1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

Endpoint summary

Description of key information

Repeated dose toxicity - oral: In a combined 28-day repeated dose toxicity study with the reproduction/ developmental toxicity screening test, the test substance was administered daily to rats up to a dose level of 0, 11, 33 and 90 mg/kg body weight/day (OECD 422; Peter B., 2017). The NOAEL is established to be 33 mg/kg bodyweight.

Repeated dose toxicity - inhalation: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

Repeated dose toxicity - dermal: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-15 to 2019-11-04

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developm ental Toxicity Screening Test

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA Guideline OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)

Deviations:

no

Principles of method if other than guideline:

No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I18IB2522
- Expiration date of the lot/batch: 2020-03-17 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Solubility and stability of the test substance in the solvent/vehicle: in Propylene glycol, the stability is insured for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 1 to 200 mg/mL (Test Facility Study No. 20164353)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity/composition of the test item. A correction factor of 1.00 was used

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Suspension (Groups 2-4).

OTHER SPECIFICS: correction factor is 1

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 10-12 weeks (at start F0-treatment); females approx. 12-14 weeks
- Weight at study initiation: 268-295 g (males) and 204-235g (females)
- Fasting period before study: No
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to tap-water
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21°C
- Humidity (%): 46 to 62%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: 2018-10-15 to 2019-05-24

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage using a plastic feeding tube

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity/composition of the test item. A correction factor of 1.00 was used.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch
- Amount of vehicle (if gavage): 10 mL/kg, Actual dose volumes were calculated according to the latest body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (19 Mar 2019, Day 1 of treatment) according to a validated method (Test Facility Study No. 20164353). Sextuplicate samples (i.e. 3 sets of duplicate samples) Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature. In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of +/-10.00% compared to those obtained during the method validation.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was <=10.00%. Formulations were considered stable if the relative difference before and after storage was maximally 10.00%.
Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study (Test Facility Study No. 20164353).

Duration of treatment / exposure:

29 days (males); 50-64 days (females that delivered); 41-52 days (females with total litter loss). Female Nos. 61, 63 and 73 (group 3 and 4) were left out from treatment for one day and Female No. 50 (Group 1) for two days as they were littering at the moment of dosing. Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

11 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

33 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

90 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 animals/sex/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a dose range finding study (Test Facility Study No. 20164350) in which animals were dosed for 10 days at 40, 90, 100 and 500 mg/kg/day.
- Rationale for animal assignment (if not random): randomized

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (early in the morning and close to the end of the working day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals after treatment. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first 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.
Body weight gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.
Relative food consumption was calculated and reported.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group, between 7.00 and 10.30 am
- Anaesthetic used for blood collection: Yes, isoflurane (Abbott B.V., Hoofddorp, The Netherlands)
- Animals fasted: yes, the animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- How many animals: 5 animals/sex/group ; blood samples were drawn from the retro-orbital sinus ans collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL) and with citrate for clotting tests (0.45 mL)
- Parameters examined: white blood cells, differential white blood counts (neutrophils, lymphocytes, monocytes, eosinophiles, basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, coagulation parameters (prothrombin time, activated partial thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group
- Animals fasted: yes, the animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- Anaesthetic used for blood collection: Yes, isoflurane (Abbott B.V., Hoofddorp, The Netherlands)
- How many animals:
5 animals/sex/group ; blood samples were drawn from the retro-orbital sinus ans collected into tubes prepared with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.
- Thyroid hormone analysis:
T4: F0 males: after at least 28 days of treatment

FUNCTIONAL OBSERVATIONS:
The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
hearing ability, pupillary reflex, and static righting reflex,fore- and hind-limb grip strength, locomotor activity. Total movements and ambulations are reported. The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs.

Sacrifice and pathology:

SACRIFICE
All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetized using isoflurane and subsequently exsanguinated.
- Males: all surviving animals; Following completion of the mating period (a minimum of 28 days of dose administration).
- Females which delivered: PND 14-16.
- Females which failed to deliver with evidence of mating: Post-coitum Day 25 (Nos. 41, 76 and 80) or postcoitum Day26 (No. 51) and without evidence of mating: 24 days after the last day of the mating period (No. 78).
- Females with total litter loss (Nos. 68, 69) within 24 hours of litter loss.
- Spontaneous deaths (No. 42) as soon as possible after death and always within 24 hours
- Euthanized in extremis (Nos. 40 and 62): When pain, distress or discomfort was considered not transient in nature or was likely to become more severe

GROSS PATHOLOGY: Yes
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur with bone marrow including joint (M/F), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve ( M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin:
Cervix (F), Clitoral gland (F), Coagulation gland (M), Epididymides (M), Mammary gland area inguinal region with skin (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F).

HISTOTECHNOLOGY
All organ and tissue samples as defined under 'Histopathology' were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxyin.
In addition, the heads (without pituitary gland) of the selected males and females of all 4 groups were decalcified. From the decalcified heads of selected animals of group 4, the nasal cavity at different levels were processed to slides.

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
- From selected animals of Groups 2 and 3, stomach and brain (level 1, including olfactory bulbs, only) of males and female, cecum of females and bone marrow-femur and sternum of males.
- From selected animals of Group 4, nasal cavity/skull at levels 3, 4 and 8 to further investigate the microscopic findings in the brain/olfactory bulbs.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were euthanized in extremis.
- The mammary gland of all females with total litter loss.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.

Other examinations:

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported
- The following organ weights and terminal body weight were recorded from all animals on the scheduled day of necropsy:
- selected 5 animals/sex/group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, including parathyroid if detectable, Uterus, including cervix
- all remaining animals: Epididymides, Prostate, Seminal vesicles, including coagulation glands, Testes, Thyroid

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in orde to select the best fitting statistical model.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Piloerection was noted in 2 females at 33 mg/kg/day for 1 to 6 consecutive days, and up to 6 females at 90 mg/kg/day on most days from Day 22 of treatment onwards. Hunched posture was noted in 3 females at 90 mg/kg/day on several days. Salivation seen after dosing among animals of all dose groups during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). Incidental findings that were noted included rales, alopecia, uncoordinated movements and lean appearance. These findings 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. At the incidence observed, these were not considered to be signs of toxicological relevance. No findings were noted during the arena observations in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Male No. 40 (90 mg/kg/day) was euthanized for ethical reasons after 22 days of treatment. Slight rales were noted 2 days before its death, and slight body weight loss compared to Day 15 (0.94x) was noted. Diffuse granulocytic inflammation of the meninges of brain (moderate) and spinal cord (mild) was regarded the cause of moribundity for this animal. Additional findings were present in the thymus as moderate decreased lymphoid cellularity, most likely resulting from the poor health condition of this animal. No reflux-related inflammatory findings were recorded for the additionally prepared levels of the nasal cavity which could have explained the pathogenesis of the meningitis. It is however unlikely the granulocytic inflammation of the meninges is related to the test item, but this cannot be fully excluded.
Female No. 42 (control) was found dead after 47 days of treatment (Day 10 of lactation) without prior signs of bad condition. At necropsy, the lungs were not collapsed, indicative of an obstruction of the trachea and a dark red discoloration of the lungs was observed. Cause of death for this animal was the severe erosion/ulceration with inflammation of the trachea, which was most likely related to the gavage procedure.
Female No. 62 (33 mg/kg/day) was euthanized for ethical reasons on Day 5 post-coitum. Slight lethargy, flat posture, piloerection and ptosis were noted on the day of preterm sacrifice and prior to that slight body weight loss on Day 3 post-coitum compared to Day 0 post-coitum (0.95x) was noted. Cause of moribundity for this animal was a malignant lymphoma. Infiltration of the lymphoma was present in a variety of organs (including adrenal glands, lung, spleen, liver, kidneys and bone marrow) and was the microscopic correlate for the enlarged liver and spleen recorded at necropsy. Malignant lymphomas can be seen as a spontaneous neoplasia in young Wistar rats and this single incidence in the mid dose was regarded to be unrelated to the treatment with the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males up to 33 mg/kg/day and in females at 11 mg/kg/day, body weight (gain) was similar between treated and control animals. In females at 33 mg/kg/day, decreased body weight (0.94x control, not statistically significant) and body weight gain (0.74x control) were noted on Day 20 post-coitum. Although this could partly be contributed to individual females with smaller litters or pups with lower body weight at birth, body weight gain for all females in this group was below the average body weight gain in concurrent controls. Subsequent body weight on Day 1 of lactation was within the same range as concurrent controls, whereas body weight (up to 0.93x control) and body weight gain (between 0.2 to 0.53x control) was again reduced throughout the lactation period. This could be partly contributed to Female No. 67, which showed severe body weight loss on Day 4 of lactation and subsequent limited body weight gain. However, overall body weight gain in 33 mg/kg/day treated females remained low compared to concurrent controls during lactation. In males at 90 mg/kg/day, decreased body weight gain was observed (from Day 8 onwards, ranging from 0.56x to 0.73x control), resulting in statistically significantly decreased body weights on Day 22 (0.95x control) and at termination (0.94x control). In females at 90 mg/kg/day, overall slight body weight loss was observed after one week of treatment and absent body weight gain in the subsequent week, resulting in decreased body weight on Day 8 (0.96x control, not statistically significant), on Day 1 of the mating period (0.91x control), during the post coitum period (up to 0.88x control), and during lactation (up to 0.87x control) and at termination (0.84x control).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males at all dose levels and in females at 11 mg/kg/day, absolute and relative food consumption was similar between treated and control animals. In females at 33 mg/kg/day, decreased absolute food consumption was noted during the lactation period (up to 0.83x control, not always statistically significant). For relative food consumption a similar trend (up to 0.88x control, not statistically significant) was noted. In females at 90 mg/kg/day, decreased absolute food consumption was noted during the premating period (up to 0.73x control, not statistically significant), and during the postcoitum and lactation periods (up to 0.74x control, not always statistically significant). A similar trend was noted ror relative food consumption (up to 0.84x control, not always statistically significant).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes were noted in treated males at 90 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased absolute monocyte levels (3.0x; exceeding historical control range).
- Increased haemoglobin levels (1.06x; within historical control range).
- Increased platelet levels (1.25x; within historical control range).
See section 'Any other information on results' for historical control data

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes were noted in treated males at 33 and 90 mg/kg/day and females at 90 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased albumin levels in males (1.05x and 1.08x at 33 and 90 mg/kg/day, respectively; exceeding historical control range).
- Increased bilirubin levels in males at 90 mg/kg/day (1.62x; exceeding historical control range).
- Increased urea levels in males at 90 mg/kg/day (1.44x; exceeding historical control range).
- Increased glucose levels in males at 90 mg/kg/day (1.32; within historical control range).
- Increased cholesterol levels in males at 90 mg/kg/day (1.41x; within historical control range).
- Increased bile acid levels in males at 90 mg/kg/day (2.05x; exceeding historical control range).
- Increased calcium levels in males at 90 mg/kg/day (1.05x; within historical control range).
- Increased inorganic phosphate levels in males at 90 mg/kg/day (1.15x; exceeding historical control range).
- Increased sodium levels in females at 90 mg/kg/day (1.02x; within historical control range).
-Thyroid hormone analyses: Serum levels of T4 in F0 males were considered to be unaffected by treatment.
See section 'Any other information on results' for historical control data

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals. The motor activity in treated animals was comparable to controls. All groups showed a similar habituation profile with a decreasing trend of activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights in females.
Test item-related lower prostate gland weights (absolute and relative to body weights) were noted in the 90 mg/kg/day treated males. The observed decrease in absolute and relative weight had no microscopic correlate and mean values remained within historical control range. There were no other test item-related organ weight changes. Some absolute organ weight differences were statistically significant when compared to the control group (epididymides in males; brain, heart spleen, adrenal glands, ovaries and uterus in females). These decreases in absolute organ weights were considered to be the result of a test item-related lower terminal body weight and not directly test item-related.
See section 'Any other information on results' for historical control data

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related macroscopic findings. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with the test substance were noted in the stomach of both sexes, olfactory bulbs (brain level 1) and bone marrow (sternum and femur) of males and cecum of females of the 90 mg/kg/day groups.
A combination of findings were recorded for the stomach (glandular mucosa) and were seen in males at 33 and 90 mg/kg/day and in females at 90 mg/kg/day. Test item-related alterations consisted of an increased incidence and severity (minimal-slight) of mixed inflammatory cell infiltrate in the glandular mucosa in males at 33 and 90 mg/kg/day and in females at 90 mg/kg/day, eosinophilic globules (minimal to moderate) in the glandular mucosa of a few males at 33 and 90 mg/kg/day and a single female at 90 mg/kg/day and increased mitosis in a few males (minimal) and a single female (slight).
Minimal inflammatory infiltrates of the glandular mucosa can be seen as a spontaneous finding and the minimal severity recorded in two males at 11 mg/kg/day was regarded within background pathology. Minimal or slight mucosal hypertrophy was recorded in the cecum of 3 females at 90 mg/kg/day.
Mononuclear inflammatory infiltrate surrounding the olfactory bulbs was recorded at a minimal degree in all males at 90 mg/kg/day. An increased number of adipocytes in the bone marrow was recorded in two surviving males at 90 mg/kg/day in femur and/or sternum at minimal degree.
Except for the unusual microscopic findings recorded in the three premature decedents described under mortality, the remainder of the recorded microscopic findings 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.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Analysis of Dose Preparations:
Accuracy of preparation:
The corrected mean accuracies analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 75.00% and 125.00%). The accuracies of the formulations were corrected with the mean accuracy of the QC samples to account for any bias introduced by inaccurate calibration solutions. No test item was detected in the Group 1 formulations.

Homogeneity:
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Parental results:
There were two unscheduled deaths (one control female and one female at 33 mg/kg/day) that were regarded to be unrelated to the treatment with the test item. For one high dose male that was euthanized in extremis, it is unlikely that the observed granulocytic inflammation of the meninges is related to the test item, but this could not be fully excluded.
General toxicity was observed parental females treated at 90 mg/kg/day. A significant decrease in body weight (gain) and food consumption were noted from start treatment onwards and piloerection observed in the majority of females from Day 22 onwards. A similar trend in body weight observed in males was considered non-adverse, as the effect was only slight and no effect on food consumption was observed. No parental toxicity was observed in rats treated at 11 or 33 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

33 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Critical effects observed:

no

Historical control data for Wistar Han rats (2017 -2019):

Monocytes (10E9/L) - males: mean = 0.1; P5 -P95 = 0.09-0.22 (n=259)

Haemoglobin (mmol/L) - males: mean = 10.1; P5 -P95 = 9.4 -10.75 (n=260)

Platelets (10E9/L) - males: mean = 735; P5 -P95 = 577 -890 (n=260)

Albumin (g/L) - males: mean = 32.2; P5 -P95 = 30.2 -34.0 (n=270)

Total bilirubin (umol/L) - males: mean = 2.3; P5 -P95 = 1.6 -3.0 (n=270)

Urea (mmol/L) - males: mean = 6.7; P5 -P95 = 4.8 -9.2 (n=270)

Glucose (mmol/L) - males = 9.05; P5 -P95 = 6.40 -12.22 (n=270)

Cholesterol (mmol/L) - males: mean = 1.95; P5 -P95 = 1.41 -2.59 (n=270)

Bile acids (umol/L) - males: mean = 25.6; P5 -P95 = 9.7 -53.7 (n=264)

Calcium (mmol/L) - males: mean = 2.62; P5 -P95 = 2.48 -2.75 (n=270)

Inorganic phosphate (mmol/L) - males: mean = 2.04; P5 -P95 = 1.70 -2.37 (n=270)

Sodium (mmol/L) - males: mean = 139.0; P5 -P95 = 135.6 -142.3 (n=210)

Historical control data for Wistar Han rats (2017 -2018):

prostate weight (gram): mean = 0.802; P5 -P95 = 0.5590 -1.0530 (n=318)

prostate weight (organ/bodyweight ratio): mean = 0.238; P5 -P95 = 0.1653 -0.3128 (n=318)

Conclusions:

In conclusion, treatment with the test substance by oral gavage in male and female Wistar rats at dose levels of 11, 33 and 90 mg/kg revealed parental toxicity at 90 mg/kg/day. The No Observed Adverse Effect Levels (NOAEL) of 33 mg/kg was derived based on reduced body weight (gain) and food consumption in 90 mg/kg/day treated females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

33 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated toxicity: oral

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats were exposed to 0 (vehicle), 11, 33, and 90 mg/kg bw/day via gavage (OECD 422; Hartman-Van Dycke K., 2019).

The vehicle used was propylene glycol and the test solutions were prepared daily within 6 hours prior to dosing.

Treatment related effects were observed on food consumption, body weight and weight change on females treated with 90 mg/kg/day dose level. No treatment related effects were observed in other parental parameters investigated in this study (clinical appearance, functional observations, clinical chemistry, haematology, macroscopic examination, organ weights and microscopic examinations).

Based on these results, the NOAEL was concluded to be 33 mg/kg/day.

Repeated toxicity: inhalation

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

Repeated toxicity: dermal

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure.

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP regulation (EC) No. 1272/2008, the test substance should not be classified as STOT RE.