Registration Dossier

Administrative data

Description of key information

28 Day repeat dose oral: NOAEL > 1000 mg/kg/day

90 Day repeat dose oral: NOAEL > 1000 mg/kg/day

28 Day repeat dose dermal read-across: NOAEL > 1000 mg/kg/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 June 2015 to 24 June 2016
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: E00031-541
- Expiration date of the lot/batch: 01 November 2016
- Purity test date: > 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature, in the dark
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Kent, UK.
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: Males: 190-233 g; females: 136-178 g
- Housing: Prior to stratification, animals were randomly housed up to 5 per cage per sex. After stratification, animals were housed randomly 2 per cage by sex with similar group mean body weights
- Diet (e.g. ad libitum): ad libitum, except during designated procedures
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 °C (There were occasions where the target environmental conditions were not maintained. However these were considered to be transient, the animals didn't show any clinical signs and, therefore, did not have any effect on the outcome or integrity of the study).
- Humidity (%): 40-70%
- Air changes (per hr): 10 or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 07 July 2015 To: 06 October 2015
Route of administration:
oral: gavage
Details on route of administration:
The test and control items were administered to the appropriate animals once daily by oral gavage from Day 1 to Day 90 and up to the day before scheduled euthanasia.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared at weekly intervals. The formulations were split into suitable volume aliquots for daily dispensing, and stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator for at least 30 minutes before dosing.
The control formulation, Arachis Oil BP, was dispensed as received and was stored in a refrigerator set to maintain 4°C, and dispensed daily. The control formulation was removed from the refrigerator at least 30 minutes prior to dosing.

VEHICLE
- Concentration in vehicle: 0, 25, 75 and 250 mg/mL of EXP0700332 in vehicle
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no: A0344960
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared at weekly intervals. The dosing formulations were split into suitable volume aliquots for daily dispensing, and stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator for at least 30 minutes before dosing.
Duplicate top, middle and bottom (duplicate middle only for control) samples (1 mL) for each sampling time point were sent to the analytical laboratory. In addition, triplicate top, middle, and bottom (triplicate middle only for control) samples (1 mL) were maintained at the Test Facility as backup samples. For concentration, the criterion for acceptability was mean sample concentration results within or equal to ± 10% of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of = 10% for each group.
Duration of treatment / exposure:
90 Days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
The animals were allowed to acclimate for 2 weeks before dosing. The test and control items were administered to the appropriate animals once daily by oral gavage from Day 1 to Day 90 and up to the day before scheduled euthanasia. The doses were given using a syringe with attached gavage cannula. The first day of dosing was designated as Day 1. The dosing formulations were stirred for at least 30 minutes before the start of administration and continuously during dose administration.
The oral route of administration was selected for this study as this route was defined by the Sponsor as a possible route of human exposure. The dose levels were agreed with the Sponsor following evaluation of a previous 28-day repeat dose oral (gavage) toxicity study in the rat (OECD Guideline 407) and a reproduction / oral gavage developmental toxicity screening study in the rat (OECD Guideline 421; Charles River Study No. 496381) performed on behalf of the Sponsor, where no significant signs of toxicity were noted at dosage levels of 30, 300 or 1000 mg/kg/day.
The animals were observed twice daily, once at the start and once towards the end of the working day, for general health/mortality and moribundity. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings. The animals were removed from the cage and examined weekly commencing in Week -1 and continuing throughout the course of the study for a detailed clinical observation. Bosy weights, food consumption, water consumption, ophthalmic examinations, functional and cageside observations were performed throughout the study.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Posture/condition on first approach (animal undisturbed), checking for:
Prostration, Lethargy, Writhing, Circling, Breathing abnormalities, Gait abnormalities, Tremor, Fasciculation, Convulsions, Biting (of cage components or self mutilating), Vocalisations and Piloerection.
Ease of removal from the cage
Body temperature: A rectal temperature was taken.
Condition of the eyes, checking for:
Pupillary function, Miosis, Mydriasis, Exophthalmos, Encrustation and Lacrimation.
Condition of the coat.
Presence of salivation.
Overall ease of handling.

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were examined for reaction to treatment regularly throughout the day. The onset, intensity and duration of these signs were recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

BODY WEIGHT: Yes
All animals were weighed individually once during pretreatment and daily during the dosing period. Body weights were also recorded on the first day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was quantitatively measured weekly, commencing at the start of Week -1

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Water consumption was visually assessed throughout the study. There were no differences seen that were considered to be related to treatment.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined during pretreatment and in Groups 1 and 4 during Week 13 using an indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl®). The anterior, lenticular and fundic areas were examined.

HAEMATOLOGY: Yes
Red blood cell count, Haemoglobin, Haematocrit, Mean cell volume, Mean cell haemoglobin concentration, Mean cell haemoglobin, Reticulocytes, Reticulocyte count (absolute), Red blood cell distribution width, Platelets, White blood cell count, Neutrophils, Lymphocytes, Monocytes. Eosinophils, Basophils, Large unstained cells, Other cells (as appropriate)

CLINICAL CHEMISTRY: Yes
Blood samples (approximately 1.0 mL) were collected into lithium heparin, processed for
plasma, and analysed for the parameters:
Urea, Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatine phosphokinase, Lactate dehydrogenase, Sodium, Potassium, Chloride, Blood Urea Nitrogen, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium, Inorganic phosphate, Triglycerides

URINALYSIS: Yes
Urine was collected from animals individually housed in metabolism cages over approximately 18 h with absence of food/presence of water. After collection, samples were transferred to the appropriate laboratory for processing. Urine samples were processed and analysed for the parameters:
Microscopic evaluation of spun deposit, Colour, Turbidity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Leukocytes, Blood Pigments, Urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
Battery of functions tested: sensory activity / grip strength / motor activity

OTHER:
Morality/Moribundity Checks:
Animals were observed twice daily, once at the start and once towards the end of the working day, for general health/mortality and moribundity. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.
Sacrifice and pathology:
All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Scheduled necropsy examinations were conducted by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
All animals were weighed, and euthanised by exposure to a rising concentration of carbon dioxide, followed by exsanguination. When possible, the animals were euthanised rotating across dose groups such that similar numbers of animals from each group, including controls were necropsied at similar times throughout the day. Animals were not fasted before their scheduled necropsy.
Histopathological evaluation was performed by the Principal Investigator at Charles River Pathology Associates, who is a board-certified veterinary pathologist.
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately.
Body weight, food consumption, selected functional observational battery and motor activity data, haematology, coagulation, clinical chemistry and selected urinalysis data were analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e. pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
In circumstances where it was not possible to perform the F Max test due to zero standard deviation in at least one group, the non-parametric ANOVA resultswere reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above as an exploratory analysis.
In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data was subjected to a rank transformation prior to analysis.
In the ANOVA and ANCOVA summary tables, the results of the analysis were reported indicating the level of statistical significance (p<0.05, p<0.01 and p<0.001) of each pairwise comparison. Actual p-values were not reported in the summary tables for these analyses.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical Observations:
Excess salivation was noted immediately post dose on Days 59, 70-73 in up to 3 animals given 100 mg/kg/day or males given 300 or 1000 mg/kg/day. Due to the isolated incidences, it was considered that the clinical observations were due to the dosing technique rather than the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gain at the end of the treatment period was statistically significantly lower in females given 100 mg/kg/day when compared with controls. However, due to the relatively small magnitude of change and the lack of a dose relationship this was considered to be incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was slightly higher throughout the study in males given 1000 mg/kg/day when compared with controls, with statistical significance being achieved on Day 42 only. However, due to all values, with the exception of Day 42, being within 10% variation of the control values this was considered not to be toxicologically significant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Coagulation - There were no differences in any coagulation parameter that were considered to be treatment related.
Prothrombin time was longer in animals given 1000 mg/kg/day and males given 100 mg/kg/day when compared with controls. In addition, activated partial thromboplastintime was longer in males given 1000 mg/kg/day when compared with controls. However, due to the small magnitude of change and the lack of a correlating change in fibrinogen this was considered not to be treatment related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Creatine phosphokinase was lower females given 1000 mg/kg/day and animals given 100 mg/kg/day when compared with controls. However, due to the small magnitude of difference and the direction of the change this was considered not to be treatment related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional Observations:
Hind grip values were statistically significantly higher and tail flick values were statistically significantly lower at Week 12 in males given 1000 mg/kg/day when compared with controls. However, due to the small magnitude of difference from pre-treatment values, the lack of any other neurotoxicity signs and larger differences in control animals these differences were considered to be incidental.

Motor Activity:
There were isolated incidences of statistical significance noted in the X and Y-ambulation when compared to controls. However, due to the small magnitude of difference, the lack of consistency and this was not seen in any other parameters this was considered to be incidental.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute and covariate spleen weight were statistically significantly lower in females given 100 mg/kg/day when compared with controls. However due to the small magnitude of change and the lack of any dose relationship this was considered incidental and unrelated to EXP0700332 administration.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of EXP0700332.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test item-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of EXP0700332.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Urinalysis - no differences in any urinalysis parameter that were observed were considered treatment related
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Conclusions:
In conclusion, administration of EXP0700332 by once daily oral gavage was well tolerated in rats at levels of 100, 300 and 1000 mg/kg/day. There were no treatment-related clinical observations or target organ effects. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.
Executive summary:

The objective of this study was to determine the potential toxicity of EXP0700332 when given orally for 91 consecutive days to rats.

40 male and 40 female rats were randomly organized into 4 (1-4) dose groups of 10 animals per sex per dose with dosage levels of 0, 100, 300 and 1000 mg/kg/day.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, ophthalmology, detailed functional observations, clinical pathology parameters (haematology, coagulation, clinical chemistry, and urinalysis), gross necropsy findings, organ weights, and histopathologic examinations.

All analysed formulation concentrations were found to be within the acceptable range and homogeneous.

There were no unscheduled deaths during the study.

There were no clinical observations noted that were considered to be treatment related. Excess salivation was noted in 3 animals, however, these were isolated incidences and were considered to be related to the dosing technique.

Any differences seen were considered unrelated to the administration of EXP0700332.

In conclusion, administration of EXP0700332 by once daily oral gavage was well tolerated in rats at levels of 100, 300 and 1000 mg/kg/day. There were no clinical observations or target organ effects. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 January 2009 and 11 May 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern GLP study conducted in accordance with OECD test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Ltd, Blackthorn, Bicester, Oxon
- Age at study initiation: approximately 6 to 8 weeks
- Weight at study initiation: males -218 to 258g; females 167 to 194g
- Fasting period before study: no data
- Housing: 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): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days

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

IN-LIFE DATES: no data
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg/day of Arachis oil BP. Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to initiation of dose administration, the test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom strata of the 7.5 and 250 mg/kg of the dosing formulation for homogeneity determination. Sampling was performed in triplicate. For stability determination samples were collected and stored at approximately +4ºC in the dark for fourteen days. The results shown the formulations to be
stable for at least 14 days. Therefore, formulations were prepared weekly and stored at approximately 4ºC in the dark under nitrogen.
In addition, for verification of test material formulation concentrations, samples were analysed within three days of preparation. The results indicate that the prepared formulations were within ± 7% of the nominal concentration. All analyses were conducted by the Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory.
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
4 ml/kg vehicle only (control & recovery control group)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000mg/kg/day ( recovery group)
Basis:
actual ingested
No. of animals per sex per dose:
Control group - 5 males and 5 females
Recovery control- 5 males and 5 females
Low treatment group ( 30mg/kg) - 5 males and 5 females
Intermediate treatment group ( 300 mg/ kg) - 5 males and 5 females
High treatment group ( 1000 mg/ kg) - 5 males and 5 females
Recovery high treatment group ( 1000 mg/kg)- 5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
Preliminary 14 day repeated dose oral (gavage) range-finder in the Wistar Han™:HsdRccHan™:WIST strain rat was performed to establish the maximum tolerated dose level (up to1000 mg/kg/day) of the test material and to provide information for selection of dose levels for use in the twenty-eight day oral toxicity phase. The dose levels were chosen, as: High dose: 1000 mg/kg/day, Intermediate dose: 300 mg/kg/day, Low dose: 30 mg/kg/day
plus a control group treated with vehicle alone.
The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories. A total of sixty animals (thirty males and thirty females) were accepted into the study and all were examined for signs of ill-health or injury. Prior the testing animals were acclimatised for nine days.
The test material was administered by gavage to three groups, each of five male and five female rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 1000 mg/kg/day. A control group of five males and five 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/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.
Clinical signs, functional observations, bodyweight development, 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.











Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS:
- Time schedule: up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays.

BODY WEIGHT:
Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights 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 AND COMPOUND INTAKE (if feeding study):
Food consumption was recorded for each cage group at weekly intervals throughout the study.

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

WATER CONSUMPTION AND COMPOUND INTAKE :
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically.

HAEMATOLOGY:
Measured parameters included ( blood tubes contained the 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) - Stained slides were prepared and reticulocytes were assessed
Prothrombin time (CT) and Activated partial thromboplastin time (APTT) were assessed


BLOOD CHEMISTRY:
Measured parameters on plasma from blood samples ( tubes contained lithium heparin anti-coagulant ) included:
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 (¿GT), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Triglycerides (Tri), Total cholesterol (Chol), Total bilirubin (Bili)

URINALYSIS:
Measured parameters included: Volume, Specific Gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Reducing Substances, Blood

NEUROBEHAVIOURAL EXAMINATION:
- Battery of functions tested: sensory activity, forelimb/ hindlimb grip strength, motor activity

BEHAVIOURAL ASSESSMENTS:
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
Sacrifice and 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 plasma from each animal was stored frozen at -20°C. As no treatment-related effect on the pituitary-thyroid axis was identified these samples were discarded.

Oestrus Cycle Assessment- At termination, a vaginal smear was taken from all female animals and the stage of oestrus was recorded.

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, Liver, Ovaries, Prostate, Pituitary (post fixation), Prostate, Seminal vesicles (with coagulation glands and fluids), Spleen, Testes, Thymus, Thyroid/Parathyroid glands (post fixation), Uterus & 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 Rectum pons) , Caecum, Colon, Duodenum, Epididymides, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Mammary gland, Muscle (skeletal), Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (with coagulating gland and fluids), Skin (hind limb), Spinal cord (cervical, mid thoracic and lumbar), Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Trachea, Urinary bladder, Uterus & Cervix, Vagina







Statistics:
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 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).

Clinical signs:
no effects observed
Description (incidence and severity):
no mortality
Mortality:
no mortality observed
Description (incidence):
no mortality
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY- No clinically observable signs of toxicity were detected and there were no deaths during the study.

FUNCTIONAL OBSERVATIONS-
Behavioural Assessments-There were no treatment-related changes detected in the behavioural parameters measured. All remaining inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and are considered to be of no toxicological importance.

Functional Performance Tests- There were no adverse changes in the functional performance parameters measured. The incidental findings were co nsidered to be of no toxicological significance.

Sensory Reactivity Assessments-There were no treatment-related changes in sensory reactivity. Statistical analysis of the data revealed no significant inter-group differences.

BODY WEIGHT AND WEIGHT GAIN- No treatment-related effect on bodyweight change was detected for treatment animals, in comparison to controls.
Statistical analysis of the data revealed no significant inter-group differences.


FOOD CONSUMPTION- No treatment-related effect on dietary intake or food efficiency was detected for treatment animals, in comparison to controls.

WATER CONSUMPTION- No treatment-related effects on water consumption were detected for treatment animals, in comparison to controls.

HAEMATOLOGY- There were no toxicologically significant changes in the haematological parameters measured. The statistically significant inter-group differences detected were considered to be of no toxicological importance.

BLOOD CHEMISTRY-There were no toxicologically significant changes detected in the blood chemical parameters measured. The statistically significant inter-group differences detected were considered to be of no toxicological importance.

URINALYSIS- No adverse effect on urinalytical parameters was detected for treatment animals, in comparison to controls. Statistical analysis of the data revealed no significant inter-group differences.

ORGAN WEIGHTS- There were no toxicologically significant changes in organ weight measurements. The statistically significant inter-group differences detected were considered to be of no toxicological importance.

GROSS PATHOLOGY- No treatment-related abnormalities were detected at terminal kill. The incidental findings seen were of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC- No treatment-related changes were observed.




Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Episodes of increased salivation, scab formation and generalised fur loss were observed in males treated with 1000 mg/kg/day. These findings were considered incidental and not indicative of systemic toxicity.
Critical effects observed:
not specified
Conclusions:
The oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day did not result in any treatment-related effects. The No Observable Effect Level (NOEL) for animals of either sex was therefore considered to be 1000 mg/kg/day.
Executive summary:

The study was designed to investigate the systemic toxicity of the test material, by repeated oral administration to the Wistar Han™:HsdRccHan™:WIST strain rat for a period of twenty- eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day. A control group 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/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations, bodyweight development, 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.

The oral administration of the test material to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day did not result in any treatment-related effects.The No Observable Effect Level (NOEL) for animals of either sex was therefore considered to be 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Modern GLP study conducted in accordance with OECD test guideline, Klimisch score 1.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
27.01. 1997- 19-07.1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guidelines followed but no statement of GLP. Restriction due to the fact that the study was conducted on read-across substance.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Crl:CD®BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston Facility, Stone Ridge, New York
- Age at study initiation: Males: Approximately 7-8 weeks, Females: Approximately 9-10 weeks
- Weight at study initiation: Males: 246.3 to 270.2 grams, Females: 197.9 to 245.9 grams
- Fasting period before study: no data
- Housing: Single housed during the test period, Caging: Suspended stainless steel and wire mesh with absorbent paper below cages
- Diet (e.g. ad libitum): PMI Certified Rodent Diet Meal 5002, ad libitum
- Water (e.g. ad libitum): Automatic Watering System, ad libitum
- Acclimation period: 13 days; animals were examined for viability at least once daily

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-22.3
- Humidity (%): 30 to 70 percent relative humidity
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light (0700 to 1900 Hours) and 12 hours dark (1900 to 0700 Hours) by automatic timer.

IN-LIFE DATES: From: January 28, 1997 To March 11, 1997
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: the dorsal surface from the shoulder region to the lumbar region
- % coverage: approximately 10% of the body surface
- Type of wrap if used: COBAN wrap
- Time intervals for shavings or clipplings: at least once weekly

REMOVAL OF TEST SUBSTANCE
- Washing : gentle wipe of the exposure site with peanut oil and a paper towel
- Time after start of exposure: approximately 6 hours after exposure

TEST MATERIAL
- Amounts applied :100mg/kg, 300mg/kg, 1000 mg/kg


USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, The animal was wrapped with COBAN to prevent ingestion of the test material.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
1000 mg/kg (satellite group)
Basis:
nominal per unit area
No. of animals per sex per dose:
Group 1 ( control group)- 0 mg/kg (sham dose )- 5 males and 5 females
Group 2- 100 mg/kg- 5 males and 5 females
Group 3- 300 mg/kg- 5 males and 5 females
Group 4- 1000 mg/kg- 5 males and 5 females
Group 5 (satellite recovery group) - 1000 mg/kg- 5 males and 5 females
Control animals:
yes, sham-exposed
Details on study design:
Dose levels were selected based on findings of previously conducted studies with this material including an acute dermal toxicity study (LD50 > 2000 mg/kg) and 28-day oral toxicity study (no signs of overt toxicity at the limit dose level of 1000 mg/kg).

All animals were examined for abnormalities prior to dose initiation and those determined to be unsuitable for inclusion on this study because of poor health, outlying body weights, or other abnormalities were excluded from selection. Study animals were selected from the suitable animals by a computer generated sorting program and their weight variation within their sex was within ±20% of the mean body weight by sex.

Fifty Crl:CD®BR rats were divided into 5 groups of 5 male and 5 female rats each. The test material was applied to the clipped, unabraded dorsal surface of the rats 7 days per week for 28 days. Group 1 served as a control group. Groups 2, 3, and 4 received 100, 300, and 1000 mg/kg of the test material, respectively. Group 5 served as a satellite recovery group. The satellite animals were treated with test material at 1000 mg/kg and then observed for 14 days after treatment for reversibility, persistence, or delayed occurrence of toxic effects. Individual doses were adjusted weekly based on the most recent body weights.

Clinical observations were performed daily throughout the study and dermal responses were evaluated at specified intervals according to the Draize method. Body weight was recorded pretest, at dose initiation (Day 0), on Days 7, 14, 21 and 27, on the day of death, and on Days 35 and 41 for the satellite animals. Food consumption was measured weekly. Hematology and serum chemistry samples were taken from all animals on Day 28 and from all surviving satellite animals on Day 42. A full macroscopic postmortem examination was performed on all animals and required organs were preserved. Selected organs were weighed at study termination. A range of tissues was examined microscopically.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were made as to the nature, onset, severity, and duration of toxicological signs daily during the study.

DERMAL IRRITATION : Yes
- Time schedule for examinations: Dermal evaluations were performed prior to dosing on Days 0, 1, 3, 7, 10, 14, 17, 21 and 24. Dermal evaluations also were performed after sleeve removal on Day 0 and prior to blood collection on Day 28. Additional dermal evaluations were performed for the satellite animals on Days 31, 35, 38 and 42. All evaluations were performed according to the Draize method.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded prior to initiation of dosing for group allocation. Body weights also were recorded on the day of initiation of dosing (Day 0), on Days 7, 14, 21, and 27, and on the day of death. Additionally, body weights were recorded for the satellite animals on Days 35 and 41.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured on Days 7, 14, 21 and 27 during the test period. Food consumption also was measured for the satellite animals on Days 35 and 41. The Weeks 4 and 6 food consumption values were based on 6 days due to fasting of the animals on Days 27 and 41 for blood collection on Days 28 and 42, respectively.

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: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: samples were taken from all animals on Day 28 and from all satellite animals on Day 42
- Anaesthetic used for blood collection: Yes - methoxyflurane
- Animals fasted: Yes, overnight fast
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:samples were taken from all animals on Day 28 and from all satellite animals on Day 42
- Animals fasted: Yes, overnight fast
- How many animals: all animals

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
All animals were sacrificed via exsanguination following methoxyflurane anesthesia and subjected to a gross necropsy.

The following organs were weighed prior to placement in fixative from all animals in Groups 1, 2, 3, and 4 on Day 28 and all animals from Group 5 on Day 42 : liver, testes, brain, kidneys and adrenals.

The following tissues were removed from all animals and preserved in 10% neutral buffered formalin: adrenals, aorta, brain (3 levels), esophagus,
epididymides, exorbital lacrimal gland, eyes, femoris muscle with sciatic nerve, femur with articular surfaces, Harderian gland, heart, kidneys, large intestine (sections from colon and cecum), liver, lung (with mainstem bronchi), mammary gland (female), mesenteric lymph node, ovaries and oviducts, pancreas, pituitary, prostate, rectum, salivary glands/mandibular lymph node, seminal vesicles, skin (treated and untreated), small intestine (sections from duodenum, jejunum, and ileum), spinal cord at 3 levels (cervical, mid-thoracic, and lumbar), spleen, sternum with marrow, stomach, testes, thymus, thyroid (parathyroids), trachea, urinary bladder, uterus (corpus, cervix), all gross lesions.

The following tissues from animals in Groups 1, 4, and 5 were processed, sectioned, stained (hematoxylin and eosin) and examined microscopically: liver, kidneys, testes, adrenals, skin (treated and untreated) gross lesions. The liver, kidneys, skin (treated and untreated), and gross lesions from the low and mid-dose group animals also were processed and examined microscopically.






Other examinations:
No data
Statistics:
Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test for ordered response in the dose groups. First, Bartlett's Test was performed to determine if the dose groups had equal variance. If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used.

For the parametric procedures, a standard one way ANOVA using the F distribution to assess significance was used. If significant differences among the means were indicated, Dunnett's Test was used to determine which treatment groups differed significantly from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed. The regression also tested for linear lack of fit in the model.

For the nonparametric procedures the test of equality of means was performed using the Kruskal-Wallis Test. If significant differences among the means were indicated, Dunn's Summed Rank Test was used to determine which treatment groups differed significantly from the control. In addition to the Kruskal-Wallis Test, Jonckheere's Test for monotonic trend in the dose response was performed.

Bartlett's Test for equal variance was conducted at the 1% level of significance. All other tests were conducted at the 5% and 1% level of significance.

The statistical t-test was used to compare the satellite group's main study termination and recovery termination hematology and clinical chemistry values. The t-test was used to compare the satellite group's Day 42 relative organ weight with the control group's Day 28 relative organ weight and also to compare the satellite group's Day 42 relative organ weight with the high dose group's Day 28 relative organ weight.

The statistical t-test also was used to compare the high dose and satellite animals to substantiate their equivalence in order to accurately evaluate the recovery effect.
Clinical signs:
no effects observed
Description (incidence and severity):
incidental findings of occasional sores or scabs among males and one mid dose female with an umbilical hernia but these signs were concerned as a no treatment- related. Most animals survived to terminal sacrifice on Day 28 or 42.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
transient dermal irritation observed in several treated females
Mortality:
no mortality observed
Description (incidence):
incidental findings of occasional sores or scabs among males and one mid dose female with an umbilical hernia but these signs were concerned as a no treatment- related. Most animals survived to terminal sacrifice on Day 28 or 42.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
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 specified
Details on results:
CLINICAL SIGNS AND MORTALITY
Most animals survived to terminal sacrifice on Day 28 or 42 (satellite) and there were no treatment-related clinical signs of toxicity in the animals at any dose level. Incidental findings were limited to occasional sores or scabs among males, and one mid dose female with an umbilical hernia.
One control group female was found dead on Day 27. She was not observed with any abnormalities during the study.

DERMAL OBSERVATIONS
All males were free of erythema and/or edema throughout the study with the exception of one satellite male observed with very slight erythema on Day 28. Additionally, desquamation was observed in one 300 mg/kg male on Day 7 and in one satellite animal on Day 28. [See attached Table 2 ( males)].
Very slight to slight edema was noted in several treated females on Day 3 and very slight edema was noted in one satellite female on Day 21 [See attached Table 2 (females- edema)]. Very slight to well defined erythema was noted in several animals on Day 3 and/or Day 7, and in one satellite animal on Days 10, 17, 21, and 24 [ See attached Table 2 (females- erythema)].

BODY WEIGHT AND WEIGHT GAIN
All animals displayed increases in body weight over their initial values. There were no statistically significant differences in mean body weight or mean body weight change between treated and control animals of either sex. Mean body weight of the satellite animals (not included in statistical analysis) appeared comparable to controls and other treated groups.

FOOD CONSUMPTION
There were no statistically significant differences in mean food consumption between treated and control animals of either sex. Mean food consumption of the satellite animals (not included in statistical analysis) appeared comparable to controls and other treated groups.

HAEMATOLOGY
There were no statistically significant differences in quantitative or semi-quantitative hematological parameters between treated and control animals of either sex at main study termination (Day 28). Mean hematological values of the satellite animals (not included in statistical analysis) also appeared comparable to controls and/or to other treated groups on Day 28.

Following the recovery period, there were several statistically significant differences in semi-quantitative hematological parameters in satellite group animals There was a statistically significant decrease in mean corpuscular hemoglobin concentration in the male satellite from Day 28 to Day 42. This small difference (<4%) was not considered clinically significant.

SERUM CHEMISTRY
There were no statistically significant differences in serum chemistry parameters between treated and control animals of either sex at main study termination, with the exception of a small increase (p¿0.05) in mean potassium of the high dose males compared with controls. This minor difference (<11%) were not considered clinically significant. Mean serum chemistry values of the satellite animals (not included in statistical analysis) appeared comparable to controls and/or other treated groups on Day 28.

Following the recovery period, there were several statistically significant differences in serum chemistry parameters of the satellite group animals from Day 28 to Day 42. These included statistically significant increases in mean glucose and calcium in the males; mean total protein and albumin in the females; and a decrease in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase in males; and mean sodium in the females. These differences were not considered clinically significant since they were limited to the recovery phase, and they occurred in the absence of other corroborating clinical or histopathological effects. The absence of significant findings at main study termination further supports the conclusion that these findings were spurious and unrelated to the test material.

ORGAN WEIGHTS
There were no statistically significant differences in mean absolute organ weight, organ to body weight ratio, or organ to brain weight ratio between the treated and control males at main study termination.

There were statistically significant decreases in mean absolute liver weights for the low, mid, and high dose females when compared with the controls. However, there were no statistically significant differences in mean organ to body weight ratio or organ to brain weight ratio between the treated and control females at main study termination. Thus, the decreased absolute liver weights were attributed to differences in terminal body weight rather than an adverse effect. Additionally, following recovery, the female satellite animals did not show any statistically significant differences in liver weight when compared with the controls or the high dose group.

Following recovery, there were several small, but statistically significant differences in relative organ weights. These included a statistically significant decrease in mean testes to body weight and brain to body weight ratios of the satellites on Day 42 compared with controls on Day 28 or the high dose group on Day 28. Additionally, there was a statistically significant decrease in mean brain to body weight ratio of the satellite females compared to the high dose. These differences were not considered biologically significant since they were limited to the recovery phase, and they occurred in the absence of other corroborating clinical or histopathological effects. The absence of significant findings at main study termination further supports the conclusion that these findings were spurious and unrelated to the test material.

GROSS PATHOLOGY
There were no gross postmortem observations which were judged to be related to the test material, and the majority of animals were free of observable abnormalities at termination. Postmortem findings were limited to single or low incidences of sores/scabs; folder, adhered liver; discolored liver, thymus, or mandibular lymph nodes; distended uterus; dilated renal pelvis; and/or a mass in the liver.

HISTOPATHOLOGY: NON-NEOPLASTIC

There were no microscopic changes observed in any of the organs or tissues examined to indicate any systemic toxicity or cutaneous irritation from the dermal application of the test material.

Microscopic examination of the treated area of the skin revealed acanthosis and hyperkeratosis of the epidermis in rats of all groups, including controls. Other accompanying changes were variable amounts of hyperplasia of sebaceous glands and focal dermal inflammation. There was essentially no difference in incidence or severity of these changes in the skin of the control rats or 100, 300, or 1000 mg/kg of the test material. As the changes occurred at similar incidences and severity in both control and treated animals, these changes were considered secondary to the repeated shaving of the skin, and/or wrapping and taping of the treatment site. The untreated skin also displayed microscopic changes but this was considered to be due to migration of the test material from the treated skin and/or the repeated shaving and wrapping of this area of the skin. The test material did not increase the severity of any changes and thus, was not considered to have caused any cutaneous irritating effects. Following recovery, the treated skin showed similar types of changes, but the severity and/or incidence was decreased indicating reversibility.

All other microscopic changes observed were considered to have occurred spontaneously and to have been incidental and unrelated to treatment.

The most common incidental finding was a minimal amount of multifocal mononuclear inflammatory cell infiltrations in the liver. The incidence and severity of this common incidental finding was not affected by the test material. Additionally, focal subcapsular necrosis was observed in the liver of a few rats of the various groups, including two control rats. This type of necrosis is consistent with that which has been associated with wrapping of the abdomen for dermal studies.

Incidental findings in the kidneys included single or low incidences per affected group of focal cortical tubular degeneration, pelvic dilation, increased cytoplasmic eosinophilia in cortical tubules, pyelonephritis, multifocal mineralization, focal chronic nephritis, a medullary cyst, and focal mononuclear-cell infiltrations.


Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse systemic findings were recorded up to the limit dose of 1000 mg/kg/day
Critical effects observed:
not specified
Conclusions:
In conclusion, topical application of the test material under conditions of this study elicited no signs of systemic toxicity. The only significant finding was transient dermal irritation observed in several treated females. There were no adverse effects with respect to clinical signs, body weights, body weight changes, food consumption, clinical parameters, organ weights, postmortem observations, or microscopic changes. Accordingly, a No Observable Adverse Effect Level (NOAEL) was established at 1000 mg/kg of the test material in both male and female rats.
Executive summary:

The comparison of overall physicochemical and toxicity profiles for tested and analogue materials indicates it is appropriate to apply read-across data from the structural surrogate when considering the repeated dose toxicity data.

This study was conducted to evaluate the potential toxicity of the test material following repeated dermal application to fifty Crl:CD®BR rats for a minimum of 28 days. Animals were divided into 5 groups: Group 1 -control group, Groups 2, 3, and 4 received 100, 300, and 1000 mg/kg of the test material, respectively, Group 5- satellite recovery group that was treated with test material at 1000 mg/kg and then observed for 14 days after treatment for reversibility, persistence, or delayed occurrence of toxic effects. Clinical observations were performed daily throughout the study and dermal responses were evaluated at specified intervals according to the Draize method. Body weight was recorded pre-test, at dose initiation (Day 0), on Days 7, 14, 21 and 27, on the day of death, and on Days 35 and 41 for the satellite animals. Food consumption was measured weekly. Haematology and serum chemistry samples were taken from all animals on Day 28 and from all surviving satellite animals on Day 42. A full macroscopic post-mortem examination was performed on all animals and required organs were preserved. Selected organs were weighed at study termination. A range of tissues was examined microscopically.

Most animals survived to terminal sacrifice on Day 28 or 42 (satellite) and were free of treatment-related clinical signs of toxicity. Slight transient dermal irritation was the only significant finding observed, and was limited to very slight to well-defined erythema, very slight to slight oedema, and/or desquamation in several treated group females primarily during Week 1. There were no biologically meaningful differences among the groups with respect to mean body weights, body weight gain, food consumption, haematological parameters, serum chemistry parameters, absolute organ weights, organ-to-body weight ratios, or organ-to-brain weight ratios. Similarly, there were no meaningful changes in these parameters following the recovery period. 

There were no gross post-mortem observations or microscopic changes which were judged to be related to the test material. There were no microscopic changes considered to be the result of any irritating effect or systemic toxicity of the topically applied test material. The most notable microscopic changes consisted of acanthosis (thickening) and hyperkeratosis of the epidermis, sebaceous gland hyperplasia and focal dermal inflammation in the treated areas of skin of male and female animals from all groups, including the control. As the changes occurred at similar incidences and severity in both control and treated animals, these changes were considered secondary to the repeated shaving of the skin, and/or wrapping and taping of the treatment site and not test material-related. All other microscopic changes were considered typical to those that occur spontaneously in young laboratory rats of this age and strain and not treatment-related.

 

In conclusion, topical application of the test material under conditions of this study elicited no signs of systemic toxicity. The only significant finding was transient dermal irritation observed in several treated females. There were no adverse effects with respect to clinical signs, body weights, body weight changes, food consumption, clinical pathology parameters, organ weights, post-mortem observations, or microscopic changes. Accordingly, a No Observable Adverse Effect Level (NOAEL) was established at 1000 mg/kg of the test material in both male and female rats. 

 

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse systemic findings were recorded up to the limit dose (1000 mg/kg bw/day).
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD guidelines followed but no statement of GLP. Restriction due to the fact that the study was conducted on read- across substance. Klimisch score 2.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
27.01. 1997- 19-07.1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guidelines followed but no statement of GLP. Restriction due to the fact that the study was conducted on read-across substance.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Crl:CD®BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston Facility, Stone Ridge, New York
- Age at study initiation: Males: Approximately 7-8 weeks, Females: Approximately 9-10 weeks
- Weight at study initiation: Males: 246.3 to 270.2 grams, Females: 197.9 to 245.9 grams
- Fasting period before study: no data
- Housing: Single housed during the test period, Caging: Suspended stainless steel and wire mesh with absorbent paper below cages
- Diet (e.g. ad libitum): PMI Certified Rodent Diet Meal 5002, ad libitum
- Water (e.g. ad libitum): Automatic Watering System, ad libitum
- Acclimation period: 13 days; animals were examined for viability at least once daily

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-22.3
- Humidity (%): 30 to 70 percent relative humidity
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light (0700 to 1900 Hours) and 12 hours dark (1900 to 0700 Hours) by automatic timer.

IN-LIFE DATES: From: January 28, 1997 To March 11, 1997
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: the dorsal surface from the shoulder region to the lumbar region
- % coverage: approximately 10% of the body surface
- Type of wrap if used: COBAN wrap
- Time intervals for shavings or clipplings: at least once weekly

REMOVAL OF TEST SUBSTANCE
- Washing : gentle wipe of the exposure site with peanut oil and a paper towel
- Time after start of exposure: approximately 6 hours after exposure

TEST MATERIAL
- Amounts applied :100mg/kg, 300mg/kg, 1000 mg/kg


USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, The animal was wrapped with COBAN to prevent ingestion of the test material.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal per unit area
Remarks:
Doses / Concentrations:
1000 mg/kg (satellite group)
Basis:
nominal per unit area
No. of animals per sex per dose:
Group 1 ( control group)- 0 mg/kg (sham dose )- 5 males and 5 females
Group 2- 100 mg/kg- 5 males and 5 females
Group 3- 300 mg/kg- 5 males and 5 females
Group 4- 1000 mg/kg- 5 males and 5 females
Group 5 (satellite recovery group) - 1000 mg/kg- 5 males and 5 females
Control animals:
yes, sham-exposed
Details on study design:
Dose levels were selected based on findings of previously conducted studies with this material including an acute dermal toxicity study (LD50 > 2000 mg/kg) and 28-day oral toxicity study (no signs of overt toxicity at the limit dose level of 1000 mg/kg).

All animals were examined for abnormalities prior to dose initiation and those determined to be unsuitable for inclusion on this study because of poor health, outlying body weights, or other abnormalities were excluded from selection. Study animals were selected from the suitable animals by a computer generated sorting program and their weight variation within their sex was within ±20% of the mean body weight by sex.

Fifty Crl:CD®BR rats were divided into 5 groups of 5 male and 5 female rats each. The test material was applied to the clipped, unabraded dorsal surface of the rats 7 days per week for 28 days. Group 1 served as a control group. Groups 2, 3, and 4 received 100, 300, and 1000 mg/kg of the test material, respectively. Group 5 served as a satellite recovery group. The satellite animals were treated with test material at 1000 mg/kg and then observed for 14 days after treatment for reversibility, persistence, or delayed occurrence of toxic effects. Individual doses were adjusted weekly based on the most recent body weights.

Clinical observations were performed daily throughout the study and dermal responses were evaluated at specified intervals according to the Draize method. Body weight was recorded pretest, at dose initiation (Day 0), on Days 7, 14, 21 and 27, on the day of death, and on Days 35 and 41 for the satellite animals. Food consumption was measured weekly. Hematology and serum chemistry samples were taken from all animals on Day 28 and from all surviving satellite animals on Day 42. A full macroscopic postmortem examination was performed on all animals and required organs were preserved. Selected organs were weighed at study termination. A range of tissues was examined microscopically.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were made as to the nature, onset, severity, and duration of toxicological signs daily during the study.

DERMAL IRRITATION : Yes
- Time schedule for examinations: Dermal evaluations were performed prior to dosing on Days 0, 1, 3, 7, 10, 14, 17, 21 and 24. Dermal evaluations also were performed after sleeve removal on Day 0 and prior to blood collection on Day 28. Additional dermal evaluations were performed for the satellite animals on Days 31, 35, 38 and 42. All evaluations were performed according to the Draize method.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded prior to initiation of dosing for group allocation. Body weights also were recorded on the day of initiation of dosing (Day 0), on Days 7, 14, 21, and 27, and on the day of death. Additionally, body weights were recorded for the satellite animals on Days 35 and 41.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured on Days 7, 14, 21 and 27 during the test period. Food consumption also was measured for the satellite animals on Days 35 and 41. The Weeks 4 and 6 food consumption values were based on 6 days due to fasting of the animals on Days 27 and 41 for blood collection on Days 28 and 42, respectively.

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: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: samples were taken from all animals on Day 28 and from all satellite animals on Day 42
- Anaesthetic used for blood collection: Yes - methoxyflurane
- Animals fasted: Yes, overnight fast
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:samples were taken from all animals on Day 28 and from all satellite animals on Day 42
- Animals fasted: Yes, overnight fast
- How many animals: all animals

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
All animals were sacrificed via exsanguination following methoxyflurane anesthesia and subjected to a gross necropsy.

The following organs were weighed prior to placement in fixative from all animals in Groups 1, 2, 3, and 4 on Day 28 and all animals from Group 5 on Day 42 : liver, testes, brain, kidneys and adrenals.

The following tissues were removed from all animals and preserved in 10% neutral buffered formalin: adrenals, aorta, brain (3 levels), esophagus,
epididymides, exorbital lacrimal gland, eyes, femoris muscle with sciatic nerve, femur with articular surfaces, Harderian gland, heart, kidneys, large intestine (sections from colon and cecum), liver, lung (with mainstem bronchi), mammary gland (female), mesenteric lymph node, ovaries and oviducts, pancreas, pituitary, prostate, rectum, salivary glands/mandibular lymph node, seminal vesicles, skin (treated and untreated), small intestine (sections from duodenum, jejunum, and ileum), spinal cord at 3 levels (cervical, mid-thoracic, and lumbar), spleen, sternum with marrow, stomach, testes, thymus, thyroid (parathyroids), trachea, urinary bladder, uterus (corpus, cervix), all gross lesions.

The following tissues from animals in Groups 1, 4, and 5 were processed, sectioned, stained (hematoxylin and eosin) and examined microscopically: liver, kidneys, testes, adrenals, skin (treated and untreated) gross lesions. The liver, kidneys, skin (treated and untreated), and gross lesions from the low and mid-dose group animals also were processed and examined microscopically.






Other examinations:
No data
Statistics:
Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test for ordered response in the dose groups. First, Bartlett's Test was performed to determine if the dose groups had equal variance. If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used.

For the parametric procedures, a standard one way ANOVA using the F distribution to assess significance was used. If significant differences among the means were indicated, Dunnett's Test was used to determine which treatment groups differed significantly from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed. The regression also tested for linear lack of fit in the model.

For the nonparametric procedures the test of equality of means was performed using the Kruskal-Wallis Test. If significant differences among the means were indicated, Dunn's Summed Rank Test was used to determine which treatment groups differed significantly from the control. In addition to the Kruskal-Wallis Test, Jonckheere's Test for monotonic trend in the dose response was performed.

Bartlett's Test for equal variance was conducted at the 1% level of significance. All other tests were conducted at the 5% and 1% level of significance.

The statistical t-test was used to compare the satellite group's main study termination and recovery termination hematology and clinical chemistry values. The t-test was used to compare the satellite group's Day 42 relative organ weight with the control group's Day 28 relative organ weight and also to compare the satellite group's Day 42 relative organ weight with the high dose group's Day 28 relative organ weight.

The statistical t-test also was used to compare the high dose and satellite animals to substantiate their equivalence in order to accurately evaluate the recovery effect.
Clinical signs:
no effects observed
Description (incidence and severity):
incidental findings of occasional sores or scabs among males and one mid dose female with an umbilical hernia but these signs were concerned as a no treatment- related. Most animals survived to terminal sacrifice on Day 28 or 42.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
transient dermal irritation observed in several treated females
Mortality:
no mortality observed
Description (incidence):
incidental findings of occasional sores or scabs among males and one mid dose female with an umbilical hernia but these signs were concerned as a no treatment- related. Most animals survived to terminal sacrifice on Day 28 or 42.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
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 specified
Details on results:
CLINICAL SIGNS AND MORTALITY
Most animals survived to terminal sacrifice on Day 28 or 42 (satellite) and there were no treatment-related clinical signs of toxicity in the animals at any dose level. Incidental findings were limited to occasional sores or scabs among males, and one mid dose female with an umbilical hernia.
One control group female was found dead on Day 27. She was not observed with any abnormalities during the study.

DERMAL OBSERVATIONS
All males were free of erythema and/or edema throughout the study with the exception of one satellite male observed with very slight erythema on Day 28. Additionally, desquamation was observed in one 300 mg/kg male on Day 7 and in one satellite animal on Day 28. [See attached Table 2 ( males)].
Very slight to slight edema was noted in several treated females on Day 3 and very slight edema was noted in one satellite female on Day 21 [See attached Table 2 (females- edema)]. Very slight to well defined erythema was noted in several animals on Day 3 and/or Day 7, and in one satellite animal on Days 10, 17, 21, and 24 [ See attached Table 2 (females- erythema)].

BODY WEIGHT AND WEIGHT GAIN
All animals displayed increases in body weight over their initial values. There were no statistically significant differences in mean body weight or mean body weight change between treated and control animals of either sex. Mean body weight of the satellite animals (not included in statistical analysis) appeared comparable to controls and other treated groups.

FOOD CONSUMPTION
There were no statistically significant differences in mean food consumption between treated and control animals of either sex. Mean food consumption of the satellite animals (not included in statistical analysis) appeared comparable to controls and other treated groups.

HAEMATOLOGY
There were no statistically significant differences in quantitative or semi-quantitative hematological parameters between treated and control animals of either sex at main study termination (Day 28). Mean hematological values of the satellite animals (not included in statistical analysis) also appeared comparable to controls and/or to other treated groups on Day 28.

Following the recovery period, there were several statistically significant differences in semi-quantitative hematological parameters in satellite group animals There was a statistically significant decrease in mean corpuscular hemoglobin concentration in the male satellite from Day 28 to Day 42. This small difference (<4%) was not considered clinically significant.

SERUM CHEMISTRY
There were no statistically significant differences in serum chemistry parameters between treated and control animals of either sex at main study termination, with the exception of a small increase (p¿0.05) in mean potassium of the high dose males compared with controls. This minor difference (<11%) were not considered clinically significant. Mean serum chemistry values of the satellite animals (not included in statistical analysis) appeared comparable to controls and/or other treated groups on Day 28.

Following the recovery period, there were several statistically significant differences in serum chemistry parameters of the satellite group animals from Day 28 to Day 42. These included statistically significant increases in mean glucose and calcium in the males; mean total protein and albumin in the females; and a decrease in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase in males; and mean sodium in the females. These differences were not considered clinically significant since they were limited to the recovery phase, and they occurred in the absence of other corroborating clinical or histopathological effects. The absence of significant findings at main study termination further supports the conclusion that these findings were spurious and unrelated to the test material.

ORGAN WEIGHTS
There were no statistically significant differences in mean absolute organ weight, organ to body weight ratio, or organ to brain weight ratio between the treated and control males at main study termination.

There were statistically significant decreases in mean absolute liver weights for the low, mid, and high dose females when compared with the controls. However, there were no statistically significant differences in mean organ to body weight ratio or organ to brain weight ratio between the treated and control females at main study termination. Thus, the decreased absolute liver weights were attributed to differences in terminal body weight rather than an adverse effect. Additionally, following recovery, the female satellite animals did not show any statistically significant differences in liver weight when compared with the controls or the high dose group.

Following recovery, there were several small, but statistically significant differences in relative organ weights. These included a statistically significant decrease in mean testes to body weight and brain to body weight ratios of the satellites on Day 42 compared with controls on Day 28 or the high dose group on Day 28. Additionally, there was a statistically significant decrease in mean brain to body weight ratio of the satellite females compared to the high dose. These differences were not considered biologically significant since they were limited to the recovery phase, and they occurred in the absence of other corroborating clinical or histopathological effects. The absence of significant findings at main study termination further supports the conclusion that these findings were spurious and unrelated to the test material.

GROSS PATHOLOGY
There were no gross postmortem observations which were judged to be related to the test material, and the majority of animals were free of observable abnormalities at termination. Postmortem findings were limited to single or low incidences of sores/scabs; folder, adhered liver; discolored liver, thymus, or mandibular lymph nodes; distended uterus; dilated renal pelvis; and/or a mass in the liver.

HISTOPATHOLOGY: NON-NEOPLASTIC

There were no microscopic changes observed in any of the organs or tissues examined to indicate any systemic toxicity or cutaneous irritation from the dermal application of the test material.

Microscopic examination of the treated area of the skin revealed acanthosis and hyperkeratosis of the epidermis in rats of all groups, including controls. Other accompanying changes were variable amounts of hyperplasia of sebaceous glands and focal dermal inflammation. There was essentially no difference in incidence or severity of these changes in the skin of the control rats or 100, 300, or 1000 mg/kg of the test material. As the changes occurred at similar incidences and severity in both control and treated animals, these changes were considered secondary to the repeated shaving of the skin, and/or wrapping and taping of the treatment site. The untreated skin also displayed microscopic changes but this was considered to be due to migration of the test material from the treated skin and/or the repeated shaving and wrapping of this area of the skin. The test material did not increase the severity of any changes and thus, was not considered to have caused any cutaneous irritating effects. Following recovery, the treated skin showed similar types of changes, but the severity and/or incidence was decreased indicating reversibility.

All other microscopic changes observed were considered to have occurred spontaneously and to have been incidental and unrelated to treatment.

The most common incidental finding was a minimal amount of multifocal mononuclear inflammatory cell infiltrations in the liver. The incidence and severity of this common incidental finding was not affected by the test material. Additionally, focal subcapsular necrosis was observed in the liver of a few rats of the various groups, including two control rats. This type of necrosis is consistent with that which has been associated with wrapping of the abdomen for dermal studies.

Incidental findings in the kidneys included single or low incidences per affected group of focal cortical tubular degeneration, pelvic dilation, increased cytoplasmic eosinophilia in cortical tubules, pyelonephritis, multifocal mineralization, focal chronic nephritis, a medullary cyst, and focal mononuclear-cell infiltrations.


Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse systemic findings were recorded up to the limit dose of 1000 mg/kg/day
Critical effects observed:
not specified
Conclusions:
In conclusion, topical application of the test material under conditions of this study elicited no signs of systemic toxicity. The only significant finding was transient dermal irritation observed in several treated females. There were no adverse effects with respect to clinical signs, body weights, body weight changes, food consumption, clinical parameters, organ weights, postmortem observations, or microscopic changes. Accordingly, a No Observable Adverse Effect Level (NOAEL) was established at 1000 mg/kg of the test material in both male and female rats.
Executive summary:

The comparison of overall physicochemical and toxicity profiles for tested and analogue materials indicates it is appropriate to apply read-across data from the structural surrogate when considering the repeated dose toxicity data.

This study was conducted to evaluate the potential toxicity of the test material following repeated dermal application to fifty Crl:CD®BR rats for a minimum of 28 days. Animals were divided into 5 groups: Group 1 -control group, Groups 2, 3, and 4 received 100, 300, and 1000 mg/kg of the test material, respectively, Group 5- satellite recovery group that was treated with test material at 1000 mg/kg and then observed for 14 days after treatment for reversibility, persistence, or delayed occurrence of toxic effects. Clinical observations were performed daily throughout the study and dermal responses were evaluated at specified intervals according to the Draize method. Body weight was recorded pre-test, at dose initiation (Day 0), on Days 7, 14, 21 and 27, on the day of death, and on Days 35 and 41 for the satellite animals. Food consumption was measured weekly. Haematology and serum chemistry samples were taken from all animals on Day 28 and from all surviving satellite animals on Day 42. A full macroscopic post-mortem examination was performed on all animals and required organs were preserved. Selected organs were weighed at study termination. A range of tissues was examined microscopically.

Most animals survived to terminal sacrifice on Day 28 or 42 (satellite) and were free of treatment-related clinical signs of toxicity. Slight transient dermal irritation was the only significant finding observed, and was limited to very slight to well-defined erythema, very slight to slight oedema, and/or desquamation in several treated group females primarily during Week 1. There were no biologically meaningful differences among the groups with respect to mean body weights, body weight gain, food consumption, haematological parameters, serum chemistry parameters, absolute organ weights, organ-to-body weight ratios, or organ-to-brain weight ratios. Similarly, there were no meaningful changes in these parameters following the recovery period. 

There were no gross post-mortem observations or microscopic changes which were judged to be related to the test material. There were no microscopic changes considered to be the result of any irritating effect or systemic toxicity of the topically applied test material. The most notable microscopic changes consisted of acanthosis (thickening) and hyperkeratosis of the epidermis, sebaceous gland hyperplasia and focal dermal inflammation in the treated areas of skin of male and female animals from all groups, including the control. As the changes occurred at similar incidences and severity in both control and treated animals, these changes were considered secondary to the repeated shaving of the skin, and/or wrapping and taping of the treatment site and not test material-related. All other microscopic changes were considered typical to those that occur spontaneously in young laboratory rats of this age and strain and not treatment-related.

 

In conclusion, topical application of the test material under conditions of this study elicited no signs of systemic toxicity. The only significant finding was transient dermal irritation observed in several treated females. There were no adverse effects with respect to clinical signs, body weights, body weight changes, food consumption, clinical pathology parameters, organ weights, post-mortem observations, or microscopic changes. Accordingly, a No Observable Adverse Effect Level (NOAEL) was established at 1000 mg/kg of the test material in both male and female rats. 

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Repeated dose toxicity oral

Modern GLP studies conducted in accordance with the OECD 407 and 408 guidelines and the EU Method B.7 were designed to investigate the systemic toxicity of the test material, by repeated oral administration to the Wistar Han™:HsdRccHan™:WIST strain rat for a period of twenty- eight and ninety consecutive days repectively at dose levels of 30, 300 and 1000 mg/kg/day did not result in any treatment-related effects.The No Observable Effect Level (NOEL) for animals of either sex was therefore considered to be 1000 mg/kg/day.

Repeated dose toxicity dermal

A study on an appropriate read-across material conducted in accordance with the OECD 410 guideline and the EU Method B.9 was designed to investigate the potential toxicity of the analogue test material following repeated dermal application to fifty Crl:CD BR rats for a minimum of 28 days.Most animals survived to terminal sacrifice on Day 28 or 42 (satellite) and were free of treatment-related clinical signs of toxicity. Slight transient dermal irritation was the only significant finding observed, and was limited to very slight to well-defined erythema, very slight to slight oedema, and/or desquamation in several treated group females primarily during Week 1. There were no gross post-mortem observations or microscopic changes which were judged to be related to the test material. There were no microscopic changes considered to be the result of any irritating effect or systemic toxicity of the topically applied test material.

The No Observable Adverse Effect Level (NOAEL) was established at 1000 mg/kg of the test material in both male and female rats.

The result on the analogue material was considered to be appropriate for the Registration material.

 

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable studies for a submission substance, GLP compliant with a Klimisch score 1.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Reliable study for applicable material available.

Justification for classification or non-classification

Repeated dose toxicity oral

Results from modern GLP studies conducted in accordance with the OECD 407 and OECD 408 guidelines & the EU Method B.7 reported no treatment- related effects. The test substance is not considered to require classification under the terms of CLP Regulation (EC) No 1272/ 2008.

Repeated dose toxicity dermal

Results from a study on an appropriate read-across material conducted in accordance with the OECD 410 guideline & the EU Method B.9 reported no treatment-related effects. The analogue test substance is not considered to require classification under the terms of CLP Regulation (EC) No 1272/ 2008.