Methyl 2-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)ethylidene]-1,3,3-trimethylindoline-5-carboxylate

Endpoint summary

Description of key information

The oral (gavage) administration of Macrolex Orange R to Wistar Han™:RccHan™:WIST strain rats, for twenty-eight days at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated and did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was 1000 mg/kg bw/day (highest dose tested) within the confines of this study type.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 14 September 2016. Experimental Completion Date: 12 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: Macrolex Orange R
Physical State/Appearance: Red powder
CAS Number : 5718-26-3
Date Received: 03 June 2016
Storage Conditions: Stored in darkness; under ambient conditions, used/formulated in the light
Expiry Date: 18 February 2018

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for six days during which time their health status was assessed. A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 222 to 248g, the females weighed 116 to 141g, and were approximately six to eight weeks old.

Animal Care and Husbandry
The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) was used. A certificate of analysis of the batch of diet used is given in Annex 5. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly
temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Vehicle:

arachis oil

Details on oral exposure:

The dose levels were chosen in consultation with the Study Monitor and were based on available toxicity data and the results of a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Envigo Study Number BC27TR).
The dose levels were selected as 250, 500 or 1000 mg/kg bw/day.
The test item was administered once daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analyzed for concentration of Macrolex Orange R at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 96 to 99% of the nominal concentration confirming the accuracy of the preparation procedure.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females at 250 mg/kg bw/day
5 males and 5 females at 500 mg/kg bw/day
5 males and 5 females at 1000 mg/kg be/day

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing during the working week. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

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

Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 25, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).
Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28); see deviations from Study Plan. Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)

Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++) Triglycerides (Tri)

Sacrifice and pathology:

Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -80 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples will be discarded after finalization of the report.

Organ Weights
The following organs, removed from animals that were killed at the end of the dosing period, were dissected free from fat and weighed before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus
Pituitary (post-fixation) Thyroid/Parathyroid (post fixation)
Prostate and Seminal Vesicles Uterus with Cervix
(with coagulating glands and fluids)

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals~ Ovaries~
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint) Pituitary~
Bone & bone marrow (sternum)~ Prostate~
Brain (including cerebrum, cerebellum and Rectum~
pons)~ Salivary glands (submaxillary)
Caecum~ Sciatic nerve~
Colon~ Seminal vesicles (with coagulating
Duodenum~ glands and fluids)~
Epididymides ♦~ Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes *~ and lumbar)~
Gross lesions~ Spleen~
Heart~ Stomach~
Ileum~ Testes ♦~
Jejunum~ Thymus~
Kidneys~ Thyroid/Parathyroid~
Liver~ Trachea~
Lungs (with bronchi)#~ Urinary bladder~
Lymph nodes (mandibular and mesenteric) Uterus & Cervix~
Mammary gland~ Vagina~
Muscle (skeletal)~
* Eyes fixed in Davidson’s fluid
♦ Preserved in modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The tissues with a ~ from all control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
A number of tissues from the 500 or 250 mg/kg bw/day animals showed orange discoloration; however, as this was considered to be due to the strong color of the test item and as there were no treatment-related histopathology findings in animals of either sex treated with 1000 mg/kg bw/day, microscopic examination of these tissues was not extended to these dose groups.

Pathology
Microscopic examination was conducted by the Study Pathologist (M Gregori at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS). A histopathology peer review was conducted at the Test Facility.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTMTables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical observations considered to be related to the toxicity of the test item at any dose level.
At all dose levels, animals of either sex receiving the test item showed orange staining of fur with the test item during the latter half of the treatment period. The color of fur staining after dosing appeared to be yellow on Day 26 in one male from the 250 mg/kg bw/day dose group. Orange/red faeces were observed for test item-treated animals from Day 3 which generally persisted throughout the dosing period. These findings were deemed to be due to the strong color of the test item and not an indication of its systemic toxicity.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 500 or 1000 mg/kg bw/day, males showed slightly higher group mean body weight gains than control over the first two weeks of dosing. There was no dose-relationship and statistical significance was only achieved during the first week. Overall body weight gains in these males were slightly higher than controls but without any dose-dependence. An increase in body weight gain is generally considered not to be an adverse effect and as such these observations were deemed unlikely to be of any toxicological importance. Occasional fluctuations in periodic body weight gains were also noted for males treated with 250 mg/kg bw/day but without achieving statistical significance and overall body weight gains for these animals was comparable with controls.
At all dose levels, body weight development in females was generally comparable with controls throughout the dose administration period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no adverse effect of treatment with the test item at any dose level on dietary intake for animals of either sex.
Throughout the treatment period, dietary intake for males treated with 500 mg/kg bw/day was marginally higher than controls but the corresponding values for males treated with 250 or 1000 mg/kg bw/day were generally similar to controls. There was no effect of treatment with the test item at any dose level on food consumption for the females.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no adverse effect of treatment with the test item at any dose level on food conversion efficiency for animals of either sex.
There was no effect of dosing with the test item on food conversion efficiency for animals of either sex. Minor variations were deemed to be reflective of small intergroup differences in body weight gains and/or food intake.

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

no effects observed

Description (incidence and severity):

There was no obvious effect of treatment with the test item at any dose level on water consumption for animals of either sex.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.
At the end of the treatment period, the mean corpuscular haemoglobin concentrations in males from all dose groups and females given 500 or 1000 mg/kg bw/day were statistically significantly lower than controls. No dose-relationship was evident in either sex and all individual values were within the historical controls data ranges. Males treated with 500 or 1000 mg/kg bw/day also showed statistically significantly higher reticulocyte counts in comparison with controls. A dose-relationship was noted, however, with the exception of one high dose male marginally exceeding the historical control data range, the remaining individual values remained within these ranges. In the absence of any changes in the related hematology parameters or histopathology correlates, these differences were considered unlikely to be of any toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.
At all dose levels, statistically significant intergroup differences in males included higher albumin and lower triglyceride levels in relation to controls. There was no dose-relationship for either parameter and all individual values were within the historical control data ranges. When compared with controls, males from the 500 or 1000 mg/kg bw/day dose groups also exhibited statistically significantly higher calcium concentration whilst females from these dose groups showed statistically significantly higher phosphorus levels. There was no dose-dependence for either parameter and most individual values from the test item-treated animals were within the historical control data ranges. In the absence of any associated microscopic findings, these observations were deemed unlikely to be of any toxicological importance.
Any other statistically significant intergroup differences were confined to 500 mg/kg bw/day males showing higher sodium concentration and 250 mg/kg bw/day males showing lower phosphorus levels, which were considered likely to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes in the behavioral parameters considered to be related to treatment with the test item at any dose level.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in animals of either sex receiving the test item at any dose level.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, macroscopic examination identified orange discoloration of the adipose tissue and mammary glands in most animals of either sex treated with the test item up to a dose level of 1000 mg/kg bw/day. Some of these animals also exhibited orange discoloration of the sternum (1/5 and 3/5 males treated with 250 or 1000 mg/kg bw/day, respectively), lymph nodes (mesenteric and/or mandibular), stomach and/or caecum; orange colored contents were also noted in the stomach and/or caecum for some of these animals. Microscopic examination of these tissues from the 1000 mg/kg bw/day animals did not reveal any treatment-related observation and as such these findings were considered likely to be due to the strong color of the test item rather than an indication of its systemic toxicity.
Incidental findings included red discoloration of the thymus in one female treated with 500 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic changes related to treatment with the test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Functional Performance Tests
There were no intergroup differences considered to be related to treatment with the test item at any dose level.
Functional performance evaluations during the last week of dosing identified males treated with 500 or 1000 mg/kg bw/day showing statistically significantly higher hindlimb strength in relation to controls. All individual values were within the historical control data ranges. As these differences were only evident in 1/3 tests with the corresponding values in females being comparable with controls and there were no apparent signs of neurotoxicity throughout the study, this finding was considered likely to be incidental.

Sensory Reactivity Assessments
Sensory reactivity scores across all test item-treated dose groups were similar to controls

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Critical effects observed:

no

Conclusions:

The oral (gavage) administration of Macrolex Orange R to Wistar Han™:RccHan™:WIST strain rats, for twenty-eight days at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated and did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was 1000 mg/kg bw/day (highest dose tested) within the confines of this study type.

Executive summary:

Introduction

The purpose of this study was to establish the effects of repeated oral administration of the test item to rats over a period of twenty-eight consecutive days. The study is compatible with the following regulatory guidelines:

•  The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).
• Commission Regulation (EC) No 440/2008 of 30 May 2008, test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 250, 500 or 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP).

Clinical signs,functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from the 1000 mg/kg bw/day and control animals was performed.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

Throughout the dose administration period, there were no clinical signs considered to be related to the toxicity of the test item.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests

There was no effect of treatment with the test item at any dose level on functional performance parameters tested in animals of either sex.

Sensory Reactivity Assessments

Sensory reactivity scores across all test item-treated dose groups were similar to controls.

Body Weight

There was no adverse effect of treatment with the test item on body weight development in animals of either sex.

Food Consumption

There was no adverse effect of treatment with the test item on food consumption or food conversion efficiency for animals of either sex.

Water Consumption

There was no obvious effect of treatment with the test item on water consumption in animals of either sex.

Hematology

No toxicologically significant effects were detected in animals of either sex at any dose level.

Blood Chemistry

No toxicologically significant effects were detected in animals of either sex at any dose level.

Necropsy

Neither the type, incidence or distribution of macroscopic observations in animals of either sex indicated any adverse effect of treatment up to a dose level of 1000 mg/kg bw/day.

Organ Weights

No treatment-related effects were detected in animals of either sex at any dose level.

Histopathology

Histopathological examination of the selected tissues from the 1000 mg/kg bw/day animals of either sex did not reveal any treatment-related abnormalities.

Conclusion

The oral (gavage) administration of Macrolex Orange R to Wistar Han™:RccHan™:WIST strain rats, for twenty-eight days at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated and did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was 1000 mg/kg bw/day (highest dose tested) within the confines of this study type.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Justification for classification or non-classification

The oral (gavage) administration of Macrolex Orange R to Wistar Han™:RccHan™:WIST strain rats, for twenty-eight days at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated and did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was 1000 mg/kg bw/day (highest dose tested) within the confines of this study type. No classification is warranted.