[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2018 to May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

according to OECD TG 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2018 to May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 Health Effects Test Guideline OPPTS 870.3650

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

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. 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 nulliparous and non-pregnant: yes
- Age at study initiation: Males: 10-11 weeks; Females: 13-14 weeks
- Weight at study initiation: Males: 272-304 g; Females: 203-240 g
A health inspection was performed before the initiation of dosing.
- Housing: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 cageenrichment, bedding material, food and water.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

Application volume: 5 mL/kg

Vehicle:

propylene glycol

Details on oral exposure:

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 solution and dosed within 6 hours after adding the vehicle to the test item.

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.

Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

Rationale for vehicle:
Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment is complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.
Stability of the test item in polyethylene glycol was confirmed analytically following GLP for at least 24 hours at room temperature under normal laboratory light conditions and stability for at least 3 weeks in the freezer (≤ -15 °C) is confirmed over the concentration range 1 to 200 mg/mL (solutions), Project 20157759.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Trial formulations were prepared in propylene glycol at 1 mg/g (1 mg/mL) and 195 mg/g (200 mg/mL). Formulations were solutions.
The concentrations analyzed in the trial formulations were in agreement with target concentrations (i.e. mean accuracies between 90 % and 110 %). The formulations were homogeneous (i.e. coefficient of variation ≤ 10 %).
Formulations were stable when stored at room temperature under normal laboratory light conditions for at least 24 hours and in a freezer (≤ -15 °C) for at least 21 days (3 weeks).

Duration of treatment / exposure:

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 (most males) or 33 days (two males used for re-mating), 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 51-55 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 13 to15 days after delivery, up to and including the day before scheduled necropsy. One female at 500 mg/kg/daymg/kg bw/day (No. 80) which failed to deliver was treated for 48 days.
The first day of dosing was designated as Day 1.

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

500 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
The dose levels were selected to be 0, 50, 150, 500 mg/kg bw/day based on the results of a dose range finder with oral gavage administration of the test substance in rats (Test Facility Study No. 20157757, see Appendix 6), and in an attempt to produce graded responses to the test item.

The dose levels for the main study were selected based on the results of the dose range finder with doses of 1000 and 500 mg/kg bw/day for 23 days and 3 female rats per dose. In this dose range finder no mortality was observed. All animals showed hunched posture from day 12 onwards, uncoordinated movements (between days 1-14) and piloerection (starting days 12 in the 500 mg group and on day 14 at 1000 mg). In the first 3 days of treatment marginally reduced food consumption was observed. Weight loss was shown in one animal of both groups over days 1-3, followed by normal weight gain. Increased liver weights were recorded for all animals.
The peak effect of occurrence of clinical signs was considered to occur between 1 and 3 hours after dosing. This time point was taken into account to set a time range within which clinical observations were conducted after dosing in the Main Study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed 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 3 hours (±30 min) post-dose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually 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.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 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.

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

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: Functional tests (hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity) 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 11-13).

ESTROUS CYCLE DETERMINATION - F0 Generation
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
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.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that died spontaneously.

COHABITATION/MATING PROCEDURE - F0 Generation
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.
Detection of mating was not confirmed in first instance for Female No. 43. Evidence of mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of this animal animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

SPERM PARAMETERS - F0 Generation
For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

GENERATL REPRODUCTION DATA - F0 Generation
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
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.


CLINICAL PATHOLOGY: Yes
Blood of F0-animals (except for animals which were found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis.
F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

- Hematology parameters: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophil (absolute), Haemoglobin, Lymphocyte (absolute), Haematocrit, Monocyte (absolute), Mean corpuscular volume (MCV), Eosinophil (absolute), Mean corpuscular haemoglobin (MCH), Basophil (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelet, Reticulocyte (absolute).
- Coagulation parameters: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- Clinical chemistry parameters: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos).
- Thyroid hormone: Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4). Measurement of T4 was conducted for F0-males.
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 since thyroid weight was considered not affected by treatment.

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

Sacrifice and pathology:

SACRIFICE
- Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
- Scheduled necropsies were conducted on the following days: Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration);
Females which delivered: PND 14-16; Female No. 80 which failed to deliver (with evidence of mating): Post-coitum Day 30.
All males were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0- females were not fasted.

GROSS NECROPSY
- 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
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.
Organs Weighed at Necropsy for all selected animals: Brain, Cervixa, Epididymis, Gland adrenal, Gland coagulation, Gland parathyroid, Gland prostate, Gland seminal vesicle, Gland thyroid, Heart, Kidney, Liver, Ovaries, Spleen, Testes, Thymus, Uterus
Organs Weighed at Necropsy for all remaining animals (incl. Male No. 40 that failed to sire and female No. 80 that failed to deliver pups): Epididymis, Gland Coagulationa, Gland parathyroidc, Gland prostate, Gland seminal vesicle, Gland thyroid, Testes


HISTOPATHOLOGY / ORGAN WEIGHTS
- Representative samples of the tissues identified in the tables below were collected from all animals and preserved in 10 % neutral buffered formalin (neutral phosphate buffered 4 % formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

Tissue Collection and Preservation for all selected animals, and all animals that died spontaneously or were sacrificed in extremis: Animal identification, artery, aorta, body cavity, nasopharynx, bone marrow, bone (femur and sternum), brain (seven levels), cervix, epididymis, esophagus, eye, adrenal gland, coagulation gland, harderian gland, lacrimal gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (cecum, colon, rectum), larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, pancreas, skin, small intestine (duodenum, ileum, jejunum), spinal cord,spleen, stomach, testes, thymus, tongue, trachea, urinary bladder, uterus, vagina.

Tissue Collection and Preservation for all remaining animals (incl. males that failed to sire females that failed to deliver pups; non-pregnant, implantation sites only):
Animal identification, cervix, epididymis, coagulation gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, gross lesions/masses, ovaries, testes, uterus, vagina.

Other examinations:

For examinations on reproductive function/performance of parental animals as well as on developmental toxicity of pups see IUCLID section 7.8.1.

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 3 observations.

The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1 and Group 4 vs. Group 1.

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric: 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.

Incidence: 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 of toxicity were noted during daily clinical observations or during weekly arena observations.
Piloerection was recorded for one Female (No. 75) at 500 mg/kg/daymg/kg bw/day on several days of treatment, and for two surviving females at 150 mg/kg/daymg/kg bw/day (Nos. 65 and 67) on a single day only. Given that this finding occurred incidentally and without a dose-related trend, it was considered not to represent a treatment-related finding.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One Female (No. 61) at 150 mg/kg/daymg/kg bw/day was found dead after dosing on Day 5. On the day of death, this animal showed flat posture and gasping. Main microscopic findings consisted of the presence of amorphous material with inflammatory cells in the thoracic cavity and on the surface of the heart, correlating to watery-clear fluid in the thoracic cavity and friable thickened pleura recorded at necropsy. Overall, these findings indicated that this was gavage-related death. Other necropsy findings for this animal consisted of dark red discolouration of the trachea and red foci on the thymus..
One Control Female (No. 47) died at blood sampling; this death was unrelated to treatment with the test item, and considered to be related to the blood sampling procedure.
No further mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered not affected by treatment.
A statistically significant lower body weight and body weight gain was recorded for males at 150 mg/kg/daymg/kg bw/day over Weeks 3 and 4 of treatment. These concerned slight differences from the control means, and in absence of a dose-related trend these variations were considered not related to treatment.
Body weights and body weight gain of other groups remained in the same range as controls.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower mean reticulocyte counts were recorded for both males and females at 150 mg/kg bw/day (0.74x control mean for both sexes), and to a lesser extent for males at 500 mg/kg bw/day (0.85x control mean, not statistically significant). For males at 150 mg/kg bw/day, the mean reticulocyte count was outside the historical control range, while for females (and males at 500 mg/kg bw/day) the mean was just within this range. Although treatment-related changes would typically be expected to follow a dose-related trend, it could not be excluded that these changes represented an effect of the test item, given the pronounced degree and consistency of the change across both sexes. However, since a dose-related trend was absent, and there were no supportive changes in red blood cell parameters or morphological correlates, these lower reticulocyte counts were considered not to represent an adverse effect of treatment with the test item.
Other haematological parameters of treated rats were considered not affected by treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered not affected by treatment.

Thyroid hormone analyses: Serum levels of T4 in F0 males were considered not affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 50, 150 and 500 mg/kg, forelimb grip strength of males was statistically significantly lower than the control mean (0.55x, 0.59x and 0.70x control mean, respectively). Mean values did not show a dose-related trend, and remained within the historical control range (although bordering the lower margin of this range) . Values of 2/5 animals each at 50 and 500 mg/kg, and 1/5 animals at 150 mg/kg were outside this range. The control mean was similar to the mean expected based on this historical control range. These recordings were not supported by clinical observations or other functional observation tests (including hindlimb grip strength), and had no supportive morphological correlates in examined neuronal/muscle tissues. Therefore, this recorded lower forelimb grip strength of males was considered not to represent an adverse effect of treatment with the test item.
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with 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, non-treatment-related

Description (incidence and severity):

The following (statistically significant) test item-related organ weight changes, observed in males and at 500 mg/kg/kg/day only, were shown in Table 1 with the relevant historical control data of the lab.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic findings.
All observed necropsy findings among surviving animals remained within the range of background lesions to be expected for rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the thyroid glands of both sexes at 500 mg/kg bw/day, the kidneys of males starting at 50 mg/kg bw/day, adrenal glands of males at 500 mg/kg bw/day, see Tables 2 and 3.

Adverse parental findings in this study were present in the kidneys of male rats at 500 mg/kg bw/day and consisted of a combination of an increased incidence and severity in hyaline droplet accumulation, tubular basopbilia and granular casts with correlating increased kidney weights.
The hyaline droplet accumulation recorded in the male kidneys was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats nor in higher mammals, including man (Ref. 1). At 50 and 150 mg/kg bw/day the increased incidence and/or severity of hyaline droplet accumulation with low degrees of tubular basophilia occurred in the absence of indicators of renal tubular damage and the renal findings in these treatment groups were therefore considered to be non-adverse.
Other (non-adverse) morphologic alterations were recorded in the adrenal gland of males (slight increase in incidence and severity of vacuolation of the zona fasciculata, correlating to a minor increase in adrenal gland weight) and thyroid glands (slightly increased incidence/severity of hypertrophy of the follicular epithelium) of both sexes at 500 mg/kg bw/day. Compared to the incidences and severities recorded for the concurrent controls, these findings were regarded to be non-adverse.
Higher (relative) liver and (absolute and relative) prostate gland weights were recorded for males at 500 mg/kg bw/day, which occurred without microscopic correlates. As such, these changes were considered not adverse.
There were no adverse test item-related morphologic alterations in males at 50 and 150 mg/kg bw/day and females up to 500 mg/kg bw/day.

There were no other test item-related histologic changes. 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


Ref. 1: Sahota, P.S., Popp, J.A., Hardisty, J.F., Gopinath, C. (2013). Toxicologic Pathology - Nonclinical Safety Assessment. CRC Press, Tailor& Francis Group, Boca Raton, pp 450-451, 472.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

ESTROUS CYCLE:
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 one Control Female No. 46 (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.

SPERMATOGENESIS:
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: hyaline droplet accumulation in the kidney, considered to represent male rat specific alpha2µ nephropathy

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Since the renal findings in male rats are regarded as a male rat specific response, a NOAEL of 500 mg/kg/daymg/kg bw/day may be indicated for humane risk assessment purposes.

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 1: Organ weights F0 Generation: males (There were no other test item-related organ weight changes.)

dose level (mg/kg bw/day)	50	150	500
kidneys (absolute)	-3	-1	11
kidneys (relative to body weight)	0	5	14**
liver (absolute)	-5	-3	10
liver (relative to body weight)	-2	3	12*
prostate gland (absolute)	0	5	27*
prostate gland (relative to body weight)	4	12	30**
adrenal glands (absolute)	8	8	19
adrenal glands (relative to body weight)	6	12	18

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

Historical control data for Wistar Han rats; F0-animals (period 2017-2018):

Kidney (relative-%): mean = 0.66; P5 – P95 = 0.590 - 0.745 (n=255, males).

Liver (relative-%): mean = 2.51; P5 – P95 = 2.233 - 2.891 (n=255, males).

Prostate (absolute-gram): mean = 0.804; P5 – P95 = 0.5760 - 1.0580 (n=538, males).

Prostate (relative-%): mean = 0.237; P5 – P95 = 1.1689 - 0.3109 (n=538, males).

Adrenal gland (absolute-gram): mean = 0.060; P5 – P95 = 0.0470 - 0.0750 (n=245, males).

Table 2: Summary of test item related microscopic findings - thyroid gland

dose level (mg/kg bw/day	male 0	male 50	male 150	male 500	female 0	female 50	female 150	female 500
thyroid, number of tissues examined	5	4	5	5	5	5	6	5
hypertrophy follicular cell, minimal	2	2	3	3	1	-	1	2
hypertrophy follicular cell, slight	-	-	-	1	-	-	-	1

Thyroid glands: An increased incidence and severity of follicular cell hypertrophy was recorded at 500 mg/kg bw/day.  A background level of this finding was recorded for male and female rats of the remaining dose groups including controls.

Table 3: Summary of test item-related microscopic findings - kidney and adrenal gland males

dose level (mg/kg bw/day)	0	50	150	500
kidney (number of tissues examined from each group	5	5	5	5
kidney - hyaline droplet accumulation minimal	2	4	-	-
kidney - hyaline droplet accumulation slight	-	1	5	1
kidney - hyaline droplet accumulation moderate	-	-	-	4
kidney - basophilia, tubular minimal	1	3	5	3
kidney - basophilia, tubular slight	-	-	-	2
kidney - granular casts slight	-	-	-	2
adrenal glands (number of tissues examined from each group)	5	5	5	5
adrenal glands - vacuolation zona fasciculata minimal	1	1	-	2
adrenal glands - vacuolation zona fasciculata slight	-	-	-	1

Kidneys: An increased incidence and severity of hyaline droplet accumulation (minimal-slight) was recorded in males starting at 50 mg/kg bw/day.  This was accompanied by an increased incidence of minimal tubular basophilia at 50 and 150 mg/kg bw/day.  At 500 mg/kg bw/day hyaline droplet accumulation was seen up to moderate degrees and was accompanied by an increased incidence and severity (up to slight) of tubular basophilia and slight granular cast in two males.

Adrenals glands:  An increased incidence and severity (up to slight) of vacuolation of the zona fasciculata was recorded in males at 500 mg/kg bw/day.

Dose Formulation Analysis:

Accuracy:

For the formulation of Group 3 prepared for use in Week 4, the mean accuracy was below the target concentration (i.e. 84 % of target).  However, no (analytical) cause could be found for the lower accuracy.  The concentrations analyzed in the formulations of all other groups prepared for use in Week 4, and of all groups prepared for use in Week 1 were in agreement with target concentrations (i.e. mean accuracies between 90 % and 110 %). Therefore, it was considered that these results were acceptable for the purpose of this study.

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Week 1 and Week 4.  The maximum contribution to the Group 2 samples was 1.7 % for the Week 1 samples and 1.9 % for the Week 4 samples.  Since a response was detected in all the analytical blanks, it is conceivable that the actual maximum contribution from test item in Group 1 is lower than calculated. Therefore, these results were accepted.

Homogeneity:

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

Executive summary:

Ten male and ten female Wistar Han rats per group were treated with the test substance by daily oral gavage at dose levels of 50, 150 and 500 mg/kg according to OECD TG 422. The control group received the vehicle propylene glycol alone. Males were treated for > 28 days, including at least 2 weeks prior to mating, during mating, and up to termination (29 to 33 days). Females that delivered were treated for 51-55 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 13 to15 days after delivery, up to and including the day before scheduled necropsy.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels for the purpose of this study.

Adverse parental findings in this study were present in the kidneys of male rats at 500 mg/kg bw/day and consisted of a combination of an increased incidence and severity in hyaline droplet accumulation, tubular basopbilia and granular casts with correlating increased kidney weights.

The hyaline droplet accumulation recorded in the male kidneys was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia.  This male rat specific protein is not present in female rats nor in higher mammals, including man.  At 50 and 150 mg/kg bw/day the increased incidence and/or severity of hyaline droplet accumulation with low degrees of tubular basophilia occurred in the absence of indicators of renal tubular damage and the renal findings in these treatment groups were therefore considered to be non-adverse.

Other (non-adverse) morphologic alterations were recorded in the adrenal gland of males (slight increase in incidence and severity of vacuolation of the zona fasciculata, correlating to a minor increase in adrenal gland weight) and thyroid glands (slightly increased incidence/severity of hypertrophy of the follicular epithelium) of both sexes at 500 mg/kg bw/day. Compared to the incidences and severities recorded for the concurrent controls, these findings were regarded to be non-adverse (Ref. 2, Ref. 3).

Higher (relative) liver and (absolute and relative) prostate gland weights were recorded for males at 500 mg/kg bw/day, which occurred without microscopic correlates. As such, these changes were considered not adverse.

There were no adverse test item-related morphologic alterations in males at 50 and 150 mg/kg bw/day and females up to 500 mg/kg bw/day.

Potentially treatment-related (but non-adverse) in-life findings were confined to a reduced forelimb grip strength of males at 50, 150 and 500 mg/kg bw/day. Means did not show a dose-related trend, but were at the lower margin of the historical control range. These recordings were not supported by clinical observations or other functional observation tests (including hindlimb grip strength), and had no supportive morphological correlates in examined neuronal/muscle tissues. Therefore, this recorded lower forelimb grip strength of males was considered not to represent an adverse effect of treatment with the test item.

Haematological changes consisted of non-adverse lower mean reticulocyte counts for both sexes at 150 mg/kg bw/day, and to a lesser extent at 500 mg/kg bw/day in males. Mean values were outside or just within the historical control range. Although typically treatment-related changes would be expected to follow a dose-related trend, it could not be excluded that these changes represented an effect of the test item, given the pronounced degree and consistency of the change across both sexes. However, since a dose-related trend was absent, and there were no supportive changes in red blood cell parameters or morphological correlates, these lower reticulocyte counts were considered not to represent an adverse effect of treatment with the test item.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, clinical appearance, body weight, food consumption, functional observations (motor activity, hearing ability, pupillary reflex and static righting reflex), clotting parameters, clinical biochemistry (including male T4 thyroid hormone levels) and macroscopic examination).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental NOAEL of the test substance was 150 mg/kg bw/day for males (based on increased incidence and severity in hyaline droplet accumulation, tubular basophilia and granular casts in male kidneys at 500 mg/kg bw/day) and 500 mg/kg bw/day for females. Since the male renal findings are regarded as a male rat specific response, a NOAEL of 500 mg/kg bw/day may be indicated for humane risk assessment purposes.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guideline OPPTS 870.3650

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of adipic acid and neodecanoic acid, 2-​oxiranylmethyl ester (C31H56O10)+(C32H58O10)+(C33H60O10)

Molecular formula:

C32H58O10

IUPAC Name:

Reaction product of adipic acid and neodecanoic acid, 2-​oxiranylmethyl ester (C31H56O10)+(C32H58O10)+(C33H60O10)

Constituent 2

Reference substance name:

not yet assigned

Molecular formula:

C50H88O16 C51H90O16 C52H92O16

IUPAC Name:

not yet assigned

Constituent 3

Reference substance name:

2,3-epoxypropyl neodecanoate

EC Number:

247-979-2

EC Name:

2,3-epoxypropyl neodecanoate

Cas Number:

26761-45-5

Molecular formula:

C13H24O3

IUPAC Name:

(oxiran-2-yl)methyl 2,2-dimethyloctanoate

Test material form:

liquid: viscous

Details on test material:

Former EC 615-318-0 / CAS: 716336-43-5: changed to EC 825-846-5 / CAS 876528-25-5

Test animals

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. 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 nulliparous and non-pregnant: yes
- Age at study initiation: Males: 10-11 weeks; Females: 13-14 weeks
- Weight at study initiation: Males: 272-304 g; Females: 203-240 g
A health inspection was performed before the initiation of dosing.
- Housing: 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 cageenrichment, bedding material, food and water.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

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 solution and dosed within 6 hours after adding the vehicle to the test item.
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.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

Duration of treatment / exposure
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 d ays a week for a minimum of 28 days. Males were treated for 29 days (most males) or 33 days (two males used for re-mating), 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 51-55 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 13 to15 days after delivery, up to and including the day before scheduled necropsy.
One female at 500 mg/kg/daymg/kg bw/day (No. 80) which failed to deliver was treated for 48 days.
The first day of dosing was designated as Day 1.

Frequency of treatment
Once daily at approximately the same time each day.

The dose volume for each animal was based on the most recent body weight measurement (5 mL/kg bw). 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.

Details on mating 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.

Detection of mating was not confirmed in first instance for Female No. 43. Evidence of mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of this animal animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Rationale for vehicle:
Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment is complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.
Stability of the test item in polyethylene glycol was confirmed analytically following GLP for at least 24 hours at room temperature under normal laboratory light conditions and stability for at least 3 weeks in the freezer (≤ -15 °C) is confirmed over the concentration range 1 to 200 mg/mL (solutions), Project 20157759.

Trial formulations were prepared in propylene glycol at 1 mg/g (1 mg/mL) and 195 mg/g (200 mg/mL). Formulations were solutions.
The concentrations analyzed in the trial formulations were in agreement with target concentrations (i.e. mean accuracies between 90 % and 110 %). The formulations were homogeneous (i.e. coefficientof variation ≤ 10 %).
Formulations were stable when stored at room temperature under normal laboratory light conditions for at least 24 hours and in a freezer (≤ -15 °C) for at least 21 days (3 weeks).

Duration of treatment / exposure:

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 51-55 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 13 to15 days after delivery, up to and including the day before scheduled necropsy. One female at 500 mg/kg/daymg/kg bw/day (No. 80) which failed to deliver was treated for 48 days.
The first day of dosing was designated as Day 1.
A total of 9 animals (2/10 Group 1 animals (Nos. 43 and 45), 3/10 Group 3 animals (Nos. 65, 69 and 70) and 4/10 Group 4 animals (Nos. 74, 75, 76 and 79) were not dosed during two consecutive days (postnatal Days 14 and 15; see Appendix 9, Study Plan Deviations). These animals underwent scheduled necropsy on postnatal Day 16. This was considered not to have adversely affected interpretation of the study results, since animals were already dosed for 53 days, and since it was not anticipated that an additional 2 days of dosing would significantly influence the outcome of the study. Furthermore, all animals were dosed during the “sensitive periods”, i.e. premating, mating, post-coitum and early postnatal development.
Female Nos. 53 and 57 (Group 2), and 71 and 74 (Group 4), were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day over a period of several weeks was not considered to affect the toxicological evaluation.

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.

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

500 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
The dose levels were selected to be 0, 50, 150, 500 mg/kg bw/day based on the results of a dose range finder with oral gavage administration of the test substance in rats (Test Facility Study No. 20157757, see below), and in an attempt to produce graded responses to the test item.

The dose levels for the main study were selected based on the results of the dose range finder with doses of 1000 and 500 mg/kg bw/day for 23 days and 3 female rats per dose. In this dose range finder no mortality was observed. All animals showed hunched posture from day 12 onwards, uncoordinated movements (between days 1-14) and piloerection (starting days 12 in the 500 mg group and on day 14 at 1000 mg). In the first 3 days of treatment marginally reduced food consumption was observed. Weight loss was shown in one animal of both groups over days 1-3, followed by normal weight gain. Increased liver weights were recorded for all animals.
The peak effect of occurrence of clinical signs was considered to occur between 1 and 3 hours after dosing. This time point was taken into account to set a time range within which clinical observations were conducted after dosing in the Main Study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed 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 3 hours (±30 min) post-dose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually 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.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 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.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation was in troduced as no effect was suspected.

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: Functional tests (hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity) 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 11-13).

CLINICAL PATHOLOGY: Yes
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. When non-serum samples were clotted, additional blood samples were obtained in the necropsy room (if possible). After collection all samples were transferred to the appropriate laboratory for analysis. F0-females were not fasted prior to blood sampling and necropsy. F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

- Hematology parameters: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophil (absolute), Haemoglobin, Lymphocyte (absolute), Haematocrit, Monocyte (absolute), Mean corpuscular volume (MCV), Eosinophil (absolute), Mean corpuscular haemoglobin (MCH), Basophil (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelet, Reticulocyte (absolute).
- Coagulation parameters: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- Clinical chemistry parameters: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos).
- Thyroid hormone: Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4). Measurement of T4 was conducted for F0-males.
For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no toxicologically relevant changes were noted in T4 in F0-males, both sexes showed a similar degree of thyroid follicular hypertrophy and since thyroid weight was considered not affected by treatment.

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

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. 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. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.

Sperm parameters (parental animals):

For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Clinical observations were performed at least once daily for all pups.
Live pups were weighed individually on PND 1, 4, 7 and 13. Sex was externally determined for all pups on PND 1 and 4. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
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):

SACRIFICE
- Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
- Scheduled necropsies were conducted on the following days: Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration);
Females which delivered: PND 14-16; Female No. 80 which failed to deliver (with evidence of mating): Post-coitum Day 30.
All males were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0- females were not fasted.

GROSS NECROPSY
- 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.
Organs Weighed at Necropsy for all selected animals: Brain, Cervixa, Epididymis, Gland adrenal, Gland coagulation, Gland parathyroid, Gland prostate, Gland seminal vesicle, Gland thyroid, Heart, Kidney, Liver, Ovaries, Spleen, Testes, Thymus, Uterus
Organs Weighed at Necropsy for all remaining animals (incl. Male No. 40 that failed to sire and female No. 80 that failed to deliver pups): Epididymis, Gland Coagulationa, Gland parathyroidc, Gland prostate, Gland seminal vesicle, Gland thyroid, Testes

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

- Table 1: Tissue Collection and Preservation for all selected animals, and all animals that died spontaneously or were sacrificed in extremis: Animal identification, artery, aorta, body cavity, nasopharynx, bone marrow, bone (femur and sternum), brain (seven levels), cervix, epididymis, esophagus, eye, adrenal gland, coagulation gland, harderian gland, lacrimal gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (cecum, colon, rectum), larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, pancreas, skin, small intestine (duodenum, ileum, jejunum), spinal cord,spleen, stomach, testes, thymus, tongue, trachea, urinary bladder, uterus, vagina.

- Table 2: Tissue Collection and Preservation for all remaining animals (incl. males that failed to sire females that failed to deliver pups; non-pregnant, implantation sites only):
Animal identification, cervix, epididymis, coagulation gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, gross lesions/masses, ovaries, testes, uterus, vagina.

Postmortem examinations (offspring):

METHOD OF EUTHANASIA
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 14-16), except for the two pups per litter 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 were anesthetized using isoflurane followed by exsanguination.

UNSCHEDULED AND SCHEDULED DEATHS
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. For details see also section 4.11.1.
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.
In addition, blood was collected from two pups per litter (see also section 4.11.1), and the thyroid from two pups per litter (one male and one female pup) was preserved in 10 % buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

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 3 observations.

The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1 and Group 4 vs. Group 1.

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric: 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.

Incidence: 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.

Reproductive indices:

Mating index (%): Number of females mated / Number of females paired x 100

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): Number of pregnant females / Number of females mated x 100

Gestation index (%): Number of females with living pups on Day 1 / Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%): Total number of offspring born / Total number of uterine implantation sites x 100

Live birth index (%): Number of live offspring on Day 1 after littering / Total number of offspring born x 100

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check / Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check / Number of live pups at First Litter Check x 100

Viability index (%): Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering x 100

Lactation index (%): Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations.
Piloerection was recorded for one Female (No. 75) at 500 mg/kg/daymg/kg bw/day on several days of treatment, and for two surviving females at 150 mg/kg/daymg/kg bw/day (Nos. 65 and 67) on a single day only. Given that this finding occurred incidentally and without a dose-related trend, it was considered not to represent a treatment-related finding.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One Female (No. 61) at 150 mg/kg/daymg/kg bw/day was found dead after dosing on Day 5. On the day of death, this animal showed flat posture and gasping. Main microscopic findings consisted of the presence of amorphous material with inflammatory cells in the thoracic cavity and on the surface of the heart, correlating to watery-clear fluid in the thoracic cavity and friable thickened pleura recorded at necropsy. Overall, these findings indicated that this was gavage-related death. Other necropsy findings for this animal consisted of dark red discolouration of the trachea and red foci on the thymus. One Control Female (No. 47) died at blood sampling; this death was unrelated to treatment with the test item, and considered to be related to the blood sampling procedure.
No further mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered not affected by treatment.
A statistically significant lower body weight and body weight gain was recorded for males at 150 mg/kg/daymg/kg bw/day over Weeks 3 and 4 of treatment. These concerned slight differences from the control means, and in absence of a dose-related trend these variations were considered not related to treatment.
Body weights and body weight gain of other groups remained in the same range as controls.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower mean reticulocyte counts were recorded for both males and females at 150 mg/kg bw/day (0.74x control mean for both sexes), and to a lesser extent for males at 500 mg/kg bw/day (0.85x control mean, not statistically significant). For males at 150 mg/kg bw/day, the mean reticulocyte count was outside the historical control range, while for females (and males at 500 mg/kg bw/day) the mean was just within this range. Although treatment-related changes would typically be expected to follow a dose-related trend, it could not be excluded that these changes represented an effect of the test item, given the pronounced degree and consistency of the change across both sexes. However, since a dose-related trend was absent, and there were no supportive changes in red blood cell parameters or morphological correlates, these lower reticulocyte counts were considered not to represent an adverse effect of treatment with the test item.
Other haematological parameters of treated rats were considered not affected by treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered not affected by treatment.
Thyroid hormone analyses: Serum levels of T4 in F0 males were considered not affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 50, 150 and 500 mg/kg, forelimb grip strength of males was statistically significantly lower than the control mean (0.55x, 0.59x and 0.70x control mean, respectively). Mean values did not show a dose related trend, and remained within the historical control range (although bordering the lower margin of this range) . Values of 2/5 animals each at 50 and 500 mg/kg, and 1/5 animals at 150 mg/kg were outside this range. The control mean was similar to the mean expected based on this historical control range. These recordings were not supported by clinical observations or other functional observation tests (including hindlimb grip strength), and had no supportive morphological correlates in examined neonal/muscle tissues. Therefore, this recorded lower forelimb grip strength of males was considered not to represent an adverse effect of treatment with the test item.
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with 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, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the thyroid glands of both sexes at 500 mg/kg bw/day, the kidneys of males starting at 50 mg/kg bw/day, adrenal glands of males at 500 mg/kg bw/day, see IUCLID chapter 7.5.1, Table 2.
Adverse parental findings in this study were present in the kidneys of male rats at 500 mg/kg bw/day and consisted of a combination of an increased incidence and severity in hyaline droplet accumulation, tubular basopbilia and granular casts with correlating increased kidney weights.
The hyaline droplet accumulation recorded in the male kidneys was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats nor in higher mammals, including man (Ref. 1). At 50 and 150 mg/kg bw/day the increased incidence and/or severity of hyaline droplet accumulation with low degrees of tubular basophilia occurred in the absence of indicators of renal tubular damage and the renal findings in these treatment groups were therefore considered to be non-adverse.

Other (non-adverse) morphologic alterations were recorded, see IUCLID chapter 7.5.1, Table 2.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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 one Control Female No. 46 (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 function: sperm measures:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating index not affected by treatment. All paired females showed evidence of mating.
Precoital time was considered not affected by treatment. All paired females showed evidence of mating within 4 days.
Number of implantation sites was considered not affected by treatment.
Fertility index was considered not affected by treatment. The fertility indices were 100 % for the control group and for the 50 and 150 mg/kg bw/day groups, and 90 % for the 500 mg/kg bw/day group.
One female at 500 mg/kg bw/day (No. 80) was not pregnant. Since this single case remained within the historical control data range , and here were no indications of reproductive/developmental toxicity across the dose groups, this was considered not to be related to treatment.

Details on results (P0)

- Gestation index and duration of gestation were considered not affected by treatment. All pregnant females had live offspring.
- No signs of difficult or prolonged parturition were noted among the pregnant females.
- Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- 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 was 89, 87, 98 and 92 % for the control, 50, 150 and 500 mg/kg groups, respectively.
- Litter size was considered not affected by treatment.
A statistically significantly higher mean number of living pups was recorded at 150 mg/kg bw/day (14.3 vs 11.8 in the control group). As a dose-related response was absent, and since the opposite effect would be expected in case of toxicity, this was considered not to be related to 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 50 mg/kg bw/day groups, and 98 and 100 % for the 150 and 500 mg/kg bw/day groups, respectively.
One pup each in the control group and at 50 mg/kg bw/day, and two pups at 150 mg/kg bw/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 (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 98 % for the control and 50 mg/kg bw/day groups, and 96 and 99 % for the 150 and 500 mg/kg bw/day groups, respectively.
Two pups each of the control group and at 50 mg/kg bw/day, five pups at 150 mg/kg bw/day and one pup at 500 mg/kg bw/day were found dead or missing on PND 2, 3 or 4. Pups missing were most likely cannibalised. 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.
- The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not 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.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: hyaline droplet accumulation in the kidney, considered to represent male rat specific alpha2μ nephropathy

Remarks on result:

other: not relevant for reproductive performance

2

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: highest dose tested

3

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Since the renal findings in male rats are regarded as a male rat specific response, a NOAEL o f 500 mg/kg/daymg/kg bw/day may be indicated for humane risk assessment purposes.

Remarks on result:

other: highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

other: (males only)

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment. All pups of three dams at 150 mg/kg/daymg/kg bw/day (Nos. 65, 67 and 68) showed alopecia during the last 3-4 days of the study. As the incidence of this finding did not show a dose-related trend and since this finding was not observed for other pups in this dose group, this was considered not to be related to treatment.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
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.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Sex ratio
Sex ratio was considered not to be affected by treatment.

- Anogenital distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

- Areola/nipple retention
Treatment up to 500 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

- Clinical biochemistry (T4 levels)
There were no treatment-related changes in serum T4 levels in male and female PND 14-16 pups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Executive summary:

The objectives of this study were to determine the potential toxic effects of the test substance when given orally by gavage for > 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, 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.

Ten male and ten female Wistar Han rats per group were treated by daily oral gavage at dose levels of 50, 150 and 500 mg/kg according to OECD TG 422. The rats of the control group received the vehicle propylene glycol alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination. Females that delivered were treated for 51-55 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 13 to15 days after delivery, up to and including the day before scheduled necropsy.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels for the purpose of this study.

Parental results:

Adverse parental findings in this study were present in the kidneys of male rats at 500 mg/kg bw/day and consisted of a combination of an increased incidence and severity in hyaline droplet accumulation, tubular basopbilia and granular casts with correlating increased kidney weights.

The hyaline droplet accumulation recorded in the male kidneys was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia.  This male rat specific protein is not present in female rats nor in higher mammals, including man (Ref. 1).  At 50 and 150 mg/kg bw/day the increased incidence and/or severity of hyaline droplet accumulation with low degrees of tubular basophilia occurred in the absence of indicators of renal tubular damage and the renal findings in these treatment groups were therefore considered to be non-adverse.

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg bw/day).

No treatment-related 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).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (500 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

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 level (NOAEL) of the test substance were established:

Parental NOAEL (males):  150 mg/kg (based on increased incidence and severity in hyaline droplet accumulation, tubular basophilia and granular casts in male kidneys at 500 mg/kg bw/day). Since these renal findings are regarded as a male rat specific response, a NOAEL of 500 mg/kg bw/day may be indicated for humane risk assessment purposes.

Parental NOAEL (females):  at least 500 mg/kg bw/day

Reproduction NOAEL:  at least 500 mg/kg bw/day.

Developmental NOAEL:  at least 500 mg/kg bw/day.