1,4-diamino-2,3-diphenoxyanthraquinone

Endpoint summary

Description of key information

A valid Repeated Dose 28 Day Oral Toxicity Study in Rodents according to OECD TG 407 in the Han Wistar rat by oral gavage administration is available. The test item was administered by gavage to three groups, each of five male and five female rats, for twenty-eight consecutive days, at dose levels of 100, 300 or 1000 mg/kg bw/day.  A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). The oral (gavage) administration of to rats, for twenty-eight days at dose levels of 100, 300 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) of Macrolex Rotviolet R for systemic toxicity was 1000 mg/kg bw/day (high dose) 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: 20 July 2016 Experimental Completion Date: 18 October 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)

Deviations:

no

Remarks:

no deviations that affected the scientific integrity of the study or the results obtained

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: Macrolex Rotviolett R
Physical State/Appearance: Violet solid
Date Received: 17 March 2016
Storage Conditions: Ambient temperature (approximately 10 to 30°C) in the dark, used/formulated in the light
Expiry Date: 28 July 2017
No correction for purity was made.

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

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 nine 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 218 to 263g, the females weighed 161 to 189g, 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. Certificates of analysis of the batches of diet used are 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:

For the purpose of this study the test item was prepared at the appropriate concentrations in Arachis oil BP. On each day of formulation preparation, for each concentration the required amount of Test Item was weighed out and added to the required volume of vehicle and shaken/mixed to give a homogeneous bulk formulation. These bulk formulations were subsequently divided into the required daily aliquots and stored at approximately 4°C, in the dark, until the day of use.

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: LY77PH).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services as part of another study (Envigo Study Number: LY77PH), where formulations were shown to be stable for at least eleven days when stored at approximately 4°C, in the dark. Formulations for this study were made and used within the known stability period.

Samples of test item formulations were taken and analyzed on two occasions for concentration of Macrolex Rotviolett R at Envigo Research Limited, Shardlow, UK, Analytical Services. The results showed that the prepared formulations were within 94 to 100% of the nominal concentration confirming the accuracy of the preparation procedure.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

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.

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 and approximately one hour after dosing. Any staining of the cage/bedding and/or discoloration of the faeces was also 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 the last two weeks of dosing where water intake was measured gravimetrically; see deviations from Study Plan.

Special Evaluations
Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 27, 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). 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+) T
otal cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-) B
ile acids
Calcium (Ca++)
Triglycerides (Tri)

Sacrifice and pathology:

Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent 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 (with coagulating glands and fluids)
Uterus with Cervix


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 glands and fluids)
#Duodenum
#Epididymides ♦
Skin
Esophagus Spinal cord (cervical, mid thoracic and lumbar)
#Eyes *
#Gross lesions
#Spleen
#Heart
#Stomach
I#leum
#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 marked with # 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. 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.
As there were no treatment-related findings in the 1000 mg/kg bw/day dose group, microscopic examination was not extended to animals from the low and intermediate dose groups.

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 ProvantisTM Tables 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.
Isolated instances of increased post-dose salivation were noted for individual animals of either sex treated with 1000 mg/kg bw/day with 2/5 males receiving 100 mg/kg bw/day also showing similar observations. As these clinical signs were observed on a few occasions only, they were deemed likely to be due to the dosing procedure rather than an indication of systemic toxicity.
Animals of either sex receiving 1000 mg/kg bw/day showed purple staining of fur with the test item during the latter half of the treatment period with one male from the 300 mg/kg bw/day also showing an isolated instance of fur staining. Dark faeces were also observed in cages containing animals treated with 300 or 1000 mg/kg bw/day from Day 3, which 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):

There was no adverse effect of treatment with the test item at any dose level on body weight development in animals of either sex.
At 100 or 300 mg/kg bw/day, males showed some instances of slightly lower group mean body weight gains but without achieving statistical significance. This resulted in slightly lower overall body weight gains in these males. No dose-relationship was evident and the corresponding values in males from the 1000 mg/kg bw/day dose group were comparable with controls. Females treated with 100 or 1000 mg/kg bw/day also showed some instances of slightly lower mean body weight gains, with the latter achieving statistical significance during the last week of dosing. Overall mean body weight gains in these animals were slightly lower than controls albeit without any evidence of a true dose-relationship and these observations were considered likely to be due to biological variation.

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 or food conversion efficiency for animals of either sex.
Throughout the treatment period, dietary intake for males treated with 100 or 300 mg/kg bw/day and females receiving 100 or 1000 mg/kg bw/day remained marginally lower than controls. No dose-relationship was evident in either sex and these differences were deemed likely to be due to normal biological variation. Any minor variations in food conversion efficiency were considered to be reflective of small intergroup differences in body weight gains and/or food intake.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water 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 water consumption for animals of either sex.
When compared with controls, gravimetric measurement over the last two weeks of dosing identified sporadic instances of slightly lower water intake for females treated with the test item at all dose levels. There was no evidence of a persistent true dose-relationship and as water consumption values for males from these dose groups were generally comparable with controls, any differences for females were deemed unlikely to be related to treatment with the test item.

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, group mean eosinophil counts in females treated with 300 or 1000 mg/kg bw/day were statistically significantly higher than controls. A dose-relationship was noted but with the exception of 1/5 high dose females, individual values for the remaining animals were within the historical control data ranges. The corresponding values in males were also comparable with controls and this observation was considered unlikely to be of any toxicological significance.
Any other intergroup differences were minor and did not achieve statistical significance.

Clinical biochemistry 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 1000 mg/kg bw/day, females showed statistically significantly higher urea and creatinine concentrations in relation to controls with individual values for 3/5 females exceeding the historical control data ranges. The corresponding values in males from this dose group were also slightly higher than controls but statistical significance was not attained. Additionally, these females showed a statistically significant increase in group mean phosphorus levels, but individual values for most females remained within the historical control data ranges. In the absence of any histopathology correlates, these observations were deemed to be of no toxicological relevance.
At 300 or 1000 mg/kg bw/day, females showed statistically significantly higher cholesterol concentrations in relation to controls albeit without exhibiting any dose-dependence. The mean values in the corresponding males were similar to controls whilst all individual values in females remained within the historical control data ranges. Group mean albumin/globulin ratios in females from all test item-treated dose groups were statistically significantly lower than controls but without any dose-relationship and all individual values were within the historical control data ranges. There were no associated microscopic findings and these observations were considered unlikely to be of any toxicological importance.
Other intergroup differences achieving statistical significance included higher albumin/ globulin ratio in males receiving 100 mg/kg bw/day. This was, however, not evident in the 300 or 1000 mg/kg bw/day males and this finding was deemed to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

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 treatment period, females receiving 1000 mg/kg bw/day showed statistically significantly lower absolute and body weight-related thyroid/parathyroid weights in relation to controls. There was no dose-relationship and with the exception of one control female with a value exceeding the historical control data ranges, the remaining individual values were within these ranges. Histopathological examinations of the relevant tissues did not identify any treatment-related abnormalities and this finding was deemed unlikely to be of any toxicological relevance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, macroscopic examination identified violet discoloration of the skin, mammary gland and adipose tissue 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 violet discoloration of mesenteric lymph node, with 2/5 high dose males showing a similar observation for sternum. Microscopic examination of the tissues from the 1000 mg/kg bw/day animals did not reveal any treatment-related observations and 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 increased pelvic space in the right kidney in one control male and female and one female treated with 300 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:

effects observed, non-treatment-related

Description (incidence and severity):

Behavioral Assessments
There were no changes in the behavioral parameters considered to be related to treatment with the test item at any dose level.
Arena assessment during the last week of dosing identified one female from the 100 mg/kg bw/day dose group showing signs of exophthalmos. This was not evident in any of the remaining animals on the study and as such this observation was considered unlikely to be related to treatment with the test item.

Functional Performance Tests
There were no intergroup differences considered to be related to treatment with the test item at any dose level.

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:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Critical effects observed:

no

Conclusions:

The oral (gavage) administration of to Wistar Han™:RccHan™:WIST strain rats, for twenty-eight days at dose levels of 100, 300 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 (high dose) 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 daysand this study is compatible with the following regulatory guidelines:

·        Commission Directive 96/54/EC (Method B7).

·        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 100, 300 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 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 adverse effect of treatment with the test item on water consumption for 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 toxicologically significant 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 to Wistar Han™:RccHan™:WIST strain rats, for twenty-eight days at dose levels of 100, 300 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 (high dose) 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

Quality of whole database:

GLP guideline study

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

Justification for classification or non-classification

In a Repeated Dose 28 Day Oral Toxicity Study in Rodents according to OECD TG 407 Macrolex Rotviolet R was administered by gavage to three groups, each of five male and five female rats, for twenty-eight consecutive days, at dose levels of 100, 300 or 1000 mg/kg bw/day.  The dose levels of 100, 300 or 1000 mg/kg bw/day were well tolerated and did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) of Macrolex Rotviolet R for systemic toxicity was 1000 mg/kg bw/day (highest applied dose). According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is therefore not justified.