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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 1000 mg/kg/day for parental, reproduction and developmental toxicity.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 September 2017 to 11 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same 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 OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
One of the rodent species acceptable to the regulatory authorities. Historical control data for the strain are available at the testing facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Males approximately 12 weeks and females approximately 13 weeks
- Weight at study initiation: males 277 to 311 g and females 194 to 263 g
- Fasting period before study: No
- Housing: Animals were housed in one air-conditioned room in a barrier protected unit. Males were housed in groups of 5 during the pre-mating and gestation periods. Females were housed in groups of 5 during the pre-mating period. Males and females were housed together during mating (one per sex per cage) and subsequently females were housed individually during gestation and with their litter during lactation. Paper was provided as enrichment for all animals.
- Diet: Pelleted rodent diet ad libitum
- Water: Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 8 days between animal arrival and start of treatment (males) or start of pre-test oestrous cycle smears (females).

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 21 °C
- Humidity: 47 - 64 %
- Air changes: At least 10 air changes per hour with 100 % fresh air (no air recirculation)
- Photoperiod: 12 hours of light (artificial)/12 hours of darkness
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test material dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements.
- Dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light for use within 9 days after preparation. On the day of usage, the dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing.
- Test material dosing formulations were kept at room temperature protected from light until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test material.
- No correction was needed for the purity/composition of the test material (100 % purity).
- Any residual volumes were discarded.

DOSE VOLUME: 5 mL/kg/day

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLE COLLECTION
- Samples were collected on the first day of dosing for all groups (concentration) and groups 2 and 4 (homogeneity).
- In total, 16 samples were included in this study, distributed over 4 dose groups. Group 1 was the vehicle control group, Groups 2, 3 and 4 were dosed at 100, 300 and 1000 mg/kg bw/day at a dose volume of 5 mL/kg bw, respectively (test material concentrations 20, 60 and 200 mg/mL). The samples of the control Group 1 and Group 3 were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment Groups 2 and 4 were taken in duplicate from the top, middle and bottom position of the container and immediately stored on dry ice.
- All samples to be analysed were shipped on dry ice to ABL BV 4 days after preparation. The analytical laboratory was notified before shipment of the samples.
- Upon receipt at the analytical laboratory, the samples were stored in the ultra-low freezer ≤ - 70 °C until analysis.

- Stability Analysis: Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

ANALYTICAL METHOD
LC-DAD: Nexera/ Prominence (Shimadzu)
Rinsing volume: 100 µm
Needle stroke: 49 mm
Rinsing speed: 35 µL/sec
Sampling speed: 1.0 µL/sec
Rinse dip time: 1 sec
Rinse mode: After aspiration
Cooler enabled: yes
Cooler temperature: 10 °C
Injection volume: 3 µL
Needle wash: R0
Column oven temperature: 40 °C
Flow: 1.00 mL/min LC-A (UP Water): 70 %, LC-B(50 mM PFPA + 50 mM FA in ACN/UP water (5/95): 20 %, LC-C (ACN): 10 %
Acquisition time: 3.0 min
Wavelength acquisition: 240-250 nm
Wave step: 2 nm
Time constant: 0.64 s
Slit width: 1.2 nm
Sampling frequency: 3.125 Hz
Cell temperature: 40 °C

- The lower limit of quantification was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
- Sample injections: The analytical run for the determination of the accuracy of preparation and homogeneity of the test material in formulations (AB001) was acquired in the following order: Analytical blank sample, Calibration curve, Analytical blank sample, Analytical blank sample, Procedural recovery sample high and low (replicate 1), Analytical samples and Procedural recovery samples high and low (replicate 2). Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

FORMULAE
- Response (Y): Peak area test material [mAU]
- Calibration curve: Y = a + bX + cX^2
where:
X = nominal concentration [mg/L]
c = curvature
b = slope
a = intercept
- Analysed concentration (X): X = [(-b+√ (b^2 - 4c(a - Y))) / 2c] x [(V x d) / w] [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
- Recovery = (Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]
- Accuracy = (Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]

SPECIFICATIONS
- Run Acceptance: Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85-115 % were accepted. The mean recovery of the procedural recovery samples at each level had to be in the range 90-110 %. The response in all analytical blank samples at retention time of the test material should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).
- Acceptance criteria formulations: Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 – 110 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10 %.

RESULTS
- Calibration curves: Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Regression analysis was performed using the least squares quadratic regression with a 1/concentration^2 weighting factor. The coefficient of correlation (r) was equal to 0.9999. The analytical blank samples injected before the calibration curve and after the highest calibrator showed responses at the retention time of the test material which were within the accepted range (lower than 30.0 % of the response of the lowest calibration standard).
- Samples:
Procedural recovery samples: The mean recoveries of the procedural recovery samples fell within the criterion of 90-110 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.
Test samples: In the Group 1 formulation, no test material was detected. The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).
Duration of treatment / exposure:
- Males were treated for 29 days, up to and including the day before scheduled necropsy. This included 14 days prior to mating and during the mating period.
- Females that delivered were treated for 50-63 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable
time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females with no healthy offspring were treated for 40-42 days.
- Female nos. 44, 46, 47, 48 (Group 1), 51, 52, 54 (Group 2), 66, 67 (Group 3), and 80 (Group 4), were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
- Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube.
- Pups were not treated directly but were potentially exposed to the test material in utero, via maternal milk, or from exposure to maternal urine/faeces.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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: The dose levels were selected based on the results of a 10-day dose range finder with oral administration of the test material in rats, and in an attempt to produce graded responses to the test material.
- Animal assignment: A total of 40 females was selected at randomisation before initiation of the pretest phase. Any selected female classified as not having regular oestrous cycles during the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular oestrous cycles. A total of 40 females with regular oestrous cycles continued in the study. The supernumerary females were removed from the study, and their oestrous cycle results were kept in the raw data but not reported. Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20 % of the sex mean. Males and females were randomised separately.
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. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed once daily, beginning on the day of first administration of the test material and lasting throughout the dosing period up to the day prior to necropsy. During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals (no peak effect of occurrence of clinical signs was observed in the dose range finder. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
- Clinical observations were conducted in a standard arena beginning before the first administration of the test material and then once weekly throughout treatment. These observations were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

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. A terminal weight was recorded on the day of scheduled necropsy.

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.

FOOD EFFICIENCY: No

WATER CONSUMPTION:
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
From 01 Dec onwards, water consumption was measured daily in control animals until the day before scheduled necropsy. This data was used exclusively for collection of historical control data only.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood of F0-animals (except for females with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retroorbital sinus under anesthesia using isoflurane in the animal facility. Due to clotting of a few non-serum samples, additional blood samples were obtained in the necropsy room. After collection all samples were transferred to the appropriate
laboratory for analysis.
- Anaesthetic used for blood collection: Yes
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5 animals/sex/group
- Parameters examined for haematology: 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 and Reticulocyte (absolute).
- A blood smear was prepared from each hematology sample. Blood smears were labeled, stained, and stored. For a few animals these blood smears were used to perform a differential leucocyte count manually, because of an abnormal plot in the automated count.
-Blood samples were processed for plasma, and plasma was analyzed for the following parameters: Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood of F0-animals (except for females with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retroorbital sinus under anesthesia using isoflurane in the animal facility. Due to clotting of a few non-serum samples, additional blood samples were obtained in the necropsy room. After collection all samples were transferred to the appropriate
laboratory for analysis.
- Anaesthetic used for blood collection: Yes
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5 animals/sex/group
- Parameters examined: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein Sodium, Albumin Potassium, Total Bilirubin Chloride, Bile Acids Calcium, Urea and Inorganic Phosphate (Inorg. Phos)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13;). These tests were performed after dosing and completion of clinical observations. The following tests were performed:
- Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
- Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
- Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
- Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal.
- Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerised monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterised by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
- 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 oestrus.
Sperm parameters (parental animals):
Parameters examined: For all males of each group, epididymides, prostate, seminal vesicles and testes were weighed. Paired organs were weighed together. During histopathological examination, the testes from the 5 randomly selected adult males of groups 1 and 4, Group 2 male no. 13 that failed to sire, and Group 2 male no. 23 a detailed qualitative examination
was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
- The following parameters were examined in F1 offspring: number of pups born (live and dead); external abnormalities of the pups; number, weight and sex of pups alive on PND’s 1, 4, 7 and 13; anogenital distance (normalised to the cube root of body weight) on PND 1; and presence of areola/nipples on PND 13 for all males in each litter.

GROSS EXAMINATION OF DEAD PUPS:
- Pups that died before scheduled termination were examined externally and sexed (both externally and internally). 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.

HORMONE ANALYSIS:
- Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room.
- On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup.
-On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.
- Blood samples were processed for serum, and serum was analysed for total Thyroxine (T4). Measurement of total T4 was conducted for PND 14-16 pups.
- Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- Unscheduled Deaths: No animals died during the course of the study.
- Scheduled Euthanasia: Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetised 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. Females which failed to deliver (No. 53): With evidence of mating: Post-coitum Day 27. Females with total litter loss (No. 51): Within 24 hours after the last pup was found dead or missing.
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. 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.
- The following organs were weighed at necropsy for all selected scheduled euthanasia animals: Brain, Cervix (weighed together with the uterus), Epididymis (paired), Gland adrenal (paired), Gland coagulation (paired, weighed with seminal vesicles), Gland parathyroid (weighed with thyroid), Gland prostate, Gland, seminal vesicle (paired), Gland thyroid, Heart, Kidney (paired), Liver, Ovaries (paired), Spleen, Testes (paired), Thymus and Uterus.
- For all remaining animals the following were weighed: Epididymis (paired), Gland coagulation (paired, weighed with seminal vesicles), Gland parathyroid (weighed with thyroid), Gland prostate, Gland, seminal vesicle (paired), Gland thyroid, and Testes (paired).
- Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes
- Representative samples of the following tissues identified in the table below were collected from all selected animals and preserved in 10% neutral buffered formalin, unless otherwise indicated: Artery aorta, Body cavity, nasopharynx, Bone marrow, femur, sternum Brain (seven levels), Cervix, Epididymis (Davidson’s fixative), Oesophagus, Eye (Davidson’s fixative), adrenal gland, coagulation gland, harderian gland (when present, Davidson’s fixative), lacrimal gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine, cecum, Large intestine, colon, Large intestine, rectum, Larynx, Liver, Lung, Lymph node (mandibular and mesenteric site), Muscle, skeletal, Nerve optic (Davidson’s fixative), Nerve sciatic, Ovaries, Pancreas, Skin, Small intestine, duodenum, Small intestine, ileum, Small intestine, jejunum , Spinal cord, Spleen, Stomach, Testes (Davidson’s fixative), Thymus, Tongue, Trachea, Urinary bladder, Uterus and Vagina.
- For all remaining animals the following were collected: Cervix, Epididymis (Davidson’s fixative), coagulation gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, Gross lesions/masses, Ovaries, Testes (Davidson’s fixative), Uterus and Vagina.
- Tissues listed above (except animal identification, aorta, nasopharynx, oesophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue) were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin. All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
Postmortem examinations (offspring):
- On PND 4, the surplus pups (> 8 pups per litter) were euthanised by decapitation. From two surplus pups per litter, blood was collected, if possible.
- All remaining pups were euthanised by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20 %) 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, and the thyroid from two pups per litter (if possible 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.
- The pups selected for blood collection on PND 14-16 were anesthetised using isoflurane followed by exsanguination.
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 analysed 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
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. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
- 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
Pre-coital 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
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Offspring viability indices:
Live birth index (in %): (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
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- Transient piloerection for 1-7 days was noted in 3 females at 1000 mg/kg (nos. 71, 74 and 80) at different time points during the lactation period. This finding was most pronounced in female no. 74 which had piloerection during lactation Days 4-10 together with transient hunched posture on lactation Days 4 and 5. Most likely she was suffering from complications shortly after she gave birth, as she also was observed with a moderate body weight loss (-4%) together with reduced food intake over Days 1-4 of lactation. Subsequently, her condition returned to normal and at necropsy on lactation Day 15 neither macroscopic nor microscopic abnormalities were observed for this female.
- One control female (no. 48) showed rales at a slight degree on a single day of the premating period. As this control female was treated with the vehicle alone, a relation to treatment with the test material could be excluded.
- Salivation seen in a single female at 100 mg/kg (no. 77) during in total 4 days of 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 (after dosing).
- Any other clinical signs (scaling and/or alopecia) 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 doserelated trend. At the incidence observed, these were considered to be unrelated to treatment.
- No findings were noted during the weekly arena observations in this study.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- No mortality occurred during the study that was considered to be related to treatment with the test material.
- One female at 100 mg/kg (no. 51) was euthanized on Day 1 of lactation due to total litter loss. There were no findings either during in-life or at necropsy that indicated a poor condition of this female.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no treatment-related changes in body weight up to 1000 mg/kg.
- At 1000 mg/kg, mean absolute body weight of females was slightly higher than controls throughout the entire treatment period (statistically significant at different time points during the post-coitum and lactation phase). This finding was caused at least in part by the fact that control females happened to be the smallest rats among all the groups (6 % of body weight difference between control and high dose-treated animals at start of treatment). Later during gestation it could also be attributed to differences in the number of foetuses per dam (4 out of 10 control females had 2-7 fetuses only, while all 10 females in the 1000 mg/kg group had 10-15 foetuses).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- Food intake (before and after correction for body weight) was not considered affected by treatment.
- At 1000 mg/kg, absolute food consumption (before correction for body weight) of females was slightly higher than controls over Days 4-7 post-coitum and Days 7-13 of lactation. This finding was considered to be related to the higher litter size in the high dose group compared to controls. Moreover, in case of toxicity a reduction of food intake rather than increase would have been expected.
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):
- Haematological parameters of treated rats were not considered to be affected by treatment.
- For Group 4 males, two out of the five samples taken in the animal facility (nos. 32 and 33) and the additional sample taken in the necropsy room (no. 33) were clotted. As a consequence, the number of plasma samples available for measurement of haematology parameters was relatively low (n=3) in the high dose group. This, however, did not affect the final conclusion. Individual values of all haematology parameters measured were within the normal range, and there were no findings on organ level indicative for a possible treatmentrelated effect on haematology.
- The slightly, but statistically significantly lower mean value for mean corpuscular haemoglobin concentration (MCHC) observed in females at 1000 mg/kg (0.96x of control) was attributed to a lower value in a single female (no. 79 = 19.66 mmol/L) which was just below the range of historical control data1. Therefore, and in the absence of any corroborative finding in any of the correlating blood parameters this change was considered to be unrelated to treatment.
- Coagulation parameters of treated rats were not considered to be affected by treatment.
- For Group 4 males, three out of the five samples taken in the animal facility (nos. 31-33) and the additional two samples taken in the necropsy room (nos. 31 and 33) were clotted. Consequently, coagulation parameters could only be measured in two high dose males. This, however, did not affect the final conclusion. Individual values of prothrombin time (PT), activated partial thromboplastin time (APTT), and number of platelets were within the normal range, and there were no histopathological findings indicative for a possible treatment-related effect on coagulation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Clinical biochemistry parameters of treated animals were not considered to be affected by treament.
- The statistically significant lower mean potassium level observed in males at 100 mg/kg was considered to be unrelated to treatment as it occurred in the absence of a dose-related trend. At the individual level, higher values of bile acids were noted for two Group 4 males (59.6 and 46.4 μmol/L in male nos. 33 and 34, respectively). Both values were (just) above the range of historical control data 2. However, in the absence of any correlating liver findings, no toxicological relevance was attached to this observation.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Functional observation parameters were not considered to be affected by treatment. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
- The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. The decreased motor activity observed in females at 100 mg/kg (statistically significant for the number of ambulations), was not considered to be related to treatment as this occurred in the absence of a dose-related trend and was partly attributed to a lower motor activity of two females (nos. 56 and 57). Individual mean values for both females and males remained within the normal range.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- There were no test item-related microscopic observations.
- The recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Serum levels of T4 in F0 males were not considered to be affected by treatment.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
- Length and regularity of the oestrous cycle were not considered to be affected by treatment.
- Most females had regular cycles of 4 days. One female (no. 54) at 100 mg/kg which had regular cycles during the pretest and premating period was noted with extended di-oestrus during the mating period. This isolated case of pseudo pregnancy in the lowest dose group was considered a chance finding and not related to treatment with the test material. Eventually, this female became pregnant on Day 14 of the mating period and had a normal litter. An irregular cycle was noted for one control female (no. 47) and one female at 300 mg/kg (no. 65) (both with normal litters). Given its incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, a relation to treatment with the test material could be excluded.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- There were no morphological findings in the reproductive organs which could be attributed to the test material and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
- Mating index was not affected by treatment. All females showed evidence of mating.
- Precoital time was not considered to be affected by treatment. All females showed evidence of mating within 5 days, except for the single female at 100 mg/kg (no. 54) with extended di-oestrus which was mated after 14 days. This slightly delayed mating was considered as a chance finding and not related to treatment with the test material.
- Fertility index was unaffected by treatment. The fertility indices were 100, 90, 100 and 100 % for the control, 100, 300 and 1000 mg/kg groups, respectively. One female at 100 mg/kg (no. 53) was not pregnant. As this non-pregnancy was an isolated finding at the lowest dose level, occurred in absence of any reproductive toxicity and remained within the range considered normal for rats of this age and strain, it was not considered to be related to treatment.

- Litter data:
Number of Implantation Sites: Numbers of implantation sites were within the normal range in all treated groups. It was noted that the mean number of implantation sites was slightly lower for the control group than all treated groups (11.4 vs 12.9, 12.4 and 13.0 in Groups 2, 3 and 4, respectively). This lower mean value could be attributed mainly to the low numbers of implantation sites in two control females (n = 4 and 7 in female nos. 41 and 46, respectively). A relation to treatment with the test material could be excluded as these females had been treated with the vehicle alone.
Gestation index and duration of gestation were not affected by treatment. The gestation indices were 100, 89, 100 and 100 % for the control, 100, 300 and 1000 mg/kg groups, respectively. All pregnant females delivered live offspring after 21 or 22 days of gestation, except for one female at 100 mg/kg (no. 51) that delivered after 23 days of gestation. All 4 pups in this latter litter were born dead. Since all other pregnant females delivered their offspring within the expected duration of gestation (21-22 days) and the delayed delivery was observed in a single female in the lowest dose group only, a possible relation to treatment was considered to be unlikely.
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.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to the highest dose level tested (1000 mg/kg).
Critical effects observed:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- No clinical signs occurred among pups that were considered to be related to treatment.
- One pup at 100 mg/kg (litter no. 58) was noted with pale skin and cold temperature on PND 1 with no further relevant/severe clinical signs throughout the lactation period. No toxicological relevance was attributed to these clinical signs due to the absence of a doserelated trend, low incidence in the group (only 1 pup affected) and their transient appearance. The nature and incidence of other clinical signs (alopecia, scabs and wounds) remained within the range considered normal for pups of this age, and were, therefore, not considered to be toxicologically relevant.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
- The total number of offspring born compared to the total number of uterine implantations was not affected by treatment. The survival indices were 87, 94, 93 and 94 % for the
control, 100, 300 and 1000 mg/kg groups, respectively. It was noted that the mean survival index for the control group (87 %) was lower than the mean expected for rats of this age and strain. This could be mainly attributed to the fact that in female no. 43 only 5 out of the 10 implanted conceptuses had developed normally. As the control group had been treated with the vehicle alone, a possible relation to the test item could be excluded. For female no. 48 (control), no. 55 (100 mg/kg) and no. 65 (300 mg/kg), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14 or 16 days of lactation. No toxicological relevance was attached to this finding in the current study.Litter size was not considered affected by treatment. It was noted that litter size for the control group was lower than the mean expected for rats of this age and strain4 (9.8 vs 11.3). This was related to the relatively low numbers of implantation sites in two females (nos. 41 and 46) and/or the relatively low post-implantation survival index in one control female (no. 43) in this group.
- Litter size was not considered affected by treatment. It was noted that litter size for the control group was lower than the mean expected for rats of this age and strain4 (9.8 vs 11.3). This was related to the relatively low numbers of implantation sites in two females (nos. 41 and 46) and/or the relatively low post-implantation survival index in one control female (no. 43) in this group.
- The number of live offspring on Day 1 after littering compared to the total number of offspring born was unaffected by treatment. The live birth indices were 99, 94, 100 and 100 % for the control, 100, 300 and 1000 mg/kg groups, respectively. The slightly lower mean live birth index in the low dose group was caused by a single female at 100 mg/kg (no. 51) that had a total litter loss on PND 1. Her litter consisted of 4 dead pups, all of them partly cannibalised and one with no milk in the stomach. Histopathological examination of the reproductive organs and mammary gland of this dam did not reveal any abnormalities that could have caused this total litter loss. In the absence of any dose-related response, this isolated case of total litter loss was considered to be unrelated to treatment with the test material. One pup of the control group (litter no. 45) and 2 pups at 100 mg/kg (litter no. 58) were found dead at first litter check. No toxicological relevance was attributed to these dead 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 4 before culling compared to the number of offspring on Day 1 was not affected by treatment. The viability index ranged between 98 and 99 % for all groups. Two pups of the control group (litter nos. 44 and 45), one pup at 100 mg/kg (litter no. 56), two pups at 300 mg/kg (litter nos. 66 and 68) and one pup at 1000 mg/kg (litter no. 74) were missing on PND 2-3. 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. The lactation index was 99 % for the 1000 mg/kg group and 100 % for the other groups. One pup at 1000 mg/kg was missing on PND 5; it was most likely cannibalised. This isolated finding was not considered to be treatment-related as the mortality incidence remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not considered to be affected by treatment.
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
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, not considered to be related to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Sex ratio was not considered to be affected by treatment.
- Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment. A slightly higher mean value for normalized anogenital distance was recorded for females in the 100, 300 and 1000 mg/kg groups as compared to the concurrent control value (statistically significant at 300 mg/kg). This finding was caused by a relative low mean control value that was at the lowest end of the available historical range.
- Treatment up to 1000 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
- Serum T4 levels in male and female PND 14-16 pups were unaffected by treatment.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to the highest dose level tested (1000 mg/kg).
Key result
Reproductive effects observed:
no
Conclusions:
Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 1000 mg/kg/day for parental, reproduction and developmental toxicity.
Executive summary:

The reproductive toxicity of the test material was investigated in accordance with the standardised guidelines OECD 422 and US EPA OPPTS 870.3650 under GLP conditions in a combined 28-day oral repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat.

The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the dose range finder. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, oestrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

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

Formulation analyses confirmed that formulations of test material in water were prepared accurately and homogenously.

No adverse parental effects were observed up to the highest dose level tested (1000 mg/kg). At 1000 mg/kg, piloerection was recorded for three females at 1000 mg/kg at different time points during the lactation period. One female in this high dose group presented also with transient hunched posture together with a moderate decrease in body weight gain and reduced food intake at the beginning of lactation. Overall, given their low frequency of occurrence and since they were transient in nature, these findings were not considered adverse.

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

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 1000 mg/kg/day for parental, reproduction and developmental toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study was performed in accordance with standardised guidelines under GLP conditions. The quality of the database is considered to be high.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The reproductive toxicity of the test material was investigated in accordance with the standardised guidelines OECD 422 and US EPA OPPTS 870.3650 under GLP conditions in a combined 28-day oral repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the dose range finder. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, oestrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

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

Formulation analyses confirmed that formulations of test material in water were prepared accurately and homogenously.

No adverse parental effects were observed up to the highest dose level tested (1000 mg/kg). At 1000 mg/kg, piloerection was recorded for three females at 1000 mg/kg at different time points during the lactation period. One female in this high dose group presented also with transient hunched posture together with a moderate decrease in body weight gain and reduced food intake at the beginning of lactation. Overall, given their low frequency of occurrence and since they were transient in nature, these findings were not considered adverse.

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

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 1000 mg/kg/day for parental, reproduction and developmental toxicity.

Effects on developmental toxicity

Description of key information

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 1000 mg/kg/day for parental, reproduction and developmental toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study was performed in accordance with standardised guidelines under GLP conditions. The quality of the database is considered to be high.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The developmental toxicity of the test material was determined in accordance with the standardised guidelines OECD 421, OECD 422, US EPA OPPTS 870.3650 and US EPA OPPTS 870.3050 under GLP conditions in a combined 28-day oral repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the dose range finder. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, oestrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

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

Formulation analyses confirmed that formulations of test material in water were prepared accurately and homogenously.

No adverse parental effects were observed up to the highest dose level tested (1000 mg/kg). At 1000 mg/kg, piloerection was recorded for three females at 1000 mg/kg at different time points during the lactation period. One female in this high dose group presented also with transient hunched posture together with a moderate decrease in body weight gain and reduced food intake at the beginning of lactation. Overall, given their low frequency of occurrence and since they were transient in nature, these findings were not considered adverse.

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

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 1000 mg/kg/day for parental, reproduction and developmental toxicity.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive and developmental toxicity.

Additional information