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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-09-24 to 2014-03-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Deviations from Study Plan
Deviation No.1 (17 March 2014)
Six animals from the first lactation set of females and twelve animals from the second lactation set of females were assessed using activity monitors for 30 minutes over which five data acquisitions were recorded for each animal. The acquired data was printed out, signed and dated, it was also saved in excel format at the end of the assessment. When the printed data was checked at a later date, it was discovered that some of the data was missing from the printout; only twenty six data acquisition points were present rather than the thirty that was expected for the first lactation set and only fifty one data acquisition points were present rather than the sixty that was expected for the second lactation set of females.
The missing data was recovered by transferring the saved file to a PC at which point it was printed, signed and dated. The retention of the data and file transfer has not been validated so this is considered to be a GLP exception. This data has been reported although no claim of GLP compliance will be made for this part of the work. This deviation was considered not to affect the scientific integrity of the study.
Deviation No. 2 (19 March 2014)
High relative humidities were noted for this study from 2nd October 2013. Initial examination of the system did not reveal any obvious fault with the system but subsequent monitoring revealed a fault which was rectified on 4th October 2013. During the time of this fault, the highest relative humidity recorded was 94% RH. There were also other periods of the study where high humidity was apparent during the study and for these periods the highest relative humidity recorded was 80% RH. While the periods of high humidity observed on the study was not ideal, overall it was considered that they had no effect on the scientific integrity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Extremely pale tan colored solid
- Expiration date of the lot/batch: 31 July 2014
- Stability under test conditions: 18 days. Formulations were therefore prepared twice monthly and stored at approximately 4 °C in the dark.
- Storage condition of test material: Stored in darkness; may be used/formulated in light
- Other: No correction for purity was made.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: approximately twelve weeks
- Weight at study initiation: males weighed 315 to 358g, the females weighed 197 to 231g
- Fasting period before study: None
- Housing: Initially, all animals were housed in groups of four or five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
During the pairing phase, the non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of mating, the males were returned to their original cages.
Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Recovery group animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.
- Diet (e.g. ad libitum): ad libitum - A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 2013-09-26 To: 2013-11-21

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0, 25, 125, 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC using an external standard.

Samples and standards extracted in tetrahydrofuran to give a concentration of 1.0 mg/mL

GC system: Agilent Technologies 5890, 6890 or 7890
Column: RXI 1MS (15 x 0.25 mm id x 1 µm film)
Oven temperature: Initial 150°C for 0 mins, rate 10°C/min, final 350°C for 5 mins
Injection temp: 300°C
Flame ionisation detector temp: 300°C
Injection volume: 1 µL

Specificity: No interference
Linearity: R = 0.997
Accuracy: 91 - 99%

Stability determination: analysed initally and after 18 days in the dark at +4°C.
Stability: 93 - 95% of initial concentration after 18 days

Formulation concentrations:
Formulation: 91 - 101% of nominal
Duration of treatment / exposure:
28-days
Frequency of treatment:
1 x day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day (arachis oil BP)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg.kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Control: 12 male/ 12 female
Recovery control and Recovery high dose: 5 male/ 5 female
Dose groups: 12 male/ 12 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (Harlan Laboratories Ltd., Project Number 41302024). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
The study was performed between 16 August 2013 and 23 May 2014 (date of final histopathology report). The in-life phase of the study was conducted between 26 September 2013 (first day of treatment) and 21 November 2013 (final day of necropsy).
Chronological Sequence of Study
Non-Recovery Dose Groups
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).
The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43/44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Recovery Dose Groups
i. Groups of five male and five female rats were dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued.
ii. The males and females were maintained without treatment for a further fourteen days.
iii. Blood samples were taken for hematological and blood chemical assessment on Day 56.
iv. After fourteen days of recovery, males and females were killed and examined macroscopically.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, up to thirty minutes after dosing, and one hour after dosing, during the working week (except for females during parturition where applicable). During the treatment-free period, recovery animals were observed daily. All observations were recorded.
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each non-recovery dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
Behavioral Assessments
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 behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation
Functional Performance Tests
Motor Activity.The tests were performed at approximately the same time on each occasion (at least two hours after dosing). The evaluation period was thirty minutes for each animal.
Forelimb/Hindlimb Grip Strength
Sensory Reactivity
The following parameters were observed:
Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach
Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.
Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.
Weekly food consumptions were performed for each cage of recovery group animals throughout the study period.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for non-recovery males (except during the mating phase) and recovery group animals throughout the study period and for non-recovery females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females, during gestation and lactation.
Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.
Reproductive Performance
Mating
Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition.
Observations were carried out early morning and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Sacrifice and pathology:
Laboratory Investigations
Hematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group prior to termination (Day 42 for males and Day 4 post partum for females). In addition hematological and blood chemical investigations were performed on all recovery group animals after the fourteen day treatment free period at termination (Day 56). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
Hematology
The following parameters were measured on blood collected into tubes containing potassium
EDTA anti-coagulant:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte 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).
Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol),
Chloride (Cl-), Total bilirubin (Bili), Bile acids

Pathology
Necropsy
Adult non-recovery males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43/44. Adult non-recovery females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.
For all non-recovery females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
Recovery group animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 57.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each non-recovery dose group and from all recovery group animals.
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation)
Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated.
Ovaries, Pituitary, Prostate, Adrenals , Muscle (skeletal), Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes, Gross lesions, Spleen, Heart, Stomach, Ileum (including peyer’s patches), Thyroid/parathyroid, Jejunum, Trachea, Kidneys, Testes, Liver, Thymus, Lungs (with bronchi), Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus/Cervix, Mammary gland, Vagina
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
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.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analyzed 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 shows non-homogeneity of means, the data were analyzed 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).
Data not analyzed by Provantis were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test. Where Levene’s test was shown to be non-significant (p≥0.05), parametric analysis of the data was applied, incorporating analysis of variance (ANOVA). If this data was shown to be significant, this analysis was followed by pair-wise comparisons using Dunnett’s test. Where Levene’s test was significant, non-parametric analysis of the data was analyzed incorporating the Kruskal-Wallis test which if significant, was followed by the Mann-Whitney U test. Where the data was unsuitable for these analyses, then pair-wise tests were performed using tStudent 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
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
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:
Mortality
There were no unscheduled deaths.
Clinical Observations
Treatment at 1000 mg/kg bw/day was associated with increased post-dosing salivation for both sexes from Day 15, with all animals being affected at some stage of the treatment period.
Additionally one male treated with 1000 mg/kg bw/day had staining around the snout on Day 40 and another had noisy respiration on Day 43. At 500 mg/kg bw/day, increased post-dosing salivation was observed for four animals of either sex on Day 42 or 43 of the study.
Observations of this nature are commonly observed following the oral administration of an unpalatable test item formulation and in isolation are considered not to be of toxicological importance.
No such effects were detected in animals of either sex treated with 100 mg/kg bw/day. One control female had noisy respiration on Day 43 only. This observation can be observed following the dosing procedure and may represent the slight aspiration of the vehicle.
Functional Observations
Behavioral Assessments
There were no treatment-related changes in the behavioral parameters at 100, 500 or 1000 mg/kg bw/day.
Functional Performance Tests
There were no changes in functional performance considered to be related to treatment at 100, 500 or 1000 mg/kg bw/day.
For males, lower values for hind limb grip strength during trial three at all dosages and lower fore limb grip strength during trial three at 500 mg/kg bw/day attained statistical significance when compared with control. However, there was no dosage-relationship and, in the absence of any statistically significant differences from control in previous assessments of grip strength for these animals, these findings were considered incidental and unrelated to treatment.
Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores.
Body Weight
There were no adverse effects of treatment on body weight development for either sex throughout the study and, including for females gestation and lactation phases, at dosage up to 1000 mg/kg bw/day.
For non-recovery males, a statistically significant reduction in body weight gain was evident at 500 and 1000 mg/kg bw/day during Week 4 of treatment. Overall body weight gain of these males was only 85% by Day 43 of the study. However, when recovery animals were included, values at 1000 mg/kg bw/day were approximately 95% of the concurrent control on Day 43. It was considered that the differences from control for treated animals probably reflected normal
biological variation rather than any adverse effect of treatment on body weight gain.
Food Consumption
Food consumption for males during the pre and post-pairing phases of the study and for females during the pre-pairing, gestation and lactation phases of the study was considered to be unaffected by treatment at 100, 500 and 1000 mg/kg bw/day.
Food conversion for both sexes during the pre -pairing phase and for males during the postmating phase of the study was considered to be unaffected by treatment at 100, 500 and 1000 mg/kg bw/day.
Water Consumption
Daily visual assessment of water bottles did not reveal any significant intergroup differences between control and treated animals.
Hematology
Assessment of hematology parameters did not indicate any adverse effect of treatment at 100, 500 or 1000 mg/kg bw/day for either sex.
For males at 1000 mg/kg bw/day, lower mean cell hemoglobin concentration levels attained statistical significance when compared with control. All individual values were within historical control range for rats of the strain and age used and, in isolation, this finding was considered incidental and unrelated to treatment.
For females at 500 and 1000 mg/kg bw/day, lower mean hemoglobin levels attained statistical significance compared to control. All individual values were within the historical control ranges for rats of the strain and age used and as values showed no clear dosage relation, they were considered to be unrelated to treatment.
For females at all dosages, lower mean cell volume attained statistical significance compared to control. There was no dosage-relationship and only one value at 100 mg/kg bw/day and two values at 1000 mg/kg bw/day exceeded the historical control range, compared to three in the Control group. The differences observed were considered to reflect particularly high control values and were therefore considered to be unrelated to treatments.
For recovery males at 1000 mg/kg bw/day, lower total leukocyte count and lymphocyte count attained statistical significance when compared to controls. In the absence of a similar effect detected in non recovery males at the end of the treatment period, the intergroup differences were considered of no toxicological importance.
Blood Chemistry
Males at 1000 mg/kg bw/day showed a statistically significant increase in alanine aminotransferase compared with control. Females at this dosage showed statistically significant increases in alanine aminotransferase, aspartate aminotransferase, creatinine and bile acids compared with control. The majority of individual values were above the normal ranges for rats of the strain and age used.
No statistically significant differences from control were apparent for blood chemistry parameters in either sex at both 100 and 500 mg/kg bw/day.
Recovery males treated with 1000 mg/kg bw/day showed a statistically significant reduction in albumin/globulin ratio and bile acids whilst recovery females showed a statistically significant increase in total protein, albumin, calcium concentration and bilirubin. In the absence of a similar effect detected in non recovery males or at the end of the treatment period, the intergroup differences were considered of no toxicological importance.
Pathology
Necropsy
Neither the type, incidence or distribution of necropsy findings for adult animals indicated any adverse effects of treatment.
Organ Weights
For non-recovery males at 500 and 1000 mg/kg bw/day, higher absolute and body weight relative liver weights attained statistical significance when compared with control, although there was no consistent dosage relationship. No statistically significant differences in organ weights were apparent at the end of the recovery period for males at 1000 mg/kg bw/day.
No statistically significant differences in organ weights were apparent at the end of the treatment period for females at 100, 500 and 1000 mg/kg bw/day. For recovery females at 1000 mg/kg bw/day higher absolute and body weight-relative kidney weights did attain statistical significance when compared with control but, in the absence of any increase at the end of the treatment period, this finding was considered incidental and unrelated to treatment.
Histopathology
The following treatment related microscopic abnormality was detected: Liver: minimal centrilobular hepatocellular hypertrophy was evident in two males treated with 1000 mg/kg bw/day. No such effects were detected in females treated with 1000 mg/kg bw/day. Although treatment related liver changes were evident in two males treated with 1000 mg/kg bw/day, these were considered to be an adaptive response to the test item and was not considered to represent an adverse effect of treatment. Microscopic examination of the liver was therefore not performed in the remaining treatment groups.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects related to dose treatment which are not considered to be adaptive responses.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of the test material to rats by gavage for 28-days, at dose levels of 100, 500 and 1000 mg/kg bw/day, did not result in any adverse effects of treatment. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996). This study was also designed to 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 twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 500 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study. Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Extensive functional observations were performed on five selected non-recovery males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry assessments were performed on five non-recovery males and females per dose group prior to termination. Adult non-recovery males were terminated on Day 43/44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Following forty-two days of treatment, recovery group animals were maintained without treatment for a further fourteen days. Hematological and blood chemical assessments were performed on all recovery group animals at the end of the treatment-free period. Recovery animals were killed on Day 57. All animals were then subjected to a gross necropsy and histopathological examinations of selected tissues was performed.

Results

Adult Responses

Mortality

There were no unscheduled deaths.

Clinical Observations

Treatment at 1000 mg/kg bw/day was associated with increased post-dosing salivation for both sexes from Day 15 onwards, with all animals being affected at some stage of the study. At 500 mg/kg bw/day, isolated incidences of increased post-dosing salivation was observed for animals of either sex towards the end of the treatment period. No such effects were detected in animals of either sex treated with 100 mg/kg bw/day.

Behavioral Assessment

There were no treatment related effects detected.

Functional Performance Tests

There were no changes in functional performance considered to be related to treatment.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores.

Body Weight

There were no treatment related effects on body weights throughout the study of either treated sex.

Food Consumption

No adverse effects on dietary intake were noted for males during the study or for females during the pre-pairing, gestation or lactation phases of the study, including the recovery phase.

Water Consumption

Visual assessment of water bottles did not reveal any significant intergroup differences.

Reproductive Performance Mating

There were no treatment-related effects on mating performance.

Fertility

There were no treatment related differences in fertility.

Gestation Lengths

Gestation lengths were between 22 and 23½ days and appeared unaffected by maternal treatment.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no obvious effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 100, 500 or 1000 mg/kg bw/day. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex. Offspring Growth and Development

Offspring body weight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were essentially similar or slightly superior to controls and appeared unaffected by maternal treatment at 100, 500 or 1000 mg/kg bw/day. Offspring performance during assessment of surface righting appeared to be unaffected by maternal treatment.

Laboratory Investigations

Hematology

Assessment of hematology parameters did not indicate any adverse effect of treatment at 100, 500 or 1000 mg/kg bw/day for either sex.

Blood Chemistry

Males at 1000 mg/kg bw/day showed a statistically significant increase in alanine aminotransferase compared with control. Females at this dosage showed statistically significant increases in alanine aminotransferase, aspartate aminotransferase, creatinine and bile acids compared with control. No statistically significant differences from control were apparent for blood chemistry parameters in either sex at both 100 and 500 mg/kg bw/day.

Pathology

Necropsy

Neither the type, incidence or distribution of necropsy findings for adult animals indicated any adverse effects of treatment.

Organ Weights

For non-recovery males at 500 and 1000 mg/kg bw/day, higher absolute and body weight relative liver weights attained statistical significance when compared with control. No such effects were detected in females treated with 1000 or 500 mg/kg bw/day, animals of either sex treated with 100 mg/kg bw/day or recovery animals following fourteen days without treatment.

Histopathology

A treatment related microscopic abnormality was detected in the liver. Minimal centrilobular hepatocellular hypertrophy was evident in two males treated with 1000 mg/kg bw/day. This was considered to be an adaptive response to the test item and was not considered to represent an adverse effect of treatment. No such effects were detected in females treated with 1000 mg/kg bw/day.

Conclusion

The oral administration of test material to rats by gavage, at dose levels of 100, 500 and 1000 mg/kg bw/day, did not result in any adverse effects of treatment. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.