4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Endpoint summary

Description of key information

Oral: NOAEL = 50 ppm and 150 ppm in males and females, respectively, equivalent to dose levels of 3.9 mg/kg bw/day for males and 13.4 mg/kg bw/day for females, rats, sub-chronic, 3 months, feeding, OECD TG 408, Laidlaw 2019.

Dermal: NOAEL = 100 mg/kg bw/day in males and females, rats, sub-acute, 28 days, semi-occlusive dermal application (6 hours per day, five days per week), OECD TG 410, Cooper 2019

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Dec 2014 to 01 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This longer-term repeated dose toxicity study was generated to meet the data requirements of regulations not related to REACH in non-EEA countries.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 417. Toxicokinetics

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

1998

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approx. 7 - 8 weeks
- Weight at study initiation: range 202 - 242 g for males and 129 - 172 g for females
- Housing: Two per cage by sex from study assignment in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. Sterilised white wood shavings were provided as bedding.
- Diet: Rat and Mouse diet, ad libitum
- Water: Tap water, ad libitum.
- Acclimation period: 14 days prior.

DETAILS OF FOOD AND WATER QUALITY:
- Each batch of diet was routinely analysed by the supplier for various nutritional components and chemical and microbiological contaminants. The quality of water supply is stipulated by Water Quality (Scotland) Regulations 2001 and certificates of analysis for dissolved materials, heavy metals, pesticide residues, pH, nitrates and selected bacteria are periodically provided

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): minimum 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 02 Dec 2014 To 05 Mar 2015

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DIET FORMULATIONS
Diet formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements.
Diet formulations were prepared as often as required and dispatched to the animal room. Once in the animal room diet formulations were held at ambient temperature. Diet formulations were prepared as serial dilutions from 200 g of a more concentrated stock. The stock was prepared by first mixing the test substance with the required amount of untreated control diet in an automated mortar and pestle and was then mixed until visibly homogeneous. Diet for the high dose group was prepared by adding the more concentrated stock prepared to a suitable sized diet bin, adding an appropriate amount of untreated diet and this was then blended for 20 min in a drum mixer. The diets at lower concentrations were prepared as a serial dilution from the high dose group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLE COLLECTION AND ANALYSIS
Analysis samples were collected from diets formulated, to be fed to animals as follows. In week 1, 6 and 12 concentations and homogeneity for all groups were analysed.
Samples to be analysed were transferred at ambient temperature to the analytical laboratory at the Test Facility within two days of preparation. The samples were stored at ambient temperature in the dark prior to analysis during Week 1 and 6 and analysed on receipt during Week 12. in week 1, 6 and 12.

CONCENTRATION AND HOMOGENEITY ANALYSIS
- Samples for Analysis: Duplicate top, middle, and bottom samples (duplicate middle only for control).
- Backup Samples: Triplicate top, middle, and bottom samples (duplicate middle only for control); maintained at the Test Facility. Backup samples were not analysed.
- Sampling Containers: Appropriate sized glass container or polypropylene containers.
- Sample Volume: 10 g for analysis and 20 g for back up samples.
- Storage Conditions: Kept at ambient temperature or in a freezer set to maintain -20 °C.
- Acceptance Criteria: For concentration, the criteria for acceptability were mean sample concentration results within or equal to ± 10 % For homogeneity, the criteria for acceptability were a relative standard deviation (RSD) of concentrations of < 10 % for each group.

STABILITY ANALYSIS
Stability analysis performed previously in conjunction with a previous study demonstrated that the test substance was stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Continuously

Dose / conc.:

50 ppm

Remarks:

Group 2. Low dose, dietary concentration; equivalent to 3.9 and 4.4 mg/kg bw/day for males and females, respectively

Dose / conc.:

150 ppm

Remarks:

Group 3. Mid dose, dietary concentration; equivalent to 11.2 and 13.4 mg/kg bw/day for males and females, respectively

Dose / conc.:

300 ppm

Remarks:

Group 4. High dose, dietary concentration; equivalent to 22.0 and 24.0 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

DOSE SELECTION RATIONALE
The oral (dietary) route of administration was selected for this study as this route had been defined by the Sponsor as a possible route of human exposure. The dosage levels were agreed with the Sponsor after evaluation of the data from previous 14 and 28 day studies carried out at the performing labpratory. In these studies, dose levels of 700 ppm and above were not tolerated (mortality was seen in all females above 800 ppm, and 1 female at 700 ppm). A dose of 300 ppm, which was proposed as the high dose for this 13 week study, was expected to be tolerated, but was likely to result in a reduced overall body weight gain for both sexes (females dosed at 300 ppm in 28 day study showed reduced bodyweight gain compared to the control animals, as did the males dosed at 350 ppm). Some clinical chemistry/haematology effects were observed at 100 ppm in the second 28 day study , while at the lower dose of 50 ppm (in the first study), no effects were observed. Therefore 50 ppm has been selected as the low dose and is intended to be the No Observed Adverse Effect Level (NOAEL).

DEFINITION OF DAY
The first day of treatment (Day 1) ran from midnight before the first administration until 24 h later, subsequent day numbers (Day 2, etc) also followed this pattern. Body weight, food consumption and clinical observation data recorded on Day 1 of the study were classified as Day 0 on the in-life data collection system. Body weights food consumption and clinical observation data recorded before Day 0 were classified as Day -1, etc. Recording of laboratory investigation bleeds and terminal kill days were carried out according to treatment days, that is, Day 1 being the day treatment started. All other references to study days refer to the first day of the study being Day 1.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
- All animals were checked in their cages twice daily for general health/mortality and moribundity. All animals were observed in their cage for reaction to treatment twice daily, animals were not removed from their cage during observation, unless necessary for identification or confirmation of findings . In addition, once each week starting during pretreatment, all animals received a detailed examination, including appearance, movement and behavior patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT
- Individual body weights were recorded twice during pretreatment, daily throughout Week 1 of the dosing period and twice weekly from Week 2 to Week 13. A body weight was also recorded on the first day of scheduled necropsy

FOOD CONSUMPTION AND TEST SUBSTANCE INTAKE
- Food consumption was quantitatively measured for each cage of animals twice during pretreatment, daily throughout Week 1 of dosing and twice weekly from Week 2 to Week 13. Daily food consumption measurements were recorded over an approximate 24 hour period and reported on the day the measurement was stopped, for example, Day 1 to Day 2 was reported as Day 2. The same pattern was used in the longer food consumption measurement periods.
- The estimated achieved intake of test substance in mg/kg/day was calculated separately for each cage (Groups 2 - 4) for each recorded period of food consumption, using the following formula:
Dosage (mg/kg/day) = (Nominal dietary concentration (ppm) x cage mean food consumption (g/animal/day)) / (cage mean body weight at the middle of the period)
The cage mean body weight at the middle of the period was calculated as follows:
((Mean weight of all animals in a cage at start of interval) + (Mean weight of all animals in a cage at end of interval)) /2.
An overall estimated achieved dose for each group was calculated by averaging the group means for each period. Data are presented as a cage mean for each period of food consumption and as an overall achieved dose, which is the arithmetic mean of all calculations during the treatment period for each group.

WATER CONSUMPTION
- Water consumption was monitored regularly by visual inspection of the water bottles throughout the study.

OPHTHALMIC EXAMINATIONS
- The anterior, lenticular and fundic areas of all animals during pretreatment and Groups 1 and 4 during Week 13 were examined using an indirect ophthalmoscope after the application of a mydriatic agent .

DETAILED FUNCTIONAL OBSERVATIONS
- During Week 11/12 detailed functional observations were conducted for all animals. This was done by a technician not involved in the dosing procedures or in the collection of body weight and food consumption data, and was performed at an approximately standardised time of day (between 08.50 and 14.15). Before the independent technician entered the animal room to perform the examinations, the cage card showing the treatment group was removed from each cage, leaving the second pre-prepared card as the functional observation animal identifier. In each cage both animals had their tails marked to allow the independent technician to identify each animal.
A) CAGESIDE OBSERVATIONS
- On first approach to the cage, the technician checked the posture/condition of the animal for signs of prostration, lethargy, writhing, circling, breathing abnormalities, gait abnormalities, tremor, fasciculation, convulsions, biting (of cage components or self mutilating), vocalizations and piloerection. The technician also checked the animals body temperature (measured rectally), ease at which the animal was removed from the cage, the conditions of its eyes (for pupillary function, miosis, mydriasis, exophthalmos, encrustation and lacrimation), the condition of the coat, the presence of salivation and overall ease of handling.
B) OBSERVATIONS IN A STANDARDISED ARENA (2 MIN OBSERVATION PERIOD)
- Animals were checked for latency (time to first locomotory movement), level of mobility, rearing, grooming, urination/defecation, arousal (level of alertness), posture, temor/convulsions, vocalisation, piloerection, palpebral closure, gait abnormalities, stereotypy (excessive repetition of behaviours) and/or unusual behaviours.
C) FUNCTIONAL TESTS
- The technician observed the reactions to a sudden sound (click above the head) and the reaction to a touch on the rump with a blunt probe.
D) GRIP STRENGTH
- Grip strength was measured using a Dual/Single Channel Grip Strength Meter (Linton Instruments), to which a wire screen assembly is attached. Once the animal had gripped the screen, the body was pulled until its grasp was broken; the strain gauge recorded the force required. The procedure was repeated 3 times for the forelimbs and 3 times for the hindlimbs, and the mean fore and hind grip strengths calculated.
E) PAIN PERCEPTIONS
This was assessed by measurement of the tail flick response, using a technique based on the method devised by D`Amour and Smith (1941). The apparatus used shines a calibrated infra-red heat source onto the tail and automatically measures the reaction time of the animal (accurate to 0.1 s). It was Ensured that no visible injury to the tail was caused in this test.
F) LANDING FOOT SPLAY
- Corn oil was applied to the hind feet of each animal. The animal was then held in a horizontal, prone position with the nose ca 30 cm above a bench surface covered with absorbent paper. When the animal was calm, it was dropped. The distance between the prints of the central footpads was measured and the average measurement recorded. The procedure was repeated 3 times and a mean value calculated.
G) MOTOR ACTIVITY
- Each animal was placed in an individual cage held within a Smartfame utilising infra-red pyroelectric detectors. Movement was detected in 2 dimensions anywhere in the cage, and was differentiated into basic and fine movements, and X and Y ambulation. Each animal was monitored for one session of 1 h, activity counts were recorded over successive 12 periods of 5 min each.

HAEMATOLOGY:
- Sample collection: Non-fasted blood was collected from the orbital sinus under isoflourane anaesthesia using a capillary tube, in random order. Urine was collected over an approximately 6 h period where the animals had no access to food but had access to water. After collection, samples were transferred to the appropriate laboratory for processing. Samples were collected as follows for all animals: Week 13 - Urinalysis only; Before
euthanasia - Haematology, Coagulation and Clinical Chemistry.
- Parameters: Whole blood (0.5 mL) was collected into tubes containing K2EDTA and analysed for the following parameters:
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).
A blood smear was prepared from each haematology sample. Blood smears were labelled, stained, stored but not examined.
- Coagulation: Whole blood (0.9 mL) was collected in to 3.8% (w/v) trisodium citrate, processed to plasma and analysed for the following parameters:
Activated partial thromboplastin time, Fibrinogen, Prothrombin time.

CLINICAL CHEMISTRY
- Whole blood (1.0 mL) was collected into lithium heparin, processed to plasma and analysed for the following parameters:
Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Triglycerides, Cholesterol, Gamma glutamyltransferase, Sodium, Potassium, Chloride, Urea, Glucose, Total protein, Albumin, Globulin, Albumin/globulin ratio, Creatinine, Total bilirubin Calcium, Inorganic phosphate.

ADDITIONAL BLOOD SAMPLE
Blood (0.5 mL) was collected from all animals, with a few exceptions where no blood was obtained, into K2EDTA and stored in a refrigerator (set to maintain 4 °C) before transfer to central laboratories for processing to plasma (centrifuged at 4 °C). Where possible, 130 to 250 µL of plasma was transferred into plastic tubes and stored at - 80 °C, pending possible future analysis.

URINALYSIS
Urine was analysed for the following parameters:
Microscopic evaluation of spun deposit, Colour, Turbidity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Leukocytes, Blood Pigments, Urobilinogen.

Sacrifice and pathology:

TERMINAL PROCEDURES
- Terminal procedures are summarised in Table 1 in 'Any other information on materials and methods incl. tables'.
- Unscheduled deaths: On Day 2 of one animal (Group 3 Female) was euthanised as per Test Facility standard operating procedures after suffering convulsions in a warming cabinet. This animal was replaced. Necropsy data for this animal are retained in the study records.
- Scheduled euthanasia: All animals surviving until scheduled euthanasia had a terminal body weight recorded, and were euthanised by exposure to rising levels of carbon dioxide, followed by exsanguination. The animals were euthanised in a rotating order across dose groups such that similar numbers of animals from each group, including controls, were necropsied throughout the day. Animals were not fasted before their scheduled necropsy.
- Necropsy: 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. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist was available throughout the necropsy period.

ORGAN WEIGHTS
The organs identified for weighing in the Tissues Collection and Preservation table were weighed at necropsy for all scheduled euthanasia animals. Paired organs were reported together. Terminal body weights were used for organ weight analysis.

TISSUE COLLECTION AND PRESERVATION
Representative samples of the tissues identified in the Tissue Collection and Preservation table were collected from all animals and preserved in 10 % neutral buffered formalin (NBF), unless otherwise indicated in Table 2 in 'Any other information on materials and methods incl. tables'.

LIVER SAMPLING
For all animals surviving to scheduled necropsy, a representative section from the left lateral lobe, the right median lobe and the caudate lobe was taken and fixed in 10 % neutral buffered formalin for 36- 48 hours and then processed to paraffin wax block. Tissues were not allowed to sit in hot wax for more than 2 hours. A small piece of duodenum was incorporated into each block.
Multiple samples of liver (8 x ca 150 mg) were taken from two 5 mm sections of the left lateral lobe. The samples were snap frozen in liquid nitrogen in individual RNA-ase free tubes. All samples were taken as quickly as possible and stored in a freezer set to maintain -80 °C pending possible future analysis. Two 150 mg samples were shipped on dry ice to the Sponsor. The further investigations on these samples were covered under a separate protocol and were reported separately. The remainder of the liver was cut into chunks (approximately 2 g) and snap frozen in liquid nitrogen. Samples were stored in a freezer set to maintain -80 °C pending possible future analysis. Any future analysis will be conducted and reported as a separate study.

HISTOLOGY
Tissues in the Tissue Collection and Preservation table from animals identified in the Terminal Procedures table were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin. All tissues from all treatment groups were processed to blocks, but in the first instance, processing to slides was carried out only for tissues from control and high dose groups, and gross lesions (including from low and mid dose animals). Processing of target tissues from low and mid dose animals (from blocks to slides) was conducted for the adrenal gland (males and females), testes and epididymis (males).

HISTOPATHOLOGY
Histopathological evaluation was performed by a veterinary pathologist with training and experience in laboratory animal pathology. In the first instance, microscopic examinations were carried out only for tissues from control and high dose animals, and gross lesions (from all groups). After agreement with the Sponsor the adrenal glands from low and mid dose animals and, testes and epididymis from low and mid dose males were evaluated.

PATHOLOGY PEER REVIEW
An internal peer review was undertaken by an appropriately qualified veterinary pathologist at the Test Facility, who validated the conclusions of the Study Pathologist, by independently assessing the microscope slides.

Other examinations:

BIOANALYSIS EVALUATION:
- SAMPLE COLLECTION
Blood (0.1 mL) was collected from the tail vein, following warming for an appropriate time (ca. 15 min) in a cabinet (30 - 41 °C) using disposable plastic syringes and hypodermic needles. Sampling was carried out after careful cleaning of the sampling site to avoid any possible contamination. Samples were collected at time points 07:00, 10:00 and 16:00 on Days 2, 28 and 85 for Group No 1, 2 3 and 4. Preparation of sampling started at the times indicated in the table and all animals were bled within 2 hours 38 mins, individual bleed times have been recorded in the data.
- SAMPLE PROCESSING
Immediately following collection, tubes were placed on a roller mixer for a short time (ca 1 min). After mixing, 50 µL of whole blood from each sample was accurately measured into a plain plastic tube containing exactly 50 µL of deionised water, labelled with the Charles River study number, animal identification, timepoint and group number. Residue blood samples were discarded. Tubes were placed on a roller mixer for a short time (ca 1 min) and then frozen as soon as possible on dry ice. The blood was stored in a freezer set to maintain -80 °C until transfer for analysis.
- Sample analysis
Samples were stored immediately on receipt in the Department of Bioanalytical Chemistry in a freezer set to maintain -80 °C until analysed. The concentration of the test substance in the blood was determined using a research grade 3 (RGA3) analytical method which was established. The study samples were extracted and analysed in batches together with calibration standards and quality control samples at concentrations over the established assay range.

Statistics:

STATISTICAL APPROACHES:
- All analyses were two-tailed for significance levels of 5 % and 1 %.
- All means were presented with standard deviations.
- If the variances are clearly heterogeneous, appropriate transformations (e.g. log, square root, double arcsine) were used in an attempt to stabilise the variances. In the final report, any transformations that were utilised were indicated in the specific results tables and/or the Statistical Methods section.
- Body weights, cumulative body weight gain, food consumption, selected functional observation battery and motor activity, haematology, coagulation, clinical chemistry, selected urinalysis parameters and absolute organ weights were analysed initially by a one-way analysis of variance (ANOVA).
- Organ weights were also analysed by analysis of covariance (ANCOVA) on final body weight (Shirley 1977). This statistical analysis provided an Adjusted Organ Weight value, which was displayed in the results table in the final report along with flags for statistical significance.
- Summary values of organ to body weight ratios were presented but these were not analysed statistically.
- For all of the parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett’s test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant, and statistical flags were presented in the tables of results in the final report.
- Micropathology incidence data were analysed using Fisher’s exact test.
- Individual values were rounded before printing. All derived values that appear in the tables represent the rounded results of calculations that are based on the exact (non-rounded) raw data values

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse clinical signs considered to be associated with feeding with the test sunstance diet at 50, 150 or 300 ppm. On Day 2 of the study one animal (Low dose, Female) was noted with convulsion in the warming cabinet. This animal subsequently recovered. Convulsions in the warming in cabinet in Han Wistar rats were noted on multiple occasions as a background finding at the Test Facility and these clinical signs were considered to not be associated with feeding with the test substance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On Day 2 of the study one animal (Mid dose, Female) was euthanised after suffering severe convulsions in the warming cabinet. This animal was replaced. Another animal (High dose, Female) failed to recover from anaesthesia on the day of scheduled necropsy (Day 92) and is thus treated as it would be terminated during scheduled euthanasia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weights and cumulative body weight change were lower in males receiving diet at 300 ppm, when compared with controls, with statistical significance being achieved from Day 10 onward and Day 6 using cumulative body weight change. In these animals, at the end of the study, body weight was approximately 12 % lower and cumulative body weight change was approximately 21 % lower than controls.
Cumulative body weight change was lower in females receiving diet at 300 ppm, on Days 28 - 35 when compared with controls, with statistical significance being achieved. Although not always attaining statistical significance, a lower cumulative body weight change was observed throughout the study. In these animals, at the end of the study, body weight was approximately 6 % lower and cumulative body weight change was approximately 15 % lower than controls. Group mean body weights and cumulative body weight change in males and females receiving diet at 50 or 150 ppm were considered to be similar to that of their controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was lower in males and females receiving diet at 300 ppm throughout the treatment period, when compared with controls with statistical significance achieved on a number of occasions. Food consumption in males and females receiving diets at 50 and 150 ppm were considered to be similar to that of their controls. Statistically significant differences were observed on separated occasions, however they were considered to be result of inter-individual variation.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Visual inspection indicated no observable differences between groups

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no inter-group differences

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Higher white blood cell counts mostly consisting of lymphocytes were noted in males at 150 or 300 ppm, when compared to their controls. Higher white blood cell, lymphocytes, monocytes, basophils and large unclassified cells counts were noted in females at 300 ppm, when compared to the control animals.
There were no other haematology and coagulation parameters considered to be related to dietary exposure to test substance.
Some minor inter-group differences were observed in a few additional parameters in females including lower haematocrit values at 150 ppm and lower mean cell volume and higher fibrinogen at 300 ppm. These findings were considered to reflect biological variability because the mean values were within the Historical Control Data (HCD) ranges. Therefore, these changes were considered to be unrelated to treatment with test substance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Lower cholesterol levels were noted in males at 300 ppm, when compared to their controls with statistical significance achieved.
Higher potassium levels were noted in males and females at 300 ppm when compared to their controls with statistical significance achieved only in females.
Higher phosphate levels were noted in males and females at 300 ppm when compared to their controls with statistical significance achieved.
Lower calcium levels were observed in males at 300 ppm and females at 150 and 300 ppm when compared to their controls, with statistical significance achieved.
Some minor inter-group differences were observed in a number of additional parameters including higher alanine aminotransferase and lower total protein in females receiving 300 ppm. These were considered to be due to biological variability and not related to treatment because the group mean values were within the HCD ranges.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no notable inter-group differences in urinalysis parameters that were considered to be due to the consumption of diets containing the test substance.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

During the functional observation battery (FOB) parameters, animals were identified by a randomly allocated FOB animal identifier. There were no treatment-related inter-group differences in the functional observation battery parameters following administration of test substance at dietary inclusion levels up to 300 ppm in males and females. The mean fore grip strength value for the 300 ppm males was slightly, but statistically significantly, lower than the control mean value. In the absence of any other remarkable neurobehavioral findings, this finding was considered more likely a reflection of the treatment-related reduction in the mean body weight noted for this group (approximately 12 % lower than control) rather than a direct effect of treatment. There were no other inter-group differences in the quantitative functional observation parameters that could be attributed to treatment following administration of test substance at dietary inclusion levels up to 300 ppm in males and females.
There were no treatment-related inter-group differences in motor activity following administration of test substance at dietary inclusion levels up to 300 ppm in males and females. Isolated instances of statistically significant differences were observed within a small number of the 5-minute intervals over the 60-minute session. These differences occurred in a generally non-dose related manner. Since the total values for basic and fine movements, and for X and Y ambulation were similar across all groups, these sporadic differences are not attributed to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher Adrenal gland weights were observed in females when compared to the control group at 300 ppm with statistical significance being achieved. Adrenal gland weights were also higher in females receiving 150 ppm and in males receiving 150 and 300 ppm although statistical significance was not ascertained. The group mean absolute testes and epididymis weights for males receiving 300 ppm were statistically significantly lower than controls. However, following covariant analysis, taking into account the terminal body weight, the weight of the epididymides and testes were comparable to the controls. Statistically significant higher kidney weights were observed in males at 150 and 300 ppm following covariant analysis when compared to controls. In females at 300 ppm, ovarian weight was statistically higher than controls following covariant analysis. There were no microscopic correlates for these weight differences. No other test substance-related organ weight changes were noted. For more details see Table 3 in 'Any other information on results incl. tables'.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Abnormal content (pale yellow fluid) in the duodenum and/or jejunum of 2/10 females at 300 ppm was noted. Other gross findings observed were of similar incidence in control and treated animals, and/or of the nature commonly observed in this strain and age of rats and/or related to differences in the stage of oestrus. These findings were therefore considered incidental and unrelated to administration of test substance

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Adrenal gland: In the adrenal gland zona fasciculata there was minimal vacuolation in males and minimal hypertrophy in females at 300 ppm. Hypertrophy correlated with higher adrenal gland weights in females.
- Testes and epidiymis: In males at 150 or 300 ppm there was minimal to marked tubular degeneration in the testis, minimal or mild cellular debris and minimal or moderate reduced sperm in the epididymis, with a dose-related increase in incidence and severity. These findings were bilateral in all except one case of minimal tubular degeneration of the testis. In Animal 34, which had the most severe grades of tubular atrophy and reduced sperm, these findings correlated with the most significantly deceased organ weights in the testis and epidiymis. Other microscopic findings observed were of similar incidence in control and treated animals, and/or of the nature commonly observed in this strain and age of rats. These findings were therefore considered incidental and unrelated to administration of the test substance. For more details see Table 4 in 'Any other information on results incl. tables'.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

BIOANALYSIS
The blood samples obtained from the toxicokinetic phase of the study were analysed to determine the concentrations of test substance. The reported analyses performed relating to the test substance data were acceptable with reference to the assay acceptance criteria. All control samples were below the limit of quantification for test substance on Days 2, 28 and 85.

TOXICOKINETICS
Blood test substance concentration were variable in animals throughout the sampling period with concentrations observed for all dose groups, sampling days and males and females. Systemic exposure to test substance increased in a generally greater than proportional manner between 50 and 150 ppm and generally dose proportionally between 150 and 300 ppm, in males and females. Following repeat dosing, systemic exposure to test substance was generally comparable between Days 2 and 28 and lower on Day 85 in males whilst in females, exposure was greater on Day 28 than on Day 2 and generally comparable between Days 2 and 85. Systemic exposure to test substance was comparable between males and females on Day 2 and greater in females than males after repeat dosing

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Dietary equivalent to 3.9 mg/kg bw/day

Dose descriptor:

NOAEL

Effect level:

150 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: Dietary equivalent to 13.4 mg/kg bw/day

Key result

Critical effects observed:

no

Verification of test diets

The results of analysis for all diets samples were found to be within or equal to the acceptance criteria of ±10% of their theoretical concentrations. For homogeneity, the RSD of concentrations for all samples in each group was within the acceptance criteria of <10%. The test substance was not detected in the control diet samples.

Table 3: Estimated Achieved Dosage

Group No.	Test Substance	Dosage Level (p.p.m)	Estimated Achieved Dosage (mg/kg/day test substance)
			Males	Females
2	test substance	50	3.9	4.4
3	test substance	150	11.2	13.4
4	test substance	300	22.0	24.0

 

Table 4: Summary Group Mean Organ Weight Data – (Day 92/93)

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (ppm)	0	50	150	300	0	50	150	300
No. animals per group	10	10	10	10	10	10	10	10
TerminalBody Weight(g)	429	419	418	378^b	240	251	237	227
Adrenal gland (No. weighed)	(10)	(10)	(10)	(10)	(10)	(10)	(10)	(9)
Absolute value (g)	0.0593	0.0570	0.0647	0.0624	0.0713	0.0794	0.0845	0.0986^b
ANCOVA (g)	0.0576	0.0562	0.0641	0.0655	0.0712	0.0788	0.0846	0.0992^b
Body weight relative (%)	0.01389	0.01358	0.01553	0.01653	0.02990	0.03194	0.03605	0.04334
Epididymis (No. weighed)	(10)	(10)	(10)	(10)	–	–	–	–
Absolute value (g)	1.3344	1.3675	1.3862	1.1868	–	–	–	–
ANCOVA	1.2999	1.3522	1.3737	1..2492	–	–	–	–
Body weight relative (%)	0.31204	0.32641	0.33305	0.31459	–	–	–	–
Testis (No. weighed)	(10)	(10)	(10)	(10)	–	–	–	–
Absolute value (g)	3.70	3.70	3.79	3.46	–	–	–	–
ANCOVA (g)	3.60	3.65	3.75	3.64	–	–	–	–
Body weight relative (%)	0.864	0.885	0.911	0.912	–	–	–	–
Kidney (No. weighed)	(10)	(10)	(10)	(10)	(10)	(10)	(10)	(9)
Absolute value (g)	2.42	2.48	2.57	2.51	1.55	1.66	1.64	1.58
ANCOVA (g)	2.37	2.46	2.54^a	2.61^a	1.55	1.61	1.65^a	1.63
Body weight relative (%)	0.567	0.593	0.618	0.663	0.648	0.664	0.694	0.695
Ovary (No. weighed)	–	–	–	–	(10)	(10)	(10)	(9)
Absolute value (g)	–	–	–	–	0.099	0.107	0.115	0.120
ANCOVA (g)	–	–	–	–	0.099	0.104	0.116	0.123^a
Body weight relative (%)	–	–	–	–	0.0415	0.0432	0.0487	0.0526

 Significantly different from Group 1: a = p<0.05, b = p<0.01

Table 5: Summary Microscopic Findings – (Day 92/93)

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (ppm)	0	50	150	300	0	50	150	300
No. animals examined	10	10	10	10	10	10	10	10
Adrenal gland (No. Examined)	(10)	(10)	(10	(10)	(10)	(10)	(10)	(10)
Hypertrophy, zona fasciculata, minimal	0	0	0	0	0	0	0	4
Vacuolation, zona fasciculata, minimal	0	0	0	3	0	0	0	0
Testis (No. Examined)	(10)	(10)	(10	(10)	(–)	(–)	(–)	(–)
Tubular degeneration	1	0	3	10^b	–	–	–	–
Minimal	1	0	2	5	–	–	–	–
Mild	0	0	1	3	–	–	–	–
Moderate	0	0	0	1	–	–	–	–
Marked	0	0	0	1	–	–	–	–
Epididymis (No. Examined)	(10)	(10)	(10)	(10)	(–)	(–)	(–)	(–)
Cellular debris, luminal	0	0	2	8b	–	–	–	–
Minimal	0	0	2	2	–	–	–	–
Mild	0	0	0	6	–	–	–	–
Reduced sperm, luminal	0	0	1	3	–	–	–	–
Minimal	0	0	1	2	–	–	–	–
Moderate	0	0	0	1	–	–	–	–

Significantly different from Group 1: a = p<0.05, b = p<0.01

 

Conclusions:

Dietary administration of test substance in rats for 13 weeks was associated with reduced body weight gain and food consumption, higher adrenal gland weights and histopathological changes in the adrenal gland and testis/epidiymis. The No Observed Adverse Effect Level (NOAEL) in this study was considered to be 50 ppm in males and 150 ppm in females, equating to 3.9 mg/kg/day in males and 13.4 mg/kg/day in females.

Executive summary:

This OECD 408 and GLP compliant study was performed in order to investigate the potential of the test substance to induce repeated dose toxicity in the rat, following dietary administration for 13 weeks. In addition, the toxicokinetic characteristics of test substance were determined. Ten male and ten female Han Wistar (Crl:WI(Han)) rats were assigned to each of 4 groups and fed diet containing 0, 50, 150 or 300 ppm of test substance for 91 days. The control substance used was SDS Rat and Mouse (modified) No. 1 Diet SQC Expanded (Ground) diet. The overall mean achieved dosages were 0, 3.9, 11.2 or 22.0 mg/kg/day test substance in males and 0, 4.4, 13.4 or 24.0 mg/kg/day test substance in females corresponding to dietary inclusions levels of 0, 50, 150 or 300 ppm. Systemic exposure to test substance increased in a generally greater than proportional manner between 50 and 150 ppm and generally dose proportionally between 150 and 300 ppm, in males and females. Following repeat dosing, systemic exposure to test substance was generally comparable between Days 2 and 28 and lower on Day 85 in males whilst in females, exposure was greater on Day 28 than on Day 2 and generally comparable between Days 2 and 85. Systemic exposure to the substance was comparable between males and females on Day 2 and greater in females than males after repeat dosing. There were no adverse clinical signs or ophthalmic findings associated with administration of test substance in the diet. Group mean body weight and cumulative body weight change were lower in males receiving test substance in the diet at 300 ppm, when compared with controls. Cumulative body weight change was also lower in females receiving test substance in the diet at 300 ppm. Food consumption was lower in males and females receiving test substance in the diet at 300 ppm throughout the treatment period, when compared with controls, with statistical significance achieved on a number of occasions. In the functional observation battery the only finding of note was a slightly lower fore grip strength in males receiving 300 ppm. Given the small magnitude of this change it was considered a reflection of the treatment-related reduction in the mean body weight noted for this group (approximately 12 % lower than control) rather than a direct effect of treatment. A number of treatment-related findings in blood chemistry were noted, but given the small magnitude of these changes and in the absence of any associated microscopic changes or alterations in other clinical chemistry parameters indicative of target organ toxicity these findings are considered to be non-adverse. There were no notable inter-group treatment-related differences in urinalysis parameters. Higher, statistically significant adrenal gland weights were observed in females when compared to the control groups at 300 ppm. Adrenal gland weights were also higher in females receiving 150 ppm and in males receiving 150 and 300 ppm although statistical significance was not attained. Lower testis and epididymis weights were noted in one male at 300 ppm. Abnormal content (pale yellow fluid) in the duodenum and/or jejunum was observed at gross necropsy in 2/10 females at 300 ppm. In the absence of a microscopic correlate the toxicological significance of this was not established. In the adrenal gland zona fasciculata there was minimal vacuolation in males and minimal hypertrophy in females at 300 ppm. Hypertrophy correlated with higher adrenal gland weights in females. In males at 150 or 300 ppm tubular degeneration in the testis and cellular debris and reduced sperm in the epididymis was observed, with a dose-related increase in incidence and severity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

3.9 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

GLP compliant OECD 408 study

System:

male reproductive system

Organ:

testes

other: epididymides

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start: 13 July 2016
Experimental end (last day of pathology data recording): 14 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This short-term repeated dose toxicity study was generated to meet the data requirements of regulations not related to REACH in non-EEA countries.

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: RccHan:WIST

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHanTM;WIST) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Envigo RMS Limited
Age: 76 to 82 days
Weight: 268 to 324 g (males), 173 to 212 g (females)
Acclimatisation: 12 days
Housing: Animals were housed individually (main study) in polycarbonate cages with stainless steel mesh lid with wood bedding. Two pellets of diet were added directly to the shavings after dose administration each day as forage food. An Aspen chew block was provided to each cage throughout the study and replaced when necessary. Plastic shelter was provided to each cage throughout the study.
Diet: Teklad 2014C Diet, ad libitum
Water: potable water from public supply, ad libitum
Temperature: 20 to 24 °C
Humidity: 40 to 70%
Lighting: artificial lighting, 12 hours light to 12 hours darkness cycle

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

Approximately 24 hours before treatment commenced the dorsum between the limb girdles was clipped free of hair, as close as possible, using electric clippers. An area estimated to be at least 10% of the total body surface was clipped. The animals were clipped again as required, at a minimum frequency of weekly, avoiding damage to the site. Any re-clipping was undertaken at the end of the working day after removal of the Elizabethan collars.
The application site was moistened with approximately 0.2 mL of purified water and the dose was applied to the middle of the clipped site. Care was taken to ensure that the dose was distributed evenly across the test site. The sham control was treated similarly, but with the absence of test substance.
Unmedicated gauze dressing (2- or 4-ply) was applied immediately after dosing, held in position around the trunk with a cotton wool pad, a length of ‘Wide Open Weave (WOW)’ bandage and a Tubigrip bandage with sufficient tension to ensure dose remained in contact with the skin.
Not less than six hours (no more than six hours and 15 minutes). The semi-occlusive dressing was removed carefully and the exposed area cleansed with copious quantities of warm tap water (approximately 30-35°C) and dabbed-dry with disposable paper towels to reduce the risk of oral ingestion of the test material during grooming. After the application site had been washed and dried, an Elizabethan collar was fitted to each animal for a period of not less than 30 minutes (no more than 60 minutes).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Six hours

Frequency of treatment:

Five days per week for four weeks

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:

Ten females and ten males

Control animals:

yes, sham-exposed

Details on study design:

Three days before the start of the treatment, variations in body weight of the animals were checked to ensure that they did not exceed ±20% of the mean for the appropriate sex.

Positive control:

Not applicable

Observations and examinations performed and frequency:

Blood samples were taken from the tail vein at defined time points after dosing for investigating the toxicokinetics. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Detailed observations were recorded daily during the first week of treatment and once weekly during the following weeks of treatment at defined time points before and following dosing. The dermal application site was examined for signs of dermal irritation before each administration. A detailed physical examination was performed on each animal before treatment commenced and during each week of treatment. Body weight was recorded before treatment commenced, on the day of first treatment, on the following day and weekly thereafter. The weight of food supplied to each cage, remaining food and an estimate of spilled food was recorded for the week before treatment commenced and for each week of treatment. Ophthalmic examinations were done before treatment started and in week 4. Haematology investigations on peripheral blood were carried out a necropsy, and bone marrow smears were prepared immediately following sacrifice on completion of the scheduled treatment period. Blood chemistry was analysed at necropsy. All animals were subject to a detailed necropsy at the end of the study. Organs were weighed and tissues were routinely preserved for histological investigations.

Sacrifice and pathology:

Animals were euthanised at the end of the scheduled treatment period, and tissues were routinely preserved for histological investigations.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. The following data types were analysed at each timepoint separately:
- Body weight, using gains over appropriate study periods
- Food consumption, over appropriate study periods
- Hematology
- Blood chemistry
- Organ weights, absolute and adjusted for terminal body weight
- Pathological findings, for the number of animals with and without each finding
The following sequence of statistical tests was used for body weight, food consumption, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. Treated groups were compared to Control using Dunnett's test (Dunnett 1955, 1964).
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to Control using Steel's test (Steel 1959). For macropathology and histopathology findings, the treatment groups were compared using pairwise comparisons of each dose group against the control using one-tailed Fisher’s exact tests (Fisher, 1973) for an increase. For histopathology findings, tissues with the majority of animals examined were analysed statistically. All groups were included in the statistical analysis of adrenals, duodenum and jejunum and control and high dose only for the other tissues. For organ weight data, analysis of covariance was also performed, in addition to analysing the absolute organ weight data, using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights. Significant differences between Control and treated groups were expressed at the

Clinical signs:

no effects observed

Description (incidence and severity):

The behaviour and appearance of the animals were unaffected by treatment.
There were no findings at the dermal application sites that were attributed to treatment with the substance. Intermittent eschar-formation occurred at the dermal application site of one male and two females given 100 mg/kg/day, two males and one female given 300 mg/kg/day and one male given 1000 mg/kg/day, and erythema was recorded in one female given 100 mg/kg/day, one male given 300 mg/kg/day and one male and one female given 1000 mg/kg/day. The low incidences of these findings indicated that they were incidental.
One female given 300 mg/kg/day (No. 121) was seen to be gasping upon return to the home cage following dosing on Day 2, and on Day 17 a female given 1000 mg/kg/day (No. 114) displayed signs of gasping upon return to the home cage, which continued until 1-2 hours after dosing but was then absent at the end of the working day. Given the lack of similar findings in any other animals, this transient finding was considered incidental to treatment.

Dermal irritation:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Control female No. 139 was killed for welfare reasons on Day 1 after a traumatic skin injury was observed adjacent to the application site. Macroscopic findings confirmed the presence of an open area on the skin; this animal was subsequently replaced with a spare animal (No. 141) which was treated from Day 2 in the same manner as the other control animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gains of females given 1000 mg/kg/day were lower than those of the controls, with the extent of the reduction being 35%, and this difference from controls was statistically significant. The effect was particularly marked during the final two weeks where there was a 48% reduction, compared to controls. There was also a reduction of weight gain in the final two weeks in females given 300 mg/kg/day.
There was no clear effect of treatment upon the body weights of males. At 1000 mg/kg/day there was a 42% reduction in the first two weeks but, in contrast, the weight gains of these animals were higher than controls in Week 3 and 4. The overall (Day 1 to 28) weight gains were similar to controls at all doses. There was considered to have been no effect of treatment upon body weight at 100 mg/kg/day in both sexes and at 300 mg/kg/day in males.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was persistently low, when compared to pre-treatment and controls, in females given 300 or 1000 mg/kg/day, with males given 1000 mg/kg/day showing a similar finding in the first two weeks of treatment only.
There was considered to have been no effect of treatment upon food intake at 100 mg/kg/day in both sexes and at 300 mg/kg/day in males.

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematological examination performed after four weeks of treatment revealed, when compared with controls, high neutrophil and lymphocyte counts, attaining statistical significance at 1000 mg/kg/day in the males and at 300 and 1000 mg/kg/day in the females, but there was no clear dose-relationship in either sex. There were also minor increases of eosinophil and basophil counts in males and females given 1000 mg/kg/day and monocyte counts in males given 1000 mg/kg/day but despite these differences attaining statistical significant in most cases, the degree of increase was unlikely to be of any toxicological significance. As a consequence of these findings the total leucocyte counts of females given 300 mg/kg/day and of males and females given 1000 mg/kg/day were higher than controls. Platelet counts were high, when compared to controls, at all doses in females and at 1000 mg/kg/day in males.
All other differences from control, including those attaining statistical significance, were considered due to natural biological variation and were therefore unrelated to treatment. Such differences included the statistically significantly high reticulocyte count and red cell distribution width in males given 1000 mg/kg/day since there were no alterations of any of the other erythrocyte indices and the majority of values were within or only marginally above the background control mean ranges (0.182x1012 to 0.226x1012/L for reticulocyte count and 11.6 to 13.0% for red cell distribution width). They also included minor variations of clotting times (low prothrombin times in males, where the majority of values were within or slightly below the background control mean range (22.7 to 25.6 seconds) and low activated partial thromboplastin times in the high dose females, where 8/10 values were within the concurrent control range, although the majority (9/10) were outside the background range and was considered to be of no toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of plasma after four weeks of treatment revealed, when compared with controls, slightly high alkaline phosphatase activities in females given 300 or 1000 mg/kg/day and high alanine amino transferase activities in females given 1000 mg/kg/day. There were no similar trends in the males. Females given 300 or 1000 mg/kg/day had higher plasma urea concentrations than the controls, though statistical significance was attained only at 1000 mg/kg/day. Males were unaffected.
There was a clear and statistically significant reduction of plasma cholesterol and triglyceride concentration in females given 1000 mg/kg/day. Although the majority of individual values were within or below the background control mean range (cholesterol 1.74 to 2.20 mmol/L and triglycerides 0.25 to 0.45 mmol/L) they were generally lower than concurrent controls and these changes were probably related to treatment. Similar findings were not observed in males.
There were a few disturbances of plasma electrolyte concentrations. There was a clear and dose-related reduction of chloride concentration in males given 300 or 1000 mg/kg/day which may have associated with to the small reduction of plasma sodium concentration in these animals. Plasma potassium concentrations were high in in males given 300 mg/kg/day and in both sexes given 1000 mg/kg/day. In addition, small reductions of plasma calcium concentration occurred at all doses and in both sexes. In the absence of any corroborative histopathological findings in the liver and kidney which could account for these changes, they are considered to be incidental and unrelated to treatment.
All other differences from control, including those attaining statistical significance, were minor, lacked any evidence of dose-relationship and, consequently, were attributed to normal biological variation. Such differences included the high aspartate aminotransferase activity in males given 1000 mg/kg/day, where the difference was due primarily to one animal (No. 13) with a particularly high value (212 U/L) that was considerably above the background range. They also included the increases of albumin/globulin ratios at all doses since there was no clear dose-response, the all values were within or only marginally above the background range and there were no significant changes of total protein and albumin concentration that would account for these differences from controls.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The analysis of organs weights after four weeks of treatment revealed, when compared with controls, a dose-related increase of absolute and body weight adjusted adrenal weights in males given 100 mg/kg/day and in males and females given 300 or 1000 mg/kg/day.
In addition, slightly high body weight adjusted liver weights were recorded in males given 300 mg/kg/day and in males and females given 1000 mg/kg/day, and high body weight-adjusted kidney weights in males given 1000 mg/kg/day.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Small intestine: Epithelial vacuolation within the duodenum was observed at 100, 300 or 1000 mg/kg/day in females, with the incidence and the extent of the finding being dose-related. Similar dose-related changes within the jejunum were also observed at 300 or 1000 mg/kg/day in females. The epithelial changes were characterized by cellular swelling and pale, vacuolated cytoplasm, typically distributed around the region of the villus tip. The changes within the small intestine positively correlated with statistically significant reductions in body weight gain, amongst females treated with 1000 mg/kg/day. These findings were not considered adverse where the incidence was low within the treatment group and where there was no correlating impact on any blood biochemistry parameters or on body weight gain. Epithelial vacuolation in duodenum in females: n = 0 in controls, n = 1 in 100 mg/kg bw dose group, n = 2 in 300 mg/kg bw dose group, n = 7 in 1000 mg/kg bw dose group; epithelial vacuolation in jejunum in females: n = 0 in controls, n = 0 in 100 mg/kg bw dose group, n = 2 in 300 mg/kg bw dose group, n = 7 in 1000 mg/kg bw dose group.
Adrenal glands: Hypertrophy of the zona fasciculata, generally associated with vacuolation, was observed at 300 or 1000 mg/kg/day in both sexes, with the incidence and extent of the hypertrophy being dose-related. The morphological changes within this organ correlated with a statistically significant increase in mean absolute organ weight at 300 and 1000 mg/kg/day in both sexes. Cortical hypertrophy in Zona fasciculata in males: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 4 in 300 mg/kg dose group, n = 7 in 1000 mg/kg dose group, and in females: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 3 in 300 mg/kg dose group, n = 9 in 1000 mg/kg dose group. Cortical vacuolation in Zona fasciculata in males: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 1 in 300 mg/kg dose group, n = 7 in 1000 mg/kg dose group, and in females: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 1 in 300 mg/kg dose group, n = 9 in 1000 mg/kg dose group.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

adrenal glands

Conclusions:

The no observed adverse effect level following 4 weeks of semi-occluded dermal exposure for 6 hours per day, five days per week was 100 mg/kg bw/day.

Executive summary:

The potential systemic toxicity of the substance to female and male Han Wistar rats following sub-acute dermal exposure under semi-occlusion for a period of four weeks was studied under GLP to OECD TG 410. Animals were exposed to nominal doses of 100, 300 or 1000 mg/kg bw/day for six hours, on five days per week, during four weeks. The systemic exposure to the substance increased sub proportionally with respect to dose in male rats and proportionally in female rats. The systemic exposure to the substance was similar in males and females at 100 mg/kg/day, but higher in females at 300 and 1000 mg/kg/day. Body weight gain and food consumption were low in females given 1000 mg/kg/day, with the extent of the reduction of body weight gain being particularly marked during the final two weeks of treatment. There was also a persistent reduction of food consumption, resulting in reduced weight gain, in the final two weeks in females given 300 mg/kg/day. In males given 1000 mg/kg/day body weight gain and food consumption was low in the first two weeks of treatment. There were a number of effects with regards to haematology and blood chemistry, however there were no associated histological findings and these effects were therefore considered to be incidental and unrelated to treatment. After four weeks of treatment there was a dose-related increase of adrenal gland weight in males given 100 mg/kg/day and in males and females given 300 or 1000 mg/kg/day, slightly high liver weights in males given 300 mg/kg/day and in males and females given 1000 mg/kg/day, and high kidney weights in males given 1000 mg/kg/day. There were no treatment-related macroscopic findings.
Histopathological findings that were attributed to treatment were epithelial vacuolation in the duodenum in females given 100, 300 or 1000 mg/kg/day and in the jejunum of females given 300 or 1000 mg/kg/day, and hypertrophy and vacuolation of the adrenal gland zona fasciculata at 300 and 1000 mg/kg/day in both sexes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

GLP compliant OECD TG 410 study

System:

endocrine system

Organ:

adrenal glands

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start: 13 July 2016
Experimental end (last day of pathology data recording): 14 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This short-term repeated dose toxicity study was generated to meet the data requirements of regulations not related to REACH in non-EEA countries.

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: RccHan:WIST

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHanTM;WIST) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Envigo RMS Limited
Age: 76 to 82 days
Weight: 268 to 324 g (males), 173 to 212 g (females)
Acclimatisation: 12 days
Housing: Animals were housed individually (main study) in polycarbonate cages with stainless steel mesh lid with wood bedding. Two pellets of diet were added directly to the shavings after dose administration each day as forage food. An Aspen chew block was provided to each cage throughout the study and replaced when necessary. Plastic shelter was provided to each cage throughout the study.
Diet: Teklad 2014C Diet, ad libitum
Water: potable water from public supply, ad libitum
Temperature: 20 to 24 °C
Humidity: 40 to 70%
Lighting: artificial lighting, 12 hours light to 12 hours darkness cycle

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

Approximately 24 hours before treatment commenced the dorsum between the limb girdles was clipped free of hair, as close as possible, using electric clippers. An area estimated to be at least 10% of the total body surface was clipped. The animals were clipped again as required, at a minimum frequency of weekly, avoiding damage to the site. Any re-clipping was undertaken at the end of the working day after removal of the Elizabethan collars.
The application site was moistened with approximately 0.2 mL of purified water and the dose was applied to the middle of the clipped site. Care was taken to ensure that the dose was distributed evenly across the test site. The sham control was treated similarly, but with the absence of test substance.
Unmedicated gauze dressing (2- or 4-ply) was applied immediately after dosing, held in position around the trunk with a cotton wool pad, a length of ‘Wide Open Weave (WOW)’ bandage and a Tubigrip bandage with sufficient tension to ensure dose remained in contact with the skin.
Not less than six hours (no more than six hours and 15 minutes). The semi-occlusive dressing was removed carefully and the exposed area cleansed with copious quantities of warm tap water (approximately 30-35°C) and dabbed-dry with disposable paper towels to reduce the risk of oral ingestion of the test material during grooming. After the application site had been washed and dried, an Elizabethan collar was fitted to each animal for a period of not less than 30 minutes (no more than 60 minutes).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Six hours

Frequency of treatment:

Five days per week for four weeks

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:

Ten females and ten males

Control animals:

yes, sham-exposed

Details on study design:

Three days before the start of the treatment, variations in body weight of the animals were checked to ensure that they did not exceed ±20% of the mean for the appropriate sex.

Positive control:

Not applicable

Observations and examinations performed and frequency:

Blood samples were taken from the tail vein at defined time points after dosing for investigating the toxicokinetics. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Detailed observations were recorded daily during the first week of treatment and once weekly during the following weeks of treatment at defined time points before and following dosing. The dermal application site was examined for signs of dermal irritation before each administration. A detailed physical examination was performed on each animal before treatment commenced and during each week of treatment. Body weight was recorded before treatment commenced, on the day of first treatment, on the following day and weekly thereafter. The weight of food supplied to each cage, remaining food and an estimate of spilled food was recorded for the week before treatment commenced and for each week of treatment. Ophthalmic examinations were done before treatment started and in week 4. Haematology investigations on peripheral blood were carried out a necropsy, and bone marrow smears were prepared immediately following sacrifice on completion of the scheduled treatment period. Blood chemistry was analysed at necropsy. All animals were subject to a detailed necropsy at the end of the study. Organs were weighed and tissues were routinely preserved for histological investigations.

Sacrifice and pathology:

Animals were euthanised at the end of the scheduled treatment period, and tissues were routinely preserved for histological investigations.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. The following data types were analysed at each timepoint separately:
- Body weight, using gains over appropriate study periods
- Food consumption, over appropriate study periods
- Hematology
- Blood chemistry
- Organ weights, absolute and adjusted for terminal body weight
- Pathological findings, for the number of animals with and without each finding
The following sequence of statistical tests was used for body weight, food consumption, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. Treated groups were compared to Control using Dunnett's test (Dunnett 1955, 1964).
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to Control using Steel's test (Steel 1959). For macropathology and histopathology findings, the treatment groups were compared using pairwise comparisons of each dose group against the control using one-tailed Fisher’s exact tests (Fisher, 1973) for an increase. For histopathology findings, tissues with the majority of animals examined were analysed statistically. All groups were included in the statistical analysis of adrenals, duodenum and jejunum and control and high dose only for the other tissues. For organ weight data, analysis of covariance was also performed, in addition to analysing the absolute organ weight data, using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights. Significant differences between Control and treated groups were expressed at the

Clinical signs:

no effects observed

Description (incidence and severity):

The behaviour and appearance of the animals were unaffected by treatment.
There were no findings at the dermal application sites that were attributed to treatment with the substance. Intermittent eschar-formation occurred at the dermal application site of one male and two females given 100 mg/kg/day, two males and one female given 300 mg/kg/day and one male given 1000 mg/kg/day, and erythema was recorded in one female given 100 mg/kg/day, one male given 300 mg/kg/day and one male and one female given 1000 mg/kg/day. The low incidences of these findings indicated that they were incidental.
One female given 300 mg/kg/day (No. 121) was seen to be gasping upon return to the home cage following dosing on Day 2, and on Day 17 a female given 1000 mg/kg/day (No. 114) displayed signs of gasping upon return to the home cage, which continued until 1-2 hours after dosing but was then absent at the end of the working day. Given the lack of similar findings in any other animals, this transient finding was considered incidental to treatment.

Dermal irritation:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Control female No. 139 was killed for welfare reasons on Day 1 after a traumatic skin injury was observed adjacent to the application site. Macroscopic findings confirmed the presence of an open area on the skin; this animal was subsequently replaced with a spare animal (No. 141) which was treated from Day 2 in the same manner as the other control animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gains of females given 1000 mg/kg/day were lower than those of the controls, with the extent of the reduction being 35%, and this difference from controls was statistically significant. The effect was particularly marked during the final two weeks where there was a 48% reduction, compared to controls. There was also a reduction of weight gain in the final two weeks in females given 300 mg/kg/day.
There was no clear effect of treatment upon the body weights of males. At 1000 mg/kg/day there was a 42% reduction in the first two weeks but, in contrast, the weight gains of these animals were higher than controls in Week 3 and 4. The overall (Day 1 to 28) weight gains were similar to controls at all doses. There was considered to have been no effect of treatment upon body weight at 100 mg/kg/day in both sexes and at 300 mg/kg/day in males.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was persistently low, when compared to pre-treatment and controls, in females given 300 or 1000 mg/kg/day, with males given 1000 mg/kg/day showing a similar finding in the first two weeks of treatment only.
There was considered to have been no effect of treatment upon food intake at 100 mg/kg/day in both sexes and at 300 mg/kg/day in males.

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematological examination performed after four weeks of treatment revealed, when compared with controls, high neutrophil and lymphocyte counts, attaining statistical significance at 1000 mg/kg/day in the males and at 300 and 1000 mg/kg/day in the females, but there was no clear dose-relationship in either sex. There were also minor increases of eosinophil and basophil counts in males and females given 1000 mg/kg/day and monocyte counts in males given 1000 mg/kg/day but despite these differences attaining statistical significant in most cases, the degree of increase was unlikely to be of any toxicological significance. As a consequence of these findings the total leucocyte counts of females given 300 mg/kg/day and of males and females given 1000 mg/kg/day were higher than controls. Platelet counts were high, when compared to controls, at all doses in females and at 1000 mg/kg/day in males.
All other differences from control, including those attaining statistical significance, were considered due to natural biological variation and were therefore unrelated to treatment. Such differences included the statistically significantly high reticulocyte count and red cell distribution width in males given 1000 mg/kg/day since there were no alterations of any of the other erythrocyte indices and the majority of values were within or only marginally above the background control mean ranges (0.182x1012 to 0.226x1012/L for reticulocyte count and 11.6 to 13.0% for red cell distribution width). They also included minor variations of clotting times (low prothrombin times in males, where the majority of values were within or slightly below the background control mean range (22.7 to 25.6 seconds) and low activated partial thromboplastin times in the high dose females, where 8/10 values were within the concurrent control range, although the majority (9/10) were outside the background range and was considered to be of no toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of plasma after four weeks of treatment revealed, when compared with controls, slightly high alkaline phosphatase activities in females given 300 or 1000 mg/kg/day and high alanine amino transferase activities in females given 1000 mg/kg/day. There were no similar trends in the males. Females given 300 or 1000 mg/kg/day had higher plasma urea concentrations than the controls, though statistical significance was attained only at 1000 mg/kg/day. Males were unaffected.
There was a clear and statistically significant reduction of plasma cholesterol and triglyceride concentration in females given 1000 mg/kg/day. Although the majority of individual values were within or below the background control mean range (cholesterol 1.74 to 2.20 mmol/L and triglycerides 0.25 to 0.45 mmol/L) they were generally lower than concurrent controls and these changes were probably related to treatment. Similar findings were not observed in males.
There were a few disturbances of plasma electrolyte concentrations. There was a clear and dose-related reduction of chloride concentration in males given 300 or 1000 mg/kg/day which may have associated with to the small reduction of plasma sodium concentration in these animals. Plasma potassium concentrations were high in in males given 300 mg/kg/day and in both sexes given 1000 mg/kg/day. In addition, small reductions of plasma calcium concentration occurred at all doses and in both sexes. In the absence of any corroborative histopathological findings in the liver and kidney which could account for these changes, they are considered to be incidental and unrelated to treatment.
All other differences from control, including those attaining statistical significance, were minor, lacked any evidence of dose-relationship and, consequently, were attributed to normal biological variation. Such differences included the high aspartate aminotransferase activity in males given 1000 mg/kg/day, where the difference was due primarily to one animal (No. 13) with a particularly high value (212 U/L) that was considerably above the background range. They also included the increases of albumin/globulin ratios at all doses since there was no clear dose-response, the all values were within or only marginally above the background range and there were no significant changes of total protein and albumin concentration that would account for these differences from controls.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The analysis of organs weights after four weeks of treatment revealed, when compared with controls, a dose-related increase of absolute and body weight adjusted adrenal weights in males given 100 mg/kg/day and in males and females given 300 or 1000 mg/kg/day.
In addition, slightly high body weight adjusted liver weights were recorded in males given 300 mg/kg/day and in males and females given 1000 mg/kg/day, and high body weight-adjusted kidney weights in males given 1000 mg/kg/day.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Small intestine: Epithelial vacuolation within the duodenum was observed at 100, 300 or 1000 mg/kg/day in females, with the incidence and the extent of the finding being dose-related. Similar dose-related changes within the jejunum were also observed at 300 or 1000 mg/kg/day in females. The epithelial changes were characterized by cellular swelling and pale, vacuolated cytoplasm, typically distributed around the region of the villus tip. The changes within the small intestine positively correlated with statistically significant reductions in body weight gain, amongst females treated with 1000 mg/kg/day. These findings were not considered adverse where the incidence was low within the treatment group and where there was no correlating impact on any blood biochemistry parameters or on body weight gain. Epithelial vacuolation in duodenum in females: n = 0 in controls, n = 1 in 100 mg/kg bw dose group, n = 2 in 300 mg/kg bw dose group, n = 7 in 1000 mg/kg bw dose group; epithelial vacuolation in jejunum in females: n = 0 in controls, n = 0 in 100 mg/kg bw dose group, n = 2 in 300 mg/kg bw dose group, n = 7 in 1000 mg/kg bw dose group.
Adrenal glands: Hypertrophy of the zona fasciculata, generally associated with vacuolation, was observed at 300 or 1000 mg/kg/day in both sexes, with the incidence and extent of the hypertrophy being dose-related. The morphological changes within this organ correlated with a statistically significant increase in mean absolute organ weight at 300 and 1000 mg/kg/day in both sexes. Cortical hypertrophy in Zona fasciculata in males: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 4 in 300 mg/kg dose group, n = 7 in 1000 mg/kg dose group, and in females: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 3 in 300 mg/kg dose group, n = 9 in 1000 mg/kg dose group. Cortical vacuolation in Zona fasciculata in males: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 1 in 300 mg/kg dose group, n = 7 in 1000 mg/kg dose group, and in females: n = 0 in controls, n = 0 in 100 mg/kg dose group, n = 1 in 300 mg/kg dose group, n = 9 in 1000 mg/kg dose group.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

adrenal glands

Conclusions:

The no observed adverse effect level following 4 weeks of semi-occluded dermal exposure for 6 hours per day, five days per week was 100 mg/kg bw/day.

Executive summary:

The potential systemic toxicity of the substance to female and male Han Wistar rats following sub-acute dermal exposure under semi-occlusion for a period of four weeks was studied under GLP to OECD TG 410. Animals were exposed to nominal doses of 100, 300 or 1000 mg/kg bw/day for six hours, on five days per week, during four weeks. The systemic exposure to the substance increased sub proportionally with respect to dose in male rats and proportionally in female rats. The systemic exposure to the substance was similar in males and females at 100 mg/kg/day, but higher in females at 300 and 1000 mg/kg/day. Body weight gain and food consumption were low in females given 1000 mg/kg/day, with the extent of the reduction of body weight gain being particularly marked during the final two weeks of treatment. There was also a persistent reduction of food consumption, resulting in reduced weight gain, in the final two weeks in females given 300 mg/kg/day. In males given 1000 mg/kg/day body weight gain and food consumption was low in the first two weeks of treatment. There were a number of effects with regards to haematology and blood chemistry, however there were no associated histological findings and these effects were therefore considered to be incidental and unrelated to treatment. After four weeks of treatment there was a dose-related increase of adrenal gland weight in males given 100 mg/kg/day and in males and females given 300 or 1000 mg/kg/day, slightly high liver weights in males given 300 mg/kg/day and in males and females given 1000 mg/kg/day, and high kidney weights in males given 1000 mg/kg/day. There were no treatment-related macroscopic findings.
Histopathological findings that were attributed to treatment were epithelial vacuolation in the duodenum in females given 100, 300 or 1000 mg/kg/day and in the jejunum of females given 300 or 1000 mg/kg/day, and hypertrophy and vacuolation of the adrenal gland zona fasciculata at 300 and 1000 mg/kg/day in both sexes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Repeated dose toxicity: oral

Sub-acute repeated dose oral toxicity studies

A number of valid and reliable sub-acute repeated dose toxicity studies over a period of 28 days were conducted with rats, mice and dogs under GLP and to the relevant OECD test guidelines. The treatment-related effects in these studies, observed at higher oral doses, were consistent with those observed in the longer-term toxicity studies. Simple study summaries of the available full 28-day repeated dose toxicity studies (i.e. not range finder studies for longer-term toxicity studies) are presented as supporting studies.

3-month dietary study in rats (key study)

The potential of the test substance to induce repeated dose toxicity in the rat following oral (dietary) administration was studied under GLP to OECD TG 408. The substance was administered to test animals in the diet for 13 consecutive weeks. The toxicokinetic characteristics of test substance were determined. Ten male and ten female Han Wistar (Crl:WI(Han)) rats were assigned to each of 4 groups and fed diet containing 0, 50, 150 or 300 ppm of test substance. The control group of ten female and ten male rats received plain SDS Rat and Mouse (modified) No. 1 Diet. 

The overall mean achieved dosages corresponding to treatment levels of 0, 50, 150 and 300 ppm were 0, 3.9, 11.2 or 22.0 mg/kg/day test substance in males and 0, 4.4, 13.4 or 24.0 mg/kg/day test substance in females. Systemic exposure to the test substance increased in a generally greater than proportional manner between 50 and 150 ppm and dose proportionally between 150 and 300 ppm, in males and females. Systemic exposure to the substance was comparable between males and females on Day 2 and greater in females than males after repeat dosing. There were no adverse clinical signs or ophthalmic findings associated with administration of test substance in the diet. Group mean body weights and cumulative body weight changes were lower in males and females receiving test substance in the diet at 300 ppm, when compared with controls. Food consumption was lower in males and females receiving test substance in the diet at 300 ppm throughout the treatment period, when compared with controls, with statistical significance achieved on a number of occasions. In the functional observation battery, a slightly lower fore grip strength in males receiving 300 ppm was observed, which was considered a reflection of the treatment-related reduction in the mean body weight noted for this group (approximately 12% lower than control) rather than a direct effect of treatment.

A number of haematological and clinical chemistry parameters in males and females treated at 300 ppm were different when compared to the controls, but given the small magnitude of these changes and the absence of any associated microscopic changes or alterations in other clinical chemistry parameters indicative of target organ toxicity, these findings are considered to be non-adverse. There were no notable inter-group differences in urinalysis parameters associated with exposure to the substance. Higher adrenal gland weights were observed in females when compared to the control groups at 300 ppm, with statistical significance being achieved. Adrenal gland weights were also higher in females receiving 150 ppm and in males receiving 150 and 300 ppm although statistical significance was not attained. Lower testes and epididymis weights were noted in one male at 300 ppm. Abnormal content (pale yellow fluid) in the duodenum and/or jejunum was observed at gross necropsy in 2/10 females at 300 ppm. In the absence of a microscopic correlate the toxicological significance of this was not established. In the adrenal gland zona fasciculata there was minimal vacuolation in males and minimal hypertrophy in females at 300 ppm. Hypertrophy correlated with higher adrenal gland weights in females. In males at 150 or 300 ppm tubular degeneration in the testes and cellular debris and reduced sperm in the epididymis was observed, with a dose-related increase in incidence and severity.

Dietary administration of test substance in rats for 13 weeks was associated with reduced body weight gain and food consumption, higher adrenal gland weights and histopathological changes in the adrenal gland and testes/epidiymis. The No Observed Adverse Effect Level (NOAEL) in this study was considered to be 50 ppm in males and 150 ppm in females, equating to 3.9 mg/kg/day in males and 13.4 mg/kg/day in females.

Further sub-chronic repeated dose oral toxicity studies

Repeated dose toxicity via the oral route has also been tested under GLP in valid and reliable sub-chronic toxicity studies with mice (dietary exposure, OECD TG 408, Laidlaw 2019) and dogs (exposure via capsules, OECD TG 409, Robertson 2019) over a period of 13 weeks. Treatment related adverse effects similar to those in the available key study in the rat were observed in these species, but at higher doses. Thus, the no-adverse effect levels in mice and dogs were exceeding those in the study with rats. 

Chronic repeated dose oral toxicity studies

Furthermore, repeated dose toxicity via the (dietary) oral route has been tested under GLP in rats over a period of 52 weeks in a reliable and valid chronic toxicity study to OECD TG 453 (Strepka 2019). The adverse effects on the testes and epididymides were also observed in this chronic toxicity study. In contrast, the effects on the adrenal glands following subchronic dietary exposure did not occur following chronic exposure to the substance. 

Repeated dose toxicity: dermal

28-day semi-occluded dermal exposure study in rats

The potential of the test substance to induce repeated dose toxicity in the rat following dermal exposure was studied under GLP to OECD TG 410. Test animals were exposed by semi-occluded dermal application for 4 weeks (six hours per day, five days per week). In addition, the toxicokinetic characteristics of the test substance were determined. Ten male and ten female Han Wistar (Crl:WI(Han)) rats were assigned to each of 4 groups and treated topically with purified water (sham-exposed), or the test substance at nominal doses of 100, 300 or 1000 mg/kg bw/day. The systemic exposure to the substance was similar in males and females at 100 mg/kg/day, but higher in females at 300 and 1000 mg/kg/day. Body weight gain and food consumption were lower in females given 1000 mg/kg/day, with the extent of the reduction of body weight gain being particularly marked during the final two weeks of treatment. There was also a persistent reduction of food consumption, resulting in reduced weight gain, in the final two weeks in females given 300 mg/kg/day. In males given 1000 mg/kg/day body weight gain and food consumption was low in the first two weeks of treatment. After four weeks of treatment there was a dose-related increase of adrenal gland weight in males given 100 mg/kg/day and in males and females given 300 or 1000 mg/kg/day, slightly high liver weights in males given 300 mg/kg/day and in males and females given 1000 mg/kg/day, and high kidney weights in males given 1000 mg/kg/day. There were no treatment-related macroscopic findings.
Histopathological findings that were attributed to treatment were epithelial vacuolation in the duodenum in females given 100, 300 or 1000 mg/kg/day and in the jejunum of females given 300 or 1000 mg/kg/day, and hypertrophy and vacuolation of the adrenal gland zona fasciculata at 300 and 1000 mg/kg/day in both sexes.

Justification for classification or non-classification

Based on the available information obtained in reliable and valid repeated (dietary) dose studies over a period of 13-weeks in different mammalian species and over 52 and 104 weeks in rats, classification for specific target organ toxicity following repeated exposure is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008. The effects on the adrenal glands that were seen in the sub-chronic toxicity studies did not occur in the chronic toxicity and carcinogenicity study.