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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Study performed under GLP.

OECD 422

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 were established:

Reproduction: 1000 mg/kg/day

Developmental: 300 mg/kg/day (based on the decreased viability index and body weights of pups)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

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

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

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

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.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on 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.

Details on mating procedure:

Frequency:
Daily, after a minimum of 14 days of treatment. The mating period will consist of a maximum of 14 consecutive days.

Procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. In any case less than 9 females per group have shown evidence of mating, each non-mated female was re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating was used for re-mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Collection and Analysis:
Dose formulation samples were collected for analysis as indicated below.

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).

Accuracy
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
No test item was detected in the Group 1 formulations.

Homogeneity
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

See details on mating procedure in section above.

At initiation of dosing, males were 10-11 weeks old and weighed between 261 and 287 g and females were 13-14 weeks old and weighed between 205 and 244 g.

TERMINAL PROCEDURES – F1-GENERATION
Method of Euthanasia – F1-Generation
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 7-16), except the pups selected for blood collection, were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 14-16 will be anesthetized using isoflurane followed by exsanguination.

Unscheduled Deaths – F1-Generation
Recognizable fetuses of females that die spontaneously or are euthanized in extremis were examined externally and sexed (both externally and internally, if possible). Live fetuses were euthanized by decapitation.
Pups that die or are euthanized before scheduled termination were examined externally and sexed (both externally and internally, if possible). Pups found dead during the weekend were fixed in identified containers containing 70% ethanol if not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death will be evaluated.

Scheduled Euthanasia – F1-Generation
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. Sex was determined both externally and internally. From two surplus pups per litter, blood will be collected, if possible.
All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded.
Particular attention was paid to the external reproductive genitals to examine signs of altered development. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. In addition, the thyroid was collected from two pups per litter (if possible, from one male and one female pup and preferably from the same pups as selected for (complete) blood collection, and was preserved in 10% buffered formalin.

Dose / conc.:

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

Dose / conc.:

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

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.

Parental animals: Observations and examinations:

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.


Estrous Cycle Evaluations – F0-Generation:
Frequency: Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period (for exception, see Deviations in Appendix 8).
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous (for exception, see Deviations in Appendix 8). This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
Procedure: Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.


General Reproduction Data – F0-Generation
Frequency: Daily from the mating period onwards.
Procedure: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used when necessary to aid in confirmation of pregnancy. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Oestrous cyclicity (parental animals):

Frequency: Daily vaginal lavage were performed beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation is observed. Vaginal lavage would continue for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or had died spontaneously.
Procedure: Estrous cycles will be evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.

Litter observations:

IN-LIFE PROCEDURES, OBSERVATIONS, AND MEASUREMENTS – F1-GENERATION:
The in-life procedures, observations, and measurements listed below were performed for the pups.

Mortality/Moribundity Checks – F1-Generation
Frequency: The number of live and dead pups were determined on PND 1 and daily thereafter.
Procedure: Pups were observed for general health/mortality and moribundity. If possible, defects or cause of death were evaluated. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations – F1-Generation
Frequency: At least once daily.
Procedure: Detailed clinical observations were made for all pups. Only days on which clinical signs are present between the first and last litter check were given in the respective report tables.

Body Weights – F1-Generation
Frequency: On PND 1, 4, 7, and 13.
Procedure: Live pups were individually weighed.

Sex – F1-Generation
Frequency: On PND 1 and 4.
Procedure: Sex was externally determined for all pups.

Anogenital Distance – F1-Generation
Frequency: On PND 1.
Procedure: Anogenital distance (AGD) was measured for all live pups. The AGD was normalized to the cube root of body weight.

Areola/Nipple Retention – F1-Generation
Frequency: On PND 13.
Procedure: All males in each litter was examined for the number of areola/nipples.

Culling – F1-Generation
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

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

Statistics:

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

Reproductive indices:

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

Offspring viability indices:

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

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.

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, treatment-related

Description (incidence):

(Appendix 2)

Test item-related mortality was observed at 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 up to 0.94x compared to controls at 1000 mg/kg/day throughout the treatment period. These decreased body weights were accompanied by a decreased body weight gains noted from Day 8 of treatment onwards, reaching statistical significance at Day 15 and 29 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. As the body weight loss was slight and in absence of a dose-related trend, the body weight loss was considered not test-item related.

Food efficiency:

no effects observed

Description (incidence and severity):

Lactation Index:
(Appendix 1 and Appendix 2)
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups.

Haematological 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 (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 (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)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Parturition/Maternal Care

For female No. 76 (1000 mg/kg/day, blood was found in the cage on the expected day of delivery (see section 9.2.1 for more details).
No deficiencies in maternal care were observed.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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, although not statistically significant. For females, values were below the lower limit of the historical control range(1).

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 range3, 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).

(13) Historical control data of male Wistar Han rats (period 2017-2019):
Prostate gland (G): mean = 0.804, P5 - P95 = 0.576 – 1.058 (n=538).

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

(Appendix 2)
Length and regularity of the estrous cycle were considered not to have been affected by treatment.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for female No. 58 at 100 mg/kg/day and an acyclic cycle for female No. 72 at 1000 mg/kg/day (with normal litter). Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating Index:
(Appendix 1 and Appendix 2)
Mating index was considered not to be affected by treatment.

The mating indices were 100% for the control, 100, 300 and 1000 mg/kg groups, respectively.

Precoital Time:
(Appendix 1 and Appendix 2)
Precoital time was considered not to be affected by treatment. All females showed evidence of mating within 4 days.

Number of Implantation Sites:
(Appendix 1 and Appendix 2)
Number of implantation sites was considered not to be affected by treatment.

Noteworthy, individual data show that there was one female (no. 76) with only two implantation sites. This female was euthanized on lactation Day 1, as she had a total litter loss (see section 2.2.1 for more details). Since 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.

Fertility Index:
(Appendix 1 and Appendix 2)
Fertility index was considered not to be affected by treatment. The fertility indices were 89% for the control group, and 100% for 100, 300 and 1000 mg/kg/day.
One female in the control group was not pregnant, but as this female received vehicle only, a relationship with the test item could be excluded.

Reproductive perfromance
There was one control treated couple (male No. 7 and female No. 47) not pregnant. There was one 1000 mg/kg/day treated female with total litter loss (No. 76). There were no abnormalities recorded in the reproductive organs of these animals that could explain this.

There were no morphological findings in the reproductive organs of either sex which could be attributed tothe test item. Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Gestation Index and Duration
(Appendix 1 and Appendix 2)
Gestation index and duration of gestation were considered not to be affected by treatment.
The gestation indices were 100% for the control, 100 and 300 mg/kg/day groups, and 90% for 1000 mg/kg/day group.
Female No. 76 at 1000 mg/kg/day had a total litter loss

PARENTAL RESULTS
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 (0.85x of control within 3 days), 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 observations 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 (1000 mg/kg/day) and in females (300 and 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. 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, 300 and 1000 mg/kg/day) and cholesterol (males, 300 and 1000 mg/kg/day) and decrease in total T4 (males, 1000 mg/kg/day) were considered not adverse as values remained within normal range and in absence of correlating microscopic findings.

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
No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day).
No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Key result

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

1 000 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

gross pathology

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects at highest dose

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

(Appendix 2)
No treatment-related clinical signs were observed among pups in any dose group. Absence of milk in the stomach was noted in some of the pups that were found dead, which was probably related to their early death.

The nature and incidence of the other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not related to treatment and not to be toxicologically relevant.

Note: Only days on which clinical signs were present between first and last litter check are presented in the table.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Post-Implantation Survival Index
(Appendix 1 and Appendix 2)
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 96% for the control, and 91%, 94% and 90% for the 100, 300 and 1000 mg/kg/day groups, respectively.

Litter Size
(Appendix 1 and Appendix 2)
Litter size was considered not affected by treatment.
Mean live litter sizes were 12.4 for the control, 12.1, 12.1 and 11.0 for 100, 300 and 1000 mg/kg/day groups, respectively.

Live Birth Index
(Appendix 1 and Appendix 2)
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 99% for the control and 100%, 100% and 98% for 100, 300 and 1000 mg/kg groups, respectively.
Female no. 76 (Group 4) had a total litter loss. One pup of the control group, and two pups at 1000 mg/kg/day were found dead at first litter check. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability Index
(Appendix 1 and Appendix 2)
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 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.
Two pups of the control group were found missing on PND 2 or 4, four pups at 100 mg/kg/day were found missing on PND 2, 3 or 4 and eight pups at 1000 mg/kg/day were found missing on PND 2, 3 or 4. Pups missing were most likely cannibalised. 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 ten remaining pups (see section 2.2.1 for more details). However, upon exclusion of these ten pups 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(14).

14 Historical control data of female Wister Han rats (period 2015-2019):
Viability index (%): Mean = 98; P5 – P95 = 93 – 100 (n = 120).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Body weights of pups were considered affected by treatment with the test item at 1000 mg/kg/day.
At 1000 mg/kg/day, treatment-related lower body weights of pups were noted from PND 1 onwards (not always statistically significant). Mean pup weights at 1000 mg/kg/day ranged between 0.84 and 0.91 of concurrent controls.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Analysis of serum T4 levels in male and female PND 14-16 pups are ongoing.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Anogenital distance (absolute) in male and female pups was considered not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

(Appendix 1 and Appendix 2)
Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopy
(Appendix 2)
No macroscopic findings were noted among pups that were considered to be related to treatment.
No milk in the stomach was noted for one litter from Group 1 (litter no. 43) and two litters from Group 4 (1 pup/litter, litter no. 79 and 80). The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Sex Ratio
(Appendix 1 and Appendix 2)
Sex ratio was considered not to be affected by treatment.

DEVELOPMENTAL RESULTS
Developmental toxicity was observed at the highest dose level tested (1000 mg/kg/day).

At 1000 mg/kg/day, the viability index was decreased which was considered adverse.

At 1000 mg/kg/day, body weights of pups were decreased (up to 0.84x of controls) from PND 1 onwards. 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).

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Reproductive effects observed:

not specified

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.

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 were established:

Parental: At least 1000 mg/kg/day
Reproduction: 1000 mg/kg/day
Developmental: 300 mg/kg/day (based on the decreased viability index and body weights of pups)

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 potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

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).  

Group No.	Test Item	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Animals
					Males	Females
1	-	0 (vehicle)	5	0	10	10
2	CMTX 2-carboxymethyloxy-thioxanthone	100	5	20	10	10
3		300	5	60	10	10
4		1000	5	200	10	10

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.  

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 4 and PND 14-16 pups)).  

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.

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

Developmental toxicity was observed at the highest dose level tested (1000 mg/kg/day).

At 1000 mg/kg/day, the viability index was decreased and was considered adverse.

At 1000 mg/kg/day, body weights of pups were decreased (up to 0.84x of controls) from PND 1 onwards. Considering the magnitude of the effect, this finding was regarded as treatment-related and adverse.

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 were established:

Parental: At least 1000 mg/kg/day

Reproduction: 1000 mg/kg/day

Developmental: 300 mg/kg/day (based on the decreased viability index and body weights of pups)

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Substance does not meet the criteria for classification specified by the classification, labelling, and packaging (CLP) regulation (1272 /2008)