3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2016 to 13 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2016 to 13 April 2017

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:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3050, Repeated Dose 28-day oral toxicity study in rodents

Version / remarks:

2000

Deviations:

no

Principles of method if other than guideline:

In this study, the animals of Group 4 were dosed erroneously on a single occasion (Day 13 pre-mating) at a dose level of 150 mg/kg instead of the target high dose level of 15 mg/kg. As a result, severe toxicity was observed in these animals and it was considered appropriate that all remaining Group 4 animals were sacrificed without any further examination on Day 15 of the pre-mating period and additional control and 15 mg/kg group (Groups 5 and 6, respectively) were included in this study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (outbred, SPF-quality)

Details on species / strain selection:

This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies.
The testing facility has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

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: At the start of the pre- test females were approximately 11 weeks; at the start of F0-treatment males were approximately 10 weeks and females approximately 13 weeks.
- Weight at study initiation: Weight at start of pre- mating: Females: 206 to 247 g, Males: 278 to 315 g.
- Fasting period before study: No
- Housing: All cages were 18 cm high. During the pre-test females and during pre-mating animals were housed in groups of 5 animals/sex/cage in plastic cages. During mating males and females were cohabitated on a 1:1 basis in plastic cages. Post-mating males were housed in their home cage with a maximum of 5 animals/cage and females were individually housed in plastic cages. During lactation females were housed in plastic cages. Sterilised sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: Pelleted rodent diet ad libitum, except during motor activity measurements animals did not have access to food for a maximum of 1 hour 25 minutes.
- Water: ad libitum, except during motor activity measurements animals did not have access to water for a maximum of 1 hour 25 minutes.
- Acclimation period: At least 5 days prior to the start of pre-test (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C (actual daily mean was 22 °C)
- Humidity: 40 to 70 % (actual daily mean was 44 to 53 %)
- Air changes: at least 10 room air changes/hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES:
- From: Not specified
- To: 13 April 2017

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. No adjustment was made for specific gravity/density of the test material, vehicle, and/or formulation. No correction was made for the purity/composition of the test material. The formulations containing the test material were red solutions. The formulation was stored at room temperature and placed on a magnetic stirrer during dosing.

DOSE VOLUME: 5 mL/ kg bw

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on trial formulations performed in the testing facility.
- Concentration in vehicle: Test material concentrations 0.3, 1, 3 and 3 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The purpose of the analytical phase was to determine the accuracy of preparation, homogeneity and stability of the formulations.

SAMPLING
In total, 26 samples were included in this study, distributed over 6 dose groups. Group 1 and 5 were the vehicle control groups, Groups 2, 3, 4 and 6 were dosed at 1.5, 5, 15 and 15 mg/kg bw/day at a dose volume of 5 mL/kg bw, respectively. The samples of the control Group 1, 3 and Group 5 were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment Groups 2, 4 and 6 were taken in duplicate from the top, middle and bottom position of the container and immediately stored on dry ice. In addition, stability samples were taken from formulation Group 2 in duplicate at the middle position of the container. The stability samples were stored for 5 hours at room temperature under normal laboratory light conditions and then stored on dry ice.

ANALYTICAL CONDITIONS
The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.
Formulations containing the test material were dissolved in methanol and transferred to volumetric flasks. These solutions were further diluted depending on their concentrations and injected. Chromatographic separation was performed on an Xbridge C18 column using isocratic elution. A diode array detector was used for quantification.

- Preparation of Calibration solutions (stocks, ~ 1000 mg/L)
Weigh accurately an amount of approximately 10.0 mg directly into a 25 mL amber glass bottle using an analytical balance. Add accurately 10.0 mL (multi pipette/PD pipette) MeOH. Cap the bottle and dissolve the substance by shaking/vortexing manually. Store the solution in a 25 mL amber glass bottle at 2 to 8 °C (expiration 287 hours).

- Preparation of BT stability samples, ~ 1000 mg/L
Transfer an aliquot of the calibration stocks into 12 mL amber glass bottles. Store the samples at room temperature for at least 12 hours. After completion return the samples to a refrigerator (2 to 8 °C, expiration 22 hours).

- Preparation of calibration spike solutions and stock stability solutions
Prepare solutions according to set volumes, dispensing the correct volume of MeOH (multi pipette) into 10 mL glass tubes. Add the stock solution (PD pipette) to the tubes, cap and vortex the tubes at speed 4 to 6 for 0.5 minutes. Expiration: prepare freshly.

- Preparation of a Recovery stock (20 000 mg/L)
Weigh accurately an amount of approximately 20.0 mg (analytical balance) directly into a 12 mL amber glass bottle. Add accurately 1.00 mL (multi pipette/PD pipette) MeOH. Cap the bottle and dissolve the substance by shaking/vortexing manually. Store the solution in a 25 mL amber glass bottle at 2 to 8 °C (expiration 287 hours).

- Preparation of Accuracy/PRS High samples (~ 200 mg/g)
Weigh at least 100 mg (analytical balance) accurately into a 20 mL glass bottle. Add approximately 400 mg (analytical balance) vehicle (water) accurately to the bottle and weigh the total mass of the sample. Cap the vial and vortex gently. Incubate for at least 30 minutes at room temperature before further processing. Expiration: prepare freshly.

- Preparation of F/T & LT stability High samples (~200 mg/g)
Weigh at least 100 mg (analytical balance) accurately into a 20 mL glass bottle. Add approximately 400 mg (analytical balance) vehicle (water) accurately to the bottle and weigh the total mass of the sample. Cap the vial, vortex gently and store the samples (≤ -70 °C).

- Preparation of Accuracy/PRS Low samples (~ 1.00 mg/g)
Weigh at least 500 mg (analytical balance) vehicle (water} accurately into a 20 mL glass bottle. Add 25.0 µL (PD pipette) Recovery stock, cap the bottle and vortex gently. Incubate for at least 30 minutes at room temperature before further processing. Expiration: prepare freshly.

- Preparation of F/T & LT stability Low samples (~1.00 mg/g)
Weigh at least 500 mg (analytical balance) vehicle (water) accurately into a 20 mL glass bottle. Add 25.0 µL (PD pipette) Recovery stock, cap the bottle, vortex gently and store the samples (≤ -70 °C).

- Preparation of additional validation samples

Preparation of a Selectivity sample: Weigh at least 500 mg (analytical balance) vehicle (water) accurately into a 20 mL glass bottle. Expiration: prepare freshly.
Preparation of LOD sample (0.250 mg/L): Dispense 3000 µL (multi pipette) MeOH into a 10 mL glass tube. Add 1000 µL (PD pipette) calibration spike solution, cap the tube and vortex at speed 4 to 6 for 0.5 minutes. Expiration: prepare freshly.

- Preparation of solutions
It is allowed to prepare an adjusted amount of a solution, on the condition that the ratio of the chemicals and solvents used for the preparation is not changed.
LC Buffer (50 mM NH4FA in UP/ ACN (95:5): Weigh 3.2 g (balance) NH4FA into a 1 L glass bottle. Add 950 mL (measuring cylinder) UP water and dissolve the salt. Add 50 mL (measuring cylinder) ACN and mix (store at room temperature for a maximum of 90 days).
Needle wash (R0): Dispense 900 mL (measuring cylinder) ACN in a 1 L glass bottle. Add 100 mL (measuring cylinder) UP water. Add 1.00 mL (multi pipette) FA and mix (store at room temperature for a maximum of 90 days).

EQUIPMENT SETTINGS
- Shimadzu HPLC settings
Needle stroke (mm): 50
Sampling speed (µL/sec): 1.0
Cooler enabled: Yes
Cooler temperature (°C): 10
Rinsing speed (µL/sec): 35
Rinsing volume (µL): 200
Rinse mode: After aspiration
Rinse dip time (sec): 2
Column oven temperature (°C): 40
Pumping mode: Ternary Flow
Infection volume (µL): 2.00
Solvent: The ratio of UP water, LC buffer and MeOH (all in %) was 20:10:70, 0:10:90 and 20:10:70 at 0 and 3.20 minutes, 3.30 and 4.50 minutes and 4.60 minutes, respectively.

- DAD settings:
Acquisition time (min): 3.50
Wavelength (nm) acquisition: 525 - 531
Wave step (nm): 1
UV lamp (D2): off
Visible lamp (W): on
Time constant (s): 0.64
Slit width (nm): 1.2
Sampling frequency (Hz): 4.1667
Cell temperature (°C): 40

- MS Settings
Simulation mode

FORMULAE
Response (Y): Peak area test material [cps]
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 [%]

Relative difference (relative diff.): ((Ct – C0) / C0 ) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

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 %. Formulations were considered stable if the relative difference between the stored and freshly taken samples 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 higher than or equal to 0.9999. No response at the retention time of the test material in the analytical blank samples was detected.

- Samples
Procedural recovery samples Accuracy samples: The mean recoveries of the procedural recovery samples fell within the criterion of 90 - 110 %, in exception of the procedural recovery low (1.00 g/L) in run AB001 which was 83.1 %. Owing to the fact that the mean accuracy of the procedural recovery samples at the concentration level 0.300 g/L measured in the same analytical run was 91.5 %, which is within the criteria for the acceptance of the accuracy samples, it was decided to accept the analytical run AB001.
The mean accuracy at the level 0.300 g/L was 91.5 %, which is in the range required for the measurement of the test material in formulations (90 - 110 %). The coefficient of variation of the accuracies is 3.3 %. The coefficient of variation was acceptable and within the limits (≤ 10 %). It demonstrated that the analytical method was adequate for the determination of the test material in the test samples within the analytical range 0.300 to 200 g/L of the test material in formulations.

- Test samples
In the Group 1 and Group 5 formulations, no test material was detected. The concentrations analysed in the formulations of Groups 2, 3, 4 and 6 were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %). The formulations of Group 2, 4 and Group 6 were homogeneous (i.e. coefficient of variation ≤ 10 %). Analysis of Group 2 formulations after storage yielded a relative difference ≤ 10 %. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.

Duration of treatment / exposure:

- Group 1, 2 and 3 males were treated for 31 days, Group 4 males were treated for 14 days and Group 5 and 6 males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
- Group 1, 2 and 3 females were treated for 53-66 days, Group 4 females were treated for 14 days and Group 5 and 6 females were treated for 50-63 days,, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 42 or 43 days. Routinely, females that are littering are left undisturbed. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

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.:

1.5 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

15 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: Based on the results of a 15-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 1.5, 5 and 15 mg/kg.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons

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

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION: 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.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m.
- Animals fasted: Yes, the animals were deprived of food overnight (with a maximum of approximately 24 hours) before blood sampling, but water was available
- The following haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant: White blood cells (WBC, 10^9/L), Differential leucocyte count: Neutrophils (%WBC), Lymphocytes (%WBC), Monocytes (%WBC), Eosinophils (%WBC), Basophils (%WBC), Red blood cells (10^12/L), Reticulocytes (%RBC), Red blood cell distribution width (RDW, %) Haemoglobin (mmol/L), Haematocrit (L/L), Mean corpuscular volume (MCV, fL), Mean corpuscular haemoglobin (MCH, fmol), Mean corpuscular haemoglobin concentration (MCHC, mmol/L) and Platelets (10^9/L).
- The following clotting parameters were determined in plasma prepared with citrate as anticoagulant: Prothrombin Time (PT, s) and Activated Partial Thromboplastin Time (APTT, s).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as for haematology
- Animals fasted: as for haematology
- How many animals: as for haematology
- The following clinical biochemistry parameters were determined using the AU400. All parameters were determined in plasma, except for bile acids which were determined in serum: Alanine aminotransferase (ALAT, U/L), Aspartate aminotransferase (ASAT, U/L), Alkaline Phosphatase (ALP, U/L), Total protein (g/L), Albumin (g/L), Total Bilirubin (μmol/L), Bile acids (μmol/L), Urea (mmol/L), Creatinine (μmol/L), Glucose (mmol/L), Cholesterol (mmol/L), Sodium (mmol/L), Potassium (mmol/L), Chloride (mmol/L), Calcium (mmol/L) and Inorganic Phosphate (Inorg. Phos, mmol/L).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: a selected 5 animals/sex/group
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), and 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 computerized monitoring system). Total movements and ambulations are 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 finer movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No

OTHER: OESTROUS CYCLE DETERMINATION
- Daily vaginal lavage was performed to determine the stage of oestrous beginning 14 days prior to treatment (pre-test), 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 pre-test, this was done for all females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of oestrous.

OTHER: GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females were examined for evidence of abortion or premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

OTHER: THYROID HORMONE ANALYSIS
- Blood samples were taken at the end of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, non-mated females, non-pregnant females and all males after at least 4 weeks of treatment. No samples were collected from animals that were sacrificed in extremis or found dead and females with total litter loss.
- Blood samples were collected 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 (0.9 mL) were drawn from the retroorbital sinus and collected into serum tubes. After clotting and centrifugation, serum was used as listed below.
- Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroid stimulating hormone (TSH).
- Females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).
- Serum samples were stored at ≤ -75 °C. Under these storage conditions, the samples are stable for 6 months. Any remaining samples were discarded.
- Total T4 was measured in serum using the Immulite 1000.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Necropsy was conducted on the following days: Males: Following completion of the mating period (a minimum of 28 days of dose administration), females which delivered: PND 14 to 16, females which failed to deliver: Post-coitum Days 26-27 (females with evidence of mating), spontaneous deaths: As soon as possible after death and always within 24 hours and euthanised in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.
- After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
- Samples of the following tissues and organs were collected and fixed in 10 % buffered formalin:
- For the selected 5 animals/sex/group (see Allocation) and all animals that died spontaneously or were euthanised in extremis: Identification marks: not processed, (nasopharynx), adrenal glands (oesophagus), (aorta), ovaries, brain - cerebellum, mid-brain, cortex (7-levels), (pancreas), Caecum, Peyer's patches [jejunum, ileum] if detectable, Cervix, Pituitary gland, Clitoral gland, Preputial gland, Colon, Prostate gland, Coagulation gland, Rectum, (Cowper’s gland), (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, Eyes (with optic nerve (if detectable) and Harderian gland), Skeletal muscle, (Skin), Mammary gland area, (males and females), Spinal cord -cervical, midthoracic, lumbar, Femur including joint, Spleen, (Glans penis), Sternum with bone marrow, (Levator ani plus bulbocavernosus muscle complex (LABC)), Stomach, Heart, Testes, Ileum, Thymus, Jejunum, Thyroid including parathyroid if detectable, Kidneys, (Tongue), (Lacrimal gland, exorbital), Trachea, (Larynx), Urinary bladder, Liver, Uterus, Lung, infused with formalin, Vagina, Lymph nodes - mandibular, mesenteric and all gross lesions.
- For all remaining animals, males that failed to sire and females which failed to deliver: Identification marks: not processed, Ovaries, Cervix, Preputial gland, Clitoral gland, Prostate gland, Coagulation gland, Seminal vesicles, Cowper’s glands, Testes, Epididymides, Thyroid including parathyroid if detectable, Glans penis, Uterus, Levator ani plus bulbocavernosus muscle complex (LABC), Vagina, Mammary gland area (males and females) and all gross lesions.

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

HISTOPATHOLOGY: Yes
- All organ and tissue samples, as defined under Histopathology were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist:
The preserved organs and tissues of the selected 5 animals/sex of Groups 5 and 6, the additional slides of the testes of the selected 5 males of Groups 5 and 6 and all males that failed to sire, the preserved organs and tissues of the animals of all dose groups which died spontaneously or were euthanised in extremis, all gross lesions of all animals (all dose groups), the trachea, lungs, thymus and kidneys of all selected 5 females of Groups 2 and 3, and of the selected 5 females of the concurrent control Group 1, based on (possible) treatment-related changes in these organs and the reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Clinical signs of toxicity were noted in females at 5 and 15 mg/kg. Two females at 5 mg/kg showed rales, hunched posture, piloerection and a lean appearance on several days during the post-coitum and/or lactation period. At 15 mg/kg, all five females surviving to scheduled sacrifice showed rales during the post-coitum and/or lactation period, and one of these females showed deep respiration on two successive days during the post-coitum period. Rales were also noted in the four 15 mg/kg females which were sacrificed in extremis.
- Salivation noted after dosing at 5 mg/kg (females) and 15 mg/kg (both sexes) was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
- Red discoloration of the faeces (in all animals at 5 and 15 mg/kg) and of the skin (in a few females at 15 mg/kg) was due to the red colour of the test material and considered not toxicologically relevant.
- No additional clinical signs of toxicity were noted during the weekly arena observations. Any other clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- There were five premature decedents in the group of females treated at 15 mg/kg. The moribundity or death of four of these decedents was considered to be related to treatment as explained below.
- Three females were euthanised in extremis early in the post-coitum period (Day 0 or 4 post-coitum) and one female was euthanised in extremis on Day 17 post-coitum. Prior to their early euthanasia, these animals showed clinical signs of toxicity (including rales, gasping, a lean appearance and/or piloerection), body weight loss and reduced food consumption. One female was found dead on Day 3 of the lactation period. Prior to death, she showed neither clinical signs of toxicity nor reduced body weight gain or reduced food consumption. Main macroscopic findings observed for these premature decedents consisted of distension and purple discoloration of the gastrointestinal tract. There were no microscopic correlates to these macroscopic findings. Two females showed swollen lungs, probably representing failure to collapse, caused by (partly) obstructions in the major airways.
- At microscopic examination, main findings were seen in the respiratory tract. Marked chronic obstructive inflammation of the bronchus was seen in two females, and in four females erosion/ ulceration of the trachea and/or bronchial epithelium was noted. The findings in the respiratory tract are considered the main cause of morbidity or death for four females. The nature of the respiratory tract lesions can be related to the treatment procedure (oral gavage), but a relation to the test material cannot be completely excluded. In one female erosion/ulceration was present in vaginal epithelium, which likely attributed to the moribund state of the animal. The vagina erosion/ulceration was regarded to be procedure-related (preparation of vaginal smears) and not test material-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Body weights and body weight gain were reduced in 4/5 surviving females at 15 mg/kg during the lactation period compared to controls. Differences from control values were statistically significant at Day 13 of lactation, when the mean body weight of 15 mg/kg females was 11 % lower. During the premating, mating and post-coitum periods, body weight gain of 15 mg/kg females which survived until sacrifice was normal, except in one female which showed slightly reduced weight gain during the post-coitum period.
- At 5 mg/kg, body weights and body weight gain were normal, except for three females which showed weight loss during a few days of the post-coitum period.
- No treatment-related changes in body weight or body weight gain were observed in males treated up to 15 mg/kg and in females at 1.5 mg/kg.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- At 15 mg/kg, mean food consumption before allowance for body weight was slightly lower for females in the post-coitum and lactation periods, reaching statistical significance at Day 13 of the lactation period when mean food consumption was 18 % lower relative to controls. This was particularly due to the considerably reduced food consumption of a few females. Food consumption after allowance for body weight showed a similar pattern. During the pre-mating and mating period, food consumption of 15 mg/kg females was normal.
- At 5 mg/kg, food consumption before and after allowance for body weight were normal, except for two females which had markedly reduced food consumption between post-coitum Days 0-4. Thereafter, food consumption of these females returned to control values.
- No treatment-related changes in food consumption before or after allowance for body weight were observed in males treated up to 15 mg/kg and in females at 1.5 mg/kg.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- Haematology parameters were considered not to be adversely affected by treatment.
- Statistically significant higher mean number of white blood cells (WBC) were noted in females treated at 15 mg/kg, which was mainly caused by one female. In absence of any other WBC parameters and as all numbers remained within the range of available historical control data, this increase in number of WBC was considered not toxicologically relevant.
- In addition, females at 15 mg/kg had statistically significantly lower mean corpuscular haemoglobin concentration (MCHC). In the absence of changes in other red blood cell parameters, particularly haemoglobin concentration and number of red blood cells, slight decrease in MCHC was considered not toxicologically relevant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- No clear treatment-related changes in clinical biochemistry parameters were noted.
- On an individual basis, abnormal values were noted for one female no. 118. This female showed a low total protein, albumin and chloride level and a high urea, creatinine, cholesterol and inorganic phosphate level, which were all outside the range considered normal for rats of this age and strain. This resulted in statistically significantly lower mean chloride (8 % relative difference) and higher mean inorganic phosphate levels (53 % relative difference), compared to concurrent controls. The values of the other females at 15 mg/kg were within the normal range.
- Other statistically significant variations noted in clinical biochemistry parameters were considered to be unrelated to treatment as they occurred in the absence of a dose-related distribution and remained within the range considered normal for rats of this strain and age.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
- Motor activity data showed a similar habituation profile with a decreasing trend in activity over the duration of the test period for all groups. Findings of note were lower mean values for total movements and ambulations in males at 15 mg/kg (relative differences from controls: 42 and 28 %, respectively). Statistical significance was not achieved and there were no corroborative clinical signs or changes in other measures in the neuromuscular domain (including gait, air righting reflex and grip strength). Therefore, these changes in motor activity were considered not to reflect impaired neuromuscular function. Apparently lower mean values for total movements and ambulations in females at 15 mg/kg were due to high concurrent (Group 5) control means (particularly due to relatively high values in one control female). Values in 15 mg/kg females were comparable to those in females of control Group 1.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related alterations in organ weights.
- The statistically reduced thyroid gland and ovary weight (absolute only) in females at 15 mg/kg (relative differences of 20 and 16 %, respectively) were regarded to be related to the reduced body weight and not related to the test material.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related gross observations at the end of the scheduled treatment period consisted of purple discoloration of the stomach in 2/10 males at 15 mg/kg and distension with gas of the intestinal tract in 2/5 surviving females at 15 mg/kg. There was no microscopic correlate to these findings.
- The remainder of the recorded macroscopic findings in the animals surviving until scheduled sacrifice were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- There were no test material-related microscopic observations in male rats.
- In females surviving until scheduled sacrifice, test material-related microscopic findings were noted in the thymus and kidneys at 15 mg/kg. In the thymus, lymphoid atrophy was recorded in 3/5 females at 15 mg/kg, at minimal-moderate degree. In the kidneys, tubular basophilia was recorded at an increased incidence and severity in females at 15 mg/kg (4/5 females, at minimal-slight degree). A background level of tubular basophilia was recorded in the remaining dose groups including controls (up to 2/5 females at a minimal degree). These findings in the kidney are possibly related to elevated clinical biochemistry kidney biomarkers observed in one single female at 15 mg/kg.
- In addition, microscopic findings of note at the end of the treatment period were present in the respiratory tract (lung and trachea) of 3/5 surviving females at 15 mg/kg, and resembled the findings in the premature deaths at 15 mg/kg. These microscopic findings consisted of a combination of the following findings: In the lungs, erosion/ulceration of the epithelium of the main stem bronchi was recorded in 1/5 females (slight degree) and inflammation of the bronchus was recorded in 2/5 females (slight degree), in one animal combined with alveolar/bronchial basophilic contents (slight). In the trachea, erosion/ulceration was recorded in 2/5 females (minimal, moderate degree) and attenuated epithelium was recorded in 1/5 females (slight degree).
- The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test 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):

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

- Reproductive performance: There were 1/10 couple treated at 1.5 mg/kg and 1/10 couple of control Group 5 that were not pregnant. Histopathology did not reveal any changes in the reproductive organs that could explain this. At 15 mg/kg, 4/10 females were sacrificed in extremis post-coitum and one female of this dose group was found dead on Day 3 of lactation. Histopathologic examination revealed erosion/ulceration of the epithelium of the vagina of one female, which was sacrificed on Day 0 postcoitum. This finding was most likely procedure-related (daily of vaginal lavage). There were no other changes in the reproductive organs of these animals. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test material and stage aware evaluation of the testes did not show any indication of abnormal spermatogenesis.

- Oestrous Cycle: Length and regularity of the oestrous cycle were not affected by treatment. Most females had regular cycles of 4 to 5 days. Extended di-oestrus during mating occurred in two control females and another control female had an irregular cycle. One female at 15 mg/kg was acyclic during the last week of the pre-mating period and during mating. The control females had healthy offspring. The pregnancy status of the 15 mg/kg female could not be determined as she was sacrificed on Day 0 post-coitum. The incidence of these findings was within normal limits and showed no dose-related trend (the abnormalities were mostly noted in control females). The 15 mg/kg female had erosion/ulceration in the vagina which was most likely procedure-related (preparation of vaginal lavage) and not test material-related.
- Mating index: Mating index was not affected by treatment. All females (including decedents) showed evidence of mating.
- Precoital time: Precoital time was not affected by treatment. All females, except for control females, showed evidence of mating within 5 days. These two control females were mated after 10 or 14 days of cohabitation.
- Number of implantation sites: Number of implantation sites was not affected by treatment
- Fertility index: Fertility index for females surviving until scheduled sacrifice was not affected by treatment. Except for one control female and one female at 1.5 mg/kg, all mated surviving females were pregnant. The significantly lower fertility index at 15 mg/kg (60 %) was due to the high incidence of females sacrificed during the post-coitum period due to maternal toxicity and was not related to a reproductive effect. Pregnancy could not be determined for three females at 15 mg/kg that were sacrificed in extremis on Days 0 or 4 post-coitum. No implantation sites or corpora lutea were detectable at necropsy, which was due to the sacrifice in the early beginning of the post-coitum period, i.e. before implantation occurred. Except for one female which had erosion/ulceration of the epithelium of the vagina, no histopathological changes in reproductive organs were observed.

DEVELOPMENTAL DATA
- Gestation index and duration of gestation for females surviving until scheduled sacrifice were not affected by treatment. All pregnant females (except for females sacrificed during the post-coitum period) delivered live offspring.
- No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- Post-implantation survival index: The total number of offspring born compared to the total number of uterine implantation sites in females surviving until scheduled sacrifice was not affected by treatment. The postimplantation survival indices across the groups were 88 to 98 %.
For three control females, and for one female at 1.5 mg/kg, and one at 5 mg/kg, the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in this study.
- Litter size was not affected by treatment.
- Live birth index: The number of live offspring on Day 1 after littering compared to total number of offspring born for females surviving until scheduled sacrifice was not affected by treatment. The live birth indices across the groups were 96 to 100 %. At first litter check, one pup of control Group 1, one pup at 5 mg/kg, four pups of control Group 5 and one pup at 150 mg/kg were found dead. This pup mortality was considered to be unrelated to treatment because themortality incidence showed no dose-related trend and remained within normal limits.
- Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring born for females surviving until scheduled sacrifice was not affected by treatment. The viability indices across the groups were 97 to 100 %. One control pup and one pup at 1.5 mg/kg was found dead on PND 2 or 4. Two control pups, one pup at 5 mg/kg and one pup at 15 mg/kg went missing at PND 2 or 4 and were most likely cannibalised. This post-natal loss was considered to be unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age. All pups of one female at 15 mg/kg were sacrificed on PND 3 as their dam was found dead on Day 3 of lactation.
- Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) for females surviving until scheduled sacrifice was considered not to be affected by treatment. The lactation index was 95 % at 15 mg/kg and 100 % in the other groups. The mortality at 15 mg/kg (two pups of a litter went missing on PND 9 or 10) remained within the range considered normal for pups of this age.
- No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore regarded as unrelated to treatment.
- At PND 13, mean body weights of pups at 15 mg/kg were 12 % (males) or 11 % (females) lower relative to controls but statistical significance was not achieved. On an individual litter basis, pup body weight gain was considerably reduced in 2/5 litters at 15 mg/kg. No treatment-related changes in pup body weight gain were noted at the lower dose levels.
- Sex ratio was not affected by treatment.
-Anogenital distance (absolute and normalised for body weight) in male and female pups was considered not to be affected by treatment. Relative to control Group 5, female pups at 15 mg/kg showed a slight (10 %), but statistically significantly lower mean normalised anogenital distance. As values in 15 mg/kg female pups were similar to those in female pups of control Group 1, this finding was considered to be unrelated to treatment.
- Treatment up to 15 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 13-15 pups showed no changes that were considered to be toxicologically relevant. Statistically significantly higher mean T4 levels were noted in male and female pups at 15 mg/kg (relative differences from controls: 26 and 51 %, respectively). Values at 15 mg/kg remained well within normal limits. Moreover, lower rather than higher T4 levels are expected in case of compromised thyroid function. Therefore, the higher T4 levels at 15 mg/kg were considered non-adverse.
- No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of the macroscopic findings noted incidentally remained within the
range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

1.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

respiratory system: lower respiratory tract

Organ:

lungs

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Due to a formulation error, the animals of Group 4 were dosed once (Day 13 pre-mating) at a dose level of 150 mg/kg instead of at the target dose level of 15 mg/kg. As a result, severe toxicity was observed in these animals. In consultation with the Sponsor, all Group 4 animals were sacrificed without any further examination on Day 15 of the pre-mating period, and additional control and 15 mg/kg groups (Groups 5 and 6, respectively) were included in this study. The recorded animal data for Group 4 was not included in the toxicological evaluation.

Conclusions:

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is 1.5 mg/kg/day.

Executive summary:

The repeated dose oral toxicity of the test material was determined in accordance with the standardised guidelines OECD 422, and US EPA OPPTS 870.3650 under GLP conditions in a combined 28-day oral (gavage) 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 test material, formulated in water, was administered daily by oral gavage to SPF-bred Wistar Han rats at doses of 0, 1.5, 5 and 15 mg/kg. Each group consisted of 10 males and 10 females. Due to a formulation error, the animals of the original high dose were dosed once, on day 13 of pre-mating, at a dose level of 150 mg/kg instead of at the target dose level of 15 mg/kg. As a result, severe toxicity was observed in these animals. It was therefore decided to sacrifice all animals of this group without any further examination on day 15 of the pre-mating period, and additional control and 15 mg/kg/day groups were included in the study. The recorded animal data for the original high dose group were not included in the toxicological evaluation.

Males were treated for 2 weeks prior to mating, during mating, and up to termination (Groups 1-3 for 31 days, Groups 5-6 for 29 days). The females that delivered were treated for 2 weeks prior to mating, during mating, during pregnancy, and at least 13-15 days of lactation (for 50-66 days). Females which failed to deliver healthy offspring were treated for 42 or 43 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. 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 weight, anogenital distance, areola/nipple retention and macroscopy). Formulations were analysed once during the study to assess accuracy and homogeneity and stability.

Formulation analysis showed that the formulations were prepared accurately and homogenously, and confirmed stability (at 1.5 mg/kg) upon storage for at least 5 hours at room temperature under normal laboratory light conditions.

At 15 mg/kg/day, three females were euthanized in extremis early in the post-coitum period (day 0 or 4 post-coitum) and one female was euthanized in extremis on day 17 post-coitum. One female was found dead on Day 3 of lactation. Main cause of death/moribundity of three out of the four females euthanized in extremis were effects in the respiratory tract which, according to the study director, were treatment-related. For the remaining female sacrificed in extremis during the post-coitum period, necropsy revealed erosion/ulceration in the vaginal epithelium, which the study director attributed to the preparation of vaginal smears and the moribund state of the animal. Prior to their early euthanasia, the three animals showed clinical signs of toxicity including rales, gasping, a lean appearance and/or piloerection, body weight loss and reduced food consumption. At necropsy, swollen lungs were noted for two of these decedents, probably representing failure to collapse, caused by obstructions in the major airways. At microscopic examination, erosion/ulceration and/or (obstructive) inflammation of the bronchi were noted. In addition, erosion/ulceration or inflammation of the trachea and/or lung was also noted in three of the surviving females at 15 mg/kg/day. These respiratory tract findings were regarded to be related to the treatment procedure (oral gavage) and unlikely related to the test material. The study director however, noted that these findings were only recorded for the 15 mg/kg/day treated females, were not seen in any of the females of the remaining dose groups and were not recorded in male rats and on this basis concluded that although a relation to the test material is very unlikely, this cannot be completely excluded. According to the certificate of analysis included in the report and physical-chemical properties, the tested substance has a pH value of 3.95; the dosing solution also had a likely pH value of 3-4. In addition, the substance causes severe, non-reversible eye damage when testedin vivo. It is therefore considered that any of the dosing solution reaching the respiratory tract would be highly irritating. The observation of erosions and ulcerations in the respiratory tract, as well as in the trachea, strongly suggest that these lesions were due to sub-optimal gavage dosing, with some of the dosing solution reaching the trachea or even the respiratory tract rather than any specific target organ toxicity systemically mediated. This is further supported by available additional data for the animals of the original high dose group, and information from the range-finding study. Only one animal of the original high dose group, out of 10 animals/sex, showed a respiratory symptom (rales) which, in contrast to the group on study, was seen pre-mating and did not require the animal to be terminated prior to day 13, when the whole group were erroneously treated at 10x the target dose. Some respiratory symptoms were also noted in the range finding study conducted in groups of 3 females each, much earlier than the post-coital timing of any respiratory effects in the main study. One female at 30 mg/kg/day showed rales, laboured respiration and gasping on days 6-9 and was sacrificed on day 10; another female treated at 100 mg/kg/day showed gasping and squeaking on day 5. Given that these dose levels were not tolerated, and all animals treated at 30 mg/kg/day were sacrificed on day 10 while the entire group treated at 100 mg/kg/day was sacrificed on day 5 or 7, these signs could readily be attributed to excessive systemic toxicity. As already mentioned, during the post-coitum and/or lactation periods, clinical signs were observed in the surviving females at 15 mg/kg/day which showed mainly rales, and in two females at 5 mg/kg/day which showed rales, hunched posture, piloerection and a lean appearance. Moreover, reduced body weight gain and food consumption were observed in females treated at 15 mg/kg/day during the lactation phase. Mean body weights and food consumption at scheduled sacrifice were respectively 11% and 18% lower compared to controls. At 5 mg/kg/day, the two same females that showed clinical signs had body weight loss accompanied by reduced food consumption during the first four days of the post-coitum period. The clinical signs observed, in combination with the dose-related reduced body weight gain or body weight loss and reduced food consumption are considered to be adverse.

Further treatment-related findings at 15 mg/kg/day included macroscopic findings in the stomach in 2 males, consisting of purple discoloration (without microscopic correlate) and in two surviving females the intestinal tract was discoloured and/or distended with gas (without microscopic correlate). Test material-related microscopic alterations noted in females at 15 mg/kg/day consisted of lymphoid atrophy in the thymus and a slightly increased incidence and severity of tubular basophilia in the kidneys. The findings in the kidney are possibly related to elevated clinical biochemistry kidney biomarkers in one individual female at 15 mg/kg. These findings were considered to be non-adverse.

No parental toxicity was observed in males treated up to 15 mg/kg/day and females treated at 1.5 mg/kg/day.

The study director concluded that under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is 1.5 mg/kg/day. This is considered to be a NOEL (no-effect-level i.e. no difference from control) rather than a NOAEL (no–adverse-effect-level). As effects at 5 mg/kg/day were limited to rales, hunched posture, piloerection and a lean appearance on some days during the post-coitum and/or lactation period in two females, generally slight in degree, and transient effects on body weight gain and food consumption again only affecting 2 or 3 animals at most, 5 mg/kg/day is considered to be the NOAEL for both systemic toxicity and local effects.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2016 to 13 April 2017

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:

other: OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Principles of method if other than guideline:

In this study, the animals of Group 4 were dosed erroneously on a single occasion (Day 13 pre-mating) at a dose level of 150 mg/kg instead of the target high dose level of 15 mg/kg. As a result, severe toxicity was observed in these animals and it was considered appropriate that all remaining Group 4 animals were sacrificed without any further examination on Day 15 of the pre-mating period and additional control and 15 mg/kg group (Groups 5 and 6, respectively) were included in this study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (outbred, SPF-quality)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: At the start of the pre- test females were approximately 11 weeks; at the start of F0-treatment males were approximately 10 weeks and females approximately 13 weeks.
- Weight at study initiation: Weight at start of pre- mating: Females: 206 to 247 g, Males: 278 to 315 g.
- Fasting period before study: No
- Housing: All cages were 18 cm high. During the pre-test females and during pre-mating animals were housed in groups of 5 animals/sex/cage in plastic cages. During mating males and females were cohabitated on a 1:1 basis in plastic cages. Post-mating males were housed in their home cage with a maximum of 5 animals/cage and females were individually housed in plastic cages. During lactation females were housed in plastic cages. Sterilised sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: Pelleted rodent diet ad libitum, except during motor activity measurements animals did not have access to food for a maximum of 1 hour 25 minutes.
- Water: ad libitum, except during motor activity measurements animals did not have access to water for a maximum of 1 hour 25 minutes.
- Acclimation period: At least 5 days prior to the start of pre-test (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C (actual daily mean was 22 °C)
- Humidity: 40 to 70 % (actual daily mean was 44 to 53 %)
- Air changes: at least 10 room air changes/hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES:
- From: Not specified
- To: 13 April 2017

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. No adjustment was made for specific gravity/density of the test material, vehicle, and/or formulation. No correction was made for the purity/composition of the test material. The formulations containing the test material were red solutions. The formulation was stored at room temperature and placed on a magnetic stirrer during dosing.

DOSE VOLUME: 5 mL/ kg bw

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on trial formulations performed in the testing facility.
- Concentration in vehicle: Test material concentrations 0.3, 1, 3 and 3 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The purpose of the analytical phase was to determine the accuracy of preparation, homogeneity and stability of the formulations.

SAMPLING
In total, 26 samples were included in this study, distributed over 6 dose groups. Group 1 and 5 were the vehicle control groups, Groups 2, 3, 4 and 6 were dosed at 1.5, 5, 15 and 15 mg/kg bw/day at a dose volume of 5 mL/kg bw, respectively. The samples of the control Group 1, 3 and Group 5 were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment Groups 2, 4 and 6 were taken in duplicate from the top, middle and bottom position of the container and immediately stored on dry ice. In addition, stability samples were taken from formulation Group 2 in duplicate at the middle position of the container. The stability samples were stored for 5 hours at room temperature under normal laboratory light conditions and then stored on dry ice.

ANALYTICAL CONDITIONS
The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.
Formulations containing the test material were dissolved in methanol and transferred to volumetric flasks. These solutions were further diluted depending on their concentrations and injected. Chromatographic separation was performed on an Xbridge C18 column using isocratic elution. A diode array detector was used for quantification.

- Preparation of Calibration solutions (stocks, ~ 1000 mg/L)
Weigh accurately an amount of approximately 10.0 mg directly into a 25 mL amber glass bottle using an analytical balance. Add accurately 10.0 mL (multi pipette/PD pipette) MeOH. Cap the bottle and dissolve the substance by shaking/vortexing manually. Store the solution in a 25 mL amber glass bottle at 2 to 8 °C (expiration 287 hours).

- Preparation of BT stability samples, ~ 1000 mg/L
Transfer an aliquot of the calibration stocks into 12 mL amber glass bottles. Store the samples at room temperature for at least 12 hours. After completion return the samples to a refrigerator (2 to 8 °C, expiration 22 hours).

- Preparation of calibration spike solutions and stock stability solutions
Prepare solutions according to set volumes, dispensing the correct volume of MeOH (multi pipette) into 10 mL glass tubes. Add the stock solution (PD pipette) to the tubes, cap and vortex the tubes at speed 4 to 6 for 0.5 minutes. Expiration: prepare freshly.

- Preparation of a Recovery stock (20 000 mg/L)
Weigh accurately an amount of approximately 20.0 mg (analytical balance) directly into a 12 mL amber glass bottle. Add accurately 1.00 mL (multi pipette/PD pipette) MeOH. Cap the bottle and dissolve the substance by shaking/vortexing manually. Store the solution in a 25 mL amber glass bottle at 2 to 8 °C (expiration 287 hours).

- Preparation of Accuracy/PRS High samples (~ 200 mg/g)
Weigh at least 100 mg (analytical balance) accurately into a 20 mL glass bottle. Add approximately 400 mg (analytical balance) vehicle (water) accurately to the bottle and weigh the total mass of the sample. Cap the vial and vortex gently. Incubate for at least 30 minutes at room temperature before further processing. Expiration: prepare freshly.

- Preparation of F/T & LT stability High samples (~200 mg/g)
Weigh at least 100 mg (analytical balance) accurately into a 20 mL glass bottle. Add approximately 400 mg (analytical balance) vehicle (water) accurately to the bottle and weigh the total mass of the sample. Cap the vial, vortex gently and store the samples (≤ -70 °C).

- Preparation of Accuracy/PRS Low samples (~ 1.00 mg/g)
Weigh at least 500 mg (analytical balance) vehicle (water} accurately into a 20 mL glass bottle. Add 25.0 µL (PD pipette) Recovery stock, cap the bottle and vortex gently. Incubate for at least 30 minutes at room temperature before further processing. Expiration: prepare freshly.

- Preparation of F/T & LT stability Low samples (~1.00 mg/g)
Weigh at least 500 mg (analytical balance) vehicle (water) accurately into a 20 mL glass bottle. Add 25.0 µL (PD pipette) Recovery stock, cap the bottle, vortex gently and store the samples (≤ -70 °C).

- Preparation of additional validation samples

Preparation of a Selectivity sample: Weigh at least 500 mg (analytical balance) vehicle (water) accurately into a 20 mL glass bottle. Expiration: prepare freshly.
Preparation of LOD sample (0.250 mg/L): Dispense 3000 µL (multi pipette) MeOH into a 10 mL glass tube. Add 1000 µL (PD pipette) calibration spike solution, cap the tube and vortex at speed 4 to 6 for 0.5 minutes. Expiration: prepare freshly.

- Preparation of solutions
It is allowed to prepare an adjusted amount of a solution, on the condition that the ratio of the chemicals and solvents used for the preparation is not changed.
LC Buffer (50 mM NH4FA in UP/ ACN (95:5): Weigh 3.2 g (balance) NH4FA into a 1 L glass bottle. Add 950 mL (measuring cylinder) UP water and dissolve the salt. Add 50 mL (measuring cylinder) ACN and mix (store at room temperature for a maximum of 90 days).
Needle wash (R0): Dispense 900 mL (measuring cylinder) ACN in a 1 L glass bottle. Add 100 mL (measuring cylinder) UP water. Add 1.00 mL (multi pipette) FA and mix (store at room temperature for a maximum of 90 days).

EQUIPMENT SETTINGS
- Shimadzu HPLC settings
Needle stroke (mm): 50
Sampling speed (µL/sec): 1.0
Cooler enabled: Yes
Cooler temperature (°C): 10
Rinsing speed (µL/sec): 35
Rinsing volume (µL): 200
Rinse mode: After aspiration
Rinse dip time (sec): 2
Column oven temperature (°C): 40
Pumping mode: Ternary Flow
Infection volume (µL): 2.00
Solvent: The ratio of UP water, LC buffer and MeOH (all in %) was 20:10:70, 0:10:90 and 20:10:70 at 0 and 3.20 minutes, 3.30 and 4.50 minutes and 4.60 minutes, respectively.

- DAD settings:
Acquisition time (min): 3.50
Wavelength (nm) acquisition: 525 - 531
Wave step (nm): 1
UV lamp (D2): off
Visible lamp (W): on
Time constant (s): 0.64
Slit width (nm): 1.2
Sampling frequency (Hz): 4.1667
Cell temperature (°C): 40

- MS Settings
Simulation mode

FORMULAE
Response (Y): Peak area test material [cps]
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 [%]

Relative difference (relative diff.): ((Ct – C0) / C0 ) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

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 %. Formulations were considered stable if the relative difference between the stored and freshly taken samples 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 higher than or equal to 0.9999. No response at the retention time of the test material in the analytical blank samples was detected.

- Samples
Procedural recovery samples Accuracy samples: The mean recoveries of the procedural recovery samples fell within the criterion of 90 - 110 %, in exception of the procedural recovery low (1.00 g/L) in run AB001 which was 83.1 %. Owing to the fact that the mean accuracy of the procedural recovery samples at the concentration level 0.300 g/L measured in the same analytical run was 91.5 %, which is within the criteria for the acceptance of the accuracy samples, it was decided to accept the analytical run AB001.
The mean accuracy at the level 0.300 g/L was 91.5 %, which is in the range required for the measurement of the test material in formulations (90 - 110 %). The coefficient of variation of the accuracies is 3.3 %. The coefficient of variation was acceptable and within the limits (≤ 10 %). It demonstrated that the analytical method was adequate for the determination of the test material in the test samples within the analytical range 0.300 to 200 g/L of the test material in formulations.

- Test samples
In the Group 1 and Group 5 formulations, no test material was detected. The concentrations analysed in the formulations of Groups 2, 3, 4 and 6 were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %). The formulations of Group 2, 4 and Group 6 were homogeneous (i.e. coefficient of variation ≤ 10 %). Analysis of Group 2 formulations after storage yielded a relative difference ≤ 10 %. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: Evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. Referred to as day 0 (p.c.) of pregnancy
- After successful mating each pregnant female was caged individually. Males were housed in their home cage with a maximum of 5 animals/cage.

Duration of treatment / exposure:

- Group 1, 2 and 3 males were treated for 31 days, Group 4 males were treated for 14 days and Group 5 and 6 males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
- Group 1, 2 and 3 females were treated for 53-66 days, Group 4 females were treated for 14 days and Group 5 and 6 females were treated for 50-63 days,, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 42 or 43 days. Routinely, females that are littering are left undisturbed. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
- Pups were not treated directly but were potentially exposed to the test material in utero, via maternal milk or from exposure to maternal urine/feces.

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.:

1.5 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

15 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: Based on the results of a 15-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 1.5, 5 and 15 mg/kg.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons

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

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION: Yes
- Weekly, except for females which were housed together with males for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m.
- Animals fasted: Yes, the animals were deprived of food overnight (with a maximum of approximately 24 hours) before blood sampling, but water was available
- The following haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant: White blood cells (WBC, 10^9/L), Differential leucocyte count: Neutrophils (%WBC), Lymphocytes (%WBC), Monocytes (%WBC), Eosinophils (%WBC), Basophils (%WBC), Red blood cells (10^12/L), Reticulocytes (%RBC), Red blood cell distribution width (RDW, %) Haemoglobin (mmol/L), Haematocrit (L/L), Mean corpuscular volume (MCV, fL), Mean corpuscular haemoglobin (MCH, fmol), Mean corpuscular haemoglobin concentration (MCHC, mmol/L) and Platelets (10^9/L).
- The following clotting parameters were determined in plasma prepared with citrate as anticoagulant: Prothrombin Time (PT, s) and Activated Partial Thromboplastin Time (APTT, s).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as for haematology
- Animals fasted: as for haematology
- How many animals: as for haematology
- The following clinical biochemistry parameters were determined using the AU400. All parameters were determined in plasma, except for bile acids which were determined in serum: Alanine aminotransferase (ALAT, U/L), Aspartate aminotransferase (ASAT, U/L), Alkaline Phosphatase (ALP, U/L), Total protein (g/L), Albumin (g/L), Total Bilirubin (μmol/L), Bile acids (μmol/L), Urea (mmol/L), Creatinine (μmol/L), Glucose (mmol/L), Cholesterol (mmol/L), Sodium (mmol/L), Potassium (mmol/L), Chloride (mmol/L), Calcium (mmol/L) and Inorganic Phosphate (Inorg. Phos, mmol/L).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: a selected 5 animals/sex/group
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), and 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 computerized monitoring system). Total movements and ambulations are 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 finer movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No

OTHER: GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females were examined for evidence of abortion or premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

OTHER: THYROID HORMONE ANALYSIS
- Blood samples were taken at the end of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, non-mated females and non-pregnant females after at least 4 weeks of treatment. No samples were collected from animals that were sacrificed in extremis or found dead and females with total litter loss.
- Blood samples were collected 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 (0.9 mL) were drawn from the retroorbital sinus and collected into serum tubes. After clotting and centrifugation, serum was used as listed below.
- Females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).
- Serum samples were stored at ≤ -75 °C. Under these storage conditions, the samples are stable for 6 months. Any remaining samples were discarded.

GROSS PATHOLOGY: Yes
- Necropsy was conducted on the following days: Females which delivered: PND 14 to 16, females which failed to deliver: Post-coitum Days 26-27 (females with evidence of mating), spontaneous deaths: As soon as possible after death and always within 24 hours and euthanised in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.
- After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
- Samples of the following tissues and organs were collected and fixed in 10 % buffered formalin:
- For the selected 5 animals/sex/group and all animals that died spontaneously or were euthanised in extremis: Identification marks: not processed, (nasopharynx), adrenal glands (oesophagus), (aorta), ovaries, brain - cerebellum, mid-brain, cortex (7-levels), (pancreas), Caecum, Peyer's patches [jejunum, ileum] if detectable, Cervix, Pituitary gland, Clitoral gland, Preputial gland, Colon, Rectum, (Cowper’s gland), (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Eyes (with optic nerve (if detectable) and Harderian gland), Skeletal muscle, (Skin), Mammary gland area, Spinal cord -cervical, midthoracic, lumbar, Femur including joint, Spleen, Sternum with bone marrow, (Levator ani plus bulbocavernosus muscle complex (LABC)), Stomach, Heart, Ileum, Thymus, Jejunum, Thyroid including parathyroid if detectable, Kidneys, (Tongue), (Lacrimal gland, exorbital), Trachea, (Larynx), Urinary bladder, Liver, Uterus, Lung, infused with formalin, Vagina, Lymph nodes - mandibular, mesenteric and all gross lesions.
- For all remaining animals and females which failed to deliver: Identification marks: not processed, Ovaries, Cervix, Preputial gland, Clitoral gland, Thyroid including parathyroid if detectable, Uterus, Levator ani plus bulbocavernosus muscle complex (LABC), Vagina, Mammary gland area and all gross lesions.

- The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
- Selected 5 animals/sex/group: Adrenal glands, Brain, Heart, Kidneys, Liver, Ovaries, Spleen, Thymus, Thyroid (including parathyroid if detectable), Uterus (including cervix).
- All remaining animals: Thyroid.
- Absolute organ weights and organ to body weight ratios are reported.

HISTOPATHOLOGY: Yes
- All organ and tissue samples, as defined under Histopathology were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin.
- The following slides were examined by a pathologist:
The preserved organs and tissues of the selected 5 animals/sex of Groups 5 and 6, the preserved organs and tissues of the animals of all dose groups which died spontaneously or were euthanised in extremis, all gross lesions of all animals (all dose groups), the trachea, lungs, thymus and kidneys of all selected 5 females of Groups 2 and 3, and of the selected 5 females of the concurrent control Group 1, based on (possible) treatment-related changes in these organs.

Ovaries and uterine content:

- The ovaries and uterine content was examined after termination: Yes
- Examinations included:
- Gravid uterus weight: Yes
- Number of implantations: Yes
- Descriptions of all macroscopic abnormalities were recorded.
- The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

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.
- 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 normalised 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.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

HORMONE ANALYSIS
- PND 4 pups: From 2 surplus pups per litter at culling (if possible). If only 1 surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. Blood samples were collected by decapitation, between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter (0.4 mL in total) were collected into one serum tube for possible future measurement of thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤- 75 °C. Under these storage conditions, the samples are stable for 6 months. Any remaining samples were discarded.
- PND 13-15 pups: From 2 pups per litter (if possible from one male and one female) at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane, between 7.00 and 10.30 a.m. Blood was collected into serum tubes. After clotting and centrifugation, serum from each sample was divided into 2 aliquots: 150 μL serum for measurement of thyroxine (T4) and the remaining volume of serum for possible future measurement of thyroid-stimulating hormone (TSH). Serum samples were stored at ≤ -75 °C. Under these storage conditions, the samples are stable for 6 months. Any remaining samples were discarded.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- 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.

Indices:

- Mating index (%)= (Number of females mated/ Number of females paired) x 100
- 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
- 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
- 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
- Group mean values were calculated from individual litter values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Clinical signs of toxicity were noted in females at 5 and 15 mg/kg. Two females at 5 mg/kg showed rales, hunched posture, piloerection and a lean appearance on several days during the post-coitum and/or lactation period. At 15 mg/kg, all five females surviving to scheduled sacrifice showed rales during the post-coitum and/or lactation period, and one of these females showed deep respiration on two successive days during the post-coitum period. Rales were also noted in the four 15 mg/kg females which were sacrificed in extremis.
- Salivation noted after dosing at 5 mg/kg (females) and 15 mg/kg (both sexes) was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
- Red discoloration of the faeces (in all animals at 5 and 15 mg/kg) and of the skin (in a few females at 15 mg/kg) was due to the red colour of the test material and considered not toxicologically relevant.
- No additional clinical signs of toxicity were noted during the weekly arena observations. Any other clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- There were five premature decedents in the group of females treated at 15 mg/kg. The moribundity or death of four of these decedents was considered to be related to treatment as explained below.
- Three females were euthanised in extremis early in the post-coitum period (Day 0 or 4 post-coitum) and one female was euthanised in extremis on Day 17 post-coitum. Prior to their early euthanasia, these animals showed clinical signs of toxicity (including rales, gasping, a lean appearance and/or piloerection), body weight loss and reduced food consumption. One female was found dead on Day 3 of the lactation period. Prior to death, she showed neither clinical signs of toxicity nor reduced body weight gain or reduced food consumption. Main macroscopic findings observed for these premature decedents consisted of distension and purple discoloration of the gastrointestinal tract. There were no microscopic correlates to these macroscopic findings. Two females showed swollen lungs, probably representing failure to collapse, caused by (partly) obstructions in the major airways.
- At microscopic examination, main findings were seen in the respiratory tract. Marked chronic obstructive inflammation of the bronchus was seen in two females, and in four females erosion/ ulceration of the trachea and/or bronchial epithelium was noted. The findings in the respiratory tract are considered the main cause of morbidity or death for four females. The nature of the respiratory tract lesions can be related to the treatment procedure (oral gavage), but a relation to the test material cannot be completely excluded. In one female erosion/ulceration was present in vaginal epithelium, which likely attributed to the moribund state of the animal. The vagina erosion/ulceration was regarded to be procedure-related (preparation of vaginal smears) and not test material-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Body weights and body weight gain were reduced in 4/5 surviving females at 15 mg/kg during the lactation period compared to controls. Differences from control values were statistically significant at Day 13 of lactation, when the mean body weight of 15 mg/kg females was 11 % lower. During the premating, mating and post-coitum periods, body weight gain of 15 mg/kg females which survived until sacrifice was normal, except in one female which showed slightly reduced weight gain during the post-coitum period.
- At 5 mg/kg, body weights and body weight gain were normal, except for three females which showed weight loss during a few days of the post-coitum period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- At 15 mg/kg, mean food consumption before allowance for body weight was slightly lower for females in the post-coitum and lactation periods, reaching statistical significance at Day 13 of the lactation period when mean food consumption was 18 % lower relative to controls. This was particularly due to the considerably reduced food consumption of a few females. Food consumption after allowance for body weight showed a similar pattern. During the pre-mating and mating period, food consumption of 15 mg/kg females was normal.
- At 5 mg/kg, food consumption before and after allowance for body weight were normal, except for two females which had markedly reduced food consumption between post-coitum Days 0-4. Thereafter, food consumption of these females returned to control values.
- No treatment-related changes in food consumption before or after allowance for body weight were observed in males treated up to 15 mg/kg and in females at 1.5 mg/kg.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
- Motor activity data showed a similar habituation profile with a decreasing trend in activity over the duration of the test period for all groups. Findings of note were lower mean values for total movements and ambulations in males at 15 mg/kg (relative differences from controls: 42 and 28 %, respectively). Statistical significance was not achieved and there were no corroborative clinical signs or changes in other measures in the neuromuscular domain (including gait, air righting reflex and grip strength). Therefore, these changes in motor activity were considered not to reflect impaired neuromuscular function. Apparently lower mean values for total movements and ambulations in females at 15 mg/kg were due to high concurrent (Group 5) control means (particularly due to relatively high values in one control female). Values in 15 mg/kg females were comparable to those in females of control Group 1.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related alterations in organ weights.
- The statistically reduced thyroid gland and ovary weight (absolute only) in females at 15 mg/kg (relative differences of 20 and 16 %, respectively) were regarded to be related to the reduced body weight and not related to the test material.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related gross observations at the end of the scheduled treatment period consisted of distension with gas of the intestinal tract in 2/5 surviving females at 15 mg/kg. There was no microscopic correlate to these findings.
- The remainder of the recorded macroscopic findings in the animals surviving until scheduled sacrifice were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- In females surviving until scheduled sacrifice, test material-related microscopic findings were noted in the thymus and kidneys at 15 mg/kg. In the thymus, lymphoid atrophy was recorded in 3/5 females at 15 mg/kg, at minimal-moderate degree. In the kidneys, tubular basophilia was recorded at an increased incidence and severity in females at 15 mg/kg (4/5 females, at minimal-slight degree). A background level of tubular basophilia was recorded in the remaining dose groups including controls (up to 2/5 females at a minimal degree). These findings in the kidney are possibly related to elevated clinical biochemistry kidney biomarkers observed in one single female at 15 mg/kg.
- In addition, microscopic findings of note at the end of the treatment period were present in the respiratory tract (lung and trachea) of 3/5 surviving females at 15 mg/kg, and resembled the findings in the premature deaths at 15 mg/kg. These microscopic findings consisted of a combination of the following findings: In the lungs, erosion/ulceration of the epithelium of the main stem bronchi was recorded in 1/5 females (slight degree) and inflammation of the bronchus was recorded in 2/5 females (slight degree), in one animal combined with alveolar/bronchial basophilic contents (slight). In the trachea, erosion/ulceration was recorded in 2/5 females (minimal, moderate degree) and attenuated epithelium was recorded in 1/5 females (slight degree).
- The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- Number of implantation sites: Number of implantation sites was not affected by treatment

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

- All pregnant females (except for females sacrificed during the post-coitum period) delivered live offspring.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

- Gestation index and duration of gestation for females surviving until scheduled sacrifice were not affected by treatment.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): - Gestation index and duration of gestation for females surviving until scheduled sacrifice were not affected by treatment.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

- Fertility index for females surviving until scheduled sacrifice was not affected by treatment. Except for one control female (no. 110) and one female at 1.5 mg/kg (no. 55), all mated surviving females were pregnant.
- The significantly lower fertility index at 15 mg/kg (60%) was due to the high incidence of females sacrificed during the post-coitum period due to maternal toxicity and was not related to a reproductive effect.
- Pregnancy could not be determined for three females at 15 mg/kg that were sacrificed in extremis on Days 0 or 4 post-coitum. No implantation sites or corpora lutea were detectable at necropsy, which was due to the sacrifice in the early beginning of the post-coitum period, i.e. before implantation occurred. Except for one female which had erosion/ulceration of the epithelium of the vagina, no histopathological
changes in reproductive organs were observed.

Other effects:

no effects observed

Description (incidence and severity):

- Length and regularity of the oestrous cycle were not affected by treatment.
- Most females had regular cycles of 4 to 5 days.
- Extended di-oestrus during mating occurred in two control females and another control female had an irregular cycle.
- One female at 15 mg/kg was acyclic during the last week of the pre-mating period and during mating.
- The control females had healthy offspring. The pregnancy status of the 15 mg/kg female could not be determined as she was sacrificed on Day 0 post-coitum. The incidence of these findings was within normal limits and showed no dose-related trend (the abnormalities were mostly noted in control females).
- The 15 mg/kg female had erosion/ulceration in the vagina which was most likely procedure-related (preparation of vaginal lavage) and not test material-related.

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- At PND 13, mean body weights of pups at 15 mg/kg were 12 % (males) or 11 % (females) lower relative to controls but statistical significance was not achieved. On an individual litter basis, pup body weight gain was considerably reduced in 2/5 litters at 15 mg/kg.
- No treatment-related changes in pup body weight gain were noted at the lower dose levels.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - At PND 13, mean body weights of pups at 15 mg/kg were 12 % (males) or 11 % (females) lower relative to controls but statistical significance was not achieved. On an individual litter basis, pup body weight gain was considerably reduced in 2/5 litters at 15 mg/kg.
- No treatment-related changes in pup body weight gain were noted at the lower dose levels.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

- The number of live offspring on Day 1 after littering compared to total number of offspring born for females surviving until scheduled sacrifice was not affected by treatment. The live birth indices across the groups were 96 to 100 %.
- At first litter check, one pup of control Group 1, one pup at 5 mg/kg, four pups of control Group 5 and one pup at 150 mg/kg were found dead. This pup mortality was considered to be unrelated to treatment because the mortality incidence showed no dose-related trend and remained within normal limits.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was not affected by treatment.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

- The number of live offspring on Day 4 before culling compared to the number of offspring born for females surviving until scheduled sacrifice was not affected by treatment. The viability indices across the groups were 97 to 100 %.
- One control pup and one pup at 1.5 mg/kg was found dead on PND 2 or 4. Two control pups, one pup at 5 mg/kg and one pup at 15 mg/kg went missing at PND 2 or 4 and were most likely cannibalized. This post-natal loss was considered to be unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
- All pups of one litter from the 15 mg/kg group were sacrificed on PND 3 as their dam was found dead on Day 3 of lactation.
- The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) for females surviving until scheduled sacrifice was considered not to be affected by treatment. The lactation index was 95 % at 15 mg/kg and 100 % in the other groups. The mortality at 15 mg/kg (two pups went missing on PND 9 or 10) remained within the range considered normal for pups of this age.

External malformations:

no effects observed

Description (incidence and severity):

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

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- No clinical signs occurred among pups that were considered to be related to treatment.
- Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment. Relative to control Group 5, female pups at 15 mg/kg showed a slight (10%), but statistically significantly lower mean normalised anogenital distance. As values in 15 mg/kg female pups were similar to those in female pups of control Group 1, this finding was considered to be unrelated to treatment.
-Treatment up to 15 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 13-15 pups showed no changes that were considered to be toxicologically relevant. Statistically significantly higher mean T4 levels were noted in male and female pups at 15 mg/kg (relative differences from controls: 26 and 51 %, respectively). Values at 15 mg/kg
remained well within normal limits. Moreover, lower rather than higher T4 levels are expected in case of compromised thyroid function. Therefore, the higher T4 levels at 15 mg/kg were considered non-adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material to offspring development is 5 mg/kg/day.

Executive summary:

Additional information:

The repeated dose oral toxicity of the test material was determined in accordance with the standardised guidelines OECD 422, and US EPA OPPTS 870.3650 under GLP conditions in a combined 28-day oral (gavage) 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 test material, formulated in water, was administered daily by oral gavage to SPF-bred Wistar Han rats at doses of 0, 1.5, 5 and 15 mg/kg. Each group consisted of 10 males and 10 females. Due to a formulation error, the animals of the original high dose were dosed once, on day 13 of pre-mating, at a dose level of 150 mg/kg instead of at the target dose level of 15 mg/kg. As a result, severe toxicity was observed in these animals. It was therefore decided to sacrifice all animals of this group without any further examination on day 15 of the pre-mating period, and additional control and 15 mg/kg/day groups were included in the study. The recorded animal data for the original high dose group were not included in the toxicological evaluation.

Males were treated for 2 weeks prior to mating, during mating, and up to termination (Groups 1-3 for 31 days, Groups 5-6 for 29 days). The females that delivered were treated for 2 weeks prior to mating, during mating, during pregnancy, and at least 13-15 days of lactation (for 50-66 days). Females which failed to deliver healthy offspring were treated for 42 or 43 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. 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 weight, anogenital distance, areola/nipple retention and macroscopy). Formulations were analysed once during the study to assess accuracy and homogeneity and stability.

Formulation analysis showed that the formulations were prepared accurately and homogenously, and confirmed stability (at 1.5 mg/kg) upon storage for at least 5 hours at room temperature under normal laboratory light conditions.

At 15 mg/kg/day, three females were euthanized in extremis early in the post-coitum period (day 0 or 4 post-coitum) and one female was euthanized in extremis on day 17 post-coitum. One female was found dead on Day 3 of lactation. Main cause of death/moribundity of three out of the four females euthanized in extremis were effects in the respiratory tract which, according to the study director, were treatment-related. For the remaining female sacrificed in extremis during the post-coitum period, necropsy revealed erosion/ulceration in the vaginal epithelium, which the study director attributed to the preparation of vaginal smears and the moribund state of the animal. Prior to their early euthanasia, the three animals showed clinical signs of toxicity including rales, gasping, a lean appearance and/or piloerection, body weight loss and reduced food consumption. At necropsy, swollen lungs were noted for two of these decedents, probably representing failure to collapse, caused by obstructions in the major airways. At microscopic examination, erosion/ulceration and/or (obstructive) inflammation of the bronchi were noted. In addition, erosion/ulceration or inflammation of the trachea and/or lung was also noted in three of the surviving females at 15 mg/kg/day. These respiratory tract findings were regarded to be related to the treatment procedure (oral gavage) and unlikely related to the test material. The study director however, noted that these findings were only recorded for the 15 mg/kg treated females, were not seen in any of the females of the remaining dose groups and were not recorded in male rats and on this basis concluded that although a relation to the test material is very unlikely, this cannot be completely excluded. According to the certificate of analysis included in the report and physical-chemical properties, the tested substance has a pH value of 3.95; the dosing solution also had a likely pH value of 3-4. In addition, the substance causes severe, non-reversible eye damage when testedin vivo. It is therefore considered that any of the dosing solution reaching the respiratory tract would be highly irritating. The observation of erosions and ulcerations in the respiratory tract, as well as in the trachea, strongly suggest that these lesions were due to sub-optimal gavage dosing, with some of the dosing solution reaching the trachea or even the respiratory tract rather than any specific target organ toxicity systemically mediated. This is further supported by available additional data for the animals of the original high dose group, and information from the range-finding study. Only one animal of the original high dose group, out of 10 animals/sex, showed a respiratory symptom (rales) which, in contrast to the group on study, was seen pre-mating and did not require the animal to be terminated prior to day 13, when the whole group were erroneously treated at 10x the target dose. Some respiratory symptoms were also noted in the range finding study conducted in groups of 3 females each, much earlier than the post-coital timing of any respiratory effects in the main study. One female at 30 mg/kg/day showed rales, laboured respiration and gasping on days 6-9 and was sacrificed on day 10; another female treated at 100 mg/kg/day showed gasping and squeaking on day 5. Given that these dose levels were not tolerated, and all animals treated at 30 mg/kg/day were sacrificed on day 10 while the entire group treated at 100 mg/kg/day was sacrificed on day 5 or 7, these signs could readily be attributed to excessive systemic toxicity. As already mentioned, during the post-coitum and/or lactation periods, clinical signs were observed in the surviving females at 15 mg/kg/day which showed mainly rales, and in two females at 5 mg/kg/day which showed rales, hunched posture, piloerection and a lean appearance. Moreover, reduced body weight gain and food consumption were observed in females treated at 15 mg/kg/day during the lactation phase. Mean body weights and food consumption at scheduled sacrifice were respectively 11% and 18% lower compared to controls. At 5 mg/kg/day, the two same females that showed clinical signs had body weight loss accompanied by reduced food consumption during the first four days of the post-coitum period. The clinical signs observed, in combination with the dose-related reduced body weight gain or body weight loss and reduced food consumption are considered to be adverse.

Further treatment-related findings at 15 mg/kg/day included macroscopic findings in the stomach in 2 males, consisting of purple discoloration (without microscopic correlate) and in two surviving females the intestinal tract was discoloured and/or distended with gas (without microscopic correlate). Test material-related microscopic alterations noted in females at 15 mg/kg/day consisted of lymphoid atrophy in the thymus and a slightly increased incidence and severity of tubular basophilia in the kidneys. The findings in the kidney are possibly related to elevated clinical biochemistry kidney biomarkers in one individual female at 15 mg/kg. These findings were considered to be non-adverse.

No parental toxicity was observed in males treated up to 15 mg/kg/day and females treated at 1.5 mg/kg/day.

No reproduction toxicity was observed up to the highest dose level tested (15 mg/kg/day). Body weight gain of male and female pups was not statistically significantly reduced at 15 mg/kg/day, resulting in 11% (females) - 12% (males) lower mean body weights at PND 13; the effect was particularly evident in 2/5 litters. At birth, pup body weights were similar among the groups. This reduced pup weight gain at 15 mg/kg/day occurred in the presence of maternal toxicity (clinical signs, reduced body weight gain, reduced food consumption), but again, the study director stated that a direct developmental effect of the test material cannot be excluded. Considering that during the lactation phase dams at 15 mg/kg/day showed significant reductions of both body weight (11% lower than controls) and food consumption (18% lower than controls), and pup weight was comparable to controls at birth, it is clear that the effects on pup weight were secondary to maternal toxicity.

 

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material for offspring development is 5 mg/kg/day.

 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Principles of method if other than guideline:

In this study, the animals of Group 4 were dosed erroneously on a single occasion (Day 13 pre-mating) at a dose level of 150 mg/kg instead of the target high dose level of 15 mg/kg. As a result, severe toxicity was observed in these animals and it was considered appropriate that all remaining Group 4 animals were sacrificed without any further examination on Day 15 of the pre-mating period and additional control and 15 mg/kg group (Groups 5 and 6, respectively) were included in this study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

EC Number:

221-326-1

EC Name:

3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

Cas Number:

3068-39-1

Molecular formula:

C27H29N2O3.Cl

IUPAC Name:

3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Brown red powder
- Storage conditions of test material: At room temperature
- Stable until: 10 June 2021 (expiry date)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (outbred, SPF-quality)

Details on species / strain selection:

This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies.
The testing facility has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

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: At the start of the pre- test females were approximately 11 weeks; at the start of F0-treatment males were approximately 10 weeks and females approximately 13 weeks.
- Weight at study initiation: Weight at start of pre- mating: Females: 206 to 247 g, Males: 278 to 315 g.
- Fasting period before study: No
- Housing: All cages were 18 cm high. During the pre-test females and during pre-mating animals were housed in groups of 5 animals/sex/cage in plastic cages. During mating males and females were cohabitated on a 1:1 basis in plastic cages. Post-mating males were housed in their home cage with a maximum of 5 animals/cage and females were individually housed in plastic cages. During lactation females were housed in plastic cages. Sterilised sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: Pelleted rodent diet ad libitum, except during motor activity measurements animals did not have access to food for a maximum of 1 hour 25 minutes.
- Water: ad libitum, except during motor activity measurements animals did not have access to water for a maximum of 1 hour 25 minutes.
- Acclimation period: At least 5 days prior to the start of pre-test (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C (actual daily mean was 22 °C)
- Humidity: 40 to 70 % (actual daily mean was 44 to 53 %)
- Air changes: at least 10 room air changes/hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES:
- From: Not specified
- To: 13 April 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. No adjustment was made for specific gravity/density of the test material, vehicle, and/or formulation. No correction was made for the purity/composition of the test material. The formulations containing the test material were red solutions. The formulation was stored at room temperature and placed on a magnetic stirrer during dosing.

DOSE VOLUME: 5 mL/ kg bw

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on trial formulations performed in the testing facility.
- Concentration in vehicle: Test material concentrations 0.3, 1, 3 and 3 mg/mL

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: Evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. Referred to as day 0 (p.c.) of pregnancy
- After successful mating each pregnant female was caged individually. Males were housed in their home cage with a maximum of 5 animals/cage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The purpose of the analytical phase was to determine the accuracy of preparation, homogeneity and stability of the formulations.

SAMPLING
In total, 26 samples were included in this study, distributed over 6 dose groups. Group 1 and 5 were the vehicle control groups, Groups 2, 3, 4 and 6 were dosed at 1.5, 5, 15 and 15 mg/kg bw/day at a dose volume of 5 mL/kg bw, respectively. The samples of the control Group 1, 3 and Group 5 were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment Groups 2, 4 and 6 were taken in duplicate from the top, middle and bottom position of the container and immediately stored on dry ice. In addition, stability samples were taken from formulation Group 2 in duplicate at the middle position of the container. The stability samples were stored for 5 hours at room temperature under normal laboratory light conditions and then stored on dry ice.

ANALYTICAL CONDITIONS
The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.
Formulations containing the test material were dissolved in methanol and transferred to volumetric flasks. These solutions were further diluted depending on their concentrations and injected. Chromatographic separation was performed on an Xbridge C18 column using isocratic elution. A diode array detector was used for quantification.

- Preparation of Calibration solutions (stocks, ~ 1000 mg/L)
Weigh accurately an amount of approximately 10.0 mg directly into a 25 mL amber glass bottle using an analytical balance. Add accurately 10.0 mL (multi pipette/PD pipette) MeOH. Cap the bottle and dissolve the substance by shaking/vortexing manually. Store the solution in a 25 mL amber glass bottle at 2 to 8 °C (expiration 287 hours).

- Preparation of BT stability samples, ~ 1000 mg/L
Transfer an aliquot of the calibration stocks into 12 mL amber glass bottles. Store the samples at room temperature for at least 12 hours. After completion return the samples to a refrigerator (2 to 8 °C, expiration 22 hours).

- Preparation of calibration spike solutions and stock stability solutions
Prepare solutions according to set volumes, dispensing the correct volume of MeOH (multi pipette) into 10 mL glass tubes. Add the stock solution (PD pipette) to the tubes, cap and vortex the tubes at speed 4 to 6 for 0.5 minutes. Expiration: prepare freshly.

- Preparation of a Recovery stock (20 000 mg/L)
Weigh accurately an amount of approximately 20.0 mg (analytical balance) directly into a 12 mL amber glass bottle. Add accurately 1.00 mL (multi pipette/PD pipette) MeOH. Cap the bottle and dissolve the substance by shaking/vortexing manually. Store the solution in a 25 mL amber glass bottle at 2 to 8 °C (expiration 287 hours).

- Preparation of Accuracy/PRS High samples (~ 200 mg/g)
Weigh at least 100 mg (analytical balance) accurately into a 20 mL glass bottle. Add approximately 400 mg (analytical balance) vehicle (water) accurately to the bottle and weigh the total mass of the sample. Cap the vial and vortex gently. Incubate for at least 30 minutes at room temperature before further processing. Expiration: prepare freshly.

- Preparation of F/T & LT stability High samples (~200 mg/g)
Weigh at least 100 mg (analytical balance) accurately into a 20 mL glass bottle. Add approximately 400 mg (analytical balance) vehicle (water) accurately to the bottle and weigh the total mass of the sample. Cap the vial, vortex gently and store the samples (≤ -70 °C).

- Preparation of Accuracy/PRS Low samples (~ 1.00 mg/g)
Weigh at least 500 mg (analytical balance) vehicle (water} accurately into a 20 mL glass bottle. Add 25.0 µL (PD pipette) Recovery stock, cap the bottle and vortex gently. Incubate for at least 30 minutes at room temperature before further processing. Expiration: prepare freshly.

- Preparation of F/T & LT stability Low samples (~1.00 mg/g)
Weigh at least 500 mg (analytical balance) vehicle (water) accurately into a 20 mL glass bottle. Add 25.0 µL (PD pipette) Recovery stock, cap the bottle, vortex gently and store the samples (≤ -70 °C).

- Preparation of additional validation samples

Preparation of a Selectivity sample: Weigh at least 500 mg (analytical balance) vehicle (water) accurately into a 20 mL glass bottle. Expiration: prepare freshly.
Preparation of LOD sample (0.250 mg/L): Dispense 3000 µL (multi pipette) MeOH into a 10 mL glass tube. Add 1000 µL (PD pipette) calibration spike solution, cap the tube and vortex at speed 4 to 6 for 0.5 minutes. Expiration: prepare freshly.

- Preparation of solutions
It is allowed to prepare an adjusted amount of a solution, on the condition that the ratio of the chemicals and solvents used for the preparation is not changed.
LC Buffer (50 mM NH4FA in UP/ ACN (95:5): Weigh 3.2 g (balance) NH4FA into a 1 L glass bottle. Add 950 mL (measuring cylinder) UP water and dissolve the salt. Add 50 mL (measuring cylinder) ACN and mix (store at room temperature for a maximum of 90 days).
Needle wash (R0): Dispense 900 mL (measuring cylinder) ACN in a 1 L glass bottle. Add 100 mL (measuring cylinder) UP water. Add 1.00 mL (multi pipette) FA and mix (store at room temperature for a maximum of 90 days).

EQUIPMENT SETTINGS
- Shimadzu HPLC settings
Needle stroke (mm): 50
Sampling speed (µL/sec): 1.0
Cooler enabled: Yes
Cooler temperature (°C): 10
Rinsing speed (µL/sec): 35
Rinsing volume (µL): 200
Rinse mode: After aspiration
Rinse dip time (sec): 2
Column oven temperature (°C): 40
Pumping mode: Ternary Flow
Infection volume (µL): 2.00
Solvent: The ratio of UP water, LC buffer and MeOH (all in %) was 20:10:70, 0:10:90 and 20:10:70 at 0 and 3.20 minutes, 3.30 and 4.50 minutes and 4.60 minutes, respectively.

- DAD settings:
Acquisition time (min): 3.50
Wavelength (nm) acquisition: 525 - 531
Wave step (nm): 1
UV lamp (D2): off
Visible lamp (W): on
Time constant (s): 0.64
Slit width (nm): 1.2
Sampling frequency (Hz): 4.1667
Cell temperature (°C): 40

- MS Settings
Simulation mode

FORMULAE
Response (Y): Peak area test material [cps]
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 [%]

Relative difference (relative diff.): ((Ct – C0) / C0 ) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

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 %. Formulations were considered stable if the relative difference between the stored and freshly taken samples 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 higher than or equal to 0.9999. No response at the retention time of the test material in the analytical blank samples was detected.

- Samples
Procedural recovery samples Accuracy samples: The mean recoveries of the procedural recovery samples fell within the criterion of 90 - 110 %, in exception of the procedural recovery low (1.00 g/L) in run AB001 which was 83.1 %. Owing to the fact that the mean accuracy of the procedural recovery samples at the concentration level 0.300 g/L measured in the same analytical run was 91.5 %, which is within the criteria for the acceptance of the accuracy samples, it was decided to accept the analytical run AB001.
The mean accuracy at the level 0.300 g/L was 91.5 %, which is in the range required for the measurement of the test material in formulations (90 - 110 %). The coefficient of variation of the accuracies is 3.3 %. The coefficient of variation was acceptable and within the limits (≤ 10 %). It demonstrated that the analytical method was adequate for the determination of the test material in the test samples within the analytical range 0.300 to 200 g/L of the test material in formulations.

- Test samples
In the Group 1 and Group 5 formulations, no test material was detected. The concentrations analysed in the formulations of Groups 2, 3, 4 and 6 were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %). The formulations of Group 2, 4 and Group 6 were homogeneous (i.e. coefficient of variation ≤ 10 %). Analysis of Group 2 formulations after storage yielded a relative difference ≤ 10 %. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.

Duration of treatment / exposure:

- Group 1, 2 and 3 males were treated for 31 days, Group 4 males were treated for 14 days and Group 5 and 6 males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
- Group 1, 2 and 3 females were treated for 53-66 days, Group 4 females were treated for 14 days and Group 5 and 6 females were treated for 50-63 days,, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 42 or 43 days. Routinely, females that are littering are left undisturbed. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
- Pups were not treated directly but were potentially exposed to the test material in utero, via maternal milk or from exposure to maternal urine/feces.

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

15 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: Based on the results of a 15-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 1.5, 5 and 15 mg/kg.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons

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

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION: 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.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m.
- Animals fasted: Yes, the animals were deprived of food overnight (with a maximum of approximately 24 hours) before blood sampling, but water was available
- The following haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant: White blood cells (WBC, 10^9/L), Differential leucocyte count: Neutrophils (%WBC), Lymphocytes (%WBC), Monocytes (%WBC), Eosinophils (%WBC), Basophils (%WBC), Red blood cells (10^12/L), Reticulocytes (%RBC), Red blood cell distribution width (RDW, %) Haemoglobin (mmol/L), Haematocrit (L/L), Mean corpuscular volume (MCV, fL), Mean corpuscular haemoglobin (MCH, fmol), Mean corpuscular haemoglobin concentration (MCHC, mmol/L) and Platelets (10^9/L).
- The following clotting parameters were determined in plasma prepared with citrate as anticoagulant: Prothrombin Time (PT, s) and Activated Partial Thromboplastin Time (APTT, s).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as for haematology
- Animals fasted: as for haematology
- How many animals: as for haematology
- The following clinical biochemistry parameters were determined using the AU400. All parameters were determined in plasma, except for bile acids which were determined in serum: Alanine aminotransferase (ALAT, U/L), Aspartate aminotransferase (ASAT, U/L), Alkaline Phosphatase (ALP, U/L), Total protein (g/L), Albumin (g/L), Total Bilirubin (μmol/L), Bile acids (μmol/L), Urea (mmol/L), Creatinine (μmol/L), Glucose (mmol/L), Cholesterol (mmol/L), Sodium (mmol/L), Potassium (mmol/L), Chloride (mmol/L), Calcium (mmol/L) and Inorganic Phosphate (Inorg. Phos, mmol/L).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: a selected 5 animals/sex/group
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), and 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 computerized monitoring system). Total movements and ambulations are 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 finer movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No

OTHER: GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females were examined for evidence of abortion or premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

OTHER: THYROID HORMONE ANALYSIS
- Blood samples were taken at the end of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, non-mated females, non-pregnant females and all males after at least 4 weeks of treatment. No samples were collected from animals that were sacrificed in extremis or found dead and females with total litter loss.
- Blood samples were collected 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 (0.9 mL) were drawn from the retroorbital sinus and collected into serum tubes. After clotting and centrifugation, serum was used as listed below.
- Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroid stimulating hormone (TSH).
- Females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).
- Serum samples were stored at ≤ -75 °C. Under these storage conditions, the samples are stable for 6 months. Any remaining samples were discarded.
- Total T4 was measured in serum using the Immulite 1000.

Oestrous cyclicity (parental animals):

- Daily vaginal lavage was performed to determine the stage of oestrous beginning 14 days prior to treatment (pre-test), 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 pre-test, this was done for all females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of oestrous.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

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.
- 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 normalised 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.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

HORMONE ANALYSIS
- PND 4 pups: From 2 surplus pups per litter at culling (if possible). If only 1 surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. Blood samples were collected by decapitation, between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter (0.4 mL in total) were collected into one serum tube for possible future measurement of thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤- 75 °C. Under these storage conditions, the samples are stable for 6 months. Any remaining samples were discarded.
- PND 13-15 pups: From 2 pups per litter (if possible from one male and one female) at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane, between 7.00 and 10.30 a.m. Blood was collected into serum tubes. After clotting and centrifugation, serum from each sample was divided into 2 aliquots: 150 μL serum for measurement of thyroxine (T4) and the remaining volume of serum for possible future measurement of thyroid-stimulating hormone (TSH). Serum samples were stored at ≤ -75 °C. Under these storage conditions, the samples are stable for 6 months. Any remaining samples were discarded.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
- Necropsy was conducted on the following days: Males: Following completion of the mating period (a minimum of 28 days of dose administration), females which delivered: PND 14 to 16, females which failed to deliver: Post-coitum Days 26-27 (females with evidence of mating), spontaneous deaths: As soon as possible after death and always within 24 hours and euthanised in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.
- After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
- Samples of the following tissues and organs were collected and fixed in 10 % buffered formalin:
- For the selected 5 animals/sex/group and all animals that died spontaneously or were euthanised in extremis: Identification marks: not processed, (nasopharynx), adrenal glands (oesophagus), (aorta), ovaries, brain - cerebellum, mid-brain, cortex (7-levels), (pancreas), Caecum, Peyer's patches [jejunum, ileum] if detectable, Cervix, Pituitary gland, Clitoral gland, Preputial gland, Colon, Prostate gland, Coagulation gland, Rectum, (Cowper’s gland), (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, Eyes (with optic nerve (if detectable) and Harderian gland), Skeletal muscle, (Skin), Mammary gland area, (males and females), Spinal cord -cervical, midthoracic, lumbar, Femur including joint, Spleen, (Glans penis), Sternum with bone marrow, (Levator ani plus bulbocavernosus muscle complex (LABC)), Stomach, Heart, Testes, Ileum, Thymus, Jejunum, Thyroid including parathyroid if detectable, Kidneys, (Tongue), (Lacrimal gland, exorbital), Trachea, (Larynx), Urinary bladder, Liver, Uterus, Lung, infused with formalin, Vagina, Lymph nodes - mandibular, mesenteric and all gross lesions.
- For all remaining animals, males that failed to sire and females which failed to deliver: Identification marks: not processed, Ovaries, Cervix, Preputial gland, Clitoral gland, Prostate gland, Coagulation gland, Seminal vesicles, Cowper’s glands, Testes, Epididymides, Thyroid including parathyroid if detectable, Glans penis, Uterus, Levator ani plus bulbocavernosus muscle complex (LABC), Vagina, Mammary gland area (males and females) and all gross lesions.

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

HISTOPATHOLOGY: Yes
- All organ and tissue samples, as defined under Histopathology were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist:
The preserved organs and tissues of the selected 5 animals/sex of Groups 5 and 6, the additional slides of the testes of the selected 5 males of Groups 5 and 6 and all males that failed to sire, the preserved organs and tissues of the animals of all dose groups which died spontaneously or were euthanised in extremis, all gross lesions of all animals (all dose groups), the trachea, lungs, thymus and kidneys of all selected 5 females of Groups 2 and 3, and of the selected 5 females of the concurrent control Group 1, based on (possible) treatment-related changes in these organs and the reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.

Postmortem examinations (offspring):

- Pups, younger than 7 days were euthanised by decapitation. All remaining pups (PND 7-15), except those used for blood sampling on PND 13-15, were sacrificed using Euthasol® 20% by intraperitoneal (ip) injection.
- On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter were collected into one serum tube. Pups found dead during the weekend were fixed in an identified container containing 70 % ethanol as they were not necropsied on the same day. On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane, between 7.00 and 10.30 a.m., followed by exsanguination. Blood was collected into serum tubes.
- Necropsy was conducted on the following days: Culling: PND 4, Terminal sacrifice: PND 13-15 and Spontaneous deaths: As soon as possible after death and always within 24 hours.
- All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
- At terminal sacrifice (PND 13-15), 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.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- 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 mated) x 100
- Gestation index (%) = (Number of females with living pups on Day 1/ Number of pregnant females) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine implantation sites) x 100
- 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
- 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
- Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Clinical signs of toxicity were noted in females at 5 and 15 mg/kg. Two females at 5 mg/kg showed rales, hunched posture, piloerection and a lean appearance on several days during the post-coitum and/or lactation period. At 15 mg/kg, all five females surviving to scheduled sacrifice showed rales during the post-coitum and/or lactation period, and one of these females showed deep respiration on two successive days during the post-coitum period. Rales were also noted in the four 15 mg/kg females which were sacrificed in extremis.
- Salivation noted after dosing at 5 mg/kg (females) and 15 mg/kg (both sexes) was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
- Red discoloration of the faeces (in all animals at 5 and 15 mg/kg) and of the skin (in a few females at 15 mg/kg) was due to the red colour of the test material and considered not toxicologically relevant.
- No additional clinical signs of toxicity were noted during the weekly arena observations. Any other clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- There were five premature decedents in the group of females treated at 15 mg/kg. The moribundity or death of four of these decedents was considered to be related to treatment as explained below.
- Three females were euthanised in extremis early in the post-coitum period (Day 0 or 4 post-coitum) and one female was euthanised in extremis on Day 17 post-coitum. Prior to their early euthanasia, these animals showed clinical signs of toxicity (including rales, gasping, a lean appearance and/or piloerection), body weight loss and reduced food consumption. One female was found dead on Day 3 of the lactation period. Prior to death, she showed neither clinical signs of toxicity nor reduced body weight gain or reduced food consumption. Main macroscopic findings observed for these premature decedents consisted of distension and purple discoloration of the gastrointestinal tract. There were no microscopic correlates to these macroscopic findings. Two females showed swollen lungs, probably representing failure to collapse, caused by (partly) obstructions in the major airways.
- At microscopic examination, main findings were seen in the respiratory tract. Marked chronic obstructive inflammation of the bronchus was seen in two females, and in four females erosion/ ulceration of the trachea and/or bronchial epithelium was noted. The findings in the respiratory tract are considered the main cause of morbidity or death for four females. The nature of the respiratory tract lesions can be related to the treatment procedure (oral gavage), but a relation to the test material cannot be completely excluded. In one female erosion/ulceration was present in vaginal epithelium, which likely attributed to the moribund state of the animal. The vagina erosion/ulceration was regarded to be procedure-related (preparation of vaginal smears) and not test material-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Body weights and body weight gain were reduced in 4/5 surviving females at 15 mg/kg during the lactation period compared to controls. Differences from control values were statistically significant at Day 13 of lactation, when the mean body weight of 15 mg/kg females was 11 % lower. During the premating, mating and post-coitum periods, body weight gain of 15 mg/kg females which survived until sacrifice was normal, except in one female which showed slightly reduced weight gain during the post-coitum period.
- At 5 mg/kg, body weights and body weight gain were normal, except for three females which showed weight loss during a few days of the post-coitum period.
- No treatment-related changes in body weight or body weight gain were observed in males treated up to 15 mg/kg and in females at 1.5 mg/kg.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- At 15 mg/kg, mean food consumption before allowance for body weight was slightly lower for females in the post-coitum and lactation periods, reaching statistical significance at Day 13 of the lactation period when mean food consumption was 18 % lower relative to controls. This was particularly due to the considerably reduced food consumption of a few females. Food consumption after allowance for body weight showed a similar pattern. During the pre-mating and mating period, food consumption of 15 mg/kg females was normal.
- At 5 mg/kg, food consumption before and after allowance for body weight were normal, except for two females which had markedly reduced food consumption between post-coitum Days 0-4. Thereafter, food consumption of these females returned to control values.
- No treatment-related changes in food consumption before or after allowance for body weight were observed in males treated up to 15 mg/kg and in females at 1.5 mg/kg.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- Haematology parameters were considered not to be adversely affected by treatment.
- Statistically significant higher mean number of white blood cells (WBC) were noted in females treated at 15 mg/kg, which was mainly caused by one female. In absence of any other WBC parameters and as all numbers remained within the range of available historical control data, this increase in number of WBC was considered not toxicologically relevant.
- In addition, females at 15 mg/kg had statistically significantly lower mean corpuscular haemoglobin concentration (MCHC). In the absence of changes in other red blood cell parameters, particularly haemoglobin concentration and number of red blood cells, slight decrease in MCHC was considered not toxicologically relevant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- No clear treatment-related changes in clinical biochemistry parameters were noted.
- On an individual basis, abnormal values were noted for one female no. 118. This female showed a low total protein, albumin and chloride level and a high urea, creatinine, cholesterol and inorganic phosphate level, which were all outside the range considered normal for rats of this age and strain. This resulted in statistically significantly lower mean chloride (8 % relative difference) and higher mean inorganic phosphate levels (53 % relative difference), compared to concurrent controls. The values of the other females at 15 mg/kg were within the normal range.
- Other statistically significant variations noted in clinical biochemistry parameters were considered to be unrelated to treatment as they occurred in the absence of a dose-related distribution and remained within the range considered normal for rats of this strain and age.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
- Motor activity data showed a similar habituation profile with a decreasing trend in activity over the duration of the test period for all groups. Findings of note were lower mean values for total movements and ambulations in males at 15 mg/kg (relative differences from controls: 42 and 28 %, respectively). Statistical significance was not achieved and there were no corroborative clinical signs or changes in other measures in the neuromuscular domain (including gait, air righting reflex and grip strength). Therefore, these changes in motor activity were considered not to reflect impaired neuromuscular function. Apparently lower mean values for total movements and ambulations in females at 15 mg/kg were due to high concurrent (Group 5) control means (particularly due to relatively high values in one control female). Values in 15 mg/kg females were comparable to those in females of control Group 1.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- There were no test material-related microscopic observations in male rats.
- In females surviving until scheduled sacrifice, test material-related microscopic findings were noted in the thymus and kidneys at 15 mg/kg. In the thymus, lymphoid atrophy was recorded in 3/5 females at 15 mg/kg, at minimal-moderate degree. In the kidneys, tubular basophilia was recorded at an increased incidence and severity in females at 15 mg/kg (4/5 females, at minimal-slight degree). A background level of tubular basophilia was recorded in the remaining dose groups including controls (up to 2/5 females at a minimal degree). These findings in the kidney are possibly related to elevated clinical biochemistry kidney biomarkers observed in one single female at 15 mg/kg.
- In addition, microscopic findings of note at the end of the treatment period were present in the respiratory tract (lung and trachea) of 3/5 surviving females at 15 mg/kg, and resembled the findings in the premature deaths at 15 mg/kg. These microscopic findings consisted of a combination of the following findings: In the lungs, erosion/ulceration of the epithelium of the main stem bronchi was recorded in 1/5 females (slight degree) and inflammation of the bronchus was recorded in 2/5 females (slight degree), in one animal combined with alveolar/bronchial basophilic contents (slight). In the trachea, erosion/ulceration was recorded in 2/5 females (minimal, moderate degree) and attenuated epithelium was recorded in 1/5 females (slight degree).
- The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test 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):

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- Length and regularity of the oestrous cycle were not affected by treatment.
- Most females had regular cycles of 4 to 5 days.
- Extended di-oestrus during mating occurred in two control females and another control female had an irregular cycle.
- One female at 15 mg/kg was acyclic during the last week of the pre-mating period and during mating.
- The control females had healthy offspring. The pregnancy status of the 15 mg/kg female could not be determined as she was sacrificed on Day 0 post-coitum. The incidence of these findings was within normal limits and showed no dose-related trend (the abnormalities were mostly noted in control females).
- The 15 mg/kg female had erosion/ulceration in the vagina which was most likely procedure-related (preparation of vaginal lavage) and not test material-related.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage aware evaluation of the testes did not show any indication of abnormal spermatogenesis.

Reproductive performance:

no effects observed

Description (incidence and severity):

- There were 1/10 couples treated at 1.5 mg/kg and 1/10 couples of control Group 5 that were not pregnant. Histopathology did not reveal any changes in the reproductive organs that could explain this.
- At 15 mg/kg, 4/10 females were sacrificed in extremis post-coitum and one female of this dose group was found dead on Day 3 of lactation. Histopathologic examination revealed erosion/ulceration of the epithelium of the vagina of one female, which was sacrificed on Day 0 postcoitum. This finding was most likely procedure-related (daily of vaginal lavage).
- There were no other changes in the reproductive organs of these animals. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test material and stage aware evaluation of the testes did not show any indication of abnormal spermatogenesis.
- Mating index: Mating index was not affected by treatment. All females (including decedents) showed evidence of mating.
- Precoital time: Precoital time was not affected by treatment. All females, except for control females, showed evidence of mating within 5 days. These two control females were mated after 10 or 14 days of cohabitation.
- Number of implantation sites: Number of implantation sites was not affected by treatment
- Fertility index: Fertility index for females surviving until scheduled sacrifice was not affected by treatment. Except for one control female and one female at 1.5 mg/kg, all mated surviving females were pregnant. The significantly lower fertility index at 15 mg/kg (60 %) was due to the high incidence of females sacrificed during the post-coitum period due to maternal toxicity and was not related to a reproductive effect. Pregnancy could not be determined for three females at 15 mg/kg that were sacrificed in extremis on Days 0 or 4 post-coitum. No implantation sites or corpora lutea were detectable at necropsy, which was due to the sacrifice in the early beginning of the post-coitum period, i.e. before implantation occurred. Except for one female which had erosion/ulceration of the epithelium of the vagina, no histopathological changes in reproductive organs were observed.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

5 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

gross pathology

2

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

- No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore regarded as unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- Live birth index: The number of live offspring on Day 1 after littering compared to total number of offspring born for females surviving until scheduled sacrifice was not affected by treatment. The live birth indices across the groups were 96 to 100 %. At first litter check, one pup of control Group 1, one pup at 5 mg/kg, four pups of control Group 5 and one pup at 150 mg/kg were found dead. This pup mortality was considered to be unrelated to treatment because themortality incidence showed no dose-related trend and remained within normal limits.
- Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring born for females surviving until scheduled sacrifice was not affected by treatment. The viability indices across the groups were 97 to 100 %. One control pup and one pup at 1.5 mg/kg was found dead on PND 2 or 4. Two control pups, one pup at 5 mg/kg and one pup at 15 mg/kg went missing at PND 2 or 4 and were most likely cannibalised. This post-natal loss was considered to be unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age. All pups of one female at 15 mg/kg were sacrificed on PND 3 as their dam was found dead on Day 3 of lactation.
- Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) for females surviving until scheduled sacrifice was considered not to be affected by treatment. The lactation index was 95 % at 15 mg/kg and 100 % in the other groups. The mortality at 15 mg/kg (two pups of a litter went missing on PND 9 or 10) remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At PND 13, mean body weights of pups at 15 mg/kg were 12 % (males) or 11 % (females) lower relative to controls but statistical significance was not achieved. On an individual litter basis, pup body weight gain was considerably reduced in 2/5 litters at 15 mg/kg. No treatment-related changes in pup body weight gain were noted at the lower dose levels.

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

- No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Sex ratio was not affected by treatment.
- Anogenital distance (absolute and normalised for body weight) in male and female pups was considered not to be affected by treatment. Relative to control Group 5, female pups at 15 mg/kg showed a slight (10 %), but statistically significantly lower mean normalised anogenital distance. As values in 15 mg/kg female pups were similar to those in female pups of control Group 1, this finding was considered to be unrelated to treatment.
- Treatment up to 15 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 13-15 pups showed no changes that were considered to be toxicologically relevant. Statistically significantly higher mean T4 levels were noted in male and female pups at 15 mg/kg (relative differences from controls: 26 and 51 %, respectively). Values at 15 mg/kg remained well within normal limits. Moreover, lower rather than higher T4 levels are expected in case of compromised thyroid function. Therefore, the higher T4 levels at 15 mg/kg were considered non-adverse.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction toxicity was observed up to the highest dose tested.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 15 mg/kg/day for reproduction toxicity.

Executive summary:

The reproductive and developmental toxicity of the test material was determined in accordance with the standardised guidelines OECD 422, and US EPA OPPTS 870.3650 under GLP conditions in a combined 28-day oral (gavage) 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 test material, formulated in water, was administered daily by oral gavage to SPF-bred Wistar Han rats at doses of 0, 1.5, 5 and 15 mg/kg. Each group consisted of 10 males and 10 females. Due to a formulation error, the animals of the original high dose were dosed once, on day 13 of pre-mating, at a dose level of 150 mg/kg instead of at the target dose level of 15 mg/kg. As a result, severe toxicity was observed in these animals. It was therefore decided to sacrifice all animals of this group without any further examination on day 15 of the pre-mating period, and additional control and 15 mg/kg/day groups were included in the study. The recorded animal data for the original high dose group were not included in the toxicological evaluation.

Males were treated for 2 weeks prior to mating, during mating, and up to termination (Groups 1-3 for 31 days, Groups 5-6 for 29 days). The females that delivered were treated for 2 weeks prior to mating, during mating, during pregnancy, and at least 13-15 days of lactation (for 50-66 days). Females which failed to deliver healthy offspring were treated for 42 or 43 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. 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 weight, anogenital distance, areola/nipple retention and macroscopy). Formulations were analysed once during the study to assess accuracy and homogeneity and stability.

Formulation analysis showed that the formulations were prepared accurately and homogenously, and confirmed stability (at 1.5 mg/kg) upon storage for at least 5 hours at room temperature under normal laboratory light conditions.

At 15 mg/kg/day, three females were euthanized in extremis early in the post-coitum period (day 0 or 4 post-coitum) and one female was euthanized in extremis on day 17 post-coitum. One female was found dead on Day 3 of lactation. Main cause of death/moribundity of three out of the four females euthanized in extremis were effects in the respiratory tract which, according to the study director, were treatment-related. For the remaining female sacrificed in extremis during the post-coitum period, necropsy revealed erosion/ulceration in the vaginal epithelium, which the study director attributed to the preparation of vaginal smears and the moribund state of the animal. Prior to their early euthanasia, the three animals showed clinical signs of toxicity including rales, gasping, a lean appearance and/or piloerection, body weight loss and reduced food consumption. At necropsy, swollen lungs were noted for two of these decedents, probably representing failure to collapse, caused by obstructions in the major airways. At microscopic examination, erosion/ulceration and/or (obstructive) inflammation of the bronchi were noted. In addition, erosion/ulceration or inflammation of the trachea and/or lung was also noted in three of the surviving females at 15 mg/kg/day. These respiratory tract findings were regarded to be related to the treatment procedure (oral gavage) and unlikely related to the test material. The study director however, noted that these findings were only recorded for the 15 mg/kg treated females, were not seen in any of the females of the remaining dose groups and were not recorded in male rats and on this basis concluded that although a relation to the test material is very unlikely, this cannot be completely excluded. According to the certificate of analysis included in the report and physical-chemical properties, the tested substance has a pH value of 3.95; the dosing solution also had a likely pH value of 3-4. In addition, the substance causes severe, non-reversible eye damage when testedin vivo. It is therefore considered that any of the dosing solution reaching the respiratory tract would be highly irritating. The observation of erosions and ulcerations in the respiratory tract, as well as in the trachea, strongly suggest that these lesions were due to sub-optimal gavage dosing, with some of the dosing solution reaching the trachea or even the respiratory tract rather than any specific target organ toxicity systemically mediated. This is further supported by available additional data for the animals of the original high dose group, and information from the range-finding study. Only one animal of the original high dose group, out of 10 animals/sex, showed a respiratory symptom (rales) which, in contrast to the group on study, was seen pre-mating and did not require the animal to be terminated prior to day 13, when the whole group were erroneously treated at 10x the target dose. Some respiratory symptoms were also noted in the range finding study conducted in groups of 3 females each, much earlier than the post-coital timing of any respiratory effects in the main study. One female at 30 mg/kg/day showed rales, laboured respiration and gasping on days 6-9 and was sacrificed on day 10; another female treated at 100 mg/kg/day showed gasping and squeaking on day 5. Given that these dose levels were not tolerated, and all animals treated at 30 mg/kg/day were sacrificed on day 10 while the entire group treated at 100 mg/kg/day was sacrificed on day 5 or 7, these signs could readily be attributed to excessive systemic toxicity. As already mentioned, during the post-coitum and/or lactation periods, clinical signs were observed in the surviving females at 15 mg/kg/day which showed mainly rales, and in two females at 5 mg/kg/day which showed rales, hunched posture, piloerection and a lean appearance. Moreover, reduced body weight gain and food consumption were observed in females treated at 15 mg/kg/day during the lactation phase. Mean body weights and food consumption at scheduled sacrifice were respectively 11% and 18% lower compared to controls. At 5 mg/kg/day, the two same females that showed clinical signs had body weight loss accompanied by reduced food consumption during the first four days of the post-coitum period. The clinical signs observed, in combination with the dose-related reduced body weight gain or body weight loss and reduced food consumption are considered to be adverse.

Further treatment-related findings at 15 mg/kg/day included macroscopic findings in the stomach in 2 males, consisting of purple discoloration (without microscopic correlate) and in two surviving females the intestinal tract was discoloured and/or distended with gas (without microscopic correlate). Test material-related microscopic alterations noted in females at 15 mg/kg/day consisted of lymphoid atrophy in the thymus and a slightly increased incidence and severity of tubular basophilia in the kidneys. The findings in the kidney are possibly related to elevated clinical biochemistry kidney biomarkers in one individual female at 15 mg/kg. These findings were considered to be non-adverse.

No parental toxicity was observed in males treated up to 15 mg/kg/day and females treated at 1.5 mg/kg/day.

No reproduction toxicity was observed up to the highest dose level tested (15 mg/kg/day). Body weight gain of male and female pups was not statistically significantly reduced at 15 mg/kg/day, resulting in 11% (females) - 12% (males) lower mean body weights at PND 13; the effect was particularly evident in 2/5 litters. At birth, pup body weights were similar among the groups. This reduced pup weight gain at 15 mg/kg/day occurred in the presence of maternal toxicity (clinical signs, reduced body weight gain, reduced food consumption), but again, the study director stated that a direct developmental effect of the test material cannot be excluded. Considering that during the lactation phase dams at 15 mg/kg/day showed significant reductions of both body weight (11% lower than controls) and food consumption (18% lower than controls), and pup weight was comparable to controls at birth, it is clear that the effects on pup weight were secondary to maternal toxicity.

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material for