1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

Endpoint summary

Description of key information

- Oral: NOAEL = 14 and 18 mg/kg bw/day for males and females, respectively, rats, sub-chronic, 90 days, dietary, Robertson 2013

           NOAEL = 33 and 34 mg/kg bw/day, for males and females, respectively, dog, 1 year, dietary, Varney 1986

- Dermal: NOAEL > 1000 mg/kg bw/day for males and female, rabbits, sub-acute, 28 days, occlusive dressing, Robertson 2013

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:

30 Jul 2012 to 19 Nov 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2001

GLP compliance:

yes (incl. QA statement)

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 Animal Information
- Females nulliparous and non-pregnant: not specified
- Age and weight at study initiation: 8-9 weeks old / 225 to 262 g for males and 152 to 184 g for females
- Housing: 2 or 3 per cage by sex in suspended polycarbonate cages (dimensions 61 x 43.5 x 24 cm) with stainless steel grid tops, solid bottoms and a separate stainless steel food hopper. Sterilised white wood shavings were used as bedding. For environmental enrichment, wooden chewsticks and cardboard play tunnels were also used.
- Diet: Rat and Mouse (modified) No. 1 Diet SQC Expanded (Ground). Ad libitum
- Water: Public water. Ad libitum
- Acclimation period: 14 days

Environmental Conditions
- Temperature: 19-23°C
- Humidity: 39-64%
- Air changes: minimum of 10 air changes per hour
- Photoperiod: Artificial giving 12 hours light, 12 hours dark

In-life Dates: From: 31 July 2012 To: 19 Nov 2013

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Method: Diet formulations were prepared from an initial high level stock premix. This stock was prepared by making a 400 g premix, which contained the total weight of test substance required to produce the final concentration at the weight requested, and untreated control diet. Before the test substance was weighed it was sieved through a 0.5 mm mesh sieve to remove any lumps.
- Rate of preparation of diet: Formulated diets were prepared every 2 weeks
- Storage temperature of food: Room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from diets were collected for analysis for concentration and homogeneity on Day 1, Week 6 and Week 13. Duplicate (2 x 10 g) samples from the top, middle and bottom were taken immediately after preparations from all test diets prepared for use. Samples obtained for use on Day 1 and during Weeks 6 and 13 were analysed for concentration and homogeneity within 7 days after preparation. Analyses were performed by reverse phase HPLC with uv detection using a validated analytical procedure.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously

Dose / conc.:

200 ppm

Remarks:

Group 2. Dietary equivalent to 14 and 18 mg/kg bw/day for males and females, respectively.

Dose / conc.:

3 000 ppm

Remarks:

Group 3. Dietary equivalent to 213 and 272 mg/kg bw/day for males and females, respectively.

Dose / conc.:

10 000 ppm

Remarks:

Group 4. Dietary equivalent to 704 and 868 mg/kg bw/day for males and females, respectively.

No. of animals per sex per dose:

Treatment groups: 10, Recovery groups (control and high dose group): 5

Control animals:

yes, plain diet

Details on study design:

Groups of 10 male and 10 female Han Wistar Crl:WI(Han) rats were assigned to the study and fed diets containing 0, 200, 3000 or 10000 ppm of the test item for a minimum period of up to 91 consecutive days. A further 5 male and 5 female animals were assigned as a recovery group to those receiving 0 or 10000 ppm of the test item and were retained for a further 28 days after the end of treatment to evaluate reversibility of any findings.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS
All animals were checked early morning and as late as possible each day for viability. Once each week, beginning during the week of the pre-trial period, each animal was removed from the cage and received a detailed clinical examination including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT
Body weights were recorded twice during the pretrial period, and weekly throughout the dosing and recovery periods

FOOD CONSUMPTION AND COMPOUND INTAKE
The quantity of food consumed by each cage of animals was measured and recorded twice during the pretrial period, and weekly throughout the dosing and recovery periods. The amount of experimental diet ingested was calculated at regular intervals during treatment using the following formula: Achieved intake (mg/kg/day) = ((dose (ppm) x cage mean food consumption (g/animal/day))/cage mean body weight at the middle of the period

FOOD UTILISATION
Food utilisation was calculated for each cage as follows: (cage mean weight gain x 100)/cage total food consumption
Values were calculated for intervals of Weeks 1-4, 5-8, 9-13 and also an overall value for Weeks 1-13.

WATER CONSUMPTION
Water consumption was qualitatively monitored by visual inspection of the water bottles on a weekly basis during the study.

OPHTALMOSCOPY
The eyes of all animals (including extras) were examined during the pretrial period using an indirect ophthalmoscope after the application of mydriatic agent (1% Tropicamide). Anterior, lenticular and fundus areas were evaluated. All animals in the control and high dose group (10000 ppm) were also examined during Week 13 of treatment. All recovery animals were examined during Week 4 of the recovery period.

FUNCTIONAL OBSERVATION BATTERY
Once during the treatment period (Week 12/13) a more detailed examination was made of all animals. The examinations were performed at an approximately standardised time of day. Before to the independent technician entering the room, cage cards showing treatment groups were removed and only the functional observation cards were shown. The assessor was then allowed to enter the room. One or two animals from each cage had their tail marked for identification purposes.

CAGESIDE OBSERVATIONS
- Posture/condition on first approach (animal undisturbed), checking for: Prostration, Lethargy, Writhing, Circling, Breathing abnormalities, Gait abnormalities, Tremor, Fasciculation, Convulsions, Biting (of cage components or self mutilating), Vocalisations, Piloerection
- Ease of removal from the cage.
- Body temperature was taken from the implanted electronic identification chip. On several occasions a rectal temperature was taken and recorded.
- Condition of the eyes, checked for: Pupillary function, Miosis, Mydriasis, Exophthalmos, Encrustation, Lacrimation.
- Condition of the coat.
- Presence of salivation.
- Overall ease of handling.

OBSERVATIONS IN A STANDARDISED AREA (2 MIN OBSERVATION PERIOD)
- Parameters assessed: Latency (time to first locomotory movement), Level of mobility, Rearing, Grooming, Urination/defecation, Arousal (level of alertness), Posture, tremor/convulsions, vocalisation, piloerection – recorded as for cageside observations, Palpebral closure, Gait abnormalities, Stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

FUNCTIONAL TESTS
Once during the treatment period (Week 12/13), the following additional functional tests were performed. These assessments were performed at an approximately standardised time of day.
- Reaction to sudden sound (click above the head in the arena)
- Reaction to touch on the rump with a blunt probe (in the arena)
- Grip strength: This was measured using a Linton Grip Strength dual/single channel meter (provided by Linton Instruments) to which was attached a wire-screen assembly. 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.
- Pain perception: This was assessed by measurement of the tail flick response. The apparatus used shone a calibrated infrared heat source onto the tail and automatically measured the reaction time of the animal (accurate to 0.1 s). It was ensured that no visible injury to the tail was caused during this test.
- Landing foot splay: Corn oil was applied to the hind paws of each animal. The animal was then held in a horizontal, prone position with the nose approximately 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. The procedure was repeated 3 times and the average measurement recorded. If the rat did not land properly on its feet, this was recorded.
- Motor activity (recorded at approximately standardised time of day): Each animal was placed in an individual monitoring cage, scanned by a motion sensor utilising infra-red pyroelectric detectors. Movement was detected in 3 dimensions anywhere in the cage, and was differentiated into large and small movements. Each animal was monitored for one session of 1 h, activity counts were recorded over successive periods of 5 min.
- Any other abnormality not already recorded in the above screening battery.

CLINICAL CHEMISTRY
Approximately 1.0 mL of whole blood was collected and transferred into tubes containing lithium heparin which was then centrifuged and the plasma assayed for the following parameters: Urea (Urea), Total Protein (TP), Glucose (Glu), Albumin (Alb), Aspartate Aminotransferase (AST), Globulin (Glob), Alanine Aminotransferase (ALT), AG Ratio (AG-R), Alkaline Phosphatase (ALP), Cholesterol (Chol), Creatine Phosphokinase (CPK), Creatinine (Crea), Lactate Dehydrogenase (LDH), Total Bilirubin (T.Bil), Sodium (Na), Calcium (Ca), Potassium (K), Phosphate (Phos), Chloride (Cl), Triglycerides (Trig).

HAEMATOLOGY
Blood samples for haematology, coagulation and clinical chemistry were obtained from all animals, via the orbital sinus under isoflurane anaesthesia before scheduled euthanasia of main study animals on Days 91 or 92. Blood samples were also collected from recovery study animals before scheduled euthanasia on Day 119 after completion of the recovery period. Animals were not deprived of food overnight and samples were collected in a random order.
Approximately 0.5 mL of blood was taken into tubes containing EDTA and assayed for the following parameters: Red Blood Cell Count (RBC), White Blood Cell Count (WBC), Haemoglobin (Hb), Differential White Blood Cell Count Haematocrit (Hct), Neutrophils (Neut), Mean Cell Volume (MCV), Lymphocytes (Lymph), Mean Cell Haemoglobin, Concentration (MCHC), Monocytes (Mono), Mean Cell Haemoglobin (MCH), Eosinophils (Eos), Reticulocytes (Reti), Basophils (Baso), Reticulocyte Count (Ret), Large Unstained Cells (LUC), Red Cell Distribution Width (RDW), Platelet Count (Plat). A blood smear was prepared from each haematology specimen. Blood smears were labelled, stained, stored and archived.
-Coagulation
Approximately 0.9 mL of blood was collected into tubes containing 0.1 mL 3.8% (w/v) trisodium citrate. The final sample volume was as close as possible to 1.0 mL to give a final concentration of 0.38% (blood to citrate ratio of 9:1). The citrated blood samples were then centrifuged and the plasma separated into plain plastic tubes and analysed for the following parameters: Activated Partial Thromboplastin Time (APTT) Prothrombin Time (PT) Fibrinogen (Fib)

URINALYSIS
Urine samples from all Main and Recovery study animals were collected over an approximate 4 h period during Week 13 (Days 86 and 90) and from all Recovery study animals during Week 17 (Day 114). The animals were housed individually in metabolism collection cages and were deprived of food and water. The following parameters were evaluated: Microscopy of the Spun Deposit Glucose (UGl), Colour (UCol) Bilirubin (U.Bi), Turbidity (UTb), Ketones (UKet), Specific gravity (S.G.), Leukocytes (Leu) Volume, (UVol) Blood Pigments (BP), pH (UpH), Urobilinogen (Uro), Protein (UProt).
The limit of quantification (LOQ) for the following assays was observed and reported as follows with the LOQ used to calculate means and standard deviations. Assay: Total Bilirubin; Limit of Quantification: 1.7 μmol /L; Non-detectable values reported as <1.7 μmol /L. Assay: Urinary Volume; Limit of Quantification: 0.1 mL; Non-detectable values reported as <0.1 mL.

Sacrifice and pathology:

TERMINATION
All Main study animals were euthanized on Days 92 or 93 after completion of at least 91 days of treatment by exposure to a rising concentration of carbon dioxide and had their terminal body recorded followed by severance of major blood vessels. The animals were euthanized in random order throughout the dose groups. All Recovery study animals were subsequently euthanized as above following completion of a 28 day recovery period.

NECROPSY
Each animal was subject to a detailed necropsy conducted by a trained technician. A veterinary pathologist was available for consultation for the duration of the necropsy sessions. The necropsy consisted of a complete external and internal examination including body orifices (ears, nostrils, mouth, anus and vulva) and cranial, thoracic and abdominal organs and tissues. All gross findings were recorded in descriptive terms, including location(s), size (in mm), shape, colour, consistency and number.

ORGAN WEIGHTS
The organs that were removed and weighed from all animals before sampling and preservation were: Brain, Epididymis x2, Gland, adrenal x2, Heart, Kidney x2, Liver, Ovary x2, Spleen, Testis x2, Thymus, Uterus. Paired organs were weighed separately and the sum of the individual organs used for reporting purposes. Representative samples of the tissues were taken from all animals and fixed in 10% neutral buffered formalin, unless otherwise stated. Carcasses were discarded following sampling of these tissues.

HISTOLOGY
Tissues were processed to paraffin wax block. Sections 4-6 μm thick, were cut and stained with haematoxylin and eosin (H&E). The examined tissues were: Artery, aorta, Bone marrow, femur, Bone marrow, sternum, Bone, femur, Bone, sternum, Brain, Cervix, Epididymis x2, Eye x2, Gland, adrenal (x2), Gland, harderian, (x2), Gland, lacrimal, (x2), Gland, mammary, Gland, parathyroid (x2), Gland, pituitary, Gland, prostate, Gland, salivary, (x2), Gland, seminal vesicle, Gland, thyroid, (x2), Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney (x2), Large intestine, caecum, Large intestine, colon, Large intestine, rectum, Liver, Lung, Lymph node, mandibular, Lymph node, mesenteric, Muscle, skeletal, Nerve, optic (x2), Nerve, sciatic, Oesophagus, Ovary, (x2), Oviduct (x2), Pancreas, Pharynx, Skin, Small intestine, duodenum, Small intestine, ileum, Small intestine, jejunum, Spinal cord, Spleen, Stomach, Testis (x2), Thymus, Tongue, Trachea, Urinary, bladder, Uterus, Vagina

BONE MARROW SMEARS
Duplicate bone marrow smears were taken at necropsy and stained using May-Grunwald-Giemsa. No evaluation of the smears was undertaken; smears were retained as a contingency
until completion of the study when they were archived.

HISTOPATHOLOGY AND PEER REVIEW
Histopathological evaluation was performed by a veterinary pathologist with training and experience in laboratory animal pathology. An internal peer review was undertaken by an appropriately qualified and experienced veterinary pathologist who validated the conclusions of the study pathologist by independently assessing the microscope slides. Internal peer review notes are archived with the pathology data. An external peer review was performed.

Statistics:

The following statistical approaches were used in this study: All analyses were two-tailed for significance levels of 5% and 1%. All means are presented with standard deviations. Body weights, cumulative body weight change, food consumption, selected functional observatory battery and motor activity data, haematology, coagulation, clinical chemistry and selected urinalysis parameters and 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. This statistical analysis provided Adjusted Organ Weight values. Summary values of organ to body weight ratios are presented but these were not analysed statistically. For all 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. Micropathology incidence data were analysed using Fisher’s Exact Test. Incidences of multiple severities were also analysed using Mann-Whitney U Test. Functional observational battery parameters that yield discontinuous or descriptive data were analysed using Fisher’s Exact Test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs which could be attributed to treatment with the test item. All signs were considered to be typical of those recorded on studies of this type for rats of this age and strain conducted at the Test Facility.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was an effect on body weights in animals receiving 3000 or 10000 ppm of the test item, when compared with controls. For both sexes receiving 10000 ppm of the test item, body weights were lower throughout the treatment period; achieving statistical significance in males and females from Day 7. There was a statistically significant lower group mean body weight change over the treatment period (Day 0-91) when compared with the control group (p<0.01). The difference from the controls was more marked at this dosage with the male body weight gain being 43% lower and the females body weight gain being 30% lower than the controls. Group mean body weights for animals that received 3000 ppm were generally slightly lower throughout the treatment period, when compared with controls, achieving statistical significance on Days 7 and 14 in males (p<0.05). There was a statistically lower group mean body weight change over the treatment period (Days 0-91) in males and females (p<0.05) when compared with the control group, with the male body weight gain being 18% lower and the female body weight gain being 14% lower than the controls. Group mean body weights and body weight gain of animals receiving 200 ppm of the test item were similar to controls. During the recovery period, group mean body weight change for animals that received 10000 ppm was similar or higher than the controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was an effect on food consumption in animals receiving 10000 ppm of the test substance, particularly in males, with a lower amount of food consumed throughout the treatment period when compared with controls; achieving statistical significance on Day 7 and from Day 49 to the end of the treatment period (p<0.05 or p<0.01). In females at 10000 ppm the food consumption was considered to be similar to the controls, however, there was an initial statistical significant reduction noted on Day 7 (p<0.01). In males receiving 3000 ppm there was lower food consumption during the first week of treatment, when compared with controls (p<0.01 or p<0.05). For the rest of the treatment period the food consumed was similar to the controls. Food consumption values for both sexes receiving 200 ppm and females receiving 3000 ppm was similar to the controls throughout the treatment period, however, there was minor, transient inter-group differences which achieved statistical significance, but were not considered to be treatment-related or toxicologically relevant. In the recovery period food consumption in animals that received 10000 ppm was similar to controls

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Differences in food consumption for animals receiving 3000 or 10000 ppm were reflected in the food utilisation, which was statistically significantly lower in males receiving 3000 ppm overall for the duration of the study (Weeks 1-13). Due to the differences in body weight gain in females receiving 3000 ppm, food utilisation was statistically lower over the period Week 1-4, 5-8 and overall during Week 1-13. At 10000 ppm, food utilisation was statistically significantly lower when compared with the controls over the periods Weeks 1-4, 9-13 and overall for Weeks 1-13 for males and during the period Weeks 1-4, 5-8 and overall for females.

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

no effects observed

Description (incidence and severity):

Visual inspection indicated no observable differences between groups throughout the treatment period.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no findings during ophthalmoscopic examinations that could be attributed to the test item.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related inter-group differences in haematology or coagulation parameters following administration of the test substance at dose levels up to 10000 ppm. At the end of the treatment period lower red blood cell counts and haematocrit were observed in males receiving 10000 ppm (p<0.05), and lower haemoglobin (p<0.01) and haematocrit (p<0.05) were observed in females receiving 10000 ppm, when compared with controls. However, inspection of the data indicated that although there was a high degree of inter- animal variation, broadly the values for the animals receiving the test item were similar to the controls. There were also no differences in associated parameters or indices, therefore, these differences were considered not to be related to treatment. No noteworthy differences were noted at the end of the recovery period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related inter-group differences in clinical chemistry parameters following administration of the test item at dose levels up to 10000 ppm. Minor inconsistent differences which achieved statistical significance at the end of the treatment period were observed in males (lower triglyceride and higher creatinine levels at 10000 ppm) and females (higher alanine aminotransferase activity, lower total protein and lower albumin levels at 10000 ppm) when compared to the controls. Inspection of the data indicated that although there was variation within the data, broadly the values for the animals receiving test item were similar to controls and there were also no differences in associated parameters or indices. These differences were considered not to be related to treatment. On completion of the recovery period, with the exception of the alanine aminotransferase activity, the differences described were still evident. Other minor differences which achieved statistical significance were observed in males (higher urea, lower cholesterol and lower total protein levels) and females (lower albumin and calcium levels). As these findings, with the exception of the lower albumin in females, were not seen at the end of the treatment period, they were considered toxicologically irrelevant.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

In the parameters that could be assessed there was no evidence of an effect on urine composition. To evaluate all urinary parameters, a urine volume of ≥ 0.4 mL is required; a volume of 0.1-0.3 mL will allow assessment of all parameters with the exception of specific gravity. No assessment can be made with a volume of <0.1 mL. At the end of the treatment period, there were several animals, for which no urine, or a very small volume of urine, was excreted over the collection period. Whilst this occurred in all groups, including controls, this result was more noticeable in males receiving 10000 ppm. Of the 15 animals in this group, only one animal produced a sample of a high enough volume (>0.4 mL) for all parameters to be measured. In the control group only one animal failed to provide an adequate sample volume (>0.1 mL) to assess the majority of parameters. In females this effect was slightly more difficult to interpret, however, fewer urine samples were produced in animals receiving the test item over the collection period. Six of the 15 control animals failed to produce an adequate volume (>0.1 mL), whereas 6/10 and 7/10 animals receiving 200 or 3000 ppm, respectively, and 9 of the 15 animals receiving 10000 ppm failed to produce an adequate volume (>0.1 mL) to assess the majority of parameters. At the end of the recovery period all male control animals and 4 out 5 control females produced a sample, whereas only two males and one female that received the test item produced an adequate sample.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FUNCTIONAL OBSERVATION BATTERY

There were no treatment-related differences in the functional observation battery parameters following administration of the test substance at dose levels up to 10000 ppm. A difference in the general behaviour of the animals in their home cage was observed in males receiving 200 or 10000 ppm was where the number of animals that were asleep/at rest was increased in comparison to controls. As there were no other abnormal observations or adverse observations noted on the behaviour of animals throughout treatment or in the females these differences were considered to be incidental and not treatment related. All of the behaviours exhibited and observations were considered to be typical for rats of this age and strain on this type of study conducted at the Test Facility



QUANTITATIVE FUNCTIONAL OBSERVATIONS

There were no treatment-related differences in the quantitative functional observation parameters following administration of the test substance at dose levels up to 10000 ppm. Minor differences in landing foot splay that achieved statistical significance were observed in males at 3000 and 10000 ppm when compared with the controls. This isolated difference had no association with any other indices measuring effects on movement, the values recorded were broadly similar to controls, and as such was considered to be incidental and not treatment related



MOTOR ACTIVITY

There were no notable inter-group differences in motor activity following administration of the test substance at dose levels up to 10000 ppm. The number of movements of males receiving 200 ppm was statistically significantly higher, when compared with controls during the 21-25 minute time period and at no other timepoint. A statistically significant higher number of movements were also recorded for males receiving 10000 ppm during the 31-35 minute time period and at no other timepoint. These isolated differences, in the absence of a dose-response, only present in one sex, and with no difference in the overall number of movements, was therefore considered not to be treatment related.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher liver weights (covariance analysis and relative to body weight) were noted in males and females given 10000 ppm (adjusted means higher than control by 9% and 13%, respectively), when compared with controls, with only the females attaining statistical significance. Higher kidney weights (covariance analysis and relative to body weight) were observed in males and females across all dose groups and showed a dose relationship, but statistical significance was not attained. No other noteworthy test substance-related organ weight changes were noted.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross findings were noted in both the main study animals and recovery animals. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no histopathology findings that correlated with the higher liver and kidney weights recorded at necropsy. Increased hyaline droplets was observed in the kidney in 2/10 males given 10000 ppm. There was an increased incidence of accumulation of brown pigment in the renal cortical tubules in females given 10000 ppm (4/10 animals), when compared with controls (1/10 animals). The toxicological significance of these findings was considered equivocal. Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance. No test substance-related findings were noted also in the recovery animals after a 28 day recovery period. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test item.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Dietary equivalent to 14 and 18 mg/kg bw/day for males and females, respectively.

Key result

Critical effects observed:

no

ANALYSIS OF DIET FORMULATIONS

The diets containing the test item were considered to have been prepared accurately as the found concentrations were all within 10% of the theoretical concentration. The low coefficient of variation (7.2% or lower) indicated satisfactory homogeneity of the formulated diets.

 

ACHIEVED DOSAGE

The overall mean achieved dosages were 0, 14, 213, and 704 mg test item/kg/day in males and 0, 18, 272, and 868 mg test item/kg/day for females corresponding to dietary inclusion levels of 0, 200, 3000 and 10000 ppm.

 

 

 

Conclusions:

Under the conditions of this study, the daily oral (dietary) administration of the test item to Han Wistar rats for up to 91 consecutive days was associated with lower body weights, body weight gain and food consumed at 3000 and 10000 ppm. These findings recovered after a 28-day treatment free period. Based on these findings the no observed adverse effect level (NOAEL) was considered to be 200 ppm.

Executive summary:

This OECD TG 408 study was performed under GLP to assess the toxicity of the test item in the rat after oral administration by the diet for up to 13 weeks (91 consecutive days) and to evaluate the reversibility of any findings. Groups of 10 male and 10 female Han Wistar Crl:WI(Han) rats were assigned to the study and fed diets containing 0, 200, 3000 or 10000 ppm of the test item for a minimum period of up to 91 consecutive days. A further 5 male and 5 female animals were assigned as a recovery group to those receiving 0 or 10000 ppm of the test item and were retained for a further 28 days after the end of treatment to evaluate reversibility of any findings. The following observations and end points were assessed: viability, clinical observations, body weights, food consumption and ophthalmoscopy examinations. All animals received a detailed functional observation battery (including motor activity) during Week 12/13 of treatment. Blood samples for haematology, coagulation and blood chemistry investigations and urine samples were collected from all animals towards the end of the treatment period and at the end of the recovery period. All main study animals were euthanised after completion of at least 91 days of treatment with all recovery study animals euthanised after completion of a subsequent 28 day treatment-free period, and underwent a detailed necropsy examination with selected organs weighed and subsequent histopathological evaluation.

During this test, there were no unscheduled deaths, and no treatment-related clinical observations noted during the observation periods. Lower body weight gain was observed in animals treated with 3000 or 10000 ppm and this was associated with reduced food consumption. There were no test substance related effects on the movement, general behaviour or the physiology of the animals and haematology, coagulation and clinical chemistry were unaffected by treatment. There were no eye changes that were related to treatment. Higher liver weights were noted in males and females given 10000 ppm, and higher kidney weights were noted in males and females that received 200, 3000 or 10000 ppm and there were no gross or microscopic findings which were considered related to treatment with the test item.

Under the conditions of this study, the daily oral (dietary) administration of the test item to Han Wistar rats for up to 91 consecutive days was associated with lower body weights, body weight gain and food consumed at 3000 and 10000 ppm. These findings recovered after a 28-day treatment free period. Based on these findings the no observed adverse effect level (NOAEL) was considered to be 200 ppm.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

14 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP compliant OECD TG 408 study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

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:

10 Jul 2012 to 14 Sep 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sex/Number ordered: 22 males, 22 females. Twenty males and 20 females were assigned to the study. The 2 remaining animals of each sex were assigned as extra animals
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 8-9 weeks old
- Weight at study initiation: 212-250 g for males and 156-194 g for females
- Housing: 2 or 3 per cage by sex in polycarbonate cages (dimensions
61 x 43.5 x 24 cm) with stainless steel grid tops, solid bottoms and an integral food hopper. Sterilised white wood shavings were used as bedding. For environmental enrichment, wooden chewsticks and cardboard play tunnels were supplied and analysis of these items was considered to indicate that there were no substances in sufficient concentration to have any influence on the outcome of the study.
- Diet: Ad libitum with the exception of during the 6 hour period of dermal application where animals had no access to food.
- Water: Water from the public water supply. Ad libitum with the exception of during the 6 hour period of dermal application where animals had no access to water.
- Acclimation period: 14 days. Prior to study commencement, animals were acclimatised to the occlusion procedures over a 5 day period, where the duration of the occlusion was increased on each occasion until the maximum of 6 hours was obtained.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: 10 air changes per hour.
- Photoperiod: 2 h light/dark cycle, light hours being 0700-1900 h

IN-LIFE DATES: From: 10 Jul 2012 To: 14 Sep 2012

Type of coverage:

semiocclusive

Vehicle:

other: Milli-Q water

Details on exposure:

TEST SITE
- Area of exposure: lumbar region
- Pre-treatment: The hair on an area of the lumbar region was clipped free of hair as necessary, and test or control substance placed directly onto the skin.
- % coverage: approximately 10 % body surface area. The total body surface area (BSA) of each animal was calculated using the following formula: Total BSA (cm2) = K x W^2/3 Where K = constant for calculating BSA in rats; where W= cubic root of the square of the body weight
- Type of wrap: wetted gauze patches covered in semi-occlusive tape (micropore) and then securely fastened with a strip of occlusive tape (sleek)

REMOVAL OF TEST SUBSTANCE
- The dose site was wiped clean of any excess material, by means of mains supply water and separate gauzes
- Time after start of exposure: 6 hours (± 30 minutes)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hours (± 30 minutes)

Frequency of treatment:

Once daily for at least 28 days and up to the day before termination

Dose / conc.:

10 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

Three groups of 5 male and 5 female Han Wistar Crl: WI(Han) rats received the test item directly onto the skin in the lumbar region, at dose levels of 10, 100 or 1000 mg/kg bw/day, daily for a period of 6 hours over 28 consecutive days. A concurrent control group was used which received Milli-Q water at a dose volume of 1 mL/kg.
Dose levels for this study, 10, 100 and 1000 mg/kg/day, were selected after evaluation of previous studies carried out by the Sponsor. Previous experience with dermal application of this test substance showed that the material was well tolerated at 1000 mg/kg/day, the limit dose.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
All animals were checked early morning and as late as possible each day for mortality or clinical signs. Once each week, beginning during the week of the pre-trial period, each animal was removed from the cage and received a detailed clinical examination including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta. Regularly throughout each day, all animals were examined for reaction to treatment. The onset, intensity and duration of any signs were recorded. Particular attention was paid to the animals for the first hour during dosing.

DRAIZE EVALUATION OF DERMAL REACTIONS
Dermal scoring was conducted 0 h (immediately before dosing) and 6 h after each administration of test substance. Skin was assessed for erythema and eschar formation, oedema formation, skin thickening, desquamation and any other reaction to treatment. The scoring system in Table 1 was used for assessing erythema, eschar and oedema formation.

BODY WEIGHT:
Body weights were recorded three times during pre-trial including a Day -1 body weight to allow test substance preparation, and then twice weekly until study completion.

FOOD CONSUMPTION:
The quantity of food consumed by each cage of animals was measured and recorded twice weekly commencing from pre-trial (Day -7) until study completion.

WATER CONSUMPTION:
Water consumption was qualitatively monitored by visual inspection of the water bottles on a weekly basis during the study.

OPHTHALMOSCOPIC EXAMINATION:
The eyes of all animals (including extras) were examined once during the pre-trial period using an indirect ophthalmoscope after the application of mydriatic agent (1% Tropicamide). Anterior, lenticular and fundus areas were evaluated. All control and high dose animals were also examined during Week 4.

HAEMATOLOGY:
Blood samples for haematology, coagulation and clinical chemistry were obtained from all animals, via the orbital sinus under isoflurane anaesthesia on Day 29. Animals were not deprived of food overnight prior to sampling in a random order. Animals were allowed to recover from anaesthesia before terminal necropsy.
-Approximately 0.5 mL of blood was taken into tubes containing EDTA and assayed for the following parameters: Red Blood Cell Count (RBC), Platelets (Plat), Haemoglobin (Hb), Blood smear, Haemoglobin, Distribution Width (HDW), White Blood Cell Count (WBC), Haematocrit (Hct), Neutrophils (Neut), Mean Cell Volume (MCV), Lymphocytes (Lymph), Mean Cell, Haemoglobin Concentration (MCHC),Monocytes (Mono), Mean Cell Haemoglobin (MCH), Eosinophils (Eos), Reticulocytes (Reti %), Basophils (Baso), Reticulocyte Count (absolute) (Ret), Large Unclassified Cells (LUC), Reticulocyte Maturity Index (low, medium and high absorption), Red Cell Distribution Width (RDW).
-Approximately 0.9 mL of blood was collected into tubes containing 3.8% (w/v) trisodium citrate to assess coagulation. The final sample volume was as close as possible to 1.0 mL to give a final concentration of 0.38% (blood to citrate ratio of 9:1). The citrated blood samples were then centrifuged and the plasma separated into plain plastic tubes and analysed for the following parameters: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY:
Approximately 1.0 mL of whole blood was collected and transferred into tubes containing lithium heparin which was then centrifuged and the plasma assayed for the following parameters: Urea (Urea), Chloride (Cl), Urea Nitrogen (BUN), Total Protein (TP), Glucose (Glu), Albumin (Alb), Aspartate Aminotransferase (AST), Globulin (Glob), Alanine Aminotransferase (ALT), Albumin/Globulin ratio (AG-R), Alkaline Phosphatase (ALP), Cholesterol (Chol), Creatine Phosphokinase (CPK), Creatinine (Crea), Lactate Dehydrogenase (LDH), Total Bilirubin (T.Bil), Glutamate Dehydrogenase (GLDH), Calcium (Ca), Gamm-glutamyl Transferase (GGT), Inorganic Phosphate (Phos), Sodium (Na), Triglycerides (Trig), Potassium (K).
The limit of quantification (LOQ) for the following assays was observed and reported as follows with the LOQ used to calculate means and standard deviations for values below the LOQ:
-Gamma-glutamyl Transferase asay; limit of detection: 3 U/L; Non-detectable values reported as: <3 U/L
-Total Bilirubin; limit of detection:1.7 µmol /L; Non-detectable values reported as: <1.7 µmol /L

Sacrifice and pathology:

Following 28 days of treatment, all animals were sacrificed in a random order by exposure to a rising concentration of carbon dioxide and had their terminal body weight recorded followed by severance of major blood vessels.

GROSS PATHOLOGY:
Each animal was subject to a detailed necropsy conducted by a trained technician. A veterinary pathologist was available for consultation for the duration of the necropsy session. The necropsy consisted of a complete external and internal examination including body orifices (ears, nostrils, mouth, anus and vulva) and cranial, thoracic and abdominal organs and tissues. All gross findings were recorded in descriptive terms, including location(s), size (in mm), shape, colour, consistency and number.
The organs marked ‘x’ in the ‘Weighed’ column in Table 2 were removed and weighed from all animals. Paired organs were weighed separately and the sum of the individual organs used for reporting purposes. Representative samples of the tissues listed in Table 2 were taken from all animals and fixed in 10% neutral buffered formalin, unless otherwise stated. Carcasses were discarded after checking the retained tissues against the protocol and a review of the necropsy report.

HISTOPATHOLOGY:
Tissues marked ‘x’ in the ‘Examined’ column in Table 2 were processed to paraffin wax block from all control and high dose animals, sectioned, mounted on glass slides, and stained with haematoxylin and eosin (H&E). Additionally, all gross lesions from low and intermediate dose animals were also processed. Histopathological evaluation of all tissues was then undertaken for all control and high dose animals and for any gross abnormalities (where appropriate) from low and intermediate dose animals.

Other examinations:

BONE MARROW SMEARS
Duplicate bone marrow smears were taken at necropsy and stained using May-Grunwald- Giemsa. No evaluation of the smears was undertaken; smears were retained as a contingency until completion of the study when they were archived.

PEER REVIEW
An internal peer review was undertaken by an appropriately qualified and experienced veterinary pathologist who validated the conclusions of the Study Pathologist by independently assessing the microscope slides. Internal peer review notes are archived with the pathology data

Statistics:

The following statistical approaches were used in this study:
- All analyses were two-tailed for significance levels of 5% and 1%. Males and females were analysed separately.
- All means are presented with standard deviations.
- Body weights, absolute organ weights, haematology, coagulation and clinical chemistry parameters 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. This statistical analysis provided Adjusted Organ Weight values.
- Summary values of organ to body weight ratios and food consumptions are presented but these were not analysed statistically.
- For all parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control (Group 1) 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.
- Micropathology incidence data were analysed using Fisher’s Exact Test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no signs indicative of systemic toxicity noted during the observation period. There were local signs recorded at the administration site of several males and females across the dose groups, including controls, where scabbing and/or areas of sparse hair were recorded. Additionally fur staining (2/5) was observed in males that were administered 1000 mg/kg/day of the test item. These local signs were considered minor, transient and were consistent with observations in the control group and as result were not considered to be related to treatment.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

An isolated incidence of very slight erythema (barely perceptible) was recorded in one male (1000 mg/kg/day) on days 5 through 10; mild desquamation (dry skin) was also noted for this animal during this time. This incidence was transient; the condition of the animal’s skin was generally noted to be normal, and this finding is considered to be of doubtful toxicological significance.

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related differences in group mean body weights or body weight gain in treated groups in comparison to controls throughout treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related inter-group differences noted in food consumption following administration of the test item at dose levels up to 1000 mg/kg/day.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no ophthalmoscopy findings which were considered to be related to the administration of the test item at dose levels up to 1000 mg/kg/day

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related inter-group differences in haematology or coagulation parameters following administration of the test item at dose levels up to 1000 mg/kg/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related differences in clinical chemistry parameters following administration of the test item at dose levels up to 1000 mg/kg/day.

Statistically significantly higher cholesterol levels were observed in females that received 1000 mg/kg/day when compared with controls. The difference was minor and observed in females only. Due to the isolated nature of the difference it was considered not to be treatment-related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related organ weight changes were noted. There were isolated organ weight values that were different from their control. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and/or related to differences of sexual maturity and unrelated to administration of the test item

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test item

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test item

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Critical effects observed:

no

Conclusions:

The test item, when administered topically for up to 6 hours daily over a 4 week period, was well tolerated in rats up to 1000 mg/ kg bw/day with no evidence of systemic toxicity. Therefore, under the conditions of this study, the NOAEL was considered to be 1000 mg/kg bw/day.

Executive summary:

This OECD TG 410 study was performed according to GLP, to determine the potential dermal toxicity of the test item when administered topically, for a period of 28 days to rats and to allow hazard classification to be evaluated. Three groups of 5 male and 5 female Han Wistar Crl: WI(Han) rats received the test item directly onto the skin in the lumbar region, at dose levels of 10, 100 or 1000 mg/kg bw/day, daily for a period of 6 hours over 28 consecutive days. A concurrent control group was used which received Milli-Q water at a dose volume of 1 mL/kg. The following parameters and end points were evaluated in this study from all animals: viability, clinical observations, administration-site dermal scoring reactions, body weights, food and water consumption and ophthalmoscopy examinations. Blood samples were collected from all animals at termination for haematology, coagulation and blood chemistry investigations. All animals were terminated after completion of 28 days of treatment and underwent a detailed necropsy examination with selected organs weighed. Tissues from all control and high dose (1000 mg/kg bw/day) animals were subjected to a comprehensive histological examination, with gross lesions (where appropriate) examined from low and intermediate dose animals.

The test item, when administered topically onto the skin for up to 6 hours daily over a 4 week period, was well tolerated in rats up to 1000 mg/ kg bw/day with no evidence of systemic toxicity. There was no impact on body weights, food consumption, blood chemistry or haematological parameters. Additionally, there were no organ weight differences or macroscopic or microscopic local reactions at the administration site. Therefore, the NOAEL was considered to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Species:

rabbit

Quality of whole database:

GLP compliant OECD TG 410 study

Additional information

Repeated dose toxicity studies: oral
This dossier is an Annex VII dossier. However, a number of studies for Annex VIII, or higher, are available. These studies have been included as supporting studies for this endpoint. All available data was assessed. Two sub-chronic, two chronic and one short-term oral toxicity studies in the rat and dog were performed to evaluate to toxicity potential of the test item.

Sub-chronic toxicity
An OECD TG 408 study (Robertson 2013) was performed under GLP to assess the toxicity of the test item in the rat after oral administration by the diet for up to 13 weeks (91 consecutive days) and to evaluate the reversibility of any findings. Groups of 10 male and 10 female Han Wistar Crl:WI(Han) rats were assigned to the study and fed diets containing 0, 200, 3000 or 10000 ppm (dietary equivalent to 14, 213, 704 mg/kg bw/day for males and 18, 272, 868 mg/kg bw/day for females) for a minimum period of up to 91 consecutive days. A further 5 male and 5 female animals were assigned as a recovery group to those receiving 0 or 10000 ppm of the test item and were retained for a further 28 days after the end of treatment to evaluate reversibility of any findings. All animals received a detailed functional observation battery (including motor activity) during week 12/13 of treatment. During this test, there were no unscheduled deaths, and no treatment-related clinical observations noted during the observation periods. Lower body weight gain was observed in animals treated with 3000 or 10000 ppm and this was associated with reduced food consumption. There were no test substance related effects on the movement, general behaviour or the physiology of the animals and haematology, coagulation and clinical chemistry were unaffected by treatment. There were no eye changes that were related to treatment. Higher liver weights were noted in males and females given 10000 ppm, and higher kidney weights were noted in males and females that received 200, 3000 or 10000 ppm and there were no gross or microscopic findings which were considered related to treatment with the test item. Under the conditions of this study, the daily oral (dietary) administration of the test item to Han Wistar rats for up to 91 consecutive days was associated with lower body weights, body weight gain and food consumed at 3000 and 10000 ppm. These findings recovered after a 28-day treatment free period. Based on these findings the no observed adverse effect level (NOAEL) was considered to be 200 ppm (dietary equivalent to 14 and 18 mg/kg bw/day for males and females, respectively).

Another oral sub-chronic toxicity study was performed in rat (Tai 1985), that confirmed the NOAEL value of the study performed by Robertson. In this study, based on OECD TG 407 and 408 and performed under GLP, the test item was administered in the feed to male and female rats at concentrations of 0, 200, 10000, or 20000 ppm for 92-93 consecutive days. Compound-induced effects were noted at levels higher than 10000 ppm. Major findings included: decreased food consumption, body weights, and body weight gain with secondary changes in clinical laboratory parameters and absolute and relative organ weights; and renal and/or urinary effects such as hematuria, chromaturia, and pollakiuria, increased BUN, creatinine and inorganic phosphorus levels, renal and urinary bladder calculi and related microscopic tissue lesions. The majority of these effects were reversible within the 4-week recovery period; although, urinary calculi and their sequelae were still present in high-dose females. Based on these results, the maximum tolerated dose of the test item is considered to be lower than 10000 ppm and NOAEL is 200 ppm (dietary equivalent to 9.8 to 17.7 mg/kg bw/day for males and 12.5 to 21.2 mg/kg bw/day for females).

Chronic Toxicity
An OECD TG 452 study was performed to assess the toxicity of the test item when administered in the diet to beagle dogs (Varney 1986). The animals were dosed via their diet for up to 52 weeks at doses of 100, 1000, 10000 ppm (dietary equivalent to 3.5, 33, 195 mg/kg bw/day for males and 4, 34, 212 mg/kg bw/day for females). The high dose level elicited reduced body weight gain and food intake and adverse haematological changes. Therefore the dose level of the group 4 animals was reduced to 5000 ppm from week 10. After 13 weeks of treatment 2 males and 2 females from each group were killed and necropsied. Histological evaluation of the major tissues was then performed. All surviving animals were killed and necropsied after 52 weeks of dosing and the major tissues examined histologically. Treatment with the test item elicited toxic changes in terms of reduced body weight gain and food intake, reduced red blood cell number, packed cell volume and haemoglobin concentration at 10000 ppm. At 5000 ppm, regression of these adverse changes with the exception of the single group 4 female which still appeared anaemic in week 13. There were no treatment related changes at either the 100 or 1000 ppm treatment levels and the latter was considered to be the NOAEL (dietary equivalent to 33 and 34 mg/kg bw/day for males and females, respectively).

In an EPA combined toxicity/oncogenicity GLP study, the test item was administered in the diet to male and female Sprague-Dawley rats at feeding levels of 0, 10, 1000, or 6000 ppm for up to 104 weeks (Morrow 1987). There were no treatment-related effects on clinical condition, ophthalmology, water consumption, or clinical laboratory parameters. Survival in the 6000 ppm males was slightly greater than controls at week 104. Dose-related reduction in body weight gain present at feeding levels of 1000 ppm were accompanied by decrease food consumption at 6000 ppm. Body weight change for the 1000 ppm group was comparable to controls at 103 weeks but was reduced by 25 and 39% in the 6000 ppm males and females, respectively. Slightly elevated organ to body weight ratios noted in the 6000 ppm group were secondary to decreased body weight.
Based on the results the NOAEL level was determined to be 10 ppm (dietary equivalent to 0.3 to 1.1 and 0.4 to 1.1 mg/kg bw/day for males and females, respectively).

Short-term toxicity
In a short-term toxicity GLP study, 10 males and 10 females RAIF (SPF) rats per dose group were used to assess the toxicity of the test item (Basler 1985). The test article was administered in the diet for 28 days at dosages of 0, 1000, 3000 and 10 000 ppm. The creatinine level was increased in both treated male and female animals of group 4 (10 000 ppm). Increased numbers of white blood cells were noted in the females of the high dose group. In treated females of group 4, red blood cell parameters (RBC, haemoglobin and haematocrit) were slightly decreased and blood was found in the urine sample of one female (group 4). It can be inferred from the observations made during this study that the NOAEL for the test item, when offered to rats continuously in their feed over a period of 28 days, is 1000 ppm (dietary equivalent to 79.4 and 77.7 mg/kg bw/day for males and females, respectively).

Repeated dose toxicity: dermal
Two short-term repeated dose toxicity studies via the dermal route are available, one in rats and one in rabbits. The rats study was performed according to OECD TG 410 following GLP, to determine the potential dermal toxicity of the test item, when administered topically, for a period of 28 days (Robertson 2013). Three groups of 5 male and 5 female Han Wistar Crl: WI(Han) rats received the test item directly onto the skin in the lumbar region, at dose levels of 10, 100, 1000 mg/kg bw/day, daily for a period of 6 hours over 28 consecutive days. A concurrent control group was used which received Milli-Q water at a dose volume of 1 mL/kg. The test item, when administered topically onto the skin for up to 6 hours daily over a 4 week period, was well tolerated in rats up to 1000 mg/ kg bw/day with no evidence of systemic toxicity. There was no impact on body weights, food consumption, blood chemistry or haematological parameters. Additionally, there were no organ weight differences or macroscopic or microscopic local reactions at the administration site. Therefore, the NOAEL was considered to be 1000 mg/kg bw/day.

The other study was performed in rabbits, and was performed according to OECD TG 410 was following GLP. A total of 40 rabbits (5 males and 5 females per dose group) were used to determine the potential dermal toxicity of the test item (Schoch 1986). The test article was administered dermally on the shaved back skin for a period of 3 weeks on a 5 day/week basis. The daily exposure period was 6 hours. The control animals were treated the same way, only with the vehicle. The doses were 0, 10, 100, 1000 mg/kg bw/day. No deaths occurred neither in the control group nor in the dose groups. Slight signs of dyspnea and ruffled fur were seen in all treated groups. These symptoms are commonly seen in dermal application, caused by the dressings, which were fastened tighter in the treated groups, to prevent loss of test substance. Additionally, in the high dose group (1000 mg/kg bw/day) occasional signs of sedation and abnormal body positions were seen. No erythema or oedema reactions were observed, neither in the control nor in the treated animals. The minimal reactive changes at the skin-application sites observed in several control and treated animals were attributed to repeated local micro traumatism. All other changes observed in some treated and control animals were only incidental in nature and unrelated to the effect of test compound. It was concluded that the NOAEL for the test item when applied dermally over a period of 21 days to rabbits is 100 mg/kg bw/day.

Justification for classification or non-classification

Based on the available information classification upon repeated exposure is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.