1,4-Dioxaspiro[4.5]decane, 8-[4-(4-methylphenyl)-3-cyclohexen-1-yl]-

Endpoint summary

Description of key information

In a repeated dose toxicity study combined with the reproduction/developmental toxicity screening test according to OECD guideline 422, the LOAEL (systemic) was found to be 100 mg/kg bw/day. Thus, the test item ist considered to be classified for special target organ toxicity, repeated dose, category 2 (reference 7.5.1-1).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Feb 2018 - 06 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

At current, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: See under "Principles of method if other than guideline"

Principles of method if other than guideline:

In addition, the procedures described in this study plan essentially conform to the following guidelines:
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016;
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000;
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008;
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008;
- EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: WI(Han)

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. The testing facility 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. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 weeks (males); 13 weeks (females)
- Weight at study initiation: 267 - 300 g (males); 209 - 248 g (females)
- Fasting period before study: No, except during motor activity measurements (maximum of 2 hours). F0-males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before blood sampling.
- Housing: Pretest (females only) and pre-mating period: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages; During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages; Post-mating phase: males were housed in their home cage with a maximum of 5 males/cage, females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. 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 without cage enrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. No known contaminants were present in the feed or in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 33-56
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 29-33 (males) or to day 52-70 (females)

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is the intended route of human exposure this is a possible route of human exposure during manufacture, handling or use of the test item.The test item and vehicle were administered to the appropriate animals once daily by oral gavage using a plastic feeding tube.

Vehicle:

other: Aqueous hydroxypropyl methylcellulose; 0.25% (w/v)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized using an ultra-turrax to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were formulated at least weekly as a suspension, prepared in daily portions which were stored in the refrigerator until use. On the day of dosing, the required formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before use. The formulations were used for dosing within 6 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on information of the sponsor and vehicle testing performed at the test facility.
- Preparation of the vehicle: For preparation of 1000 mL of 0.25% (w/v) aqueous hydroxypropyl methylcellulose, approximately 1/3 of the required volume of Elix water was heated to at least 90°C. 2.5 gram Methocel® K4M powder was added to the heated water with agitation. The mixture was agitated until the particles were thoroughly wetted and evenly dispersed. For complete solubilization, the remainder of the Elix water as cold water to lower the temperature of the dispersion was added with agitation. Once the dispersion reached the temperature at which the Methocel® K4M powder became soluble, the powder began to hydrate and viscosity increased. Agitation was continued for at least 30 minutes. Described volumes could be adjusted proportionally when larger quantities of vehicle had to be prepared. The vehicle is stable for 14 days in a refrigerator set to maintain 4°C. The prepared vehicle was removed from the refrigerator and stirred for at least 30 minutes before dosing. The vehicle was also stirred continuously during dosing.
- Concentration in vehicle: 20 mg/mL, 60 mg/mL and 200 mg/mL
- Dose volume: 5 mL/kg bw
- Batch No: 3B12012N02 (Methocel K4M Premium)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure. Dose formulation analysis were performed during week 1 of treatment, concentration was determined for all dose groups (including control group), homogeneity was determined for low and high dose groups. Duplicate sets of samples (approximately 500 mg) were analysed. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation of concentrations was ± 10%.
Stability analyses were performed in conjunction with the method development and validation study. The results demonstrated that the test item is stable
in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

Males were treated for 29-33 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 52-70 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 39 days.

Frequency of treatment:

Once daily

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

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A dose range finding study was conducted to select dose levels for the main study, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. Three females per dose group were dosed at 500 or 1000 mg/kg bw/day once daily oral gavage for 10 consecutive days. The dose levels were selected based on the information provided by the Sponsor (an acute oral toxicity study with the test item, S1 in rats; LD50 > 2000 mg/kg). No signs of toxicity were noted at any dose level.
- Fasting period before blood sampling: 24 hours (females)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily. During the dosing period, these observations were performed directly after dosing, the time of onset, grade and duration of any observed sign was recorded. In addition, clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of treatment (prior to dosing), and weekly thereafter. Terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION
Food consumption was quantitatively measured 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.

FOOD EFFICIENCY
Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group
- Parameters examined: as included in the guidance, including coagulation parameters (Prothrombin Time and Activated Partial Thromboplastin Time).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group
- Parameters as included in the guidance were examined. Serum of all F0 males and PND 14-16 pups was analyzed for total Thyroxine (T4). Assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, both sexes showed a similar degree of thyroid follicular hypertrophy and no treatment related changes in thyroid weight were recorded. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment (males), during the last week of lactation (i.e. PND 6-13, females).
- Dose groups that were examined: All groups
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring 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.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition. Organ weights were determined.

HISTOPATHOLOGY: Yes
All tissues as defined in the guidance were examined histopathologically. For the testes of all selected males of the control and the high dose group, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Other examinations:

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but
excluded semi-quantitative data, and any group with less than 2 observations. The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1, Group 4 vs. Group 1 (where group 1 = controls and groups 2, 3 and 4 = low, mid and high dose groups, respectively). Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. The clinical signs noted during the treatment period, i.e. alopecia, scabs and scales and chromodacryorrhoea, 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. Swelling in the throat region was observed in one males, which was prematurely sacrificed.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item. One male of the 300 mg/kg bw group was euthanized in extremis on Day 17. This male showed a swelling in the throat region from Day 10 onwards. Since the animal showed no evidence of pain or other discomfort related to this finding, the male was allowed to mate (starting on Day 15) before considering sacrifice. After confirmation of mating, the male was sacrificed. Macroscopic examination showed nodules in the salivary glands (grown together with the mandibular lymph nodes) and an enlarged spleen. Histopathology revealed an adenocarcinoma of the salivary gland, which was considered a spontaneous finding to be unrelated to treatment. One female of the control group was euthanized on Lactation Day 2, because of a total litter loss.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of male and female rats remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight were considered to be similar to the control level over the treatment period.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related changes in total and differential white blood cell counts were observed in male and female rats. At the lowest dose level of 100 mg/kg bw/day, the total white blood cells (WBC) counts were decreased approximately 25-30%. In males, the WBC showed a dose related decrease and the WBC value had halved at 1000 mg/kg bw/day when compared to controls. In females the WBC value had halved at 300 mg/kg bw/day and showed no further decrease at 1000 mg/kg bw/day. Statistical significance for WBC when compared to controls was achieved in males at 1000 mg/kg bw/day and in females in all treatment groups. From the results of the differential white blood cell counts it was clear that the decreases in the absolute number of lymphocytes accounted for the decreases in the total WBC in both sexes, whereas the absolute number of neutrophils, monocytes, eosinophils and basophils remained within the same range as controls at all dose levels. In females at 100 mg/kg bw/day, the absolute number of lymphocytes had halved to that in controls and had further decreased to approximately one third of the control value in females at 300 and 1000 mg/kg bw/day, and had achieved levels of statistical significance at all dose levels in comparison with controls. In males, the changes in absolute number of lymphocytes were comparable to that in the WBC, a dose related decrease from approximately 30% at 100 mg/kg bw/day to 50% at 1000 mg/kg bw/day. The depletion of lymphocytes in the peripheral blood resulted in decreases of the percentage of lymphocytes of the WBC and consequently in increases of the percentage of neutrophils in both sexes. These changes in relative numbers of lymphocytes and neutrophils reached levels of statistical significance in females for lymphocytes at all dose levels and neutrophils at 300 and 1000 mg/kg bw/day. The relative contribution of the monocytes, eosinophils and basophils remained low in all dose groups and since their absolute numbers were unaffected in peripheral blood, the statistically significant differences for the increased percentages of monocytes at 100 and 1000 mg/kg bw/day in both sexes were of no toxicological relevance. No treatment related changes were noted in red blood cell parameters in both sexes. Relatively high values for red blood cells, haemoglobin and haematocrit were observed in the males at 300 mg/kg bw/day. Since no changes were observed in the values of the red blood cell derived parameters, MCV, MCH and MCHC in these males, the high values were considered an incidental finding and of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical biochemistry parameters of treated male and female rats were considered not to have been affected by treatment. The changes in the clinical biochemistry parameters albumin and sodium in males at 300 mg/kg bw/day, that achieved statistical significance when compared to controls, were considered fortuitous findings and to be unrelated to treatment as these occurred in the absence of a dose-related trend.

The mean serum levels of T4 in treated F0 males were approximately 20-25% lower at all dose levels when compared to that in controls. No clear dose response relationship was observed and a statistically significant difference for the T4 level in males at 300 mg/kg bw/day was apparent. Moreover, all T4 values were within the historical control data range for rats of this strain an age and used in this type of studies. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, both sexes showed a similar degree of thyroid follicular hypertrophy and no treatment related changes in thyroid weight were recorded.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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. Grip strength and motor activity was similar between control and high dose animals. The animals within all groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Possible test substance-related higher relative liver weight (statistcally significant) were noted in the 1000 mg/kg bw/day males. Mean percent liver weight differences for exposed males compared to control males were -2%, -3% and 8% (absolute) and 0%, 4% and 13% (relative). For exposed females compared to control females this was -4%, -2% and 3% (absolute) and -5%, -3% and 5% (relative). In the absence of any histopathological correlates, this finding was considered to be non-adverse. There were no other test item-related alterations in organ weights. The statistically significant differences for the relative brain weight in males at 300 mg/kg bw/day and the absolute and relative spleen weight and relative pelvic weight in females at 300 mg/kg bw/day was considered incidental findings, based on the absence of a clear dose response relationship.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross
observations encountered in rats of this age and strain. Watery fluid in the uterus, found in two mid dose and one high dose female, was related to stage in the estrous cycle and is a normal finding.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dose-related microscopic findings after treatment the test item were noted in females. It is of note that 5 animals per dose were examined. Hypercellularity of medulla and loss of distinction cortex/medulla was observed in the thymus of all exposed females that were examined (not present in controls; 100 mg/kg bw/day: 1 slight, 4 moderate; 300 mg/kg bw/day: 5 moderate; 1000 mg/kg bw/day: 4 moderate, 1 marked). This increase of lymphoid was primarily in the medulla and consisted of an increase in amount of small lymphocytes (hypercellularity), which appeared to expand the medullary area and/or obscure the boundary with the cortex. This resulted in an (optic) smaller cortex filled with a normal (to minimal increased) density of lymphocytes (loss of junction cortex/medulla).
In the spleen, depletion of lymphoid tissue (PALS, T-cell area) was present in females from 100 mg/kg bw/day (not present in controls; 100 mg/kg bw/day: 4 slight; 300 mg/kg bw/day: 3 minimal, 1 slight; 1000 mg/kg bw/day: 4 minimal, 1 slight). This decrease in lymphocytes was located in the periarteriolar lymphoid sheaths of the white pulp, known to be populated largely by T cells (PALS, T-cell area). Vacuolation of the zona fasciculata of the adrenal gland was present in females from 300 mg/kg bw/day (not present in controls and 100 mg/kg bw/day; 300 mg/kg bw/day: 3 minimal; 1000 mg/kg bw/day: 2 minimal, 3 slight). The vacuolation was in the outer layer of the zona fasciculata of the cortex. There were no test item-related microscopic observations in males. The remainder of the recorde
d 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:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

spleen

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results dose range finder study:

No mortality occurred. At 1000 mg/kg bw/day, 1/3 female showed scabs on the upper lips on days 1 and 2 of treatment. No further clinical signs were observed. Body weights and food consumption were considered normal. No abnormalities were noted at macroscopic examination. Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/day. Since no treatment-related clinical signs were observed in the dose range finder, the observation of clinical signs at no specific time point after dosing was selected in the main study.

 

Results formulation analysis:

The concentrations analyzed in the test item formulations were in agreement with target concentrations (i.e. mean accuracies between 98% and 100%). No test item was detected in the control group formulation. The formulations of the low dose and the high dose groups were homogeneous (i.e. coefficient of variation ≤ 3.3%).

 

Conclusions:

In a 28-Day Repeated Dose Toxicity Study combined with a Reproduction/Developmental Toxicity Screening, performed according to OECD/EC guidelines and GLP principles, the LOAEL of the test material was found to be 100 mg/kg bw/day, based on effects adverse effects on the lymphocyte population in peripheral blood (in males and females) and histopathological alterations in the thymus and spleen at all dose levels (females only).

Executive summary:

A combined 28 -day repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. The test material was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were treated for 29-33 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 52-70 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 39 days. Formulation analyses confirmed that formulations of test item in aqueous hydroxypropyl methylcellulose were prepared accurately and homogenously. No test item-related mortality occurred, no clinical signs were observed related to test item exposure. In peripheral blood, marked decreases in lymphocytes were noted already at the lowest dose level of 100 mg/kg bw/day in both sexes, i.e. decreases of approximately 30% in males and 45% in females compared to control values. The total number of lymphocytes had further decreased at higher dose levels, reaching approximately 50% of the control values in males at 1000 mg/kg bw/day and 30% in females at 300 and 1000 mg/kg bw/day. The absolute values of the other differential white blood cells, neutrophils, monocytes, eosinophils and basophils remained unchanged at all dose levels. The decreases in white blood cell counts seen at each dose level in both sexes could be fully attributed to decreases in the number of lymphocytes. In the females, histopathology of the lymphoid organs thymus and spleen revealed hypercellularity of the thymic medulla, consisting of small sized lymphocytes, and a decrease in T-cells in the spleen. These findings are likely to be related to the lower lymphocyte count in the peripheral blood. Therefore, the combination of changes in thymus and spleen resulting in decreased circulating white blood cells and lymphocytes was considered to be adverse. Histopathological alterations in the thymus and spleen were not observed in males at all dose levels. Further histopathological changes observed as vacuolation of the zona fasciculata of the cortex of the adrenal gland in females treated at 300 and 1000 mg/kg bw/day were considered as non adverse, based on the minor severity, the fact that it can be seen as a spontaneous finding in untreated rats and the absence of any associated degenerative changes. Slightly higher liver weights (13% higher than in concurrent controls) were observed in males at 1000 mg/kg bw/day. In the absence of any histopathological correlates, this finding was considered to be non-adverse. The serum levels of T4 in treated F0 males were lower at all dose levels when compared to that in controls, but showed no dose response relationship. No corroborating morphological changes were observed in the thyroid gland in males. Based on the magnitude of the changes and the fact that all T4 values were within the historical control range, the changes in T4 were considered to be non-adverse. No treatment-related or toxicologically significant changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical biochemistry investigations (including male T4 thyroid hormone levels) and macroscopic examination). Based on the adverse effects on the lymphocyte population in peripheral blood (males and females) and histopathological alterations in the thymus and spleen (females only) at 100 mg/ kg bw/day, with a dose-related increase in severity, the Lowest Observed Adverse Effect Level (LOAEL) for the test material was established to be 100 mg/kg bw/day. The outcome of this study did not allow conclusion on the No Observed Adverse Effect Level (NOAEL).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study under GLP conditions

System:

hepatobiliary

Organ:

spleen

thymus

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral repeated-dose toxicity, key study

A combined 28 -day repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. The test material was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were treated for 29-33 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 52-70 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 39 days. Formulation analyses confirmed that formulations of test item in aqueous hydroxypropyl methylcellulose were prepared accurately and homogenously. No test item-related mortality occurred, no clinical signs were observed related to test item exposure. In peripheral blood, marked decreases in lymphocytes were noted already at the lowest dose level of 100 mg/kg bw/day in both sexes, i.e. decreases of approximately 30% in males and 45% in females compared to control values. The total number of lymphocytes had further decreased at higher dose levels, reaching approximately 50% of the control values in males at 1000 mg/kg bw/day and 30% in females at 300 and 1000 mg/kg bw/day. The absolute values of the other differential white blood cells, neutrophils, monocytes, eosinophils and basophils remained unchanged at all dose levels. The decreases in white blood cell counts seen at each dose level in both sexes could be fully attributed to decreases in the number of lymphocytes. In the females, histopathology of the lymphoid organs thymus and spleen revealed hypercellularity of the thymic medulla, consisting of small sized lymphocytes, and a decrease in T-cells in the spleen. These findings are likely to be related to the lower lymphocyte count in the peripheral blood. Therefore, the combination of changes in thymus and spleen resulting in decreased circulating white blood cells and lymphocytes was considered to be adverse. Histopathological alterations in the thymus and spleen were not observed in males at all dose levels. Further histopathological changes observed as vacuolation of the zona fasciculata of the cortex of the adrenal gland in females treated at 300 and 1000 mg/kg bw/day were considered as non adverse, based on the minor severity, the fact that it can be seen as a spontaneous finding in untreated rats and the absence of any associated degenerative changes. Slightly higher liver weights (13% higher than in concurrent controls) were observed in males at 1000 mg/kg bw/day. In the absence of any histopathological correlates, this finding was considered to be non-adverse. The serum levels of T4 in treated F0 males were lower at all dose levels when compared to that in controls, but showed no dose response relationship. No corroborating morphological changes were observed in the thyroid gland in males. Based on the magnitude of the changes and the fact that all T4 values were within the historical control range, the changes in T4 were considered to be non-adverse. No treatment-related or toxicologically significant changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical biochemistry investigations (including male T4 thyroid hormone levels) and macroscopic examination). Based on the adverse effects on the lymphocyte population in peripheral blood (males and females) and histopathological alterations in the thymus and spleen (females only) at 100 mg/ kg bw/day, with a dose-related increase in severity, the Lowest Observed Adverse Effect Level (LOAEL) for the test material was established to be 100 mg/kg bw/day. The outcome of this study did not allow conclusion on the No Observed Adverse Effect Level (NOAEL).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the test item is classified for repeated dose special organ toxicity, category 2, according to EU Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/217.