2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Endpoint summary

Description of key information

Repeated dose toxicity oral: NOAEL ≥ 100 mg/kg bw/d (similar to OECD 408, GLP, K, rel. 2)

Key value for chemical safety assessment

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:

26 August 2013 - 10 April 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no recovery group/period but no effects observed, water consumption no recorded

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Redbook 2000. Toxicological Principles for the Safety Assessment of Food Ingredients. IV.B.1. General Guidelines for the Designing and Conducting Toxicity Studies. Office of Food Additive Safety, FDA.

Version / remarks:

November 2003

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study because it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: 7 weeks old
- Weight at study initiation: 187 g to 242 g for males and 154 g to 207 g for females
- Fasting period before study: No
- Housing: The animals were housed individually throughout the study in wire mesh floor cages equipped with an automatic watering valve. Cages were arranged on the racks in group order. Housing and care were as specified in the USDA Animal Welfare Act (9 CFR, Parts 1, 2, and 3) and as described in the Guide for the Care and Use of Laboratory Animals from the National Research Council.
- Diet (e.g. ad libitum): PMI Nutrition International Certified Rodent Chow No. 5CR4 (14% protein), ad libitum
- Water (e.g. ad libitum): Municipal tap water following treatment by reverse osmosis and ultraviolet irradiation, ad libitum (except during designated procedures)
- Acclimation period: 9 days before the first day of dosing

DETAILS OF FOOD AND WATER QUALITY:
- The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the dietary analyses were provided by the manufacturer for each lot of diet and are on file at the Testing Facility. Based on the results of these analyses, there were no contaminants that would interfere with the conduct or interpretation of the study.
- The water is analyzed semi-annually for microbial contamination and for total dissolved solids, hardness, and various environmental contaminants. Results of these analyses are maintained on file at the Testing Facility. Based on the results of the most recent analyses, there were no contaminants that would interfere with the conduct or interpretation of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 23°C
- Humidity (%): 47% to 58%
- Air changes (per hr): 10 or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
- Remark: Temperatures in the animal behavioral room ranged from 61°F to 68°F (16°C to 20°C) with a relative humidity of 25% to 76%.

IN-LIFE DATES: From: 05 September 2013 (dosing initiation) To: 06 December 2013

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is the intended route of human exposure.

Vehicle:

other: 1% (w/v) Methylcellulose (400 cps) in Reverse Osmosis Deionized (RODI) Water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Prior to suspension, the test article was milled to a fine powder with a mortar and pestle. The test article was suspended in control article (1% (w/v) Methylcellulose (400 cps) in RODI water) and stirred overnight before use. Homogeneity of the suspensions was also facilitated by the use of a homogenizer and stirring bar at room temperature. The dosing formulations were prepared weekly, stored at room temperature, and dispensed daily. The dosing formulations were stirred continuously during dosing.
The test article formulations were visually inspected prior to initiation of dosing. This visual inspection was performed to ensure that the formulations were visibly homogeneous and acceptable for dosing. Test article was administered under suspension form.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration in vehicle: 1% (w/v) Methylcellulose (400 cps) in Reverse Osmosis Deionized (RODI) Water
- Lot/batch no. (if required): SLBB2809V and MKBG8210V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Dose formulation samples were collected on weeks 1, 5, 9 and 13 and analyzed by HPLC according to a validated procedure.
- Homogeneity of the test formulation was determined on weeks 2 and 4.
- Stability analysis demonstrated that the test article is stable for at least 10 days in the vehicle when prepared and stored under controlled room temperature.
- All results for sample and homogeneity analyses met the acceptance criteria [Sample analysis: mean concentrations within or equal to the acceptance criteria of ± 15% (individual values within or equal to ± 20%) of their theoretical concentrations ; Homogeneity: RSD of concentrations for all samples in each group tested was within the acceptance criteria of ≤ 5%].

Duration of treatment / exposure:

From Day 1 to Days 90/91/92

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 animals/dose/sex for the main study

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor on the basis of available data from proposed human exposure and based on a no-observed-effect level from a 28-day toxicology study with the test item in which the same concentrations of test material were administered once daily by oral gavage to rats. No test article-related mortality or evidence of any systemic toxicity was observed and there were no organ weight or microscopic changes identified after the 28-day exposure. No target organs were identified and the NOEL was >= 100 mg/kg bw/day (Charles River Study No. 200366404).
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.
- Rationale for selecting satellite groups: No satellite animals.
- Post-exposure recovery period in satellite groups: No recovery period.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, beginning Week -1, and at termination

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of randomization (Day -7), twice weekly for the first week, at least once every week thereafter, and at termination

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (quantitatively measured for each animal once pretest (Day -6), twice weekly for the first week, and at least once every week thereafter throughout the dosing phase)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to in-life initiation (Day -7) and during the last week of the treatment period (Day 89)
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 1 (Day 5/6/7), Week 6 (Daay 40/41/42) and Day 91/92/93
- Anaesthetic used for blood collection: Yes (under isoflurane anesthesia)
- Animals fasted: Yes (overnight but access to water ad libitum)
- How many animals: All
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 1 (Day 5/6/7), Week 6 (Day 40/41/42) and Day 91/92/93
- Animals fasted: Yes (overnight but access to water ad libitum)
- How many animals: All
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Day 91/92/93
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight but access to water ad libitum)
- Following parameters were examined: sodium, potassium, chloride.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Animals were euthanized by isoflurane inhalation, followed by exsanguination.

HISTOPATHOLOGY: Yes
- Histopathology of all collected tissues from animals from testing groups 1 and 4.
- In addition, histopathology examination was performed on gross lesions observed in animals from testing groups 2 and 3.
- Following organs were collected and preserved for examination: Artery, aorta, Bone marrow smear, Bone marrow (femur, sternum), Bone (femur, sternum), Brain, Cervix, Epididymis, Esophagus, Eye, Gland (adrenal, harderian, lacrimal, mammary, parathyroid, pituitary, prostate, salivary, seminal vesicle, thyroid), Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine (cecum, colon, rectum), Liver, Lung, Lymph node (mandibular, mesenteric), Skeletal muscle, Nasal structures, Nerve (optica, sciatic), Ovary, Oviduct, Pancreas, Skin, Small intestine (duodenum, ileum, jejunum), Spinal cord, Spleen, Stomach, Testis, Thymus, Tongue, Trachea, Ureter, Urinary bladder, Uterus and Vagina).
- Tissues for microscopic examination were fixed and preserved in 10 % neutral buffered formalin (except eye with optic nerve in Davidson’s fixative and testis in Modified Davidson’s fixative), processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin.

ORGAN WEIGHTS:
Brain, Epididymis, Gland, adrenal, Pituitary gland, Prostate, Thyroid (including parathyroid), Heart, Kidney, Liver, Lung, Ovary, Spleen, Testis, Thymus, Uterus

Other examinations:

None

Statistics:

Details of the statistical analysis is reported below.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Thin fur cover was observed sporadically; however, this is a common background finding in the laboratory rodent and was not test article related. Dry tail skin and broken teeth were noted infrequently and are occasionally observed in laboratory rodents. Labored breathing and abnormal breathing sounds were observed only once during the study (Day 63) in one Group 4 male and were most likely related to inadvertent aspiration during the gavage procedure; therefore, these findings were considered incidental.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled euthanasia.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A few statistically significant changes in body weight gain were noted throughout the study; however, these differences were not considered test article related due to their sporadic occurrence and the absence of test article-related changes in absolute body weights.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A few statistically significant changes in food consumption were noted throughout the study; however, these differences were not considered test article related due to their sporadic occurrence and/or the absence of a dose response.

Food efficiency:

not examined

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):

A few statistically significant differences were noted during the study; however, all values were within historical control ranges. The differences were not considered test article related either due to the absence of a dose response or due to their small magnitude.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few statistically significant differences were noted during the study; however, all values were within historical control ranges. The differences were not considered test article related either due to the absence of a dose response, their small magnitude, or the absence of dose-related trends over time.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-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 test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-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 test item.

Histopathological findings: neoplastic:

not specified

Key result

Dose descriptor:

NOEL

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

Under the test conditions, no test article-related mortality or evidence of any systemic toxicity was observed and no target organs were identified. Based on these results, the no-observed-effect level (NOEL) was considered to be ≥ 100 mg/kg/day.

Executive summary:

In a repeated dose toxicity study performed according to Redbook 2000, ICH Harmonised Tripartite Guideline S3a (similarly to OECD test Guideline No. 408 with some acceptable restrictions) and in compliance with GLP, Sprague Dawley rats (20/sex/dose) were exposed to test material suspended in a vehicle (1% (w/v) Methylcellulose (400 cps) in Reverse Osmosis Deionized (RODI) Water) by gavage at 10, 30 and 100 mg/kg bw/day, once daily for a minimum of 90 days. Control rats were given the vehicle alone. Clinical signs, body weights, body weight changes and food consumption were monitored during the study. Functional observational battery, ophthalmology, clinical pathology parameters (hematology, coagulation, clinical chemistry, urinalysis, and urine chemistry), toxicokinetic parameters, and organ weight examinations were also perfomed. All animals were subjected to gross necropsy examination and histopathological evaluation of all tissues was performed in control and high dose group and of selected tissues in the two intermediate doses.

There were no test article-related clinical signs or changes in mean body weight, body weight gain, food consumption, functional observational battery assessments, ophthalmic examinations, hematology parameters, coagulation parameters, red blood cell morphology, clinical chemistry parameters, macroscopic or microscopic urinalysis data, or urine chemistry parameters during this study.

There were no test article-related gross observations, changes in absolute or relative organ weights, or microscopic findings observed at study termination.

In conclusion, once daily oral administration of test item for a minimum of 90 days was well tolerated in rats at dose levels up to 100 mg/kg/day (highest dose tested). No test article-related mortality or evidence of any systemic toxicity was observed and no target organs were identified. Based on these results, the no-observed-effect level (NOEL) was considered to be ≥ 100 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is GLP-compliant and of high quality (Klimisch score = 2)

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A key study was identified (CRL, 2014, rel. 2). In this repeated dose toxicity study performed according to Redbook 2000, ICH Harmonised Tripartite Guideline S3a (similarly to OECD test Guideline No. 408 with some acceptable restrictions) and in compliance with GLP, Sprague Dawley rats (20/sex/dose) were exposed to test material suspended in a vehicle (1% (w/v) Methylcellulose (400 cps) in Reverse Osmosis Deionized (RODI) Water) by gavage at 10, 30 and 100 mg/kg bw/day, once daily for a minimum of 90 days. Control rats were given the vehicle alone.

There were no test article-related clinical signs or changes in mean body weight, body weight gain, food consumption, functional observational battery assessments, ophthalmic examinations, hematology parameters, coagulation parameters, red blood cell morphology, clinical chemistry parameters, macroscopic or microscopic urinalysis data, or urine chemistry parameters during this study. There were no test article-related gross observations, changes in absolute or relative organ weights, or microscopic findings observed at study termination.

In conclusion, once daily oral administration of test item for a minimum of 90 days was well tolerated in rats at dose levels up to 100 mg/kg/day (highest dose tested). No test article-related mortality or evidence of any systemic toxicity was observed and no target organs were identified. Based on these results, the no-observed-effect level (NOEL) was considered to be ≥ 100 mg/kg/day.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data, no self-classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

There were no data regarding the dermal and inhalation route.