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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 September 2017 to 05 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Guideline study, conducted to GLP and current methodology.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Section 4: Health Effects. Repeated Dose 90-day Oral Toxicity Study in Rodents (No.: 408, 21st September 1998. Organization for Economic Co-Operation and Development, Paris)
Deviations:
yes
Remarks:
See Principles of method if other than guideline below.
Principles of method if other than guideline:
Due to technical reasons, the dosing of animal #1506 was not registered in the Provantis system on Day 85 (30 November 2017). It is considered that the dosing of this control animal was performed with the vehicle, however this cannot be proven based on the available raw data.

Due to technical reasons, data of all groups were evaluated in SMART analysis instead of the evaluation of High dose and Control groups as indicated in the Study Plan. However, this fact was considered to have no impact on the results or integrity of the study.

These deviations are considered to have no impact on the outcome of the study and interpretation of the results.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Name: C.I. Solvent Yellow 124
Batch/Lot Number: S3424 / S3422
Description: Dark Brown Oily Viscous Liquid
Purity: 94.8 % / 94.5%
Expiry Date: 25 April 2018 / 06/04/2018
Storage conditions: Refrigerated
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species and strain: Crl: WI Wistar rats
Justification of species/strain: The rat is regarded as suitable species for toxicology studies. Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies.
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Crl: WI Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany
Hygienic level: SPF at arrival, standard laboratory conditions during the study.
Number of animals: 40 male and 40 female rats, 10 animals/group/sex, 4 groups. Additional 8 spare animals/sex animals were ordered.
Age of animals: Young adult rats, approx. 6 weeks old at start of treatment.
Body weight: Males: 238 – 260 g, females: 174 – 206 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
Animal receipt: 31 August 2017
Acclimation period: 5-6 days

Husbandry
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Room numbers: 608, 638
Cage type: Type II and/or III polycarbonate
Bedding: Lignocel® (produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany) Bedding for Laboratory Animals and nest building material (Arbocell crinklets natural (produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany)) were available to animals during the study.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.9 – 22.6 °C
Relative humidity: 30 – 65 %
Ventilation:15-20 air exchanges/hour
Housing/Enrichment: Rodents were housed 2 animals of the same sex and group/cage. Group housing allows social interaction and the deep wood sawdust bedding allows digging and other normal rodent activities.
The temperature and relative humidity were recorded twice daily during the study and acclimation period.

Diet and water supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice –breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal assignment to the study, randomisation and identification
Each animal was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at Citoxlab Hungary Ltd.
The animal number consisted of 4 digits, the first digit was the group number, the second was 0 for the males, and 5 for the females, and the last 2 stood for the animal number within the group.
The cages were identified by cards holding information about the study code, sex, dose group, cage number and individual animal numbers.
During the acclimation period, the animals were assigned to their respective dose groups by randomization based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; the software PROVANTIS v.9 was used in order to verify homogeneity/variation among/within groups. Males and females were randomised separately.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is one of the possible routes of human exposure.
Vehicle:
olive oil
Details on oral exposure:
Vehicle
Olive oil was selected for vehicle of the study by the Sponsor based on the formulation and analytical trials. The same vehicle was used in the Dose Range Finding (DRF) study. Identification data of the chemical used in the study are as follows:
Vehicle: Olive oil
Manufacturer: Sigma-Aldrich
Batch number: BCBT7822
Expiry date: 28 February 2019
Storage conditions: Room temperature, protected from light

Formulation and analysis of formulation
The test item was formulated at appropriate concentrations in the vehicle (as a visibly stable homogenous formulation) in the Pharmacy of Citoxlab Hungary Ltd. Formulations were prepared up to 6 days before use (formulation were kept closed, at room temperature until use in that case). Stability of the test item in the vehicle was assessed in the conditions employed on the study during the analytical method validation. In that study, the formulation samples in the 1-50 mg/mL concentration range (using olive oil as vehicle) were proven as being stable for at least 7 days when stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed using an HPLC-UV method in the Analytical Laboratory of Citoxlab Hungary Ltd. Top, middle and bottom duplicate samples were taken from test item formulations four times during the study (during first, fifth, ninth and last week of the treatment), one set to analyse (which could be collected in replicates as practical), one set to analyse (which can be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Acceptance criteria of the concentration analysis were set according to the analytical method validation, expected to be at 100 ± 15 % of the nominal concentration.

See Section 8 below for full details of the analytical method.
Duration of treatment / exposure:
The dose formulations were administered from Day 0 for 90 consecutive days by oral gavage..
Frequency of treatment:
The dose formulations were administered daily.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
40 male and 40 female rats, 10 animals/group/sex, 4 groups
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-selection and route of administration
The dose levels were selected by the Sponsor in consultation with the Study Director based on available data provided by the Sponsor and information from a Dose Range Finding study in the rat (Citoxlab study code 17/122-101PE) where test item-related mortality was seen at 150 and 200 mg/kg bw/day.
Based on the results from the preliminary study, doses of 10, 30 and 100 mg/kg bw/day were selected for the main study. The aim is to use a maximum of 100 mg/kg bw/day to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.

Dosing procedure
The dose formulations were administered daily starting from Day 0 for 90 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 5 mL/kg bw/day was administered to all animals. The actual volumes to be administered were calculated and adjusted based on the most recent individual body weight. Control animals were treated concurrently with the vehicle only.
Positive control:
Positive control not required for this study.

Examinations

Observations and examinations performed and frequency:
Mortality and clinical observations
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made at least once a day at approximately the same time with minor variations as practical.
Detailed clinical observations were made on all animals outside the home cage in a standard arena before the first treatment (on Day 0 male/female) and weekly thereafter, in the morning hours (am) and once before necropsy.
Observations were performed on the skin, fur, eyes, eyeballs and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (ex. hunchback posture, etc), gait, or response to handling and to environmental stimulation. Particular attention was directed to observations for tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Neurological assessment (Functional Observation Battery)
Towards the end of the treatment period, during Week 11/12, all animals were examined in the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
Parameters such as, body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.
To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured. The fore paws of the rat were painted for any possible additional measurements.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for a 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups

Ophthalmology evaluation
Ophthalmoscopic examination was conducted in all animals before treatment (Day -1 or -2), and in the Control group and High dose group animals, during Week 12 (Day 85/84). Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL cyclopentolate hydrochloride) into the conjunctival sac. The evaluation was performed using a Gowlland® ophthalmoscope. As in Week 12 no treatment related alterations were found in the Control group and High dose group animals, the remaining animals were not examined at termination.

Body weight measurements
Body weights were recorded with a precision of 1 g at randomisation (pre-treatment period), on the first day of treatment (Day 0, prior to start of treatment), then weekly, including on Day 89, and prior to necropsy, fasted on Day 90.

Food consumption measurements
The determination of food consumption was performed for all groups once a week.
The food was measured on Day 0 then the remaining, non-consumed food was weighed weekly from Day 7 with a precision of 1 g. Daily food consumption was calculated for reporting purposes.

CLINICAL PATHOLOGY
At the end of the treatment period, prior to scheduled necropsy on Day 90, clinical pathology investigations (haematology, coagulation, clinical biochemistry and urinalysis) were conducted in all animals. Food was withdrawn during the overnight urine collection.
After an overnight period of food deprivation of animals, 3 blood samples were collected by heart puncture under pentobarbital anaesthesia, for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Haematology and blood clotting times
The following parameters were evaluated in all animals:
RBC Red Blood Cell (erythrocyte) count, WBC White Blood Cell (leukocyte) count, Hgb Haemoglobin concentration, Hct Haematocrit (relative volume of erythrocytes), MCV Mean Corpuscular (erythrocyte) Volume, MCH Mean Corpuscular (erythrocyte) Haemoglobin, MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, RDW Red Cell (erythrocyte) volume, Plt Platelet (thrombocyte) count, MPV Mean Platelet Thrombocyte volume, RETIC % Reticulocyte count, NE % Neutrophil, LY % Lymphocyte, MO % Monocyte, BA % Basophil, EO % Eosinophil, LUC % Large Unstained Cells, APTT Activated Partial Thromboplastin Time, PT Prothrombin Time.
Blood smears were prepared for all animals but not examined, as the standard haematology evaluation was considered to be adequate.

Clinical chemistry
The following parameters were evaluated in all animals:
Glucose Blood sugar concentration, T-BIL Total Bilirubin concentration, Urea Urea concentration, Chol. Cholesterol concentration, Creat. Creatinine concentration, Phos. Phosphorus concentration, Na+ Sodium concentration, K+ Potassium concentration, Ca++Calcium concentration, Cl- Chloride concentration, Tot. Prot. Total Protein concentration, Alb. Albumin concentration, A/G Alb/glob ration, AST/GOT Aspartate Aminotransferase activity, ALT/GPT Alanine Aminotransferase activity, GGT Gamma-Glutamyl transferase activity, ALKP Alkaline Phosphatase activity, Bile acids.

Urinalysis
Urine collection was conducted over approximately 16 hours, during an overnight period of food deprivation of animals, which was placed in metabolic cages.
The evaluation of the urine samples was performed by observation (e.g. colour, appearance) or test strips as applicable. The following parameters were evaluated in all animals:
LEU / Leukocyte, NIT / Nitrite, pH, PRO / Protein, GLU / Glucose, UBG / Urobilinogen, BIL / Bilirubin, KET / Ketones, BLD / ERY Blood/Erythrocytes, SG / Specific Gravity, SED / Sediment, VOL / Volume, Colour/appearance.
Sacrifice and pathology:
Terminal procedures
Necropsy and macroscopic examination were performed on all animals, at the end of treatment period, on Day 90 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia.

Macroscopic evaluation
After exsanguination the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
In addition, bone marrow smears from the femur of each animal were prepared at necropsy. The smears were fixed, then stained but not analysed. The smears are stored/archived at Citoxlab Hungary Ltd.

Organ weight measurement
The following organs were trimmed of fat and weighed in all animals:
Brain, Epididymidesl, Heart , Kidneys, Liver, Prostate, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Uterus including cervix, Adrenal glands, Ovaries, Thyroid with parathyroid glands, Pituitary.
Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.

Histopathology
On completion of the macroscopic examination the following tissues and organs were retained from all animals:
Gross findings, Adrenals, Animal identification, Aorta, Brain, Epididymis, Eye with the optic nerve, Oesophagus, Femur with marrow, Heart, Kidney, Large intestine, Extraorbital lachrymal gland, Harderian gland, Liver, Lungs with bronchi, Lymph node, Ovary, Oviduct, Pancreas, Pituitary, Prostate, Salivary gland (including mandibular, sublingual and parotid glands), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, subcutis with mammary gland (inguinal), Skeletal muscle (quadriceps), Small intestine, Spinal cord, Spleen, Sternum with marrow, mStomach, Testis, Thymus, Thyroid with parathyroid gland, Tongue, Trachea, Urinary bladder, Uterus, Vagina
Other examinations:
Examination of vaginal smears
Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 software package (within the validated Provantis system). The following decision tree was applied automatically for statistical evaluation of continuous numeric data.
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.

For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Abnormal yellow skin colour was seen in all High dose males and females from Day 2 or 3 until the end of the observation period.
Heightened startle response and hyperactivity were seen in all High dose females from Day 26 until Day 42, and then the symptoms gradually disappeared and all animals became symptom-free from Day 48.
One High dose male had hunched back from Day 29 until Day 34.
There were no clinical findings in the Low and Mid dose groups during the study.
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males, slightly lower bodyweight values were recorded in the High dose group during the whole observation period, but without reaching statistical significance. The bodyweight gain values were often below the control values in the male High dose group, with occasional statistical significances, the overall body weight gain during the study was 12% lower than control but was not statistically different.
In females, slightly lower bodyweight values were recorded in all dose group during the whole observation period, reaching statistical significance only on Day 7 in the High dose group. The bodyweight gain values were sometimes below the control values in female dose groups, with a statistical significance only during the first week of treatment in the High dose group; the overall body weight gain during the study was 12.7% lower than control in the High dose group but was not statistically different.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no clear test item related differences in the mean daily food consumption in any test item treated group when compared to the controls. The slightly lower body weight and body weight gain values did not correlate with the food consumption values. Occasional statistically significant differences were regarded as incidental and of no toxicological significance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Most of the RBC related haematology parameters evaluated at the completion of the 90-day treatment period were significantly changed in the Mid and High dose groups. At the high dose these parameters were typically outside the Historic control range, particularly Reticulocytes which were about twice the historic control maximum in both sexes. The pattern of these changes suggested a partially compensated anaemia in these dose groups. In the Mid dose groups erythrocyte parameters were generally within the Historic control range although 50% of the Mid dose males were above the historical control range. At the High dose, the effect was clearly adverse, at the Mid dose in males, the extent of the changes also indicated that the effect of the test item was probably adverse.
Incidental statistically significant differences were seen sporadically in the white blood cell parameters. All these values were well within the historical control range. The differences in the lymphocyte, monocyte, neutrophil and Large Unclassified Cell parameters were considered to be incidental, not test item-related.

Coagulation parameters
The Prothrombin Time (PTT) was statistically (p<0.01) lower in the male Mid dose group, when compared to the control. All values were considered to be in the normal range, the statistical differences were not considered to represent an adverse effect.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Several clinical chemistry parameters were significantly different in the High dose groups, but generally there was no dose response or no consistency between the sexes; all values were within the historical change. Some of the differences at the High dose may be related to the haematology or liver weight effects in this group, but none of the changes appeared to represent a direct adverse effect of the test item. Most statistical differences were considered to be incidental.
In the Low dose groups there were no statistical differences compared to the Control.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No effects were noted on the urinalysis parameters evaluated, which were considered to be related to the test item.
The urinalysis parameters were comparable to the controls in all test treated groups. Variations occurred and consisted of minor differences to controls, these were unrelated to treatment and considered to be normal.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
During Days 26-48, heightened startle response and hyperactivity were observed in 100% of the female High dose group; the same finding was not observed in other groups. From Day 48 until the end of the observation period, the animals did not show any neurological symptoms. The detailed neurological evaluations made near the end of the study, as per OECD guidelines, showed the following neurological effects:
At the functional observation battery (FOB) performed at the end of exposure (Week 11/12), there were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
When assessing the grip strength of the hind limbs, statistically significantly lower values were recorded in the Mid and High dose animals with an apparent dose response. Grip strength of the forelimbs were also slightly below the Control values in the Mid and High dose groups, these were out of the historical range but did not reach statistical significance. Although the grip strength differences appeared to be dose related in both sexes, the differences were not clearly adverse when assessing the Historic ranges and statistical differences together; the grip strength changes were considered equivocal in their relationship with treatment.
In the foot splay test, lower values were recorded in the High dose groups, but without reaching statistical significance, all were within the historical control range, the differences were not considered to represent an adverse effect of the test item.
In conclusion, a test item-related adverse neurotoxic effect was seen in High dose females between days 26 to 48; the profile of the observation over time may be explained by an accumulation (as evidenced by adipose colour) and enhanced metabolism later in the study (suggested by the hepatic hypertrophy observed). Reduced grip strength of the High dose group in both sexes in the last week of the study was considered to be of equivocal relationship with treatment.
During the motor activity test, higher locomotor activity was seen in the male High dose group during the 60-minute observation period, reaching statistical significance at several time points. However, the High dose initial result was close to the mean Historical control data and the overall shape of the curve was considered to be normal. No test item effect was attributed to the differences in locomotor activity measured in the last week of the study.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significantly higher liver weights were seen in the male and female Mid and High dose groups. The High dose values were generally out of the historical control ranges (up to 59% above control) while the Mid dose values were within. The effect was confirmed at histopathology.
The spleen weights were also statistically significantly higher in the male Mid and High dose and in the female High dose groups. The High dose spleen values were also out of historical control range (up to 105% above control). The effect was confirmed at histopathology.
Moreover, the kidney weights were also significantly higher in the male and female High dose groups. As the observed values were within or near the historical control range and no microscopic changes were seen at histopathology, these organ weight changes were considered as adaptive, non-adverse changes.
Besides these, all other statistically significant weight differences were considered to be incidental and not test item-related or ascribed to differences in body weight .
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related enlargement of the spleen in 7/10 High dose males and in 5/10 High dose females were seen. Enlargement of all lobes of the liver in 2/10 High dose males were also considered test item-related.
Yellow diffuse discoloration of the abdominal and subcutaneous adipose tissues was seen in all dose groups treated with the test item. The test item is a similar yellow colour, so it is considered likely that the colour is from deposited test item rather than any other cause. It should be noted, that in the preliminary study at higher dose levels, the yellow discoloration was also seen in the brains of animals at necropsy.
Besides these, the following incidental or background findings were seen in the terminally euthanized animals: Dilatation with clear fluid in the body and horns of the uterine (#1501, 1506, 2505, 4505, 4509), single firm nodule in the cervical section of the oesophagus (#3010) and yellow military focus in all lobes of the lungs (#4501, 4510).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were seen in the liver, spleen and thyroid glands; these organs were examined at all dose levels.
High dose groups (100 mg/kg bw/day): Minimal to slight hepatocellular centrilobular hypertrophy was seen in the liver of 9/10 males and 8/10 females. In the spleen minimal to moderate extramedullary haematopoiesis were seen in all animals. Minimal follicular cell hypertrophy was also seen in the thyroid glands of 5/10 males and 6/10 females.
Mid dose groups (30 mg/kg bw/day): Slight to moderate extramedullary haematopoiesis were seen in 8/10 males and in 3/10 females. Besides this, there were no test item-related findings seen (the single case of one minimal thyroid hypertrophy was considered to be incidental).
Low dose groups (10 mg/kg bw/day): There were no test item-related findings. The incidence of spleen extramedullary haematopoiesis was comparable to the control groups, therefore it was not considered to be test item-related.
The adipose tissues that were seen as coloured at all dose levels at necropsy were not examined histologically (the colour is dissolved during tissue processing, so there is no remaining colour at histopathology). Besides these above, all other histopathology findings seen in the terminally euthanized animals were considered to be incidental or background findings.
The liver hypertrophy and spleen extramedullary haematopoiesis are considered to be secondary or adaptive, non-adverse changes, which correlate with the changes seen in the clinical pathology parameters. The observed thyroid gland hypertrophy is most probably secondary to liver enzyme induction (as evidenced by hepatic hypertrophy) and caused by the increased thyroxine clearance, hence it is not ascribed to a direct effect of the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (nominal)
System:
other: Blood
Organ:
other: blood
Treatment related:
yes
Dose response relationship:
yes

Any other information on results incl. tables

DOSE FORMULATION ANALYSIS

Summary of analytical results

Nominal concentration(mg/mL)

Measured concentration(mg/mL)

Percentage of the nominal concentration(%)

Analytical sampling #1(Sampling: 11 September 2017)

Control

Not detectable

-

2

2.12 ± 0.01

106

6

6.70 ± 0.09

112

201

21.4 ± 0.28

107

Analytical sampling #2(Sampling: 05 October 2017)

Control

Not detectable

-

2

2.06 ± 0.01

103

6

6.28 ± 0.05

105

20

21.3 ± 0.34

107

Analytical sampling #3(Sampling: 02 November 2017)

Control

Not detectable

-

2

1.91 ± 0.01

95

6

5.86 ± 0.04

98

20

19.7 ± 0.18

98

Analytical sampling #4(Sampling: 01 December 2017)

Control

Not detectable

-

2

1.98 ± 0.01

99

6

5.85 ± 0.22

98

20

20.6 ± 0.19

103

 

NEUROLOGICAL ASSESSMENT (FUNCTIONAL OBSERVATION BATTERY)

Grip strength means (males)

Grip strength (males)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Forelimbs (g)

1473

1387

1378

1280

Differences from control

-6%

-6%

-13%

Historical control data

1397 – 2438

Hind limbs (g)

791

726

680 *

646 **

Differences from control

-8%

-14%

-18%

Historical control data

286 – 989

Note: *= p<0.05, **= p<0.01; Dunnett two sided test

Grip strength means (females)

Grip strength (females)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Forelimbs (g)

116

1074

1029

991

Differences from control

-4%

-8%

-11%

Historical control data

1043 – 1765

Hind limbs (g)

572

534

529

462 **

Differences from control

-7%

-7%

-19%

Historical control data

189 - 824

Note: *= p<0.05, **= p<0.01; Dunnett two sided test

 

Splay test means (males)

Splay test (males)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Distance between hindpaws (mm)

94

95

99

66

Differences from control

1%

6%

-30%

Historical control data

33 - 133

Splay test means (females)

Splay test (females)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Distance between hindpaws (mm)

70

60

60

56

Differences from control

-15%

-16%

-20%

Historical control data

27 - 138

 

CLINICAL PATHOLOGY

Haematology

Significant haematology parameters in males

Haematology (males)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Red blood count (M/μL)

9.132

9.010

8.613 **

6.561 **

Differences from control

-1%

-6%

-28%

Historical control data

8.07 – 9.61

Haemoglobin concentration (g/dL)

15.70

15.71

15.05 *

13.25 **

Differences from control

0%

-4%

-16%

Historical control data

13.6 – 16.8

Haematocrit (%)

47.31

47.30

46.43

42.06 **

Differences from control

0%

-2%

-11%

Historical control data

40 – 78.6

Mean cell volume (fL)

51.85

52.55

53.96 *

64.20 **

Differences from control

1%

4%

24%

Historical control data

47.3 – 55.1

Mean cell haemoglobin (pg)

17.21

17.46

17.46

20.21 **

Differences from control

2%

2%

17%

Historical control data

15.1 – 18.6

MCH concentration (g/dL)

33.19

33.24

32.39 **

31.50 **

Differences from control

0%

-2%

-5%

Historical control data

31.4 – 36.6

Lymphocytes (%)

68.13

67.34

72.77

76.27

Differences from control

-1%

7%

12%

Historical control data

36.9 – 79.8

Monocytes (%)

3.01

3.92 *

3.28

2.03 *

Differences from control

30%

9%

-33%

Historical control data

1.2 – 8.3

Red cell distribution width (%)

12.43

12.67

13.45 **

15.16 **

Difference from control

2%

8%

22%

Historical control data

11.7 – 14.4

Reticulocytes (%)

1.95

2.23

3.17 **

7.07 **

Differences from control

14%

63%

263%

Historical control data

1.4 – 3.2

Neutrophils (%)

26.71

26.47

21.47

19.95 †

Differences from control

-1%

-20%

-25%

Historical control data

15.9 – 57.7

Notes:  *= p<0.05, **= p<0.01; Dunnett two sided test,

           †= p<0.05, ††= p<0.01; Dunn two sided test.

Values out of the historical control range are indicated with bold font.

Significant haematology parameters in females

Haematology (females)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Red blood count (M/μL)

9.640

8.310

7.938 **

5.646 **

Differences from control

-4%

-8%

-35%

Historical control data

7.41 – 9.16

White blood cell count (K/μL)

5.548

3.616 **

3.439 **

3.661 **

Differences from control

-35%

-38%

-34%

Historical control data

0.44 – 7.48

Haemoglobin concentration (g/dL)

15.57

15.23

14.70 *

11.72 **

Differences from control

-2%

-6%

-25%

Historical control data

13.4 – 16.3

Haematocrit (%)

47.07

45.84

44.72 *

38.04 **

Differences from control

-3%

-5%

-19%

Historical control data

41.1 – 46.9

Mean cell volume (fL)

54.51

55.14

56.33

67.47 **

Differences from control

1%

3%

24%

Historical control data

48.9 – 57.0

Mean cell haemoglobin (pg)

18.05

18.29

18.52

20.77 ††

Differences from control

1%

3%

15%

Historical control data

16.0 – 19.9

MCH concentration (g/dL)

33.11

33.21

32.87

30.77 ††

Differences from control

0%

-1%

-7%

Historical control data

30.9 – 36.0

Mean platelet volume (fL)

6.65

6.91

7.11

8.43 **

Differences from control

4%

7%

27%

Historical control data

6.2 – 9.4

Monocytes (%)

3.12

3.03

2.50

1.51 **

Differences from control

-3%

-20%

-52%

Historical control data

1.2 – 6.2

Red cell distribution width (%)

11.39

11.54

11.83

18.12 ††

Difference from control

1%

4%

59%

Historical control data

104. – 14.1

Reticulocytes (%)

2.32

2.56

2.90

9.84 **

Differences from control

10%

25%

324%

Historical control data

1.4 – 5.8

Large Unclassified Cells (%)

0.86

0.71

0.69

0.53 *

Differences from control

-17%

-29%

-38%

Historical control data

0.2 – 1.9

Notes:  *= p<0.05, **= p<0.01; Dunnett two sided test,

           †= p<0.05, ††= p<0.01; Dunn two sided test.

Values out of the historical control range are indicated with bold font.

 

Coagulation parameters

Significant coagulation parameters in males

Coagulation (males)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Prothrombin time (sec)

9.81

9.65

9.36 **

9.72

Differences from control

-2%

-5%

-1%

Historical control data

9.2 – 10.5

Notes:  *= p<0.05, **= p<0.01; Dunnett two sided test,

           †= p<0.05, ††= p<0.01; Dunn two sided test.

 

Clinical chemistry

Significant clinical chemistry parameters in males

Clinical chemistry (males)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Glucose (mmol/L)

8.381

8.923

9.476 *

9.599 *

Differences from control

7%

13%

15%

Historical control data

7.05 – 12.68

Phosphorus (mml/L)

2.265

2.392

2.532 **

2.436

Differences from control

6%

12%

8%

Historical control data

2.05 – 3.02

Calcium (mmol/L)

2.573

2.581

2.559

2.465 *

Differences from control

0%

-1%

-4%

Historical control data

2.35 – 2.73

Total protein (g/L)

57.33

57.07

57.16

54.21 *

Differences from control

-1%

0%

-5%

Historical control data

48.9 – 61.0

Albumin (g/L)

32.13

32.13

31.68

30.57 †

Differences from control

0%

-1%

-5%

Historical control data

25.8 -33.0

Alkaline phosphatase (U/L)

92.9

94.2

82.9

74.3 **

Differences from control

1%

-11%

-20%

Historical control data

46 – 128

Bile acid (μmol/L)

10.031

10.959

13.109 *

13.201 **

Differences from control

9%

31%

32%

Historical control data

5.53 – 35.56

Notes:  *= p<0.05, **= p<0.01; Dunnett two sided test,

           †= p<0.05, ††= p<0.01; Dunn two sided test.

Significant clinical chemistry parameters in females

Clinical chemistry (females)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Glucose (mmol/L)

7.854

8.383

9.076 *

7.736

Differences from control

7%

16%

-2%

Historical control data

4.96 – 12.78

Bilirubin (μmol/L)

3.95

3.86

3.45

5.61 **

Differences from control

-2%

-13%

42%

Historical control data

2.6 – 20.0

Sodium (mmol/L)

144.37

145.08

144.16

148.16 **

Differences from control

1%

0%

3%

Historical control data

142.9 – 150.4

Calcium (mmol/L)

2.653

2.642

2.572

2.535 **

Differences from control

0%

-3%

-4%

Historical control data

2.36 – 2.71

Chloride (mmol/L)

103.85

104.72

104.66

109.69 **

Differences from control

1%

1%

6%

Historical control data

93.7 – 104.9

Albumin/Globulin ratio

1.42

1.42

1.39

1.32

Differences from control

0%

-2%

-7%

Historical control data

1.2 – 1.7

Notes:  *= p<0.05, **= p<0.01; Dunnett two sided test,

           †= p<0.05, ††= p<0.01; Dunn two sided test.

 

Urinalysis

Significant urinalysis parameters in males

Urinalysis (males)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Urine volume (mL)

1.90

1.65

7.00 **

3.50 *

Differences from control

-13%

268%

84%

Historical control data

2.0 – 56.0

pH

7.10

6.20 ††

6.90

6.20 ††

Differences from control

-13%

-3%

-13%

Historical control data

6.0 – 8.0

Specific gravity

1.0160

1.0275 **

1.0135

1.0255 **

Differences from control

1%

0%

1%

Historical control data

1.003 – 1.020

Notes:  *= p<0.05, **= p<0.01; Dunnett two sided test,

           †= p<0.05, ††= p<0.01; Dunn two sided test.

Significant urinalysis parameters in females

Urinalysis (females)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

pH

6.00

6.90 †

6.80 †

6.50

Differences from control

15%

13%

8%

Historical control data

6.0 – 8.0

Specific gravity

1.0240

1.0145 ††

1.0195

1.0235

Differences from control

-1%

0%

0%

Historical control data

1.005 – 1.025

Notes:  *= p<0.05, **= p<0.01; Dunnett two sided test,

           †= p<0.05, ††= p<0.01; Dunn two sided test.

 

PATHOLOGY EVALUATION

Kidney, liver and spleen organ weights of the male animals

Organ weights (males)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Terminal bodyweight (g)

519.4

513.6

532.7

479.0

Differences from control

-1%

3%

-8%

Historical control data

455 – 657

Kidneys (g)

2.903

2.997

3.210

3.627 **

Differences from control

3%

11%

25%

Historical control data

2.59 – 4.22

Kidneys / Bodyweight (%)

0.560

0.589

0.603

0.758 **

Differences from control

5%

8%

35%

Historical control data

0.518 – 0.761

Kidneys / Brain weight (%)

134.84

133.72

142.91

159.48 **

Differences from control

-1%

6%

18%

Historical control data

117.500 – 189.908

Liver (g)

14.207

14.580

17.403 **

20.739 **

Differences from control

3%

23%

46%

Historical control data

11.22 – 22.55

Liver / Bodyweight (%)

2.721

2.837

3.265 **

4.324 **

Differences from control

4%

20%

59%

Historical control data

2.183 – 3.442

Liver / Brain weight (%)

658.83

650.73

775.67 *

913.07 **

Differences from control

-1%

18%

39%

Historical control data

496.281 – 939.583

Spleen (g)

0.804

0.897

0.988 *

1.527 **

Differences from control

12%

23%

90%

Historical control data

0.69 – 1.52

Spleen / Bodyweight (%)

0.155

0.175

0.187

0.319 **

Differences from control

13%

20%

105%

Historical control data

0.128 – 0.257

Spleen / Brain weight (%)

37.26

39.91

43.98

67.32 ††

Differences from control

7%

18%

81%

Historical control data

31.222 – 64.623

Notes:  *= p<0.05, **= p<0.01; Dunnett two sided test,

           †= p<0.05, ††= p<0.01; Dunn two sided test.

Values out of the historical control range are indicated with bold font.

Kidney, liver and spleen organ weights of the female animals

Organ weights (females)

Group / Concentration (mg/kg bw/day)

Control (0)

Low (10)

Mid (30)

High (100)

Terminal bodyweight (g)

180.5

269.8

271.3

254.2

Differences from control

-4%

-3%

-9%

Historical control data

254 – 352

Kidneys (g)

1.804

1.838

1.803

2.065 *

Differences from control

2%

0%

15%

Historical control data

1.45 – 2.29

Kidneys / Bodyweight (%)

0.646

0.681

0.664

0.810 **

Differences from control

6%

3%

26%

Historical control data

0.519 – 0.748

Kidneys / Brain weight (%)

89.75

90.91

87.62

98.35

Differences from control

1%

-2%

10%

Historical control data

71.429 – 109.048

Liver (g)

8.507

8.678

9.214

11.538 **

Differences from control

2%

8%

36%

Historical control data

6.37 – 10.01

Liver / Bodyweight (%)

3.041

3.206

3.397 *

4.530 **

Differences from control

5%

12%

49%

Historical control data

2.273 – 3.502

Liver / Brain weight (%)

423.48

429.34

447.61

548.86 **

Differences from control

1%

6%

30%

Historical control data

307.729 – 489.899

Spleen (g)

0.620

0.612

0.606

1.012 **

Differences from control

-1%

-2%

63%

Historical control data

0.46 – 0.86

Spleen / Bodyweight (%)

0.221

0.227

0.223

0.397 ††

Differences from control

2%

1%

79%

Historical control data

0.171 – 0.307

Spleen / Brain weight (%)

30.85

30.38

29.46

48.21 **

Differences from control

-2%

-5%

56%

Historical control data

22.439 – 43.216a

Notes:  *= p<0.05, **= p<0.01; Dunnett two sided test,

           †= p<0.05, ††= p<0.01; Dunn two sided test.

Values out of the historical control range are indicated with bold font.

 

Applicant's summary and conclusion

Conclusions:
In summary, daily administration of C.I. Solvent Yellow 124 by oral gavage to Wistar rats at dose levels of 10, 30 or 100 mg/kg bw/day during the treatment period under the conditions of this study did not result in test item related mortality. However, at the High dose, adverse neurotoxicity was seen, accompanied by slightly lower bodyweights, with a significant anaemia. Increased liver, spleen and kidney weights, with hypertrophy in livers and thyroids, plus extramedullary haematopoiesis in the spleen were considered to be secondary effects to anaemia or non-adverse adaptive changes.
In the Mid dose groups there was no clear neurotoxicity observed, but the anaemia effect was of a similar profile to the High dose with statistical differences in both sexes, although the differences were generally within the historical control ranges.
Significant yellow discoloration of the skin in High dose animals during the in-life phase, and of the adipose tissues in all treated groups was attributed to accumulation of the test item; although a significant observation, the apparent accumulation of test item was not considered to reflect a true toxic effect in the context of this study.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for C.I. Solvent Yellow 124 is considered to be 10 mg/kg bw/day.
Executive summary:

This 90-day study was performed to obtain information on the toxicity of C.I. Solvent Yellow 124 when administered by oral gavage to Wistar rats at 3 dose levels selected by the Sponsor (OECD No. 408) daily for 90 days.

 

Forty male and forty female Wistar rats were treated as follows:

 

Group No.

Group Designation

Dose level (mg/kg bw/day)

Concentration (mg/mL)

Dose volume (mL/kg bw)

Animal Number

Males

Females

1

Control

0

0

5

10

10

2

Low Dose

10

2

10

10

3

Mid Dose

30

6

10

10

4

High Dose

100

20

10

10

 

Analysis of test item formulations for concentration and homogeneity was performed four times during the treatment period (during the first, fifth, ninth and last week of treatment) using a validated HPLC-UV method. No test item was detected in the control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 95-112 % of the nominal concentrations.

 

The examinations included clinical signs, mortality, body weights, food consumption, ophthalmoscopy, neurological assessment including measurements of the landing foot splay, grip strength and motor activity assessment, clinical pathology, gross pathology, organ weights and histopathology. Full histopathology was performed in Groups 1 (Control) and 4 (High dose), and additionally the liver, spleen and thyroid glands were also processed in Groups 2 (Low dose) and 3 (Mid dose).

 

Conclusion

In summary, daily administration of C.I. Solvent Yellow 124 by oral gavage to Wistar rats at dose levels of 10, 30 or 100 mg/kg bw/day during the treatment period under the conditions of this study did not result in test item related mortality. However, at the High dose, adverse neurotoxicity was seen, accompanied by slightly lower bodyweights, with a significant anaemia. Increased liver, spleen and kidney weights, with hypertrophy in livers and thyroids, plus extramedullary haematopoiesis in the spleen were considered to be secondary effects to anaemia or non-adverse adaptive changes.

 

In the Mid dose groups there was no clear neurotoxicity observed, but the anaemia effect was of a similar profile to the High dose with statistical differences in both sexes, although the differences were generally within the historical control ranges.

 

Significant yellow discoloration of the skin in High dose animals during the in-life phase, and of the adipose tissues in all treated groups was attributed to accumulation of the test item; although a significant observation, the apparent accumulation of test item was not considered to reflect a true toxic effect in the context of this study.

 

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for C.I. Solvent Yellow 124 is considered to be 10 mg/kg bw/day.