5-chloro-1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the test substance was administered daily to rats at dose levels up to 150 mg/kg bodyw eight/day (OECD 422, Van Otterdijk, 2016). The parental and reproductive NOAEL were established as 50 mg/kg body weight/day. The substance is classified as reproductive toxicant category 2, according to CLP Regulation.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-08-24 to 2015-12-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.7 (Repeated dose (28 days) toxicity (oral))

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050

Deviations:

no

Principles of method if other than guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A14JB3414 (pilot phase), A15CB1464 (main phase)
- Retest date of the lot/batch: 2015-10-01 (pilot phase), 2016-05-18 (main phase)
- Purity test date: 2014-12-22 (pilot phase), 2016-02-23 (main phase)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was confirmed as part of the analytical method development and validation study (Test Facility Study No. 509776).

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspension

OTHER SPECIFICS:
correction factor: 1.00

Species:

rat

Strain:

Wistar

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:WI(Han) (outbred, SPF-Quality) from Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
pilot phase: females approx. 8 weeks (group 1) or 10 weeks (group 2)
main phase: females approx. 11 weeks (at start pretest) and 13 weeks (at start F0-treatment); males approx. 9 weeks (at start F0-treatment)
- Weight at study initiation: 295-331 g (males), 207-239 g (females)
- Fasting period before study: No
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Use of restrainers for preventing ingestion (if dermal): no (not applicable)
- Diet (e.g. ad libitum): Free access to pelleted rodent diet
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C,
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES:
From: 2015-10-07 (start pretest; females aged approx. 11 weeks); 2015-10-21 (start treatment; males aged approx. 9 weeks); 2015-11-27/28/29/30 and 2015-12-1/10 (delivery of litters)
To: 2015-12-11/14/15/23 (necropsy females); 2015-11-19 (necropsy males); 2015-12-9/10/11/14/22 (necropsy pups)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% aqueous carboxymethyl cellulose, specific gravity 1.00

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. A correction was made for the purity/composition of the test item. A correction factor of 1 as used.
- Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch and on information provided by the Sponsor.
- Concentration in vehicle: 0 mg/mL (group 1), 3 mg/mL (group 2), 10 mg/mL (group 3), 30 mg/mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight. (actual dose volumes were calculated according to the latest body weight)
- Lot/batch no. (if required): no data
- Purity: no data

Details on mating procedure:

- M/F ratio per cage: 1 animal/sex/cage
- Length of cohabitation: Following a minium of 14 days of exposure for the males and the females, until proof of pregnancy
- Proof of pregnancy: 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 was confirmed, the males and females were separated.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (27 October 2015; Day 21 of treatment), according to a validated method (Test Facility Study no. 509776). Sextuplicate samples (i.e. 3 sets of duplicate samples) were collected. Two sets of duplicate samples were stored as reserve samples. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10% compared to those obtained during the method validation.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration.
Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Stability of the test item under test conditions was demonstrated in the method validation study (Test Facility Study no. 509776).
Density was determined from all formulations on a single day during the treatment phase to express analytical concentrations in mg/mL.

Duration of treatment / exposure:

pilot phase: 10 days
main phase: 29 days (males), 51-63 days (females), 38-55 days (females that failed to deliver healthy offspring)
pups: were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

group 1

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

group 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

group 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

group 4

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were based on the results of a 10-day range finding study with T000990 (Test Facility Study no. 509027; pilot phase) in which doses of 100 and 300 mg/kg/day were administered. No mortality occurred. At 300 mg/kg/day, hunched posture, piloerection, lethargy and/or salivation were noted for all animals from Day 6 onwards. At 100 mg/kg/day, clinical signs were confined to the occurrence of piloerection for two out of three animals on Day 7. At 300 mg/kg/day, weight loss was recorded for two out of three animals between Days 1-5 (-2 and -5%) and for one out of three animals also between Days 5-10 (-10%), and food intake was reduced. At 100 mg/kg/day, body weight and food intake were considered to be normal for all animals. At both 100 and 300 mg/kg/day, liver weights were increased, and kidney weights were considered to be normal. No macroscopic abnormalities were noted at 100 and 300 mg/kg/day. Based on these data, the highest dose level to be administered in the main study was selected to be 150 mg/kg/day upon consultation with the Sponsor.
- Rationale for animal assignment (if not random): randomized

Positive control:

no

Parental animals: Observations and examinations:

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

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

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Body weight gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Relative food consumption was calculated and reported.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

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

FUNCTIONAL OBSERVATIONS
- Time schedule: between 1 and 3 hours after dosing on the selected 5 animals/sex/group. Selected males were tested during week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs (incl. arena observation, if applicable)
- parameters: hearing ability, pupillary reflex, static righting reflex, fore- and hindlimb grip strength recorded as the mean of three measurements per animal, locomotor activity

HAEMATOLOGY
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 aniamsl/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with K3-EDTA for hematology parameters, and with citrate for clotting tests
- parameters assessed: white blood cells, differential leukocyte counts, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothombin time, activated partial thromboplastin time

CLINICAL BIOCHEMISTRY
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 aniamsl/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with Li-heparin
- parameters assessed: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate
- thyroid hormone analysis

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
During pretest, this was done for 48 females. At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random and further used in the study. The remaining females were removed from the study, and estrous cycle results were not reported but kept in the raw data.
On the day of necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations: additional slides of the testes (to examine staging of spermatogenesis), testis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no. All pups were randomized per litter and idividually identified by means of subcutaneous injection of Indian ink on Post-natal day 1 (=day the litter was found completed)
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); Blood samples were collected from two of the surplus pups; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
- Clinical signs At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective table.
- Body weights Live pups were weighed on PND 1, 4, 7 and 13.
- Sex Sex was determined for all pups on PND 1 and 4.
- Anogenital distance Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention On PND 13, all males in each litter were examined for the number of areola/nipples.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death wasdetermined for pups born or found dead if possible
Pups found dead during the weekend were necropsied on the same day.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration)
- Maternal animals: All surviving animals, on PND 14-16 (following delivery), Post-coitum day 26-27 (females with evidence of mating, but which failed to deliver) or within 24h after total litter loss (females with total litter loss)

GROSS NECROPSY
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Necropsy was conducted as soon as possibe after spontaneous death and always within 24 hours.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin: Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F) (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin: Cervix (F), Clitoral gland (F), Coagulation gland (M), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levator ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

HISTOPATHOLOGY / ORGAN WEIGHTSORGAN WEIGHTS:
- Absolute organ weights and organ to body weight ratios were reported.
- The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/group on the scheduled day of necropsy: Adrenal glands, Brain, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus, bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix)
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavernosus muscle complex (LABC), Testes, Thyroid
HISTOPATHOLOGY:
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist: The preserved organs and tissues of 5 animals/sex of Groups 1 and 4 (for Group 4 females only reproductive organs were examined histopathologically from selected females which had implantation sites only (nos. 75 and 79) or were not pregnant (no. 80). No further females were available with live pups; Additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males that failed to sire (see table below) to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups; Histopathological examination of the mammary gland was also conducted for female nos. 71, 74, 77 and 78 (Group 4) that had a total litter loss; Thyroid glands, liver, thymus and spleen of all selected 5 animals of Groups 2 and 3 (males and females), based on (possible) treatment-related changes in these organs in Group 4.
- All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
- A peer review on the histopathology data was performed by a second pathologist.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 (by decapitation between 7.00 and 10.30 a.m.), and at PND 7-15 days of age (using Ethasol® 20% by intraperitoneal (ip) injection).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All pups were sexed both externally and internally.
- At terminal sacrifice (13-15 days of age), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin.
- 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.

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/ Number of females paired) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Survival indices:
Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) 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
Group mean values were calculated from individual litter values.
Sex ratio (percentage males) = (Number of males in litter/Total number of offspring in litter) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg, hunched posture and/or piloerection were observed in 6 out of 10 males on some occasions during Weeks 3 and 4 of treatment and among all females from Week 2 of treatment onwards. Pale faeces was additionally noted for females at 150 mg/kg from Week 5 of treatment onwards.
Salivation seen after dosing of both sexes at 50 and 150 mg/kg and for males also at 15 mg/kg during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test item.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No unscheduled mortality occurred during the study. At 150 mg/kg, 4 females (nos. 71, 74, 77 and 78) were sent for necropsy due to total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg, females had statistically significantly lower body weights and body weight gain or slight weight loss during the first week (up to 4%) over the 14-day premating period. Weight gain was lower on Days 17 and 20 of the post-coitum (statistically significant), and absolute body weights were statistically significantly lower on several occasions during post-coitum. Body weights of the 150 mg/kg group females were also statistically significantly lower on Day 1 of lactation, but weight gain during lactation (based on Day 1 lactation body weights) was similar between the groups. At 15 and 50 mg/kg (both sexes) and at 150 mg/kg (males), body weights and body weight gain remained essentially in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg, females had a lower food intake (before and after correction for body weights) over the premating period, and also during the last week of the post-coitum and during the lactation period (being statistically significant over Days 0-4 and 14-20 of the post coitum period). At 15 and 50 mg/kg (both sexes) and at 150 mg/kg (males), food intake (before and after correction for body weights) remained in the same range as controls over the treatment period.

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):

No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significantly lower activated partial thromboplastin time (APTT) for males at 150 mg/kg was considered not toxicologically relevant since the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
− Higher alanine aminotransferase activity (ALAT) in males at 150 mg/kg (approximately 28% higher than control mean). Mean ALAT levels of female dose groups also appeared to show a trend towards an increase, but no level of statistical significance was achieved.
− Higher mean aspartate aminotransferase activity (ASAT) of females at 15, 50 and 150 mg/kg (approximately 45, 28 and 16% higher than control mean, respectively). A relatively high individual variation was noted across the dose groups, no clear dose-related response was noted and no level of statistical significance was achieved.
− Higher total bilirubin in males at 150 mg/kg (approximately 17% higher than control mean), and in females at 50 and 150 mg/kg (approximately 42% higher than control mean at both dose levels), not statistically significant at 150 mg/kg.
− Higher bile acid in males at 150 mg/kg (approximately 459% higher than control mean), and in females at 15, 50 and 150 mg/kg (approximately 329, 733 and 11,336% higher than control mean, respectively); a relatively high individual variation was noted across the female dose groups, and statistical significance was only achieved at 150 mg/kg.
− Lower sodium in males at 150 mg/kg (approximately 2% lower than control mean).
− Higher potassium in males at 150 mg/kg (approximately 8% higher than control mean).
− Lower chloride in males at 150 mg/kg (approximately 2% lower than control mean.
Thyroid hormone analyses: Mean serum T4 levels in F0 males appeared lower than controls (not statistically significant; mean approximately 24% lower than control mean).
The statistically significantly lower inorganic phosphate level of males at 15 mg/kg occurred in the absence of a dose-related trend and was therefore not considered to be related to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Females at 150 mg/kg showed a statistically significantly lower grip strength of the forelimbs (approximately 29% lower than controls). This was primarily due to a low value for one female (no. 72). Without the value for this female, the mean was 846±67 gram, and not statistically different from the control mean (approximately 21% lower than controls). Hindlimb grip strength of these females also appeared slightly lower (approximately 22% lower than controls), but did not achieve a level of statistical significance.
In addition, motor activity (total movements and ambulations) of males and females at 150 mg/kg appeared lower than controls (approximately -30% and -37% for males and females, respectively). For females, motor activity showed an apparent trend towards a decrease over the dose groups. None of these variations achieved a level of statistical significance, and all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Grip strength of males was considered to have been unaffected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following test item-related microscopic findings were noted:
− Increased incidence and severity of hepatocellular vacuolation and hepatocellular hypertrophy in the liver in males starting at 50 mg/kg and in females in the 150 mg/kg group only.
− Thymus cortical atrophy in 40-50% of the animals of the males and females in the 150 mg/kg group and in 20% of the females of the 15 and 50 mg/kg group.
− Increased severity of follicular cell hypertrophy in the thyroid gland in males and females of the 50 and 150 mg/kg group.
− Increased severity of extra-medullary hematopoiesis in the spleen in males of the 150 mg/kg group.
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.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg, a total of 3 females (nos. 74, 75 and 79) had an irregular cycle, and one female (no. 80) was acyclic.
Length and regularity of the estrous cycle at 15 and 50 mg/kg were not considered to have been affected by treatment. Most females had regular cycles of 4 days. Extended di-estrus occurred in one control female (along with an irregular cycle), one female at 15 mg/kg (not pregnant) and in one female at 150 mg/kg (not pregnant). An irregular cycle was also noted for one female at 50 mg/kg which had a normal litter. The incidence of these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic staging profiles were normal for all males examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

REPRODUCTION DATA
At 150 mg/kg, a statistically significantly lower number of implantation sites was recorded (mean 10.4 vs 13.8 in the control group).
All females showed evidence of mating. A total of two females at 15 mg/kg and two females at 150 mg/kg were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was not considered to be related to treatment. Mating and fertility indices, and precoital time were not considered to have been affected by treatment.
For 15 mg/kg female no. 54 and 50 mg/kg female no. 70, the number of pups was slightly higher than the number of implantations. This was considered caused by normal resorption of these areas as these enumerations were performed on Day 16 of lactation.

DEVELOPMENTAL DATA
At 150 mg/kg, gestation index was reduced (38% vs. 100% in the control group). A total of 3 out of 8 pregnant females at this high dose had live offspring (nos. 71, 72 and 76) of which 1 (no. 71) was sacrificed due to total litter loss on Day 2. For the other two dams (nos. 72 and 76) offspring survived until the end of the scheduled lactation period. The number of pups for dam 72 (3 in total) was lower than expected for rats in this type of study. Overall, this resulted in a statistically significantly lower mean number of living pups on Days 4 and 13 at this dose. Two pregnant females (nos. 75 and 79) had implantation sites only. The other 3 pregnant females (nos. 74, 77 and 78) had total litter loss. There were no supportive morphological findings in the reproductive organs of either sex, and spermatogenic staging profiles were normal for all males examined.
At 150 mg/kg, post-implantation survival index was lower than controls (31% vs 85% in the control group), as well as the live birth index (50% vs. 97% in the control group).
At 150 mg/kg, mean pup body weight (per sex and both sexes combined) appeared lower throughout the lactation phase, achieving a level of statistical significance for female pups on Day 1 of lactation.
At 50 and 150 mg/kg, the sex ratio was increased (49/51 for the control group, vs. 63/37 and 77/23 for the 50 and 150 mg/kg groups, respectively), being statistically significant at 50 mg/kg (note: due to the low number of pups available at 150 mg/kg, the sex ratio at this dose should be interpreted with caution). At 15 mg/kg, the sex ratio appeared unaffected by treatment.
The incidence of infertility at 15 and 50 mg/kg was considered to be within the normal range encountered for this type of study, and was therefore not considered to be related to treatment. This consisted of 2 non-pregnant females at 15 mg/kg and one female at 50 mg/kg with implantation sites only. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
Other developmental parameters were not considered to have been affected by treatment. These included gestation duration, parturition, maternal care and early postnatal pup development (clinical signs, anogenital distance, areola/nipple retention, PND 13-15 pup serum T4 levels and macroscopy).
Parturition/maternal care: 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.

Parental results
- At 150 mg/kg, the combination of in-life findings was considered to be adverse and were present in clinical appearance, body weight, food intake and functional observation tests. Hunched posture and piloerection were observed primarily for females from the second week of treatment onwards. These females additionally showed pale faeces from week 5 of treatment onwards, and had slight weight loss during first week of the premating period. Body weight gain was also lower towards the end of the post-coitum period. The lower weight gain during the premating period and during the last week of the post-coitum period was accompanied by a lower food intake. Food intake remained lower during lactation, but body weight gain was comparable to controls.
- Females at 150 mg/kg also had fore- and hindlimb grip strength (approx -29% and -22%, resp), and motor activity appeared lower for males and females at 150 mg/kg (approx -30% and -37% for males and females resp). Other functional observation tests revealed no treatment-related changes in any sex. Since it could not be excluded that these findings represented an effect on neurobehaviour, and give the magnitude of change, these findings were considered to be adverse in nature.
- No treatment related changes were noted in haematology parameters and at macroscopic examination.
- histopathological findings that were considered to be adverse based on their nature and severity were noted in the liver (hepatocellular vacuolation), thymus (cortical atrophy) and thyroid gland (follicular hypertrophy).
- Hepatocellular vacuolation was recorded for males and females at 150 mg/kg (up to moderate and minimal degree, resp) was accompanied by higher liver weights for both sexes (mean relative weights were approx 37 and 22% higher than control means, for males and females resp). Hepatocellular hypertrophy noted for males at 50 and 150 mg/kg (up to minimal degree) and for females at 150 mg/kg (slight dgree) was considered to be non-adverse based on the low severity and the absence of any other degenerative and/or inflammatory findings.Hepatocellular vacuolation noted for two males at 50 mg/kg at minimal or slight degrees was not considered adverse at this dose.
- Other adverse histopathological lesions consisted of cortical atrophy of the thymus in males at 150 mg/kg (up to moderate degree) and follicular hypertrophy of the thyroid gland in males and females at 150 mg/kg (up to moderate degree). Serum T4 levels in F0 males appeared slightly lower than controls, but did not show a clear correlation to thyroid gland hypertrophy on an individual animal basis.
- Other histopathological findings (minimal cortical atrophy in the thymus in females at 50 mg/kg and higher, slight follicular cell hypertrophy of the thyroid gland in males and females at 50 mg/kg and the slightly increased extra-medullary hematopoiesis in the spleen in males at 150 mg/kg) were considered not to be adverse in nature, based on the low severity and absence of any other degenerative and/or inflammatory findings in these organs.
- clinical biochemistry changes that were considered to be related to treatment consisted of higher total bilirubin in both sexes at 150 mg/kg, and in females also at 50 mg/kg, higher alanine aminotransferase activity in males and a trend towards an increase for female dose groups, lower sodium and chloride and higher potassium and bile acid in males at 150 mg/kg, and (with a relatively high interindividual variation) higher bile acid and aspartate aminotransferase activity in females at 15, 50 and 150 mg/kg. In absence of any (correlating) treatment-related histopathological lesions, the changes in clinical biochemistry parameters at 15 and 50 mg/kg were considered not adverse in nature
- No histopathological correlating lesions were found for the higher kidney weights for males at 150 mg/kg (mean relative weight was approx 16% higher than control mean). As such, this finding was considered not adverse in nature.

Reproductive results
- At 150 mg/kg, a lower number of implantation sites was recorded (mean 10.4 vs 13.8 in the control group). This was considered adverse in nature, also taking into account the observed developmental findings.
- all females showed evidence of mating. No treatment-related changes were noted in any of the other reproductive parameters investigated in the study (i.e. mating, and fertility indices and precoital time).

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

other: lower fore- and hindlimb grip strength and lower motor activity

Remarks on result:

other: based on the effects at 150 mg/kg

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: based on effects at 150 mg/kg

Critical effects observed:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

Incidental clinical symptoms of pups consisted of blue spots on the snout, pale appearance, blue discolouration of the back and insufficient milk in the stomach. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Mortalities attributed to treatment consisted of 9 pups at 150 mg/kg (from one dam (no. 74) with total litter loss) that were found dead at first litter check, and the single pup of 150 mg/kg dam no. 71 (total litter loss) that was found missing on lactation Day 2. Two other dams at 150 mg/kg also had total litter loss on lactation Day 1; dam no. 77 had all her pups missing, and dam no. 78 had 4 dead pups.
The incidence of other dead/missing pups noted among the control group, and 15 and 50 mg/kg groups were not considered to be related to treatment. These did not show a dose-related trend and remained within the range considered normal for pups of this age. This included 3 pups of the control group and 1 pup each at 15 and 50 mg/kg that were found dead at first litter check, and a single pup of the control group that was found missing on lactation Day 3. No breeding loss occurred (i.e. between lactation Days 5 and 13). Pups missing were most likely cannibalised.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg, mean pup body weight (per sex and both sexes combined) appeared lower throughout the lactation phase, achieving a level of statistical significance for female pups on Day 1 of lactation.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in PND 13-15 pups were considered unaffected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

ANOGENITAL DISTANCE
Anogenital distance in male and female pups was not affected by treatment.
The statistically significant higher median anogenital distance for males at 50 mg/kg occurred in the absence of a dose-related and was therefore not considered related to treatment.

AREOLA/NIPPLE RETENTION
Treatment up to 150 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

MACROSCOPY
Incidental macroscopic findings of pups that were found dead included partial cannibalism, autolysis and absence of milk in the stomach (also noted for all pups of litter no. 74 at 150 mg/kg that had total litter loss; the dam had no correlating histopathological lesions in the mammary gland area). The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

SEX RATIO
At 50 and 150 mg/kg, the sex ratio was increased (49/51 for the control group, vs. 63/37 and 77/23 for the 50 and 150 mg/kg groups, respectively), being statistically significant at 50 mg/kg (note: due to the low number of pups available at 150 mg/kg, the sex ratio at this dose should be interpreted with caution). At 15 mg/kg, the sex ratio appeared unaffected by treatment.

GESTATION INDEX
At 150 mg/kg, gestation index was reduced (38% vs. 100% in the control group). A total of 3 out of 8 pregnant females at this high dose had live offspring (nos. 71, 72 and 76) of which 1 (no. 71) was sacrificed due to total litter loss on Day 2. For the other two dams (nos. 72 and 76) offspring survived until the end of the scheduled lactation period. The number of pups for dam 72 (3 in total) was lower than expected for rats in this type of study. Overall, this resulted in a statistically significantly lower mean number of living pups on Days 4 and 13 at this dose. Two pregnant females (nos. 75 and 79) had implantation sites only. The other 3 pregnant females (nos. 74, 77 and 78) had total litter loss. There were no supportive morphological findings in the reproductive organs of either sex, and spermatogenic staging profiles were normal for all males examined.

SURVIVAL INDEX
At 150 mg/kg, post-implantation survival index was lower than controls (31% vs 85% in the control group), as well as the live birth index (50% vs. 97% in the control group).

FERTILITY
The incidence of infertility at 15 and 50 mg/kg was considered to be within the normal range encountered for this type of study, and was therefore not considered to be related to treatment. This consisted of 2 non-pregnant females at 15 mg/kg and one female at 50 mg/kg with implantation sites only. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Developmental toxicity:
- At 150 mg/kg, a lower gestation index was recorded of 38% vs 100% in the control group. Of the three females that had live offspring at this dose, one was sacrificed due to total litter loss, and two dams had low litter size with offspring surviving until the end of lactation. Two females had implantation sites only, and the other three pregnant females had total litter loss. Mean pup body weight at 150 mg/kg (per sex and both sexes conbined) appeared lower throughout the lactation phase.
- In addition, post-implantation survival index and live birth index were reduced at 150 mg/kg
- At 50 mg/kg, a higher sex ratio was recorded (49/51 for the control group, vs 63/37 for the 50 mg/kg group). This sex ratio was clearly at the upper end of the range but can occassionally be seen in rats of this age and strain. Moreover, there were no accompanying changes in other parameters (primarily anogenital distance) that could support this higher sex ratio. Therefore, this variation was considered to be unrelated to treatment. No reliable sex ratio could be determined at 150 mg/kg due to the very low numbe rof pups available at this dose.
- Although the number of females that delivered live offspring was significantly decreased at 150 mg/kg, no morphological changes were observed at spermatogenic profiling and histopathological examination of reproductive organs that could explain this decrease.
- No treatment-related changes were noted in any of the other developmental parameters investigated (i.e. gestation duration, parturition, maternal care and early postnatal pup development (clinical signs, anogenital distance, areola/nipple retention, PND13-15 pup serum T4 levels and macroscopy)

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

other: lower gestation index at 150 mg/kg

Remarks on result:

other: based on effects at 150 mg/kg

Critical effects observed:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Conclusions:

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 50 mg/kg
Reproduction NOAEL: 50 mg/kg
Developmental NOAEL: 50 mg/kg
Therefore, the substance is classified as reproductive toxicant category 2, according to CLP Regulation.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats were exposed to 0 (vehicle), 15, 50, 150 mg/kg bw/d via gavage (OECD 422, van Otterdijk, 2016).

The vehicle used was 1% aqueous CMC (carboxymethyl cellulose) and the test solutions were prepared daily and administered within 6 hours after preparation.

The test substance did not cause mortality (treatment-related) during the study; however, male and female rats exposed to 150 mg/kg/day presented clinical signs (hunched posture and/or piloerection and/or pale faeces) from week 2 of treatment and onwards. Also, lower body weight, lower foor intake, hepatocellular vacuolation, cortical atrophy of the thymus, thyroid gland folliclar hypertrophy, lower fore- and hindlimb grip strength and lower motor activity were observed in male and female rats exposed to 150 mg/kg/day. Non-adverse treatment-related changes in clinical biochemistry parameters occurred essentially at 150 mg/kg/day.

At 150 mg/kg/day, a statistically significant lower number of implantation sites was recorded along with developmental effects consisting of a lower gestation index (38% vs 85% in control group), lower mean pup body weight, and lower post-implantation survival index (31% vs 85% in control group) and live birth index (50% vs 97% in control group).

Based on the abovementioned considerations, the parental, Reproduction and Developmental No Observed Adverse Effect Levels (NOAEL) were considered to be 50 mg/kg bw/day (nominal dose received).

Effects on developmental toxicity

Description of key information

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422, Van Otterdijk, 2016), a developmental NOAEL of 50 mg/kg body weight/day was established. The test substance is known to have an adverse effect on fertility and development, meeting the criteria for classification as reproductive toxicant category 2. Therefore, no further testing for fertility and developmental toxicity will be necessary, in accordance with the REACH Regulation (Annex IX, section 8.7).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results OECD 422 test, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 50 mg/kg

Reproduction NOAEL: 50 mg/kg

Developmental NOAEL: 50 mg/kg

Therefore, the substance is classified as reproductive toxicant category 2, according to CLP Regulation.