protein, albumin and calcium levels attained statistical significance when compared with control, however individual values were within the historical control range.
Females
At 600 mg/kg bw/day, at the end of the treatment period, higher total cholesterol and creatinine levels attained statistical significance when compared to control; all individual values for total cholesterol and the majority for creatinine exceeded the historical control range. Additionally lower mean triglycerides levels and albumin/globulin ratio attained statistical significance compared to control, however all individual values were within the historical control range.
At all dosages, higher levels of sodium and inorganic phosphorus at the end of the treatment period attained statistical significance. For sodium levels there was no dosage relationship and all individual values were within the historical control range. For inorganic phosphorus levels, the highest mean value occurred at 600 mg/kg bw/day and the majority of individual values exceeded the historical control range. No dosage relationship was apparent at the lower dosage groups and all individual values were within the historical control range. Additionally at 600 mg/kg bw/day, higher levels of potassium also attained statistical significance but only two individual values exceeded the historical control range.
Following the two week recovery period, at 600 mg/kg bw/day, lower albumin/globulin ratio and higher calcium and bile acid levels attained statistical significance when compared with control. Individual values for these parameters were all within the historical control range, in addition for calcium the majority of control values were below the historical range.

Urinalysis
At 300 mg/kg bw/day higher urine volume for males during assessment of urinalysis at the end of the treatment period was higher than control, with differences attaining statistical significance. No similar increase was apparent for males at 600 mg/kg bw/day at this stage of the study or at end of the two week treatment free period.
There was no obvious effect of treatment on other urine parameter for either sex at 30, 300 and 600 mg/kg bw/day at the end of the treatment period or for either sex at 600 mg/kg bw/day following the two week treatment free recovery period.

Pathology
Necropsy:
Other than one control male with small testes and epididymides, there were no macroscopic findings observed for either sex either at the end of the treatment period or at the end of the two week treatment free recovery period.

Organ Weights
At 600 mg/kg bw/day both sexes showed increased absolute and body weight-relative liver weights in comparison to control at the end of the treatment period, with differences attaining statistical significance. All body weight relative values (considered to be the more accurate indicator of effects for this organ) exceeded the historical control range. At the end of the two week recovery period, higher absolute and body weight-relative liver weights at this dosage still attained statistical significance, however all body weight relative values were within the historical control range.
For females at 30 mg/kg bw/day and both sexes at 300 mg/kg bw/day, absolute and body weight-relative liver weights were also statistically significantly higher than control at the end of the treatment period, although, particularly for females, not to the same extent as was observed at the high dosage. Only one body weight relative liver weight at these dosages (occurring at 30 mg/kg bw/day) exceeded the historical control range.
At 300 and 600 mg/kg bw/day, absolute and body weight-relative kidney weights for males were statistically significantly higher than control at the end of the treatment period; only one body weight relative value (occurring at 600 mg/kg bw/day) exceeded the historical control range. No similar increase in kidney weights was apparent for females at these dosages or for males at 600 mg/kg bw/day at the end of the two week recovery period.
At 300 and 600 mg/kg bw/day, higher absolute and body weight-relative spleen weights for males at the end of treatment attained statistical significance, when compared with control. All absolute values at these dosages were within the historical control range, although the majority of body weight relative values at 600 mg/kg bw/day did exceeded the historical range. No statistically significant differences in spleen weights were apparent for males at 600 mg/kg bw/day at the end of the two week recovery period.
At 600 mg/kg bw/day, absolute and body weight-relative adrenal weights were statistically significantly increased for females at the end of the treatment period, compared to control. tow absolute and two body weight relative values exceeded the exceeded the historical control range at this dosage. No statistically significant differences in adrenal weights were apparent for females at 600 mg/kg bw/day at the end of the two week recovery period.
At 30 and 300 mg/kg bw/day, absolute and body weight-relative thyroid weights were statistically significantly increased for males at the end of the treatment, compared to control period. At 30 mg/kg bw/day two absolute and two body weight relative values exceeded the historical control range while, at 300 mg/kg bw/day one absolute and two body weight relative values exceeded this historical range. No similar increase in thyroid weights was apparent at 600 mg/kg bw/day.
For males at 600 mg/kg bw/day, following the two week treatment free recovery period, higher absolute and body weight-relative brain weights attained statistical significance when compared with control but all individual values were within the historical control range.
Similarly, for females at 600 mg/kg bw/day, following the two week treatment free recovery period, higher absolute and body weight-relative thymus weights attained statistical significance when compared with control but all individual values were within the historical control range.

Histopathology
For both sexes at 300 and 600 mg/kg bw/day, examination of the liver revealed a dosage dependent incidence of centrilobular hypertrophy of the hepatocytes; no other indicators of liver damage was apparent. Following the two week recovery period, hypertrophy of hepatocytes was no longer present at 600 mg/kg bw/day for either sex.
For males at 300 and 600 mg/kg bw/day, examination of the kidneys revealed an increased severity of hyaline droplets in the proximal tubules, accompanied at the high dosage with sporadic tubular cell degeneration. Additionally an increased mean severity of multifocal tubular basophilia and/or interstitial mononuclear cell foci were observed in association with renal tubules where hyaline droplets were excessively deposited at both dosages. Following the treatment-free recovery period, these treatment-related findings regressed (i.e. decreased in severity) for males at 600 mg/kg bw/day.
The remainder of other histopathological findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: This represents the most appropriate dosage for any assessment of the risk to human health.

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: This represents the most appropriate dosage for any assessment of the risk to human health.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The test substance was assessed for repeated dose oral toxicity according to OECD 407. The No Observed Adverse Effect Level (NOAEL) for the female rat was considered to be 600 mg/kg bw/day but for the male rat was only 30 mg/kg bw/day. However, the adverse findings observed for males were characterised by renal changes specific to the male rat and of no toxicological relevance to man. Excluding these renal changes, the NOAEL was 600 mg/kg bw/day and this represents the most appropriate dosage for any assessment of the risk to human health.

Executive summary:

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

i)         Commission Directive 96/54/EC (Method B7).

ii)        The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004.

iii)       The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

iv)       Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

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 five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 600 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP) over the same treatment period. Two recovery groups, each of five males and five females, were treated with the high dose (600 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs,body weight change, food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

Results.

Mortality.There were no unscheduled deaths on the study.

Clinical Observations.Clinical signs were restricted to post-dosing salivation with two males and all five females at 300 mg/kg bw/day and all males and females at 1000 mg/kg bw/day being affected.

Behavioural Assessment.There were no obvious adverse effects of treatment at 30, 300 or 600 mg/kg bw/day.

Functional Performance Tests.No findings considered to represent an adverse effect of treatment were observed.

Sensory Reactivity Assessments.There were no obvious adverse effects of treatment at 30, 300 or 600 mg/kg bw/day.

Body Weight.At 600 mg/kg bw/day slightly lower mean body weight gain of males than control was observed for Week 1, differences attaining statistical significance. No adverse effect of treatment was observed for females at this dosage or either sex at 30 and 300 mg/kg bw/day.

Food Consumption.There were no obvious adverse effects of treatment at 30, 300 or 600 mg/kg bw/day.

Water Consumption.For females at 600 mg/kg bw/day water consumption was notably lower than control on the first day of treatment. Water intake for males at this dosage and for either sex at 30 or 300 mg/kg bw/day appeared unaffected by treatment.

Haematology.For males at 300 and 600 mg/kg bw/day mean platelet count at the end of the treatment period was higher than control with differences attaining statistical significance.

Blood Chemistry.There were no differences in blood chemistry parameters that were considered to represent an adverse effect of treatment.

Urinalysis:There were no obvious adverse effects of treatment at 30, 300 or 600 mg/kg bw/day.

Necropsy.There were no obvious adverse effects of treatment at 30, 300 or 600 mg/kg bw/day.

Organ Weights.At 300 and 600 mg/kg bw/day, male absolute and body weight-relative kidney weights were statistically significantly increased at the end of the treatment period compared to control. 

An statistically significant increase in absolute and body weight-relative liver weights, considered to be associated with adaptive liver changes, was observed at the end of treatment period for females at 30 mg/kg bw/day and both sexes at 300 and 600 mg/kg bw/day, in comparison to control. Higher liver weights were still apparent at the end of the recovery period and attained statistically significance in comparison to control.

Histopathology.For males at 300 and 600 mg/kg bw/day at the end of treatment, examination of the kidneys revealed an increased severity of hyaline droplets in the proximal tubules, accompanied at the high dosage with sporadic tubular cell degeneration. An increased mean severity of multifocal tubular basophilia and/or interstitial mononuclear cell foci were observed in association with renal tubules where hyaline droplets were excessively deposited was also observed at these dosages. For males at 600 mg/kg bw/day, following the treatment-free recovery period, these treatment-related findings decreased in severity.

For both sexes at 300 and 600 mg/kg bw/day, examination of the liver revealed a dosage dependent incidence of centrilobular hypertrophy of the hepatocytes; no other indicators of liver damage was apparent. Following the two week recovery period, hypertrophy of hepatocytes was no longer present at 600 mg/kg bw/day for either sex.

Conclusion.The No Observed Adverse Effect Level (NOAEL) for the female rat was considered to be 600 mg/kg bw/day but for the male rat was only 30 mg/kg bw/day. However, the adverse findings observed for males were characterised by renal changes specific to the male rat and of no toxicological relevance to man. Excluding these renal changes, the NOAEL was 600 mg/kg bw/day and this represents the most appropriate dosage for any assessment of the risk to human health.