12H-phthaloperin-12-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 4 January 2017. Experimental completion date 12 July 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

12H-phthaloperin-12-one

EC Number:

230-049-5

EC Name:

12H-phthaloperin-12-one

Cas Number:

6925-69-5

Molecular formula:

C18H10N2O

IUPAC Name:

12H-phthaloperin-12-one

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Appearance: Orange powder (with a yellow cast).
Storage conditions: At ambient temperature (15 to 25°C).
Expiry date: 30 June 2021

Test animals

Species:

rat

Strain:

other: Crl:CD(SD) rat.

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) rat (virgin) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
Strain/Species Crl:CD(SD) rat.
Supplier: Charles River (UK) Ltd.
Number of animals: 25 males and 25 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization: 14 days before commencement of treatment.
Age of the animals at start of treatment: 49 to 56 days.
Weight range of the animals at the start of treatment: Males: 243 to 326 g, Females: 170 to 217 g.

Allocation and Identification
Allocation: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Identification of animals: Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.
Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Care and Husbandry
Environmental Control
Animal facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution: Males and females were blocked by groups and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
Number of animals per cage: Five of the same sex.
Bedding: Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen chew block: Provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet: Teklad 2014C Diet.
Availability: Non-restricted (removed overnight before blood sampling for hematology or blood chemistry).

Water Supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriateintervals.
Availability: Non-restricted.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral (gavage) route of administration was chosen as an appropriate route to conduct a human risk assessment.

Vehicle:

corn oil

Details on oral exposure:

The dose levels selected for investigation in this 28-day repeat dose toxicity study (OECD407) were selected in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at these laboratories (Envigo Study No. BX18NR). In that study, dose levels of 100, 300 and 500 mg/kg/day were investigated. As part of the current study it was demonstrated in a vehicle trial prior to formulation homogeneity and stability investigations that, due to the properties of the test item, it was not possible to prepare a formulation suitable for dose administration above a concentration of 100 mg/mL in corn oil. Since the maximum dose volume for formulations using corn oil is 5 mL/kg body weight, the high dose level for investigation by oral gavage administration was limited to the maximum feasible dose of 500 mg/kg/day. In the 14-day preliminary study, there were no premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, no inter-group differences in body weight performance, food consumption or weights of the kidneys, liver or spleen, and no test item-related macroscopic abnormalities at any dose level investigated.
The high dose level for the current OECD407 study was set at 500 mg/kg/day, the maximum feasible dose. The intermediate and low dose levels of 300 and 100 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. These investigations have demonstrated that formulations in the concentration range 1 to 100 mg/mL are stable following ambient storage (15 to 25°C) for one day and following refrigerated storage (2 to 8°C) for 15 days.

Preparation of Test Samples
A representative sample of test formulation (0.5 mL and 1 mL, accurately weighed) and dissolved using ultrasonic vibration in a suitable volume of Extract Solvent. The extract was diluted using Diluent, to provide a solution containing Macrolex Orange 3G at an expected concentration within the range 4 μg/mL to 8 μg/mL. The concentration of Macrolex Orange 3G in the final solution was quantified by HPLC using UV detection as detailed in the chromatographic section.

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The homogeneity and stability was confirmed for Macrolex Orange 3G in corn oil formulations at nominal concentrations of Low nominal concentration mg/mL and High nominal concentration mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. Discrete sampling was confirmed at refrigerated storage for up to 8 days.
The mean concentrations of Macrolex Orange 3G in test formulations analyzed for the study were within 4% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Frequency: Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females at 100 mg/kg bw/day
5 males and 5 females at 300 mg/kg bw/day
5 males and 5 females at 500 mg/kg be/day

Control animals:

yes, concurrent vehicle

Details on study design:

Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg.
Volume dose: 5 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as the treated groups.
Frequency Once daily at approximately the same time each day.
Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure. A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Examinations

Observations and examinations performed and frequency:

Clinical and Behavioral Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Daily during the first week of treatment and twice weekly during Week 2 to termination (middle and end of each week), detailed observations were recorded at the following times in relation to dose administration:

Week 1 of treatment:
Pre-dose observation.
At the end of dosing each group.
One to two hours after completion of dosing of all groups.
As late as possible in the working day.

Week 2 to termination:
Pre-dose observation.
One to two hours after completion of dosing of all groups.

Cage Bedding Observation
A cage bedding observation was also made at the time of cage clean out with particular attention paid to colour of faeces.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment (mid to late week), detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 4 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Motor Activity
During Week 4 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Body Weight
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Day 1), weekly throughout the study and on the day of necropsy. More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored. These data are retained in the study data but are not reported.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

Water Consumption
Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food and water at the following occasion:
Day 29: All animals
Blood sampling was performed on the morning after overnight collection of urine. Animals were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of the blood sampling procedures. Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
* Derived values calculated in ClinAxys
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood samples were collected after overnight withdrawal of food and water at the following occasion:
Day 29: All animals
Blood sampling was performed on the morning after overnight collection of urine. Animals were, there fore, deprived of food and water overnight but were allowed access to water for a minimum period of
one hour prior to the commencement of the blood sampling procedures. Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Biomarkers
Blood samples were collected after overnight withdrawal of food on the following occasion:
Day 29: All animals
Blood sample site: Sublingual vein.
Anesthetic: Isoflurane.
Anticoagulant: EDTA
Blood volume: 0.5 mL
Treatment of samples: Samples were kept on wet ice prior to centrifugation. Microtainers used for collection of samples did not contain separator gel.
Centrifugation conditions: At 2000g for ten minutes at nominally 2 to 8°C within 30 minutes of collection.
Separation and storage of plasma: The resultant plasma was divided into two separate aliquots: 100 μL (0.1 mL) was transferred to an appropriately labelled plastic tube and was retained for potential analysis of T3 and T4.
All remaining plasma was transferred to an appropriately labelled plastic tube and was retained for potential analysis of TSH.
Final storage conditions: All resultant plasma was subsequently frozen on dry ice prior to being transferred to deep frozen storage conditions (-60 to -80°C).
Fate of plasma samples: All samples (and/or residual samples if analysis was required) will be disposed of upon finalisation of the study.

Sacrifice and pathology:

Method of Kill
Carbon dioxide asphyxiation with subsequent exsanguination.

Necropsy
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Examination included a detailed assessment and documentation of any colour changes in the internal organs, adipose tissue or skin. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
Schedule: Animals were killed following four weeks of treatment.
Sequence: To allow satisfactory inter-group comparison.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above.
Requisite organs were weighed for animals killed at scheduled termination.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid.
Eyes: In Davidson’s fluid.

Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: Terminal animals of Groups 1 and 4.
Kidneys and abnormalities only: Terminal animals of Groups 2 and 3.
Routine staining: Sections were stained with hematoxylin and eosin.
Special staining: Kidneys of all females in Groups 1 and 4 stained for lipofuscin (Schmorls) and for haemosiderin (Prussian Blue/Perls).

Light Microscopy
Tissues preserved for examination were examined as follows:
Scheduled kill:
All animals of Groups 1 and 4 - All specified in the table in section 'any other information on materials and methods'.
All animals of Groups 2 and 3 - Kidneys and Abnormalities only.
The kidney which was considered to exhibit a reaction to treatment at the high dose, were
examined for all animals.
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Statistics:

All statistical analyses were carried out separately for males and females.

The following sequence of statistical tests was used for grip strength, motor activity, body weight, food consumption, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972).
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test (Shirley 1977).

For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Treatment with Macrolex Orange 3G at doses up to and including 500 mg/kg/day for 4 weeks was generally well tolerated; there were no premature deaths, no signs observed in relation to dose administration and no test item-related changes in clinical condition. Animals in all treated groups were noted to have orange/red coloured faeces during cage bedding observations, representative of the colour of the test item formulation.
One Control female (No. 26) showed signs of irregular breathing, piloerection and elevated gait from Day 26 of treatment to scheduled termination, with chromodacyorrhea also apparent on Day 26 of study, and body weight loss of 27g was recorded during Days 22-28 of study. At macroscopic examination this animal was found to have a perforation in the oesophagus with thin/clear fluid in the thoracic cavity, numerous pale fibrous adhesions involving multiple organs in the thoracic cavity and an edematous thymus. These findings would suggest the animal had been mis-dosed. Microscopic findings for this animal included pyogranulomatous inflammation, adhesions and occasional foreign bodies (plant material), involving the thoracic cavity and associated organs. There was also increased granulopoeisis in bone marrow and atrophy of the vagina epithelium.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight performance of males given 100 mg/kg/day and of all groups of treated females was considered unaffected by treatment.
Males given 300 or 500 mg/kg/day showed low mean body weight gain during Days 1-15 of the study compared to Control (11% and 19% lower than Control, respectively), although statistical significance was not attained for these differences. The body weight gain of these males from Day 15 to scheduled termination was essentially similar to Control, such thatoverall mean body weight gain from Day 1 to Day 28 of study was 4% and 13% lower than Control at 300 and 500 mg/kg/day, respectively.
Overall body weight gain for all groups of treated females was slightly higher than Control but with no dose trend apparent and statistical significance was not attained at any dose level; differences were considered incidental and unrelated to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption remained essentially similar to Control values for males given 100 or 300 mg/kg/day and for females in all treated groups throughout the dosing period and was considered unaffected by treatment.
Males given 500 mg/kg/day showed slightly low mean food intake during Days 1-15 of study when compared to Controls, coinciding with the period of reduced body weight gain recorded for these animals.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Visual assessment of water intake did not reveal any changes in water consumption in either sex at any dose level investigated.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of hematological parameters following four weeks of treatment with Macrolex Orange 3G revealed a statistically significant non dose-dependent increase in red cell distribution width for males and females given 300 or 500 mg/kg/day. This increase in red cell distribution width occurred as a consequence of increases in the concentration of reticulocytes, which attained statistical significance in the males but not in females.
All other inter-group differences from controls were minor and were seen in one sex only and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical analysis of plasma after 4 weeks of treatment revealed a decrease in aspartate amino-transferase activity in males receiving 500 mg/kg/day. Cholesterol concentrations were increased in all groups of treated males and females (although in the absence of a clear dose response, particularly in females) with statistical significance attained for males at 500 mg/kg/day. In addition, a dose dependent reduction in glucose concentrations was evident in all groups of treated females, with statistical significance attained at 300 or 500 mg/kg/day.
Assessment of electrolytes indicated, when compared to Controls, an increase in calcium concentrations in both sexes at 500 mg/kg/day, and for males given 500 mg/kg/day sodium and chloride concentrations were low and potassium concentrations were increased.
Total protein and albumin concentrations were increased in all groups of treated males and females when compared to Controls, with statistical significance attained at 500 mg/kg/day; albumin/globulin ratio was slightly decreased in all groups of treated males, although in the absence of a dose response, but unaffected in females.
All other inter-group differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

All groups of treated males and females receiving Macrolex Orange 3G showed statistically significantly high body weight-adjusted mean liver weights compared to Controls with a dose trend apparent in males but not females.
All groups of treated males and females showed slightly high body weight adjusted mean kidney weights compared to Control; these differences attained statistical significance in all groups of treated females however there was no dose response apparent among males or females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic abnormalities were limited to orange colouration of the lower gastro-intestinal tract contents, indicative of the colour of the test item; there was no discolouration of the viscera or skin. In addition, 4/5 females given 500 mg/kg/day showed dark kidneys.
Other findings were isolated and showed no clear association with treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with Macrolex Orange 3G were seen in the kidney.
An increase in the incidence and severity of hyaline droplets in cortical tubules was seen in all males treated at 100, 300 or 500 mg/kg/day.
Yellow-brown pigment was seen in cortical tubules of females treated at 100, 300 and 500 mg/kg/day. Special staining was used to identify the pigment in Control females and females treated at 500 mg/kg/day. Perls/Prussian Blue staining to detect haemosiderin was negative. Schmorls staining for lipofuscin was positive, with the intensity and distribution of staining clearly increased in treated females compared to Controls.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Motor Activity
There was considered to be no adverse effect of treatment with Macrolex Orange 3G on locomotor activity.
Among all groups of treated females, during the majority of the 1-hour recording period group mean high and low beam breaks tended to be higher than Control, with the total number of low beam breaks attaining statistical significance in all treated groups, although in the absence of a dose response relationship. However, when compared with Historical Control Data (HCD range), the majority of Control group high and low beam scores in this study were below the HCD minimum and all of the mean scores that attained statistical significance in the treated groups were either within or marginally above the HCD range. It was therefore considered that the apparent increase in locomotor activity in all groups of treated females was actually representative of atypically low scores in the Control group, and no effect of Macrolex Orange 3G administration was inferred.

Effect levels

Dose descriptor:

NOAEL

Effect level:

500 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

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Summary of treatment related findings in the kidney for animals killed after 4 weeks of treatment:

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	500	0	100	300	500
Hyaline Droplets, Increased
Minimal	0	4	1	0	0	0	0	0
Slight	0	1	4	4	0	0	0	0
Moderate	0	0	0	1	0	0	0	0
Total	0	5	5	5	0	0	0	0
Tubular Pigment, Increased
Minimal	0	0	0	0	0	2	2	5
Total	0	0	0	5	0	0	0	0
Number of tissues examined	5	5	5	5	5	5	5	5
Schmorls Positive Staining, Increased
Present	-	-	-	-	0	-	-	5
Total	-	-	-	-	0	-	-	5
Number of tissues examined	0	0	0	0	5	0	0	5

Applicant's summary and conclusion

Conclusions:

In conclusion, oral (gavage) administration of Macrolex Orange 3G to CD rats at doses of 100, 300 or 500 mg/kg/day for 4 weeks provided clear evidence of systemic exposure but no effects which were deemed to be adverse. Test item-related histopathological changes were evident in the kidneys. In all groups of treated males there was a dose-dependent increase in severity of hyaline droplet accumulation in the cortical tubules of the kidneys, a male-rat specific phenomenon. In the kidneys of females given 500 mg/kg/day yellow-brown tubular pigment was apparent, identified as lipofuscin which was considered of limited or no functional significance and in the absence of any other histological findings in the kidneys, is not deemed adverse. Plasma biochemistry revealed some slight changes in composition which were indicative of sub-clinical adaptive changes in the liver and kidneys, and were accompanied by increases in liver and kidney weight. In the absence of any evidence of degenerative or functional change in the liver and kidneys during histopathological evaluation, the slight disturbances of biochemical parameters were considered not to be adverse. Within the context of this study, the No Observed Adverse Effect Level (NOAEL) was therefore concluded to be 500 mg/kg/day, the maximum feasible dose.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of Macrolex Orange 3G (an industrial colourant) in a 4 week oral gavage study in CD rats. The study is conducted to fulfil the data requirement according to REACH regulation Annex VIII chapter 8.6.1.

Three groups, each comprising five male and five female Crl:CD(SD) rats, received Macrolex Orange 3G at doses of 100, 300 or 500 mg/kg/day (the maximum feasible dose) in corn oil vehicle by oral gavage administration at a volume dose of 5mL/kg body weight. A similarly constituted control group received the vehicle, corn oil, at the same dosage volume as the treated groups.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by visual assessment), hematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

At study termination, blood samples were taken from all animals and stored at -60°C to -80°C for analysis of the biomarkers T3, T4 and TSH if required.

Results

Treatment with Macrolex Orange 3G at doses up to and including 500 mg/kg/day (the maximum feasible dose) was well tolerated; there were no mortalities and no

treatment-related clinical signs observed. Neurobehavioural assessments conducted during Week 4 of treatment did not reveal any adverse effects on locomotor activity, sensory reactivity or grip strength at any dose level investigated.

Males given 300 or 500 mg/kg/day showed a dose-dependent reduction in mean body weight performance during the first two weeks of treatment when compared to Controls, which was associated with slightly low food intake among males given 500 mg/kg/day during this period. From Day 15 of study, mean body weight gain and food consumption was unaffected. Visual assessment of water intake did not reveal any changes in water consumption in either sex at any dose level investigated.

The haematological investigation conducted after four weeks of treatment revealed a statistically significant non dose-dependent increase in red cell distribution width for males and females given 300 or 500 mg/kg/day, as a consequence of increased reticulocyte concentration, which attained statistical significance in the males but not in females. Test article-related biochemical changes in the plasma comprised decreased aspartate amino-transferase activity in males given 500 mg/kg/day and glucose concentrations in all groups of treated females, and elevated cholesterol concentrations in all groups of treated males and females. In addition, increased calcium concentrations were evident in both sexes given 500 mg/kg/day, and for males given 500 mg/kg/day sodium and chloride concentrations were low and potassium concentrations were increased. Increased total protein and albumin concentrations were observed in all groups of treated males and females, with slightly decreased albumin/globulin ratio in all groups of treated males.

The analysis of organ weights following 4 weeks of treatment indicated slightly high body weight-adjusted mean liver and kidney weights compared to Control in all groups of treated males and females.

Test item-related abnormalities detected at macroscopic examination were limited to orange colouration of the lower gastro-intestinal tract contents, indicative of the colour of the test item; there was no discolouration of the viscera or skin. In addition, 4/5 females given 500 mg/kg/day showed dark kidneys.

Histopathological changes that were attributable to treatment occurred in the kidneys of animals in all treated groups. An increase in the incidence and severity of hyaline droplets in cortical tubules was seen in all groups of treated males. Among all groups of treated females, yellow-brown pigment was seen in cortical tubules of the kidneys. Special staining was used to identify the pigment in Control females and females treated at 500 mg/kg/day. Perls/Prussian Blue staining to detect haemosiderin was negative. Schmorls staining for lipofuscin was positive, with the intensity and distribution of staining clearly increased in treated females compared to Controls.

Conclusion

In conclusion, oral (gavage) administration of Macrolex Orange 3G to CD rats at doses of 100, 300 or 500 mg/kg/day for 4 weeks provided clear evidence of systemic exposure but no effects which were deemed to be adverse. Test item-related histopathological changes were evident in the kidneys. In all groups of treated males there was a dose-dependent increase in severity of hyaline droplet accumulation in the cortical tubules of the kidneys, a male-rat specific phenomenon. In the kidneys of females given 500 mg/kg/day yellow-brown tubular pigment was apparent, identified as lipofuscin which was considered of limited or no functional significance and in the absence of any other histological findings in the kidneys, is not deemed adverse. Plasma biochemistry revealed some slight changes in composition which were indicative of sub-clinical adaptive changes in the liver and kidneys, and were accompanied by increases in liver and kidney weight. In the absence of any evidence of

degenerative or functional change in the liver and kidneys during histopathological evaluation, the slight disturbances of biochemical parameters were considered not to be

adverse. Within the context of this study, the No Observed Adverse Effect Level (NOAEL) was therefore concluded to be 500 mg/kg/day, the maximum feasible dose.