[(9-oxo-9H-thioxanthen-2-yl)oxy]acetic acid

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:

21 December 2018 - 12 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guideline OPPTS 870.3650

Version / remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

not applicable

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

2008

Deviations:

not applicable

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents

Version / remarks:

2000

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[(9-oxo-9H-thioxanthen-2-yl)oxy]acetic acid

EC Number:

282-803-8

EC Name:

[(9-oxo-9H-thioxanthen-2-yl)oxy]acetic acid

Cas Number:

84434-05-9

Molecular formula:

C15H10O4S

IUPAC Name:

2-[(9-oxo-9H-thioxanthen-2-yl)oxy]acetic acid

Test material form:

solid

Specific details on test material used for the study:

Appearance: Yellow powder
Purity/Composition: 98.25%
Test item storage: At room temperature protected from light desiccated

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
Condition: Outbred, SPF-Quality.
Source: Charles River Deutschland, Sulzfeld, Germany
Number of Females 6 (nulliparous and non-pregnant).
Target Age at the Initiation of Dosing: 12-14 weeks.
Target Weight at the Initiation of Dosing: 200 to 250 g.

HUSBANDRY
On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room in which the animals were kept was documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
The housing conditions were maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian.


ENVIRONMENTAL CONDITIONS
Temperature: 19 to 21°C.
Humidity: 36 to 58%.
Light Cycle: 12-hours light and 12-hours dark
Ventilation: Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days prior to start of the pretest period (females) or 7 days before the commencement of dosing (males).

FOOD
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) provided ad libitum. During motor activity measurements, animals did not have access to food for a maximum of 2 hours. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

WATER
Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals did not have access to water for a maximum of 2 hours. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.

ANIMAL ENRICHMENT
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

VETINARY CARE
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is a possible route of human
exposure during manufacture, handling or use of the test item.

Vehicle:

propylene glycol

Details on oral exposure:

VEHICLE
Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw data of these trials will be retained by the Test Facility.

ADMINISTRATION OF THE TEST MATERIAL

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 37-41 days.

The first day of dosing was designated as Day 1. Female nos. 44, 45 (Group 1), 55 (Group 2) and 68 (Group 3) were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube.

The dosing formulations were stirred continuously during dose administration. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures. Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

The dose levels were selected based on the results of a 10-day dose range finder with oral administration of CMTX 2-carboxymethyloxy-thioxanthone in rats (Test Facility Reference No. 20151968, see Appendix 6), and in an attempt to produce graded responses to the test item.


Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item.

Test item dosing formulations were kept at room temperature until dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.Any residual volumes were discarded.

Analytical verification of doses or concentrations:

yes

Remarks:

Appendix 4

Details on analytical verification of doses or concentrations:

Sample Collection and Analysis:
Dose formulation samples were collected as below.
Additional samples may be collected and analyzed at the discretion of the Study Director.

Dose Formulation Sample Collection Schedule:
Occasion: Week 1 of treatment
Concentration: All groups(a)
Homogeneity: Groups 2 and 4(a)

(a) The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.

All samples analyzed were transferred (at room temperature protected from light) to the analytical laboratory at the Test Facility.

Residual samples were discarded after completion of the sample analysis.

Analytical Method:
Analyses described were performed using a validated analytical procedure (Test Facility Study No. 20151969).

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent no treatment

Details on study design:

DOSING

A dose-range finding study (DRF) was conducted on three animals at test doses of 500 and 1000 mg/kg/day (summary of Dose Range Finder in appendix 6)

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 10 days.

A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures (Study No. 521022 was used for DCS).

The dose levels were selected based on the results of an acute oral toxicity study in rats (LD50 > 2000 mg/kg, Test Facility Study No. 20151965).

The justification of the route of administration is identical as for the main study.

JUSTIFICATION OF DOSE LEVELS

The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration of CMTX 2-CARBOXYMETHYLOXY-THIOXANTHONE in rats (Test Facility Study No. 20151968, see section 8), and in an attempt to produce graded responses to the test item.

The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Examinations

Observations and examinations performed and frequency:

Mortality/Moribundity Checks – F0-Generation:
Frequency: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
Procedure: Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

Clinical Observations – F0-Generation:
Frequency: Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing period up to the day prior to necropsy.
Procedure: During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals (no peak effect of occurrence of clinical signs was observed in the dose range finder (Test Facility Reference No. 20151968, see Appendix 6)).

The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Arena Observations – F0-Generation:
Frequency: Once before the first administration of the test item and at weekly intervals during the treatment period.
Procedure: Animals were observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

Body Weights – F0-Generation:
Frequency: Males and females will be weighed on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. In order to monitor the health status, Animal No. 74 was also weighed on PND 3. A terminal weight was recorded on the day of scheduled necropsy.
Procedure: Animals were individually weighed.

Food Consumption – F0-Generation:
Frequency: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females will be measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
Procedure: Food consumption was quantitatively measured..

Water Consumption – F0-Generation:
Frequency: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Functional Tests – F0-Generation:
Frequency:Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 9-11). These tests were performed after dosing, after completion of clinical observations. (including arena observation).
Procedure: The following tests were performed:
Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal, using a grip strength meter.
Locomotor activity (recording period: 1-hour under normal laboratory light conditions), using the Kinder Scientific Motor Monitor System. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

Sacrifice and pathology:

Clinical Pathology:
Sample Collection:
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead and females with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Additional blood samples were obtained (e.g. due to clotting of non-serum samples) if necessary in both the animal facility and in the necropsy room if permissible sampling frequency and blood volume were not exceeded. After collection, samples were transferred to the appropriate laboratory for processing.

F0-males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before blood sampling, but water was still available. F0-females were not fasted overnight.

Samples were collected according to the table ("Samples for Clinical Pathology Evaluation") below:

Hematology:
Target Volume: 0.5 mL.
Anticoagulant: K3-EDTA (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria).

Hematology Parameters:
White blood cells (WBC)
Red Blood Cell Distribution Width (RDW)
Neutrophils (absolute)
Hemoglobin
Lymphocytes (absolute)
Hematocrit
Monocytes (absolute)
Mean corpuscular volume (MCV)
Eosinophils (absolute)
Mean corpuscular hemoglobin (MCH)
Basophils (absolute)
Mean corpuscular hemoglobin concentration (MCHC)
Red blood cells
Platelets
Reticulocytes (absolute)

A blood smear was prepared from each hematology sample. Blood smears were labeled, stained, and stored. These smears were not examined.

Coagulation:
Target Volume: 0.45 mL.
Anticoagulant: Citrate (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria).

Coagulation Parameters:
Prothrombin Time (PT):
Activated Partial Thromboplastin Time (APTT)

Clinical Chemistry:
Target Volume: 0.5 mL [For bile acid measurement: 1.0 mL (same sample as for thyroid hormone measurement)]
Anticoagulant: Li-Heparin (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). Not applicable for serum tubes.
Processing: To serum (bile acids) or to plasma

Clinical Chemistry Parameters:
Alanine aminotransferase (ALAT)
Creatinine
Aspartate aminotransferase (ASAT)
Glucose
Alkaline Phosphatase (ALP)
Cholesterol
Total protein
Sodium
Albumin
Potassium
Total Bilirubin
Chloride
Bile Acids
Calcium
Urea
Inorganic Phosphate (Inorg. Phos)

Thyroid Hormone:
Target Volume: F0-animals: 1.0 mL (same sample as for bile acid measurement).

Anticoagulant: Not applicable for serum. (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria).

Thyroid Hormone Parameters:
Thyroxine (T4)
Thyroid-Stimulating Hormone (TSH; only if required)

Measurement of total T4 was conducted for F0-animals. Measurement of TSH was conducted for F0-animals.

Serum samples retained for possible future analysis were maintained by the Test Facility in the freezer (≤-75°C). Under these storage conditions, samples are stable for 6 months. Any remaining sample will be discarded.

After clotting and centrifugation, serum was used as listed below.
F0-Males: Serum from each sample was divided into 2 aliquots: 150 µL serum for measurement of total T4, and the remaining volume of serum for possible future measurement of TSH.

F0-Females: The serum was used for possible future measurement of total T4 and/or thyroid-stimulating hormone TSH.

Organ Weights – F0-Generation
The organs identified in the table below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.

Organs Weighed at Necropsy for all selected animals:
Brain
Cervix (a)
Epididymis (b)
Gland, adrenal (b)
Gland, coagulation (b,c)
Gland, parathyroid (d)
Gland, prostate
Gland, seminal vesicle (b)
Gland, thyroid (b)
Heart
Kidney (b)
Liver
Ovaries (b)
Spleen
Testes (b)
Thymus
Uterus

(a) Weighed together with the uterus.
(b) Paired organ weight.
(c) Weighed together with the seminal vesicles.
(d) Weighed together with the thyroid.

Organs Weighed at Necropsy for all remaining animals (incl. female that failed to deliver pups and female with total litter loss):

Epididymis (a)
Gland, coagulation (a,b)
Gland, parathyroid (c)
Gland, prostate
Gland, seminal vesicle (a)
Gland, thyroid
Testes (a)

(a) Paired organ weight.
(b) Weighed together with the seminal vesicles.
(c) Weighed together with the thyroid.

Tissue Collection and Preservation – F0-Generation
Representative samples of the tissues identified in the table below were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

Animal identification
Artery, aorta
Body cavity, nasopharynx
Bone marrow
Bone, femur
Bone, sternum
Brain (eight levels)
Cervix
Epididymides (a)
Esophagus
Eye (a)
Gland, adrenal
Gland, coagulation
Gland, Harderian (a,b)
Gland, lacrimal
Gland, mammary
Gland, parathyroid (c)
Gland, pituitary
Gland, prostate
Gland, salivary
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Gut-associated lymphoid tissue
Heart
Kidney
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Larynx
Liver
Lung
Lymph node (mandibular and mesenteric site)
Muscle, skeletal
Nerve, optic (a,b)
Nerve, sciatic
Ovaries
Pancreas
Skin
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testes (a)
Thymus
Tongue
Trachea
Urinary bladder
Uterus
Vagina

a Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours.
b Only collected if present in the routine section of the eye.
c Only collected if present in the routine section of the thyroid.

Tissue Collection and Preservation for all remaining animals (incl. female that failed to deliver pups and female with total litter loss).

Animal identification
Cervix
Epididymis (a)
Gland, coagulation
Gland, mammary
Gland, parathyroid (b)
Gland, pituitary
Gland, prostate
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Ovaries
Testes (a)
Uterus
Vagina

a Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours.
b Only collected if present in the routine section of the thyroid.

Histology – F0-Generation
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:

Selected animals and unscheduled deaths (sacrificed in extremis or found dead): Tissues identified in Text Table 12 (except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, parathyroid gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).

Females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, ovaries, uterus and vagina.
Females with total litter loss: Mammary gland.
Remaining animals: Gross lesions/masses.

Histopathology – F0-Generation
All tissues as defined under Histology – F0-Generation (section 4.12.6) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported.
For the testes of all selected males of Groups 1 and 4 a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. A peer review on the histopathology data was performed by a second pathologist.

Statistics:

See "Any other information on materials and methods incl. tables" below

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Test item-related clinical observations were observed starting at treatment with 100 mg/kg/day.

No additional findings were noted during the weekly arena observations in this study.

Yellow discoloration of the faeces and urine were observed in both males and females at 1000 mg/kg/day from Day 7 of treatment onwards.

Salivation seen after dosing among animals treated with 100, 300 or 1000 mg/kg/day of the test item was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

No additional findings were noted during the weekly arena observations in this study.

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post coitum and lactation phase.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

(Appendix 1 and Appendix 2)

No test item-related mortality was observed up to 1000 mg/kg/day.
One female at 1000 mg/kg/day (No. 74) was sacrificed in extremis on lactation Day 3, based on severe body weight loss (15% in 3 days). In addition, lethargy and a hunched posture were noted for this animal and at macroscopy, she was found emaciated, the stomach was distended with gas and a pale discoloration of the liver was noted. Main microscopic findings were moderate to marked ulceration in the glandular and non-glandular parts of the stomach which was considered to be main cause of moribundity. In addition, a slight ulceration in the duodenum, lymphoid depletion in the thymus and increased adipocytes with decreased
cellularity of the bone marrow (femur and sternum) were observed. The stomach findings were considered to be likely the result of an oral gavage incident.

One female of the control Group (No. 41) died shortly after dosing on Day 7 of treatment. Before her death, she had breathing difficulties and necropsy findings included a foamy content of the trachea, watery-clear fluid in the abdominal cavity, and a tan discoloration of- and several red foci on the thymus. No microscopic findings were noted that could explain the death of this animal. Based on the clinical signs and macroscopic findings, this death was considered the result of an oral gavage incident.

One female of the 1000 mg/kg/day group (No. 76) was euthanized on Lactation Day 1, as she had a total litter loss. For this animal, no pups were found and based on her appearance she was considered no longer pregnant. At macroscopy, pale discoloration of the animal, one late resorption in the right horn and dark red fluid in the vagina were noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Body weights and body weight gain of treated males were decreased by treatment with 1000 mg/kg/day.

Body weights in males were decreased with 0.97-0.98x compared with controls from Day 1 of treatment. Body weights further decreased to 0.94-0.95x compared with controls at 1000 mg/kg/day throughout the treatment period. These decreased body weights were accompanied by decreased body weight gains noted from Day 8 of treatment onwards, reaching statistical significance at Day 15 (0.80x) and 29 (0.81x) of treatment.

In females, slight (not statistically significant) body weight loss was observed at 100 and 1000 mg/kg/day during week 1 of treatment, followed by recovery to normal body weights in the following days of treatment. During post-coitum Days 17-20, a slight (not statistically significant) decrease in body weights and body weight gain was observed. As these changes in body weights and body weight gain were slight and in absence of a dose-related trend, they were considered not test-item related.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haemotology
(Appendix 1 and Appendix 2)
The following statistically significant changes distinguished treated from control animals at the end of the treatment period (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):

• In males, reticulocyte count was significantly higher at 1000 mg/kg/day (1.29x ofcontrol), although values remained within normal range(4). In females, a similar dose-dependent trend towards increased reticulocyte count was observed, althoughstatistical significance was not reached.
• In males, red blood cell distribution width (RDW) was increased at 300 (1.10x ofcontrol) and 1000 mg/kg/day (1.19x of control). At 300 mg/kg/day values remainedwithin normal range. At 1000 mg/kg/day values exceeded the normal range(5).
• Red blood cell count was decreased in females, at 1000 mg/kg/day (0.88x of control).Values were below the lower limit of the historical control range(6).
• Mean corpuscular volume (MCV) was increased in females at 300 (1.06x of control)and 1000 mg/kg/day (1.1x of control). Values remained within normal ranges at 300,but exceeded the normal range at 1000 mg/kg/day(7).
• In females, mean corpuscular hemoglobin (MCH) was increased at 1000 mg/kg/day(1.08x of control) and values were on the upper limit of the historical control range(8).

Noteworthy, in males, white blood cell (WBC) and lymphocyte counts showed a dose-dependent increase at 1000 mg/kg/day (1.30x and 1.37x of control, respectively). Changes did not reach statistical significance, but were at the upper limit of the historical control data(9).

(4) Historical control data of male Wistar Han rats (period 2017-2019): Reticulocytes (10E9/L): mean = 225.5, P5 - P95 = 165.85 – 291.05 (n=260).

(5) Historical control data of male Wistar Han rats (period 2017-2019): RDW (%): mean = 12.2, P5 - P95 = 11.1 – 13.5 (n=260).

(6) Historical control data of female Wistar Han rats (period 2017-2019): Red blood cells (10E9/L): mean = 7.28, P5 - P95 = 6.63 – 7.94 (n=206).

(7) Historical control data of female Wistar Han rats (period 2017-2019): MCV (fL): mean = 59.9, P5 - P95 = 56.3 – 64.0 (n=206).

(8) Historical control data of female Wistar Han rats (period 2017-2019): MCH (fmol): mean = 1.23, P5 - P95 = 1.16 – 1.30 (n=206).

(9) Historical control data of male Wistar Han rats (period 2017-2019): WBC (10E9/L): mean = 7.7, P5 - P95 = 5.06 – 10.77 (n=259). Lymphocytes (10E9/L): Mean = 6.4, P5 - P95 = 4.00 – 9.20 (n=259).

Coagulation:
(Appendix 1 and Appendix 2)
Coagulation parameters of treated rats were considered not to have been affected by treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

(Appendix 1 and Appendix 2)
The following statistically significant changes distinguished treated from control animals at the end of the treatment period at 300 and/or 1000 mg/kg/day (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):

• Alkaline phosphatase (ALP) was increased in males and females at 1000 mg/kg/day(4.23x of control and 3.5x of control, respectively). A similar trend was observed inmales at 300 mg/kg/day (1.42x of control), although not reaching statisticalsignificance). Values were at the upper limit or above the historical control range(10).

• Creatinine was increased in males and females at 300 (1.08x of control and 1.09x ofcontrol, respectively) and 1000 mg/kg/day (1.12x of control and 1.09x of control,respectively). In females, values remained within the normal range, but values formales were above the upper limit of the historical control range(11).

• Cholesterol was increased in males at 300 (1.39x of control) and 1000 mg/kg/day(1.47x of control). All values remained within the range of the historical controls(12).

The lower aspartate aminotransferase (ASAT) values in treated males at 300 mg/kg/day and in females at 1000 mg/kg/day achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high control values and were therefore considered to be of no toxicological significance.

Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment due to the minimal magnitude of the change and as these occurred in the absence of a dose-related trend.

Thyroid hormone analyses:
A dose dependent decrease in total T4 levels (0.76x of control) was observed in males at 1000 mg/kg/day. In females, a dose dependent decrease in total T4 levels (0.73x and 0.63x of control) was observed at 300 and 1000 mg/kg/day (0.73x and 0.63x of control, respectively).
Values remained within normal range(13).
An increase in TSH was observed in both males and females at 1000 mg/kg/day (3.21x and 3.12x of control, respectively). Values exceeded the historical control range(14).


(10) Historical control data of Wistar Han rats (period 2017-2019): ALP (U/L): Males: Mean = 164; P5 - P95 = 101.0 – 263.0 (n=270). Females: Mean = 330, P5 - P95 = 109.0 – 581.0 (n=210).

(11) Historical control data of Wistar Han rats (period 2017-2019): Creatinine (umol/L): Males: Mean = 36.5; P5 - P95 = 33.0 – 40.7 (n=270). Females: Mean = 39.7, P5 - P95 = 35.4 – 44.3 (n=210).

(12) Historical control data of male Wistar Han rats (period 2017-2019): Cholesterol (mmol/L): Mean = 1.95, P5 - P95 = 1.41 – 2.59 (n=270).

(13) Historical control data of male Wistar Han rats (period 2017-2018):
Total T4 (µg/dL): mean= 4.51, P5-P95: 2.85-6.37 (Males; n=557)
Total T4 (µg/dL): mean= 2.88, P5-P95: 1.74-4.24 (Males; n=97)

(14) Historical control data of male Wistar Han rats (period 2017-2018):
TSH (µIU/mL): mean= 0.136, P5-P95: 0.032-0.322 (Males; n=80)
TSH (µIU/mL): mean= 0.237, P5-P95: 0.060-0.633 (Females; n=78)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

(Appendix 1 and Appendix 2)

Test item-related higher liver weights were noted in the 300 mg/kg/day groups males (relative to body weights) and in the 1000 mg/kg/day females (absolute and relative to body weights) as shown in text table 1 below.

Some organ weight differences were statistically significant when compared to the control group (absolute prostate gland weight and relative kidney weight, males only) but were considered to be the result of a test item-related effect on terminal body weight.

There were no other test item-related organ weight changes.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in males and females and are summarized below in text tables 2 - 4

Liver, cytoplasmic alteration, granular was present in females starting at 300 mg/kg/day and in males at 1000 mg/k/day at minimal to slight degree.

Liver, hepatocellular hypertrophy was present in females at 1000 mg/kg/day at minimal degree.
These findings likely correlated at 1000 mg/kg/day with the increased liver weight.

Kidneys, an increased incidence and severity of hyaline droplet accumulation was present in males starting at 300 mg/kg/day up to slight degree.

Spleen, an increased incidence and severity of extramedullary hematopoiesis was present in females at 1000 mg/kg/day up to moderate degree.

The remainder of the recorded microscopic findings, including the low incidence and severity of follicular cell hypertrophy in the thyroid gland, were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Food Consumption:
(Appendix 1 and Appendix 2)
Please note that food consumption in males during the mating phase was not yet corrected for the days spend with the female (i.e. not in the home cage).

Food consumption before or after correction for body weight was decreased at 1000 mg/kg/day.

During Week 1 of treatment, absolute and relative food consumption was decreased in males and females at 1000 mg/kg/day. For females, values were below the lower limit of the historical control range(1). As food consumption levels recovered to normal values in the following days, the decreases were considered not toxicologically relevant.

During the first 4 days of lactation, absolute and relative food consumption were also statistically significantly decreased at 1000 mg/kg/day (0.80x and 0.84x of control, respectively). Values remained within normal ranges(2) and food consumption normalized to control levels during the next days and therefore this was not considered toxicologically relevant.

Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.


Functional Tests:
(Appendix 1 and Appendix 2)
Functional observation parameters were considered not to be affected by treatment.

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. In males, a (not statistically significant) trend towards decreased grip strength was observed. As statistical significance was not reached and all values remained within normal range (3), the slightly decreased grip strength levels were considered not test-item related.

The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.


(1) Historical control data of female Wistar Han rats (period 2017-2019):
Food consumption (g/animal/day): Week 1: Mean = 16; P5-P95 = 12.7 – 17.9 (n = 95).
Relative food consumption (g/kg body weight/day): Week 1: Mean = 70; P5-P95 = 58.4 – 80.5 (n = 95).

(2) Historical control data of female Wistar Han rats (period 2017-2019):
Food consumption during lactation (g/animal/day): Day 1-4: Mean = 30; P5-P95 = 19.7 – 43.0 (n = 447).
Relative food consumption during lactation (g/kg body weight/day): Day 1-4: Mean = 110; P5-P95 = 75.4 – 158.5 (n = 447).

(3) Historical control data of male Wistar Han rats (period 2015-2018):
Grip strength fore leg (gram): mean = 1158, P5 - P95 = 681 – 1606 (n=445).
Grip strength hind leg (gram): mean = 609, P5 - P95 = 371 – 883 (n=445).

Details on results:

Wistar Han rats were treated with CMTX 2-carboxymethyloxy-thioxanthone by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg. The rats of the control group received the vehicle, Propylene glycol, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 41 days. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results:

There were two test item-related mortalities at 1000 mg/kg/day: Two females at 1000 mg/kg/day (no. 76 and No. 74) were sacrificed in extremis on lactation Day 1 and 3, respectively. Female no. 76 had a total litter loss; no pups were found but blood was found in her cage and based on her appearance she was no longer pregnant. At macroscopy, pale discoloration of the animal, one late resorption in the right horn and dark red fluid in the vagina were noted. Female no. 74 had severe body weight loss (15% within 3 days). In addition, lethargy was noted for this animal and at macroscopy, she was found emaciated, the stomach was distended with gas and a pale discoloration of the liver was noted.

One female of the control Group (No. 41) died shortly after dosing on Day 8 of treatment. Before her death, she had breathing difficulties and necropsy findings included a foamy content of the trachea, watery-clear fluid in the abdominal cavity, and a tan discoloration of- and several red foci on the thymus. As such, this death was considered the result of an oral gavage incident.

Test item-related clinical observations were observed starting at treatment with 100 mg/kg/day. Yellow discoloration of the faeces and urine were observed in both males and females at 1000 mg/kg/day from Day 7 of treatment onwards. This finding was likely related to the yellow colour of the test item and in the absence of correlating findings on intestines or kidneys, was considered non-adverse, pending histopathological evaluation. Salivation seen after dosing among animals treated with 100, 300 or 1000 mg/kg/day of the test item was considered not toxicologically relevant as this sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Growth and food consumption of treated rats were not adversely affected. The slight and transient changes noted at 1000 mg/kg/day (lower body weight and body weight gain in males during treatment and lower food consumption in males and females in Week 1 of treatment) were considered of no toxicological significance.

Haematology parameters measured at the end of treatment showed several statistically significant changes, mostly at 300 and 1000 mg/kg/day. The changes consisted of higher reticulocyte count in males and females (not statistically significant) at 1000 mg/kg/day, increased red blood cell distribution width (RDW) in males at 300 and 1000 mg/kg/day, decreased red blood cell count in females at 1000 mg/kg/day, increased mean corpuscular volume (MCV) in females at 300 and 1000 mg/kg/day and increased mean corpuscular hemoglobin (MCH) in females at 1000 mg/kg/day. Noteworthy, in males, white blood cell (WBC) and lymphocyte counts showed a dose-dependent increase at 1000 mg/kg/day, although statistical significance was not reached. Except for RDW, red blood cell count and MCV at 1000 mg/kg/day, the changes remained within normal limits (within or on the limits of the historical control ranges).

Clinical laboratory investigations showed treatment-related changes, mainly at 1000 mg/kg: An increase in alkaline phosphatase (ALP) was observed in males and females at 1000 mg/kg/day (and a similar trend at 300 mg/kg/day). Moreover, creatinine was increased in males and females at 300 and 1000 mg/kg/day and cholesterol was increased in males at 300 and 1000 mg/kg/day. Except for ALP at 1000 mg/kg/day and creatinine in males, values remained within the limits of the historical control ranges. Further evaluation of the adversity of these clinical biochemistry parameters is pending histopathological evaluation.

Test-item related higher liver weights were noted in both sexes. Liver weights (absolute and relative to body weight) were increased in 1000 mg/kg males and females and in males at 300 mg/kg/day (relative to body weight only). Given the magnitude of the effect, the test-item related increased liver weights at 1000 mg/kg/day were considered adverse, pending histopathological evaluation. Test-item related higher kidney weights were noted in males at 1000 mg/kg/day (relative to body weight only), but were considered non-adverse because of the magnitude of the response (<10%), pending further histopathological evaluation. No treatment-related toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations, locomotor activity, coagulation parameters and macroscopic examination).

Reproductive results:

Please note that estrous cycle results not available for evaluation in the interim report.

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

Noteworthy, one Group 4 female (no. 76) had only two implantation sites. This female was euthanized on lactation Day 1, as she had a total litter loss. Although a relationship with treatment could not be excluded, this low number of implantation sites is occasionally observed in our historical controls and as the number of implantation sites of all other females at 1000 mg/kg/day was comparable with controls, this was considered a single finding and therefore not toxicologically relevant.

No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices and precoital time).

Developmental results:

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered affected by treatment at 1000 mg/kg/day. Viability indices were 98% for the control, 97%, 100% and 82% for the 100, 300 and 1000 mg/kg/day groups, respectively.

The low viability index at 1000 mg/kg/day could to some extend be attributed to the death of Female no.74, which was sacrificed in extremis on lactation Day 3 together with her remaining pups. However, upon exclusion of this litter from calculations, viability index at 1000 mg/kg/day is 91%, which is still decreased compared to control and outside the range of the historical control data. This finding was considered adverse.

Moreover, treatment-related lower body weights of pups were noted from PND 1 onwards at 1000 mg/kg/day (up to 16% lower than concurrent controls). Considering the magnitude of the effect, this finding was regarded as treatment-related and adverse.

No treatment-related changes toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of clinical signs, anogenital distance, areola/nipple retention and macroscopic examination).

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Text Table 1

Mean Percent Weight Differences from Control Groups

		Males			Females
Dose level (mg/kg/day):		100	300	1000	100	300	1000
LIVER
	Absolute	3	8	32**	7	9	23**
	Relative to body weight	5	12**	45**	4	8	26**
KIDNEY
	Absolute	4	3	0	-	-	-
	Relative to body weight	6	2	9*	-	-	-

*: P<0.05, **: P<0.01

Text Table 2

		Males				Females
	Dose level (mg/kg/day):	0	100	300	1000	0	100	300	1000
	Liver (a)	5	5	5	5	6	5	5	6
Cytoplasmic alteration, granular	Minimal	-	-	-	1	-	-	3	6
	Slight	-	-	-	4	-	-	1	-
Hepatocellular hypertrophy	Minimal	-	-	-	-	-	-	-	5

(a)  =  Number of tissues examined from each group.

Text Table 3

		Males
	Dose level (mg/kg/day):	0	100	300	1000
	Kidneys (a)	5	5	5	5
Hyaline droplet accumulation	Minimal	2	2	4	2
	Slight	-	-	1	3

(a)  =  Number of tissues examined from each group.

Text Table 4

		Females
	Dose level (mg/kg/day):	0	100	300	1000
	Spleen (a)	6	5	5	6
Extramedullary hematopoiesis	Minimal	3	4	3	2
	Slight	-	-	-	1
	Moderate	-	-	-	2

(a)  =  Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the interim results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of CMTX 2-carboxymethyloxy-thioxanthone was established:

Parental NOAEL: 300 mg/kg/day

Note: In this study, an approximately 3-fold increase in TSH was observed in the high dose groups (in both males and females) which was considered to be test item-related. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

Executive summary:

The objectives of this study were to determine the potential toxic effects of CMTX 2 -carboxymethyloxy-thioxanthone when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the parental ) No Observed Adverse Effect Level (NOAEL)

The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the dose range finder (Test Facility Reference No. 20151968, see Appendix 6).  

The study design was as follows:

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study:  mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0 -males), gross necropsy findings, organ weights and histopathologic examinations.

Formulation analyses confirmed that formulations of test item in Propylene glycol were prepared accurately and homogenously.

No parental toxicity was observed up to 1000 mg/kg/day.

Mortality was observed in one control female and in one female animal at 1000 mg/kg/day.

For the control female, breathing difficulties, foamy contents in the trachea and a watery-clear fluid in the thoracic cavity was found consistent with an oral gavage incident. The female treated with 1000 mg/kg/day was emaciated, had severe body weight loss, lethargy, a stomach distended with gas and a pale discoloration of the liver. At microscopic examination, the findings noted in the stomach (ulceration in the glandular and non-glandular parts of the stomach (considered to be main cause of moribundity) and a slight ulceration in the duodenum) were considered to be related to an oral gavage incident.  

Test item-related clinical signs were observed starting at treatment with 100 mg/kg/day.  

Yellow discoloration of the feces and urine were observed in both males and females at 1000 mg/kg/day from Day 7 of treatment onwards.  This finding was likely related to the yellow color of the test item and was considered non-adverse in absence of correlating microscopic findings.  Salivation seen after dosing among animals treated with 100, 300 or 1000 mg/kg/day of the test item was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.  

At 1000 mg/kg/day, a test item-related decrease in body weights and body weight gain was noted in males. This may have been partly related to the reduced food consumption observed during week 1 of treatment. As at the end of the treatment period the difference in body weight was relatively small (0.94x of control) and the decrease in body weight did not affect normal function of the animals, this finding was considered non adverse.  

In the kidneys, hyaline droplet accumulation was observed in males at 300 and 1000 mg/kg/day and was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in higher mammals, including man. The increased hyaline droplet accumulation was not accompanied by indicators of tubular damage and was therefore considered to be non-adverse.

The hepatocellular hypertrophy observed in females at 1000 mg/kg/day and granular cytoplasmic alterations in males (300 and 1000 mg/kg/day) and in females (1000 mg/kg/day) in the liver were correlated to the observed increases in alkaline phosphatase and increases in liver weights. In absence of any degenerative findings, the changes observed in the liver were considered non adverse.

The hematology changes observed in reticulocytes, white blood cells, lymphocytes and mean corpuscular hemoglobin remained within normal range and red blood cell distribution width, red blood cells, and mean corpuscular volume slightly exceeded the normal range. The changes observed in females (increased reticulocytes and low blood cell counts) may have been correlated with the observed extramedullary hematopoiesis observed in the spleen of females at 1000 mg/kg/day was considered. However, changes were small and for males, no extramedullary hematopoiesis in the spleen was observed. At the severities noted, in the absence of degenerative changes and without any organ weight changes, the observed changes in  hematology parameters and the spleen were considered non adverse.

The increases in clinical biochemistry parameters creatinine (males and females, 1000 mg/kg/day) and cholesterol (males, 300 and 1000 mg/kg/day) were considered not adverse as values remained within normal range and in absence of correlating microscopic findings.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for CMTX 2-carboxymethyloxy-thioxanthone was established:

Parental: 300 mg/kg/day.

Note: In this study, an approximately 3-fold increase in TSH was observed in the high dose groups (in both males and females) which was considered to be test item-related. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.