Carbon disulphide

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2018 - April 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

well performed and reported GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals : 10 weeks
- Basis for dose level selection : indicated by ECHA in final decision
- Inclusion/exclusion of extension of Cohort 1B: yes, F2 was produced
- Termination time for F2: when pups were ca. 3 weeks
- Inclusion of developmental neurotoxicity Cohorts 2A and 2B: yes
- Inclusion of developmental immunotoxicity Cohort 3: no
- Route of administration : oral (based on TK study in which oral and inhalation route were compared); see attachment and TK study in section 7.1

Test material

Test material information

1

Reference substance name:

Carbon disulphide

EC Number:

200-843-6

EC Name:

Carbon disulphide

Cas Number:

75-15-0

Molecular formula:

CS2

IUPAC Name:

methanedithione

Test material form:

liquid: volatile

Details on test material:

Batch number 1801042970
Purity: 100.00%

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is preferred species; Wistar strain is commonly used and historical control data are available

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of dosing, animals were 7 weeks old and weighed between 178 and 222 g (males) and between 136 and 177 g (females ).
The F0-animals were allowed to acclimate to the Test Facility toxicology accommodation for 12 days before the commencement dosing.
F0- animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.
On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages.
During the post-mating phase, males were housed in their home cage; females were individually housed in Macrolon plastic cages.
During the lactation phase, females were housed in Macrolon plastic cages; pups were housed with the dam until termination or weaning (on PND 21).
During locomotor activity monitoring, F1- Cohort 2A animals were housed individually.
The cages contained appropriate bedding and were equipped with water bottles.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
Target temperatures of 18 to 24°C and with a target relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 22°C with an actual daily mean relative humidity of 39 to 71%.
A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures.
Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, F1- Cohort 2A animals had no access to food for a maximum of 2 hours.
Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, F1- Cohort 2A animals had no access to water for a maximum of 2 hours.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

In the TK study it was investigated whether oral expsoure could be used instead of inhalation exposure (see section 7.1). See attachment for the setting of the dose levels.
The test item and vehicle are administered to the appropriate animals once daily by oral gavage 7 days a week. F0-males are treated for a minimum of 11 weeks, including 10 weeks prior to mating (with the objective of covering at least one spermatogenic cycle) and during the mating period, up to and including the day before scheduled necropsy. F0-females are treated for a minimum of 16 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females were not dosed during littering.
Animals are dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal is based on the most recent body weight measurement. The doses are given using a plastic feeding tube. The first day of dosing is designated as Day 1.
During lactation (up to PND 21), pups were not treated directly but could potentially be exposed to the test item in utero, via maternal milk of from exposure to maternal excreta. From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B, 1C and 2A are dosed up to and including the day before scheduled necropsy. The F1-animals of Cohort 2B, Cohort Surplus and Spares and F2-animals were not dosed and stayed together with their mothers until scheduled necropsy.

Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and are discarded after the assessment is complete.

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Based on stability data of the test item in vehicle, the dosing formulations were prepared as daily solutions up to 8 days prior to use.
Immediately after preparation, the containers with the test item dosing formulations were tightly closed and shaken vigorously for at least 30 seconds. Thereafter, the dosing formulations used on the day of preparation were continuously stirred and kept at room temperature until and during dosing. If not used on the day of preparation, the dosing formulations were stored in the refrigerator protected from light for a maximum of 8 days. On the day of use, the dosing formulations were taken out of the refrigerator for at least 30 minutes before start dosing (to allow acclimatizing to room temperature). While acclimatizing, the formulations were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

Details on mating procedure:

Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days is allowed for mating, after which females which had not shown evidence of mating were separated from their males without further opportunity of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples are collected for analysis in week 1, 11, 22, 33 and 34 of treatment. The concentration of the test substance was measured in all samples, the homogeneity in samples of the low and high dose groups only.
All samples to be analyzed were transferred (at room temperature protected from light) to the analytical laboratory at the Test Facility.The analyses were performed using a validated analytical procedure...................................
The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) on the first three occasions, i.e. in weeks 1, 11 and 22 of the study, and varied between ‘min’ to ‘max’ of target. On the fourth occasion in week 33, the concentrations analysed fluctuated around the lower limit of the target range of 90% and varied between ‘min’ to max’. The accuracy of concentration in the Group 3 formulation was slightly higher (i.e. 92%) and those in Groups 2 and 4 formulations were slightly exceeding the lower limit of the target range (i.e. 87% and 89%, respectively). An additional analysis was therefore performed in week 34. Accuracy of concentrations of 91%, 95% and 89% were determined in Group 2, 3 and 4 formulations, respectively. No analytical reason could be found for the slightly lower accuracies in the formulation analyses of the formulations prepared in week 33 and 34 compared to those prepared in weeks 1, 11 and 22. No test item was detected in the Group 1 (control) formulations.
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Overall , although the analyses of the dose formulations on the fourth and fifth occasion showed that the concentrations in some of the formulations were minimally outside the lower limit of 90% accuracy, whereas the results on the first three occasions were within the acceptance criteria, the dose formulations at all concentrations were considered suitable for use in the study.

Duration of treatment / exposure:

Daily gavage exposure during10 weeks prior to mating, during mating and pregnancy, up to scheduled necropsy.

Frequency of treatment:

Daily

Details on study schedule:

On PND 4, eight F1 and F2 pups from each litter of equal sex distribution (if possible) were selected to reduce variability among the litters. The non-selected pups were culled on PND 4.
On PND 21, F1 pups from available litters per group were selected and assigned to the different Cohorts. In general, pups were selected from litters in the order of delivery date. After completion of the Cohorts, any dams with pups/litters remaining were assigned as Spare animals.
On PND 21, F2 pups were sacrificed.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: set by ECHA in final decision
- Rationale for animal assignment (if not random): random (computerized)
- Fasting period before blood sampling for clinical biochemistry: The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Blood of 10 selected animals /sex/group of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anaesthesia using isoflurane.

Positive control:

Not required for this type of study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings. Clinical observations were performed at least twice daily, up to the day prior to necropsy. These observations were at least conducted prior to dosing and within 0-30 minutes after dosing. The time of onset, grade and duration of any observed sign was recorded.
DETAILED CLINICAL OBSERVATIONS
Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.
BODY WEIGHT:
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

In addition: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

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

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.
Subjective appraisal of water intake was maintained during the study, but no quantitative investigation was introduced as no effect was suspected or noted.

Blood from selected overnight fasted F0-animals (10 animals /sex/group) was collected on the day of scheduled necropsy.
Urine was collected into a specimen vial from the 10 selected animals/sex/group housed in individually in metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available. .

The following was measured in F0 animals:
- haematology: White blood cells (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Red blood cells, Reticulocytes (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets
- coagulation: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- clinical chemistry: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile Acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate (Inorg. Phos)
- thyroid hormones: Thyroxine (T4), Thyroid Stimulating Hormone (TSH)
- urinalysis: Volume, Specific gravity, Clarity, Colour, pH, Blood, White blood cells (WBC), Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite, Sediment (White blood cells (WBC-sed.), Red blood cells (RBC-sed.), Casts, Epithelial cells, Crystals, Bacteria)

Oestrous cyclicity (parental animals):

Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken.

Sperm parameters (parental animals):

Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

Litter observations:

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

PARAMETERS EXAMINED
The following parameters were examined in [F1/F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups (see further down)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: yse see further down

Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Pups showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.
Live pups were weighed individually on PND 1, 4, 7, 14 and 21 (only if necropsy on PND 21-23 was decided).
For animals of Cohorts 2B and Surplus and F2-animals of Cohort 1B (10 selected litters/group; one male and one female), a terminal weight was recorded on the day of scheduled necropsy.
Sex was externally determined for all pups on PND 1 and 4.
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
All male pups in each litter were examined for the number of areola/nipples on PND 13.

The in-life procedures, observations, and measurements listed below were performed for all F1-animals from weaning (PND 21) onwards, except for the animals of Cohort 2B, Cohort Surplus and spare F1-animals as these were terminated on PND 22-24.
* Mortality/Moribundity Checks – Cohorts 1A, 1B, 1C and 2A
Throughout the study, animals were observed for general health/mortality and moribundity twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
* Clinical Observations– Cohorts 1A, 1B, 1C and 2A
Clinical observations were performed at least twice daily, up to the day prior to necropsy. These observations were at least conducted prior to dosing and within 0-30 minutes after dosing
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.
* Arena Observations – Cohorts 1A, 1B, 1C and 2A
Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.
* Body Weights – Cohorts 1A, 1B, 1C and 2A
Animals were weekly weighed weekly.individually. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. For animals of Cohorts 1A, 1B, and 2A, a terminal weight was recorded on the day of scheduled necropsy.
* Food Consumption– Cohorts 1A, 1B, 1C and 2A
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.
* Water Consumption – Cohorts 1A, 1B, 1C and 2A
Subjective appraisal of water intake was maintained during the study, but no quantitative investigation was introduced as no effect was suspected or noted.
* Vaginal Patency – Cohorts 1A, 1B, 1C and 2A
Vaginal patency (vaginal opening) was monitored daily for all females from PND 25 onwards until vaginal patency wais present, by visual inspection of the vaginal area. Body weight was recorded on the day of acquisition of vaginal patency.
* Balanopreputial Separation – Cohorts 1A, 1B, 1C and 2A
Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area. Body weight was recorded on the day of acquisition of balanopreputial separation.
* Stage of Estrus Determination – Cohorts 1A, 1B, 1C and 2A
Estrous stages were determined by examining the cytology of vaginal lavage samples taken on the day of scheduled necropsy, and on specific time periods during treatment in particular Cohort females (see below).

Cohort 1A (20 animals/sex/group) The in-life procedures, observations, and measurements listed below were performed for the F1-females of Cohort 1A only, in addition to the procedures mentioned.
* Estrous Cycle Determination – Cohort 1A
Estrous stages were determined by examining the cytology of vaginal lavage samples taken during two periods. During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.

Cohort 1B (20 animals/sex/group) The in-life procedures, observations, and measurements listed below were performed for the F1-animals of Cohort 1B only, in addition to the procedures mentioned.
Based on the reproductive and developmental findings obtained in this study in the F0-animals and produced F1-pups, the males and females of Cohort 1B were selected for production of an F2-generation for possible clarification of these results.
* Body Weights – Cohort 1B
Mated females were weighed individually on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. The PND 21-body weight of each F1 animal that was weaned thereafter was entered to the computer for calculation of the dose volume until the next scheduled body weight measurement.
* Cohabitation/Mating Procedure – Cohort 1B
Animals were cohabitated on a 1:1 basis within the same treatment group , avoiding sibling mating after at least 10 weeks of treatment, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating. There were no couples without evidence of mating at the end of the 14 days mating period.
* Food Consumption– Cohort 1B
Food consumption was not determined for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was quantitatively measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.
* Estrous Cycle Determination – Cohort 1B
Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed from the start of the mating period until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
* General Reproduction Data – Cohort 1B
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Cohort 2A (10 animals/sex/group) The in-life procedures, observations, and measurements listed below were performed for the F1-animals of Cohort 2A only, in addition to the procedures mentioned.
* Acoustic startle response – Cohort 2A
Acoustic startle response (habituation) was assessed using the StartleMonitor System (Kinder Scientific, Poway, USA). This was performed once between PND 23-25 in a sound-attenuated room. To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose groups and sex. The animals were tested in sets of up to 3. The test sessions consisted of a five-minute acclimation period with a 65 ± 5-dB broadband background white noise. The startle stimulus for each trial was a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 milliseconds in duration). Responses were recorded during the first 250 milliseconds following onset of the startle stimulus for each trial. The test session consisted of 50 trials with an eight-second intertrial interval. Average response amplitude (AveN) was analyzed in five blocks of 10 trials each.
* Functional Observation Battery – Cohort 2A
The Functional Observation Battery (FOB) tests were conducted once between PND 63-75 in the order of sequence indicated below and were divided between several days. The detailed clinical observations and locomotor activity were conducted in separate room(s) specially equipped for these purposes. The other FOB tests were conducted in the study room.
1. Detailed clinical observations - Detailed clinical observations consisted of a number of tests conducted in- and out-side the home cage. To the extent possible, testing of animals was counterbalanced across dose groups.
2. Rectal temperature - Rectal temperature was measured immediately after the detailed clinical observations.
3. Locomotor activity was tested using the Kinder Scientific Motor Monitor System. Recording period was one hour under normal laboratory light conditions. To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose groups. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
4. Hearing ability - Score 0 = normal/present, score 1 = abnormal/absent.
5. Pupillary reflex (both eyes).
6. Fore- and hindlimb grip strength. This was recorded per animal as the mean of three measurements, using a grip strength meter (Series M4-10, Mark-10 Corporation).
7. Landing (hind) foot splay. This was recorded per animal as the mean of three measurements.

The selected Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Blood of 10 selected animals /sex/group of Cohort 1A animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anaesthesia using isoflurane.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of Cohort 1A animals housed in individually in metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available. .

F1-generation and F2-generation (PND 4) pups
On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation, between 7.00 and 10.30 a.m. in the necropsy room, and samples were pooled per litter. If available, blood was collected from one male and one female pup per litter. If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup.

F1- animals of Cohort Surplus on PND 22
On PND 22, blood was collected from all Cohort Surplus animals (max. 10/sex/group), if possible. Blood was withdrawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.

The following was measured in F1 animals (Cohort 1A):
- haematology: White blood cells (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Red blood cells, Reticulocytes (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets
- coagulation: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- clinical chemistry: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile Acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate (Inorg. Phos)
- thyroid hormones: Thyroxine (T4), Thyroid Stimulating Hormone (TSH)
- urinalysis: Volume, Specific gravity, Clarity, Colour, pH, Blood, White blood cells (WBC), Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite, Sediment (White blood cells (WBC-sed.), Red blood cells (RBC-sed.), Casts, Epithelial cells, Crystals, Bacteria)

Serum thyroid hormones Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) were also measured in the following groups:
- F1 animals (Cohort 1B) at scheduled necropsy
- F1 culled pups on PND 4
- F1 pups (Cohort 2B) and surplus pups on PND 21-22
- F2 pups on PND 21-23

Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy (Cohort 1A). Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples. One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. One pup (male or female) was selected per litter (20 litters in total). One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 µm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The following subpopulations were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy: T-cells, T-helper cells, T-cytotoxic cells, B-cells, NK-cells, Ratio T-helper cells/ T-cytotoxic cells (Th/Tc). The % lymphoid cells of peripheral blood mononuclear cells (PBMC) wasere determined using the Forward Scatter and Side Scatter.

Postmortem examinations (parental animals):

Scheduled necropsies are summarized below :
- Males (which sired and failed to sire): After successful mating and a minimum of 10 weeks of treatment.
- Females which delivered: LD 23-25.
- Females which failed to deliver:
- With evidence of mating: Post-coitum Days 26-27.
- Without evidence of mating: Approximately 26 days after the last day of the mating period.
- Females with total litter loss: Within 24 hours after the last pup was found dead or missing.
Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available.
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

Representative samples of the following tissues were collected from all F) animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated:
Artery (aorta), Body cavity (nasopharynx), Bone marrow, Bone (femur), Bone (sternum), Brain, Cervix, Epididymis, Esophagus, Eye, Gland (adrenal), Gland (clitoral), Gland (coagulation), Gland (harderian), Gland (lacrimal), Gland (mammary), Gland (parathyroid), Gland (pituitary), Gland (preputial), Gland (prostate), Gland (salivary), Gland (seminal vesicle), Gland (thyroid), Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine (cecum), Large intestine (colon), Large intestine (rectum), Larynx, Liver, Lung, Lymph node, Muscle (skeletal), Nerve (optic), Nerve (sciatic), Ovaries, Oviducts, Pancreas, Skin, Small intestine (duodenum), Small intestine (ileum), Small intestine (jejunum), Spinal cord, Spleen, Stomach, Testes,
Thymus, Tongue, Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

The following organs were weighed at necropsy for all scheduled euthanasia animals:
Brain, Epididymis, Gland (adrenal), Gland (pituitary), Gland (prostate), Gland (seminal vesicle), Gland (thyroid), Heart, Kidney, Liver, Ovaries, Spleen, Testes, Thymus, Uterus.
Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

The following tissues/organs were examined microscopically:
Bone marrow, Bone (sternum), Brain, Cervix, Epididymis, Eye, Gland (adrenal), Gland (coagulation), Gland (mammary), Gland (parathyroid), Gland (pituitary), Gland (prostate), Gland (seminal vesicle), Gland (thyroid), Gross lesions/masses, Heart, Kidney, Large intestine (cecum), Large intestine (colon), Large intestine (rectum), Liver, Lung, Muscle (skeletal), Nerve (optic), Nerve (sciatic), Ovaries, Oviducts, Small intestine (duodenum), Small intestine (ileum), Small intestine (jejunum), Spinal cord, Spleen, Stomach, Testes, Thymus, Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

Tissues were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).

Postmortem examinations (offspring):

Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally) and externally examined with emphasis on developmental morphology. For pups found dead or sacrificed in extremis from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally).
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. From two extra pups per litter, blood was collected, if possible. Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

Scheduled necropsy of the F2-animals of Cohort 1B was conducted on PND 21-23. These animals were sacrificed using Euthasol®20% by intraperitoneal (ip) injection, except the pups selected for blood collection. The pups selected for blood collection, i.e. one male and one female pups from 10 selected litters, were anesthetized using isoflurane followed by exsanguination. The animals were not deprived of food overnight. From 10 selected litters/group, terminal body weight was determined for one male and one female pup. Subsequently, these pups were deeply anaesthetized using isoflurane and subsequently exsanguinated. The animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The organs identified for weighing and representative samples of the tissues (see at parental animals) were weighed and collected. All remaining pups were sacrificed using Euthasol®20% by intraperitoneal (ip) injection. Also these pups were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded. Tissues were preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Organ weights were not recorded for animals found dead. Paired organs were weighed together. In the event of macroscopic findings, in addition to the combined weight, the weight of the aberrant organ was taken and recorded. Organ to body weight ratios (using the terminal body weight) were calculated. The organs identified for weighing and representative samples of the tissues (see at parental animals) were weighed and collected.

Cohort 1A: Scheduled necropsy of Cohort 1A was conducted on PND 89-95. Cohort 1A animals were deprived of food overnight (with a maximum of 24 hours) before necropsy, but water was available. The animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

Scheduled necropsy of the F1-Generation of Cohort 1B was conducted on the following days :
F1-Males (which sired and failed to sire): Following completion of the mating period.
F1-Females which delivered: LD 21-23
F1-Females which failed to deliver:
- With evidence of mating: Post-coitum Days 25-27.
- Without evidence of mating: Approximately 25 days after the last day of the mating period.
The F1-animals of Cohort 1B were not deprived of food overnight before necropsy. The animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites are present , nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea were recorded in addition.

Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy and no terminal body weight was recorded.
The animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Scheduled necropsy of Cohort 2A was conducted on PND 76-90. Scheduled necropsy of Cohort 2B was conducted on PND 21-22. The animals were not deprived of food overnight before necropsy and no terminal body weight was recorded.
The animals were first anaesthetized using isoflurane and subsequently sacrificed by whole body (in situ) perfusion using heparinized saline (0.9% NaCl) followed by a 4% paraformaldehyde solution (adjusted to pH 7.4; HCl, KCl, NaH2PO4 x H2O, Na2HPO4 x 2H2O, paraformaldehyde and NaOH, aqua dest.). All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. After perfusion, the cranium was removed, exposing the brain. The skull including the brain was placed in 10% buffered formalin and allowed to fix for at least 7 days prior to removal from the skull. The fixed brains were removed and weighed, and the length and maximum width of the brain was measured for all animals selected for neuropathology . Subsequently, the brain was stored in 10% buffered formalin together with selected PNS tissues until further processing. Length of the brain: on a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum. Width of the brain: pituitary region. Measurements were conducted using a digital caliper.

Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. On PND 22, blood samples (1.0 mL) were collected between 7.00 and 10.37 am from all animals by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure. Blood samples were collected into serum tubes for measurement of thyroid-stimulating hormone (TSH) and thyroxine (T4).

Tissues mentioned (see at parental animals) were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).
Cohort 1A - In addition to the procedures described above, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A animals of Group 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea. This examination was extended to females ofin the intermediate dose groups (Groups 2 and 3).
Cohort 1B - Except for the reproductive organs of infertile animals, histological processing and histopathological examination of Cohort 1B preserved tissues was not performed in first instance, but was or was not might be required based on the results of Cohort 1A.
Cohort 2A and 2B - In addition to the procedures described above, the entire brain from all Groups was processed to the block stage up front at the same time to avoid effects of fixation duration on morphometry. Sections of the brains of all Cohort 2A and 2B animals (all groups) were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet. For morphometric analysis, 3 consecutive sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections weare obtained. The brain slices were prepared using the following neuroanatomic landmarks.
Cut 1 - just caudal to the olfactory bulbs
Cut 2 - midway between the optic chiasm and the plane of the first section
Cut 3 - through the optic chiasm
Cut 4 - through the mid-infundibulum
Cut 5 - rostral to the caudal edge of the mammillary body
Cut 6 - just rostral to the edge of the pons
Cut 7 - at the caudal margin of the trigeminal nerve root
Cut 8 - through the middle of the cerebellar cortex
Cut 9 - approximately 2mm rostral to the caudal edge of the cerebellar cortex
Cut 10 - through the origin of the spinal canal at the medullary obex
All coronal brain slices were placed rostral face-downwards into the cassettes (so that these rostral faces wereas sectioned) with the exception of the olfactory bulbs, which were embedded with medial surfaces-downwards.
The block list for the brain is presented below:
Paraffin blocks, ~ 5 micron sections, stained with H&E and LFB/CV:
Block 1: Brain, level of optic nerve.
Block 2: Brain, level of infundibulum.
Block 3: Brain, level of mammillary body and midbrain.
Block 4: Brain, level of middle of the cerebellum.
Block 5: All remaining brain sections.
The time period between start of fixation in 10% buffered formalin and embedding in paraffin for brains of Cohort 2A and 2B animals was kept similar to avoid effects in morphometry that may be caused by duration of fixation.

Cohort 2A and 2B animals - Morphometric (quantitative) analyses of CNS tissues was performed for Cohort 2A and 2B animals of Groups 1 and 4. To possibly clarify the findings obtained for Group 1 and 4 (at Level 1), these examinations were extended to animals of the intermediate dose groups (Groups 2 and 3). Analyses included measurements from selected neocortical, hippocampal, and cerebellar areas selected.
Microscopic morphometric measurements were performed on sections of the brain from Blocks 1, 2 and 4 stained with LFB/CV. Microscopically, 11 linear morphometric measurements were taken from each brain. Slides were scanned using a Hamamatsu Nanozoomer whole slide scanner and imported into the Visiopharm software for measurements. These measurements were performed in a blinded random fashion for all dose groups. At the discretion of the neuropathologist, additional neuroanatomic structures may be measured (e.g., if histologic changes suggest that these regions have been affected by the treatment). The following linear measurements were taken:
1) Thickness of the frontal cortex bilaterally. This measurement is taken from the dorsal portion of the cerebral cortex within the coronal section passing through the region of the optic chiasm .
2) Thickness of the parietal cortex bilaterally. This measurement is taken from the dorsolateral portion of the cerebral cortex within the coronal section taken through the optic chiasm.
3) Diagonal width (maximum cross-sectional width) of the caudate-putamen bilaterally. This measurement is performed on the coronal section taken at the level of the optic chiasm.
4) Thickness of the corpus callosum bilaterally just lateral to its midpoint at the level of Layer 2 of the overlying cingulate gyrus. This measurement is taken within the section passing through the optic chiasm.
5) Thickness of all hippocampal layers combined, bilaterally, extending from the alveus to just lateral to the inferior blade of the dentate gyrus. This measurement is performed within the section taken at the level of the infundibulum.
6) Maximum height of the cerebellum at the level of the deep cerebellar nuclei, extending from the roof of the fourth ventricle to the dorsal surface. Lobules 1 or 10 of the cerebellum (which protrude into the fourth ventricle) may be excluded from measurement if inconsistently present on section.
Morphometric measures taken bilaterally were averaged for each rat. Statistical analysies of the morphometry data was conducted.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric - Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric - Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The motor activity data set (at least 3 groups) was compared using an overall Kruskal-Wallis.
Incidences - An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating index males (%): Number of males mated/Number of males paired x 100
Mating index females (%): Number of females mated/Number of females paired x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index males (%): Number of pregnant females/Number of males mated x 100
Fertility index females (%): 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 site x 100
Post-implantation survival index will be expressed as 100% when the number of offspring exceeds the number of implantation sites recorded.

Offspring viability indices:

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
Weaning index (%): Number of live offspring on Day 21 of lacation/Number live offspring on Day 4 (after culling) x 100
Percentage live males at weaning (%): Number of live male pups on Day 21 after littering/Number of live pups on Day 21 after littering x 100
Percentage live females at weaning (%): Number of live female pups on Day 21 after littering/Number of live pups on Day 21 after littering x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted in males and females up to 120 mg/kg/day during the daily detailed clinical observations and during the weekly arena observations. Slight salivation was predominantly noted in males and females treated at 120 mg/kg/day and occasionally among a single control and Group 3 animals from Week 2 of treatment onwards. Salivation was not observed in Group 2 males and females. Taking into account the nature, the distribution over the groups, the minor severity of the effect and its time of occurrence (i.e. generally after dosing), this sign was considered to be a physiological response related to taste of the test item, the vehicle or a combination thereof rather than being a sign of systemic toxicity.

Mortality:

no mortality observed

Description (incidence):

No treatment-related mortality occurred. All males and females were sacrificed on the day of scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gain were unaffected by treatment up to 12 mg/kg/day.
At 120 mg/kg/day, body weights and body weight gain of males and females were slightly decreased (reaching statistical significance on most occasions for the males and on some occasions for the females) from Week 5 of treatment onwards.
For high dose males, mean body weight gain was 0.86x of controls after 11 weeks of treatment (Week 2 of mating period). For high dose females, mean body weight gain was 0.89x, 0.86x and 1.88x of controls at the end of the pre-mating, post-coitum and lactation period, respectively. At termination the mean body weights of the high dose females were within the same range as controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Since the mean food consumption relative to body weight was similar for all groups at all times during treatment, the differences in absolute food consumption were considered in line with the changes in mean body weight between the treated animals and controls.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Description (incidence and severity):

not required in OECD 443

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters were unaffected by treatment up to 12 mg/kg/day in males and females .
The following changes were noted in males and lactating females at the end of treatment. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
• An increased reticulocyte count in males (1.25x ) and females (1.28x; not statistically significant) treated at 120 mg/kg/day, with values outside the historical control range .
• A dose- related increase in the number of white blood cell (1.16, 1.26 and 1.53x in 1.2, 12 and 120 mg/kg/day treated females, respectively), with values outside the historical control range .
• An increased percentage of neutrophils (1.4x) and concurrent decreased percentage lymphocytes (0.85x) relative to the number of white blood cells in high dose group females.
Coagulation parameters were unaffected by treatment up to 120 mg/kg/day.
Given the slight magnitude of these changes (compared to ranges considered normal for rats of this age and strain) and absence of corroborative alterations, all changes in haematology parameters were considered non-adverse.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes were noted in treated males and lactating females. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
- An increased alanine aminotransferase (ALAT) value in lactating females at 120 mg/kg/day (1.41x ). No toxicological relevance was attached to this finding, as mean values remained within the available historical control range and in the absence of correlating findings on liver toxicity.
- A decreased mean plasma concentration of potassium in males at 12 and 120 mg/kg/day (0 .93x and 0.91x, respectively).
- A decreased mean plasma concentration of calcium in males at 120 mg/kg/day (0.98x). No toxicological relevance was attached to this finding, given the minimal magnitude of change and as mean values remained within the available historical control range .
- An increased mean inorganic phosphate (Inorg. Phos) plasma concentration in males at 120 mg/kg/day (1.16x).
- Mean serum levels of total T4 were statistically significantly lower than those in controls in both males (0.51x) and lactating females (0.69x) at 120 mg/kg/day.
- Mean serum levels of thyroid stimulating hormone (TSH) in F0 males and females were not affected by treatment up to 120 mg/kg/day.
Given the slight magnitude of these changes (compared to ranges considered normal for rats of this age and strain) and absence of corroborative alterations, all changes in clinical biochemistry parameters were considered non-adverse.
The effects on T4 were considered to be test item-related; however, under the conditions of this study no adverse effect was observed that could be linked to the reduction of total T4.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Eyes: an increased incidence and severity of retinal atrophy of the outer nuclear layer was present at 120 mg/kg/day in males up to a moderate and in females up to a marked degree (3/25 animals).
Spleen, an increased incidence and severity of extramedullary hematopoiesis was present in males at 120 mg/kg/day up to slight degree; a minimally increased incidence was seen in males at 12 mg/kg/day.
Spleen, an increased incidence and severity of hemosiderin pigmentation was present at 120 mg/kg/day in males up to a slight degree and in females up to a moderate degree.
Thymus, an increased incidence and severity of lymphoid depletion was present in males at 120 mg/kg/day up to a slight degree. This correlated with the observed decreased mean thymus weight at 120 mg/kg/day.
See tables attached.
Except for the eyes, these changes were considered non-adverse at the severities noted and in the absence of degenerative changes. The changes in the eyes were considered adverse based on the loss of cell layers in the retina.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment up to 120 mg/kg/day.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm motility, concentration and morphology were considered to be unaffected by treatment up to 120 mg/kg/day. After treatment at 120 mg/kg/day, the percentages of motile sperm and progressive sperm were observed to be decreased (i.e. 0.86x and 0.74x, respectively), achieving a level of statistical significance for progressive sperm when compared to controls. Furthermore, the number of cells with a coiled tail (0.64x) was decreased at 120 mg/kg/day, achieving a level of statistical significance compared to controls. However, the direction of this change was in the opposite direction of what would be expected for a reproductive toxicant and this finding was therefore considered not to be related to test item administration. In addition, stage dependent qualitative evaluation of spermatogenesis in the testis was performed . The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
These changes were within the historical control range. In addition, the minimal changes in motility and progressivity of the sperm observed in the F0-generation were not observed in the F1-generation and were therefore considered an incidental finding of no toxicological significance, since it appeared not related to the changes in post-implantation survival index and litter size observed in both generations in this study.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were 3/25 couples of the control group, 3/25 couples of the 1.2 mg/kg/day group, 2/25 couples of the 12 mg/kg/day group and 4/25 couples of the 120 mg/kg/day group without healthy offspring. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
Mating index was not affected by treatment. The mating indices were 96% for the control and 1.2 mg/kg/day groups, and 100% for the 12 and 120 mg/kg/day groups.
Precoital time was considered not to be affected by treatment up to 120 mg/kg/day.
Number of implantation sites was considered not to be affected by treatment up to 120 mg/kg/day.
Fertility index was slightly lower after treatment with the test item, i.e. the fertility indices were 96, 92, 92 and 88% for the control, 1.2, 12 and 120 mg/kg/day groups, respectively. However, when taking into account the single not mated control and 1.2 mg/kg/day treated females, the number of pregnant females was comparable in all groups and varied between 22/25 and 23/25 females with offspring. Therefore, the fertility of the females was considered not to be affected by treatment.
Gestation index and duration of gestation were considered not to be affected by treatment. All pregnant females had live offspring, except for one control female (No. 102) which delivered dead pups only (total litter loss). The gestation indices were 96% for the control and 100% for the 1.2, 12 and 120 mg/kg/day groups.
No signs of difficult or prolonged parturition were noted among treated females.
Examination of cage debris of the any females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
The total number of offspring born compared to the total number of uterine implantations was slightly lower after treatment at 12 and 120 mg/kg/day. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 94, 88 and 87% for the control, 1.2, 12 and 120 mg/kg/day groups, respectively.
Litter size was slightly lower at 120 mg/kg/day. Mean litter sizes were 10.6, 11.4, 11.8 and 9.8 living pups/litter for the control, 1.2, 12 and 120 mg/kg/day groups, respectively. However, as individual values remained within the concurrent control range and mean values remained within the available historical control range, no toxicological relevance was attached to this observation.
Sex ratio was considered not to be affected by treatment.

Details on results (P0)

Overall, retinal atrophy (minimal-moderate in males and minimal-marked in females) was seen in P0 animals following treatment at 120 mg/kg/ bw/day. In addition, at this dose level reduced BW gain (up to ca. 10% compared to controls) was seen especially in males; also reduced mean brain weights (up to 6% compared to controls) were observed in males and females at 120 mg/kg/day. However, it cannot be excluded that the reduction in absolute brain weight was related to the reduction in body weight gain as the study was started with young adult animals. There was no indication of behavioural or functional changes. Based on these results, a NOAEL of 12 mg/kg/day for general toxicity was established.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced body and brain weights and retinal atrophy at the next higher level of 120 mg/kg bw

Remarks on result:

other: see at details on results (P0)

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: Reduced body and brain weight, retinal atrophy

Organ:

other: brain weight and eyes

Treatment related:

yes

Dose response relationship:

yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Slight salivation was noted for most males and females treated at 120 mg/kg/day from Week 2 of treatment onwards. Taking into account the nature and minor severity of the effect and its time of occurrence (i.e. generally after dosing), this sign was considered to be a physiological response related to taste of the test item, the vehicle or a combination thereof rather than being a sign of systemic toxicity .

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related mortality occurred during the full study period of the F1 animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight and body weight gain were statistically significantly decreased in males at 120 mg/kg/day from Day 8 of treatment onwards, with maximum differences of controls of 0.85x and 0.77x the mean control value for body weight and body weight gain, respectively. Males of the at 12 mg/kg/day group showed statistically significantly decreased mean body weight (gain) between Days 29 and 64 only, with maximum differences of controls being 0.94x and 0.90x the control value for body weight and body weight gain, respectively. At the end of the treatment period, mean body weights were 0.97x and 0.89x of controls, for males of the at 12 and 120 mg/kg/day groups, respectively.
In females of the at 120 mg/kg/day group, mean body weight and/or body weight gain were statistically significantly decreased between Days 8 and 64 of treatment, with maximum differences of controls being of 0.90x and 0.76x the control value for body weight and body weight gain, respectively. At the end of the treatment period, mean body weight was 0.95x of controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption relative to body weight was similar for all groups at all times during treatment; the differences in absolute food consumption were considered in line with the changes in mean body weight between the treated animals and controls.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Description (incidence and severity):

Not required in OECD 443

Description (incidence and severity):

The following changes were noted in males and lactating females at the end of treatment. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
• Increased mean reticulocyte count (1.19x control ) was noted in treated males at 120 mg/kg/day.
• Increased mean MCH (mean corpuscular haemoglobin, 1.03x) and MCHC (mean corpuscular haemoglobin concentration, 1.02x ) were noted in females at 120 mg/kg/day. Mean group values were within the available historical control range, however, values of one or more individual animals were slightly outside this range. As changes were slight and in the absence of any corroborating findings, these findings were considered to be isolated findings of no toxicological relevance.
Coagulation parameters of treated rats were unaffected by treatment up to 120 mg/kg/day.
Given the slight magnitude of these changes (compared to ranges considered normal for rats of this age and strain) and absence of corroborative alterations, all changes in haematology were considered non-adverse.

Description (incidence and severity):

The following statistically significant changes were noted in treated males at the end of treatment. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
• Decreased mean glucose levels (0.73x control) wasere noted in males at 120 mg/kg/day, with individual values outside the available historical control range.
• Decreased mean plasma creatinine concentration in males at 120 mg/kg/day (0.73x). As the direction of this change was in the opposite direction of what would be expected for a toxicant this finding was considered an incidental finding and of no toxicological relevance. Moreover, the majority of the individual creatinine values in high dose males were within the range of controls.
• Increased mean plasma sodium concentration in males at 120 mg/kg/day (1.01x), with individual values outside the available historical control range.
• Increased mean plasma chloride concentration in males at 120 mg/kg/day (1.02x), with individual values outside the available historical control range.
• Increase in mean plasma activity of aspartate aminotransferase (ASAT) in males at 120 mg/kg/day (1.12x). No toxicological relevance was attached to this finding, as the mean values remained within the available historical control range and in the absence of correlating liver findings.
• Mean serum levels of thyroid stimulating hormone (TSH) and T4 in F1 females of Cohort 1A and Cohort 1B were not affected by treatment. In males of Cohort 1A at 120 mg/kg/day, mean serum T4 level was lower (0.74x control) than that in controls, with individual values outside the available historical control range.
Given the slight magnitude of these changes (compared to ranges considered normal for rats of this age and strain) and absence of corroborative alterations, all changes in clinical biochemistry were considered non-adverse. The effect on T4 was seen in males only but was considered to be test item-related; however, under the conditions of this study no adverse effect was observed that could be linked to the reduction of total T4.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Acoustic startle response was considered not affected by treatment.
Detailed clinical observations revealed no symptoms that were considered to be related to treatment.
Mean rectal temperature was considered not affected by treatment.
Motor activity was considered not affected by treatment.
Functional observation parameters (hearing ability, pupillary reflex and grip strength) were considered not affected by treatment.
Hearing ability and pupillary reflex were normal in all examined animals. Grip strength was considered not affected by treatment.
Mean footsplay values were (not statistically significantly) decreased in males and females at 120 mg/kg/day (0.65x and 0.75x control, respectively). Individual values were partly outside the available historical control range. This finding might be related to the lower mean body weights observed in both sexes, consistent with a smaller body size of the high dose animals compared to controls. Moreover, the opposite effect (increased foot splay) would have been expected in case of a developmental neurotoxicant

Immunological findings:

no effects observed

Description (incidence and severity):

Splenic lymphocyte subpopulations were considered to be unaffected by treatment up to 120 mg/kg/day.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Brain weight changes were observed in the 120 mg/kg/day males and females groups. These consisted of statistically significantly decreased mean absolute brain weight in both males and females; however, mean relative brain weight was statistically significantly higher in males when compared to controls. In addition, statistically significantly lower mean thymus weights (absolute and relative to body weights) were noted in the 120 mg/kg/day group males. See tables attached.

The decreased thymic weight at 120 mg/kg/day was seen with lymphoid depletion up to a slight degree and at a similar incidence in males and females. This was considered non-adverse at the severities noted and in the absence of degenerative thymic changes in both sexes.
No histopathological alterations were found in any of the levels in the brain; a relationship between the decrease in absolute brain weight and the observed reduced growth in this high dose group is likely as the study was started with very young animals that already had a lower body weight at birth.

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 2A (Terminated PND 76-90, actively dosed for at least 8 weeks); see for cohort 2B, at F1 animals
Decreased mean fixed brain weights were noted in males of the 120 mg/kg/day group; the decrease in mean absolute weight was statistically significant.
There were no changes in test item-related brain dimension (length and width) changes.
A statistically significantly lower mean corpus callosum thickness was noted for the 120 mg/kg/day group males and a statistically significantly higher mean caudate putamen width was noted for the 120 mg/kg/day group females. Almost all (8/10) of the individual values for corpus callosum thickness in the 120 mg/kg/day group males were lower than the control group. Seven out of ten of the individual values for caudate putamen width in the 120 mg/kg/day group females were within the range of the control group.
See tables attached.

Upon morphometric analysis of the brain differences in linear measurements were observed but none of the differences exhibited the same directional trend in males and females or were observed at both time points. The changes were inconsistent across brain regions, sexes, and time points and several changes were in a direction opposite what would be expected in case of developmental neurotoxicity. Individual animal data were evaluated to investigate a correlation between lower morphometry values and lower brain weight and/or brain gross measurement values, but the correlations were inconsistent. Based on the observations, there is no evidence of the test item being a (developmental) neurotoxicant.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Eyes: retinal atrophy of the outer nuclear layer was present at 120 mg/kg/day in a few males and females up to a slight degree, which was considered to be adverse based on the loss of cell layers in the retina..
Thymus: lymphoid depletion was present at 120 mg/kg/day in males and females up to a slight degree. For males this correlated with the decreased mean thymus weight at 120 mg/kg/day.
Ovaries: Statistically significantly decreased primary and primordial follicle counts were present in the F1 120 mg/kg/day group females. There was no significant effect on corpora lutea counts in the 120 mg/kg/day group females compared to controls. In addition, this effect was not accompanied by changes in any of the other reproduction parameters assessed for the F1-females, nor for F0-females. Moreover, this change was without any qualitative histological correlate or reproductive organ weight change. Therefore, this was considered non-adverse in the present study.
See tables attached.

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed . The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
In the F1 animals (Cohort 2B), there were no test item-related effects on the H&E or Luxol Fast Blue/Cresyl Violet stained sections of brain in the control or high-dose group.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had regular cycles of 4 to 5 days as examined between PND 75 and 88.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

At 120 mg/kg/day, the mean number of cells with a detached head was statistically significantly increased (4x of controls), with the mean value being outside the available historical control range. Mean total sperm count in the epididymis, and percentage motile and progressive sperm were statistically significantly increased in males of the 12 mg/kg/day group. As the mean values for percentage motile and progressive sperm remained within the historical control range, in the absence of a dose-response relationship and given the direction of change (i.e. an increase rather than a decrease), these findings were considered to be of no toxicological relevance.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was not affected by treatment. The mating index was 100% for the control and all treatment groups.
Precoital time was considered not to have been affected by treatment. Most females showed evidence of mating within 4 days.
The number of implantation sites was considered not to be affected by treatment, i.e. the mean implantation sites were 12.6, 11.5, 11.9 and 11.1 for the control, 1.2, 12 and 120 mg/kg/day groups, respectively.
The fertility index was slightly lower after treatment at 1.2 and 120 mg/kg/day, i.e. the fertility indices were 95, 85, 95 and 90% for the control, 1.2, 12 and 120 mg/kg/day groups, respectively; a dose-response relationship was absent.
Gestation index was slightly lower at 12 and 120 mg/kg/day, i.e. gestation indices were 100, 100, 95 and 94% for the control, 1.2, 12 and 120 mg/kg/day groups, respectively. The duration of gestation was considered not to be affected by treatment up to 120 mg/kg/day.
There were 1/20 couples of the control group, 3/20 couples of the 1.2 mg/kg/day group, 2/20 couples of the 12 mg/kg/day group and 3/20 couples of the 120 mg/kg/day group without healthy offspring. No abnormalities were seen in the reproductive organs.
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
The mean total number of offspring born compared to the mean total number of uterine implantations was slightly lower after treatment at 12 and 120 mg/kg/day. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 98% for the control and 1.2 mg/kg/day groups and 93% for the 12 and 120 mg/kg/day groups.
Litter size was slightly lower at 120 mg/kg/day. Mean litter sizes were 12.2, 11.1, 11.7 and 10.8 living pups/litter for the control, 1.2, 12 and 120 mg/kg/day groups, respectively.

Details on results (P1)

Overall, retinal atrophy (minimal-slight) was seen in P1 animals following treatment at 120 mg/kg/ bw/day. In addition, at this dose level reduced BW gain (up to 10% compared to controls) was seen especially in males; also reduced mean brain weights (up to 10% when compared to controls) were observed in males and females at 120 mg/kg/day. There was no indication of behavioural or functional changes.
Based on these results, a NOAEL of 12 mg/kg/day for general toxicity was established.

Effect levels (P1)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced body and brain weights and retinal atrophy at the next higher level of 120 mg/kg bw

Remarks on result:

other: see at details on results (P1)

Target system / organ toxicity (P1)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: Reduced brain weight, retinal atrophy

Organ:

other: brain weight and eyes

Treatment related:

yes

Dose response relationship:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical signs were noted in males and females up to 120 mg/kg/day during the daily detailed clinical observations or during the weekly arena observations.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was 99, 100, 99 and 100% for the control, 1.2, 12 and 120 mg/kg/day groups, respectively.
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered not to be affected by treatment. Viability indices (number of live offspring on PND 1 as a percentage of total number of offspring born) were 100, 98, 99 and 98% for the control, 1.2, 12 and 120 mg/kg/day groups, respectively.
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At birth, the mean body weights of the male and female pups at 120 mg/kg were slightly lower in comparison with the other groups (i.e. approximately 5% lower when compared to controls). A similar difference between the body weights of the high dose and control pups was observed at weaning (PND 21), indicating the body weight gain during lactation was similar in all groups.
After weaning: see P1 animals

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Description (incidence and severity):

Not required in OECD 443

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No changes were seen in mean serum T4 levels in male and female pups, culled at PND 4. In addition, no changes were seen in mean serum T4 and TSH levels in male and female pups of Cohort Surplus and Cohort 2B at PND 21-22. .

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Vaginal patency (vaginal opening) in females and balanopreputial separation (prepuce opening) in males were unaffected by treatment.
The time between acquisition of vaginal patency and the first estrus was considered to be unaffected by treatment.The time between vaginal patency and first estrus was on average 3, 4, 3 and 4 days for the control, 1.2, 12 and 120 mg/kg/day groups, respectively. First estrus was observed on average on PND 33, 34, 33 and 36, respectively; these values were within the control range.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

In male pups at 120 mg/kg/day, the anogenital distance was slightly smaller (0.91x and 0.92x for absolute and normalized values, respectively), when compared to controls (only statistically significant when normalized for body weight). However, as all individual values remained within the concurrent control range, this minimal change was considered not to be test item-related but considered to be due to the smaller size of the pups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 120 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Lower body weights and (absolute) brain weights were observed in male and female pups at 120 mg/kg/day, achieving levels of statistical significance for the brain weights in both sexes when compared to controls. The brain/body weight ratios in the high dose pups were similar to those in controls.
The mean splenic weights (absolute) in females at 120 mg/kg/day were 0.79x control value times lower than from controls and were still 0.84x control value times lower after correction for body weight, not achieving levels of statistical significance when compared to controls in both cases. However, since all individual spleen weights and weight ratios in high dose female pups were within the range of that of controls (except for the absolute spleen weight in female pups No. 83), this difference between the mean group values for spleen weight was considered not indicative of a test item-related change and of no toxicological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment.

Description (incidence and severity):

See at P1 animals.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

See at P1 animals for behavioral testing.

Cohort 2B (Terminated PND 21-22, not actively dosed)
Statistically significantly higher mean caudate putamen width was noted in females from all treatment groups and statistically significantly higher mean frontal cortex thickness was noted in the 12 mg/kg/day group females; however, dose-response relationships were lacking and the direction of change was the opposite of what would be expected for a developmental neurotoxicant.

Cohort 2A (Terminated PND 76-90, actively dosed for at least 8 weeks)
Statistically significantly lower mean corpus callosum thickness was noted for the 120 mg/kg/day group males and statistically significantly higher mean caudate putamen width was noted for the 120 mg/kg/day group females. Almost all (8/10) of the individual values for corpus callosum thickness in the 120 mg/kg/day group males were lower than the control group. Seven out of ten of the individual values for caudate putamen width in the 120 mg/kg/day group females were within the range of the control group. The direction of change for females was the opposite of what would be expected for a developmental neurotoxicant.

Overall, upon morphometric analysis of the brain differences in linear measurements were observed, but none of the differences exhibited the same directional trend in males and females or were observed at both time points. The changes were inconsistent across brain regions, sexes, and time points and several changes were in a direction opposite what would be expected in case of developmental neurotoxicity. Individual animal data were evaluated to investigate a correlation between lower morphometry values and lower brain weight and/or brain gross measurement values, but the correlations were inconsistent. Based on the observations, there is no evidence of the test item being a (developmental) neurotoxicant.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Cohort 2B (Terminated PND 21-22, not actively dosed); see for Cohort 2A at P1 animals
Decreased mean fixed brain weights were noted in males of the 120 mg/kg/day group; the decrease in mean absolute weight was statistically significant. See tables attached.
Mean absolute brain width was statistically significantly decreased in females of the at 12 and 120 mg/kg/day group; there were no other significant differences in brain dimension (length and width) changes in animals of Cohort 2B.
A statistically significantly higher mean caudate putamen width was noted in females of all treatment groups; however, a dose- response relationship was lacking. A statistically significantly higher mean frontal cortex thickness was noted in the 12 mg/kg/day group females; however, a dose-response relationship was also lacking. See tables attached.

Overall, a slightly lower post implantation survival, resulting in a slightly lower litter size, was observed in the F1-generation following treatment at 120 mg/kg/day. These changes were not statistically significant when compared to controls and remained within the historical control data range for rats of this strain and age. Small effects on body weights of the F1--pups of the 120 mg/kg/day group were observed when compared to controls, i.e. slightly lower body weights at birth but comparable growth during lactation. Slightly lower brain weights were also observed, generally in line with the effects on body weight of these pups. These effects on body and brain weights might be the result of the same (general) toxicity as observed in the parental animals following treatment at 120 mg/kg/day, rather than being regarded as specific developmental toxicity.
Based on the effects on post implantation survival, litter size and growth of the pups, a NOAEL of 12 mg/kg/day for developmental toxicity was established.

No evidence for a developmental neurotoxic potential of the test item was obtained from the results from the Cohort 2 animals (F1). As such, a NOAEL of at least 120 mg/kg/day (the highest dose level used in this study) for developmental neurotoxicity was established.

Effect levels (F1)

1

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slightly lower post-implantation survival resulting in a slightly lower litter size, slightly reduced body and brain weight

Remarks on result:

other: see at details on results (F1)

2

Dose descriptor:

NOAEC

Generation:

other: F1 (Cohorts 2A and 2B)

Effect level:

>= 120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental neurotoxicity

Remarks on result:

other: Absence of developmental neurotoxicity at this level

Target system / organ toxicity (F1)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: Lower post-implantation survival and smaller litter size, reduced body and brain weight

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

For some pups of the 1.2, 12 and 120 mg/kg/day groups that were found dead or were killed in extremis, absence of milk in the stomach was noted. At the incidences observed and in the absence of a dose-related trend, no toxicological relevance was attributed to these signs.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was 99, 98, 100 and 99% for the control, 1.2, 12 and 120 mg/kg/day groups, respectively.
Viability indices (number of live offspring on PND 1 as a percentage of total number of offspring born) were 100, 92, 99 and 97% for the control, 1.2, 12 and 120 mg/kg/day groups, respectively.
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Pups were sacrificed 3 weeks after birth, so no food intake measured.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Mean serum T4 and TSH levels in male and female F2-pups at PND 21 were considered not to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Slightly decreased mean absolute and relative brain weight was noted in treated male pups at 120 mg/kg/day (0.95x and 0.92x control, respectively), with individual values outside the available historical control range .

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

In view of the effects seen on the eyes of the parent animals, eyes were also examined from pups at PND 21-23, i.e. from pups not directly dosed with CS2. No treatment-related effects were noted.

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

Overall, a slightly lower post implantation survival, resulting in a slightly lower litter size, was observed in the F2-generation following treatment at 120 mg/kg/day. These changes were not statistically significant when compared to controls and remained within the historical control data range for rats of this strain and age. Small effects on body weights of the F2--pups of the 120 mg/kg/day group were observed when compared to controls, i.e. comparable body weights at birth but slightly reduced growth during lactation. Slightly lower brain weights were also observed, generally in line with the effects on body weight of these pups. These effects on body and brain weights might be the result of the same (general) toxicity as observed in the parental animals following treatment at 120 mg/kg/day, rather than being regarded as specific developmental toxicity.
Based on the effects on post implantation survival, litter size and growth of the pups, a NOAEL of 12 mg/kg/day for developmental toxicity was established.

Effect levels (F2)

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slightly lower post-implantation survival resulting in a slightly lower litter size, slightly reduced body and brain weight

Remarks on result:

other: See details on results (F2)

Target system / organ toxicity (F2)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: Lower post-implantation survival and smaller litter size, reduced body and brain weight

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

General toxicity: Retinal atrophy, up to a marked degree in P0 animals and up to a slight degree in P1 animals, and reduced body and brain weights (maximally approximately 10% body weight and 5-10% brain weight reduction) compared to controls and without any indication of behavioural and functional changes, were observed in both sexes of the P0- and P1 animals following treatment at 120 mg/kg/day. Changes in body and brain weight were more pronounced in males. Based on these results, a NOAEL of 12 mg/kg/day for general toxicity was established.
Reproductive toxicity: No treatment-related effects on reproduction were observed in the P0-animals and P1-males in this study; a NOAEL of 120 mg/kg/day (highest dose tested) for reproductive toxicity was established.
Developmental toxicity: A slightly lower post implantation survival, resulting in a slightly lower litter size, was observed in the F1- and F2-generation following treatment at 120 mg/kg/day. These changes were not statistically significant when compared to controls and remained within the historical control data range for rats of this strain and age. Small effects on body weights of the F1- and F2-pups of the 120 mg/kg/day group were observed when compared to controls, i.e. slightly lower body weights at birth but similar growth during lactation for the F1-pups, and similar weight at birth but slightly lower growth during lactation for the F2-pups. Slightly lower brain weights were also observed in both F1- and F2-pups, generally in line with the effects on body weight of these pups. These effects on body and brain weights might be the result of the same (general) toxicity as observed in the parental animals following treatment at 120 mg/kg/day, rather than being regarded as specific developmental toxicity.
Based on the effects on post-implantation survival, litter size and growth of the pups, a NOAEL of 12 mg/kg/day for developmental toxicity was established.
Developmental neurotoxicity: No evidence for a developmental neurotoxic potential of the test item was obtained from the results from the Cohort 2 (F1) animals. Based on this conclusion, a NOAEL of at least 120 mg/kg/day (the highest dose level used in this study) for developmental neurotoxicity was established.