Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
Triskelion BV, Utrechtseweg 48,3704 HE Zeist
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 : range finding study (BASF 2018 03R0495/00X056)
- Inclusion of extension of Cohort 1B: yes
- Termination time for F2 : postnatal day 21
- Inclusion of developmental neurotoxicity Cohorts 2A and 2B : Yes
- Inclusion of developmental immunotoxicity Cohort 3 : No
- Route of administration : oral, via the diet

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat was used because this species is considered suitable for this type of study and is usually required by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
P: 5 weeks (males); 4 weeks (females) at animal arrival. Start of dosing approx. 2 weeks later.
F1: On postnatal day 4, litters of more than 10 pups were adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 5 males and 5 females per litter. On postnatal day 21, 65 male and 65 female pups per group were selected and placed into the cohorts
- Fasting period before study: no
- Housing:
After allocation, at or just before the start of the premating period, animals were housed 4 rats/cage (separated by sex). For mating, one male and one female were housed together. After evidence of copulation, the males were placed back in the group cages and mated females were housed individually in Makrolon cages, which were placed in another cage rack. After allocation to the cohorts at weaning at or shortly after PN day 21, the F1 animals were housed in groups of 4 or 5 per sex/cage, except for the animals of Cohort 2B that were housed per lot (animals born on the same date) per dosing group until sacrifice on or shortly after PN day 22.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: applied via feed
Details on exposure:
The test item was administered to the animals at constant concentrations in the diet.
Male animals received the test item via the diet during a 10-week premating period, during mating up to the day of sacrifice. The female animals were fed these diets during a 10-week premating period, during mating, gestation and lactation up to the day of sacrifice after lactation day 21.
The F1 generation animals were fed these diets from weaning up to sacrifice (animals of Cohort 1A were fed these diets for approx. 10 weeks, male animals of Cohort 1B for approx. 13 weeks, female animals of Cohort 1B for approx. 18 weeks and animals of Cohort 2A for approx. 8 weeks).
Since the F2-generation animals were sacrificed at weaning they were not directly exposed to the test substance.
During the lactation periods of the F0-generation and Cohort 1B F1-generation animals, the concentrations in the diet of the females were reduced to 50%. This dietary adjustment maintained the dams at the desired target doses during this period of increased food intake.

Diet preparation:
Animals of the test groups were kept on experimental diets prepared by mixing powdered diets with the appropriate amounts of test item. The diets were mixed in a mechanical blender. Fresh batches of the experimental diets were prepared about once per month and stored in a refrigerator (4-10°C) in plastic boxes or in plastic bags.
Details on mating procedure:
Male and female animals of the same dosing group were caged together until mating occurred or two weeks elapsed. Each consecutive morning during the mating period vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smears was considered as gestation day 0. Upon evidence of copulation, the positive females were housed individually for the birth and rearing of pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated conform to the following criteria:
- Linearity: the correlation coefficient of the calibration curve should be ≥ 0.996.
- Recovery: the mean recovery of the test substance from the diet should be between 85% and 115% at each of the dose levels of the study.
- Repeatability: the relative standard deviation in the percentage recovery should be ≤ 10% at each of the dose levels of the study.

The concentration of the test item at each dietary level was determined in in total five batches of test diets prepared in the study (including the batches for homogeneity determinations). The content of the test item in the diet was considered to be ‘close to intended’ if the mean measured concentration is between 90 and 110% of the intended concentration.

For homogeneity testing, five samples (taken at different locations in the feed container) of each test diet were analysed in duplicate. The test item was considered to be homogeneously distributed in the diet if p≥0.01 and/or if the relative standard deviation between the mean concentrations at the five locations is ≤ 5%.

Stabilty: In the dose-range finding study, the test compound was shown to be stable in the diet during experimental conditions for up to one week in the animal room and 5 weeks in the refrigerator (4-10°C) at concentrations of 5000 and 15000 mg/kg diet. To cover the entire concentration range, diet samples containing 375 and 750 mg/kg diet were analysed in one batch prepared in the study at t=0, after storage in the animal room for seven days and after storage in a refrigerator (4-10°C) for at least five weeks. The test item was considered to be stable in the diet if p≥0.01 and/or if the mean concentration after storage is within 90-110% of the mean concentration at t=0.
Duration of treatment / exposure:
Males: 10-week premating period, during mating up to the day of sacrifice (approx. 13 weeks).
Females:
P: 10-week premating period, during mating, gestation and lactation up to the day of sacrifice after lactation day 21.
F1: from weaning up to sacrifice (approx. 10 weeks in Cohort 1A, approx. 13 weeks (males) and approx. 18 weeks (females) in Cohort 1B; approx. 8 weeks in cohort 2A).
F2: indirectly exposed until weaning.
Frequency of treatment:
continuous via feed
Doses / concentrationsopen allclose all
Dose / conc.:
750 ppm (nominal)
Remarks:
Anticipated dose: 50 mg/kg bw/d
overall mean dose: 55 mg/kg bw/d males and 58 mg/kg bw/d females
Dose / conc.:
2 100 ppm (nominal)
Remarks:
Anticipated dose 135 mg/kg bw/d
overall mean dose: 153 mg/kg bw/d males and 163 mg/kg bw/d females
Dose / conc.:
7 000 ppm (nominal)
Remarks:
Anticipated dose 450 mg/kg bw/d
overall mean dose: 534 mg/kg bw/d males and 550 mg/kg bw/d females
No. of animals per sex per dose:
- Number of animals:
F0 generation: 28/sex/group (28 males and 27 females in control group)
F1 generation:
Cohort 1A: 20/sex/group*
Cohort 1B: 25/sex/group
Cohort 2A: 10/sex/group
Cohort 2B: 10/sex/group
*19 females instead of 20 in the mid-dose group, since one animal appeared to be a male pup instead of a female pup and was sacrificed without further examination.
Control animals:
yes, plain diet
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
-> F0-generation animals:

General clinical observations: Assessed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity; in the afternoon for dead or moribund animals.

Detailed clinical observations: Conducted outside the home cage prior to the first exposure and once weekly thereafter.

Body weight: At least once during the acclimatization period and at initiation of treatment (day 0). Subsequently, males were weighed weekly. Females were weighed once per week during the premating and mating period. Mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0, 4, 7 and 14 and 21 of lactation. Non-mated females were weighed once per week after the mating period.
Terminal body weight was determined for all F0 animals prior to necropsy.

Food consumption: Measured per cage for the same periods as the body weights, except during the mating period when food intake was not registered. The results are expressed in g per animal per day.

Intake of test item: The intake of the test item per kg body weight per day was calculated from the nominal dietary concentration, the feed consumption and the body weight at the end of the pertaining period.

Urinalysis: During the last week of premating (females); in the week before necropsy (males); 10 animals/sex/group; fasting period for 16 hours (no water for 24h). Rats were kept in stainless-steel metabolism cages (one rat per cage) and urine was collected in glass tubes. Parameters assessed:
- volume
- density
- appearance
- dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen)
- microscopic examination of the sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs)

Hematology: all animals were fasted overnight (water was freely available); blood taken from the abdominal aorta; 10 rats/sex/group (same as selected for clinical chemistry, hormone analysis) under CO2/O2 anaesthesia; Anticoagulant: EDTA and citrate (for prothrombin time). Parameters assessed:
- Hemoglobin (Hb)
- Packed cell volume (PCV)
- Red blood cell count (RBC)
- Reticulocytes
- Total white blood cell count (WBC)
- Differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes)
- Prothrombin time
- Thrombocyte count
- Activated partial thromboplastine time (APTT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)

Clinical chemistry: all animals were fasted overnight (water was freely available); blood taken from the abdominal aorta; 10 rats/sex/group (same as selected for clinical chemistry, hormone analysis) under CO2/O2 anaesthesia; Anticoagulant: heparin (used an anticoagulant). Parameters assessed:
- alkaline phosphatase activity (ALP)
- bilirubin (total)
- aspartate aminotransferase activity (ASAT)
- cholesterol (total)
- alanine aminotransferase activity (ALAT)
- triglycerides
- gamma glutamyl transferase activity (GGT)
- phospholipids
- total protein
- calcium (Ca)
- albumin
- sodium (Na)
- ratio albumin to globulin (calculated)
- potassium (K)
- urea
- chloride (Cl)
- creatinine
- inorganic phosphate (PO4).
- glucose (fasting)
- bile acids

Hormone determinations: T4 and TSH; after overnight fasting; blood taken from the aorta under CO2/O2 anaesthesia; 10 rats/sex/group; analyzed via commercially available ELISA kits of Cloud-Clone Corp.

Reproductive performance:
- number of adult females with normal or abnormal estrous cycle and cycle duration
- number of females placed with males (and vice versa)
- number of females (not) mated and number of males (not) mated
- number of females (not) pregnant
- number of males that became sire
- number of females killed moribund or found dead
- number of females completing delivery
- number of females with liveborn pups
- number of females with (all) stillborn pups
- total and mean number of pups delivered (live + stillborn pups)
- total number of liveborn pups
- total number of stillborn pups
- total and mean number of live pups(/litter) at day n
- total number of culled pups
- total number of pups lost (dead, missing, cannibalized) (period)
- number and incidence of litters lost entirely (period)
- total and mean number of live male pups at day n
- total and mean number of implantation sites
Oestrous cyclicity (parental animals):
-> F0-generation animals:
Vaginal smears to evaluate the estrus cycle length and normality were made daily during the last three weeks of the premating period and during the mating period until confirmation of mating. Smears were stained and evaluated for estrus cyclicity in all females.
An additional vaginal smear was made at the day of sacrifice but the results of the microscopic examination of the uterus and vagina did not give any indication for further examination of these smears.
Sperm parameters (parental animals):
-> F0-generation males
Epididymal sperm motility, count and morphology:
derived from the left cauda epididymis of all F0 males of each group; Cauda epididymis was dissected, weighed and minced. Sperm motility and, after sonification and DNA-staining, the cauda epididymal sperm reserves (sperm count) were measured for males of all groups. A smear of the sperm solution was prepared and stained for males of all groups, but only the smears of the control and the high-dose males were examined microscopically for morphology.

Testicular sperm count: left testis of all F0 male animals of each group were assesed. Following removal of the tunica albuginea, the testicular parenchyma was weighed, minced and homogenized. Following DNA-staining, the homogenization-resistant sperm heads were enumerated. The daily sperm production was calculated. Sperm counts were conducted in the control and the high-dose males.
Litter observations:
-> F1-generation animals (pups):

Litter evaluation (litter size, pup sex, weight and observations):
The total litter size and numbers of each sex as well as the number of stillbirths, live and dead pups were recorded on days 0, 4, 7, 14 and 21 of lactation and grossly malformed pups were examined. The pups were individually observed for clinical signs and weighed on days 0, 4, 7, 14 and 21 of lactation. Mean pup weight was calculated per sex and for both sexes combined.

Anogenital distance:
At day lactation day 4 the anogenital distance was measured of each pup before culling of the litter. The AGD is corrected for body weight and the cube root of body weight.

Nipple retention in male pups:
On postnatal day 13 and postnatal day 21 or 22 all surviving male pups were quantitatively examined (number of nipples/areolae per pup were counted) for the presence of nipples and/or areolae.


-> F2-generation animals (pups):

Litter evaluation (litter size, pup sex, weight and observations):
The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups were evaluated on days 0, 4, 7, 14 and 21 of lactation and grossly malformed pups were examined. The pups were individually observed for clinical signs and weighed on days 0, 4, 7, 14 and 21 of lactation. Mean pup weight was calculated per sex and for both sexes combined.

Anogenital distance:
At day lactation day 4 the anogenital distance was measured of each pup before culling of the litter. The AGD is corrected for body weight and the cube root of body weight.

Nipple retention in male pups:
On postnatal day 13 and postnatal day 21 or 22 all surviving male pups were quantitatively examined (number of nipples/areolae per pup were counted) for the presence of nipples and/or areolae.

Neonatal pup pathology: Grossly malformed pups were sacrificed and examined. Necropsy was performed on stillborn pups and pups dying or sacrificed in a moribund state during the study and macroscopic abnormalities were recorded.

Hormone determinations: T4 and TSH; At or shortly after weaning on PN day 21; blood taken via heart puncture under CO2/O2 anaesthesia; 10 rats/sex/group;


-> F1-generation animals (Cohort 1A + 1B):

General clinical observations (Cohort 1A & 1B): daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity; in the afternoon for dead or moribund animals.

Detailed clinical observations (Cohort 1A & 1B): Once weekly, e.g. during weighing.

Body weight (Cohort 1A & 1B): recorded weekly; Additionally, body weights were recorded on the day of attainment of vaginal patency or balano-preputial separation. The body weight of the mated female animals of Cohort 1B were also measured on days 0, 7, 14 and 21 of presumed gestation and on day 0, 4, 7, 14 and 21 of lactation. Non-mated females of Cohort 1B were weighed once per week after the mating period.
Terminal body weight was determined for all Cohort 1A and 1B animals prior to scheduled necropsy.

Food consumption (Cohort 1A & 1B): topped up when necessary and refreshed weekly. Except during the mating period, the food consumption was measured per cage over the same periods as the body weights were measured. The results are expressed in g per animal per day.

Intake of test item (Cohort 1A & 1B): per kg body weight per day; calculated from the nominal dietary concentration, the feed consumption and the body weight at the end of the pertaining period.

Sexual maturation (Cohort 1A & 1B):
The following landmarks for sexual maturation were recorded:
- males: preputial separation from postnatal day 39 onwards
- females: vaginal opening from postnatal day 31 onwards
Daily checks were discontinued after 95% of the animals reached sexual maturation. The day the animals were scored positive for these landmarks, the body weight was recorded.

Estrus cycle (Cohort 1A):
From the onset of vaginal patency, a daily vaginal smear was made for 5 successive days of all Cohort 1A females, and these smears were evaluated for the first estrus stage. In addition, vaginal smears to evaluate the estrus cycle length and normality were made daily for about 3 weeks before sacrifice for Cohort 1A females. Smears were stained and evaluated for all Cohort 1A females.

Urinalysis (Cohort 1A): During the last week of premating (females); in the week before necropsy (males); 10 animals/sex/group; fasting period for 16 hours (no water for 24h). Rats were kept in stainless-steel metabolism cages (one rat per cage) and urine was collected in glass tubes. Parameters assessed:
- volume
- density
- appearance
- dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen)
- microscopic examination of the sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs)

Hematology (Cohort 1A): all animals were fasted overnight (water was freely available); blood taken from the abdominal aorta; 10 rats/sex/group (same as selected for clinical chemistry, hormone analysis) under CO2/O2 anaesthesia; Anticoagulant: EDTA and citrate (for prothrombin time). Parameters assessed:
- Hemoglobin (Hb)
- Packed cell volume (PCV)
- Red blood cell count (RBC)
- Reticulocytes
- Total white blood cell count (WBC)
- Differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes)
- Prothrombin time
- Thrombocyte count
- Activated partial thromboplastine time (APTT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)

Clinical chemistry (Cohort 1A): all animals were fasted overnight (water was freely available); blood taken from the abdominal aorta; 10 rats/sex/group (same as selected for clinical chemistry, hormone analysis) under CO2/O2 anaesthesia; Anticoagulant: heparin (used an anticoagulant). Parameters assessed:
- alkaline phosphatase activity (ALP)
- bilirubin (total)
- aspartate aminotransferase activity (ASAT)
- cholesterol (total)
- alanine aminotransferase activity (ALAT)
- triglycerides
- gamma glutamyl transferase activity (GGT)
- phospholipids
- total protein
- calcium (Ca)
- albumin
- sodium (Na)
- ratio albumin to globulin (calculated)
- potassium (K)
- urea
- chloride (Cl)
- creatinine
- inorganic phosphate (PO4).
- glucose (fasting)
- bile acids

Hormone determinations (Cohort 1A): T4 and TSH; after overnight fasting; blood taken from the aorta under CO2/O2 anaesthesia; 10 rats/sex/group; analyzed via commercially available ELISA kits of Cloud-Clone Corp.

Sperm parameters (Cohort 1A):
- Epididymal sperm motility, count and morphology: derived from the left cauda epididymis of all male Cohort 1A animals of each group; cauda epididymis was dissected, weighed and then minced. Assessment of sperm motility and cauda epididymal sperm reserves (sperm count) after sonification and DNA-staining. A smear of the sperm solution was prepared and stained for males of all groups, but only the smears of the control and high-dose males were examined microscopically for morphology.
- Testicular sperm count: left testis of all male Cohort 1A animals of each group. Following removal of the tunica albuginea, the testicular parenchyma was weighed, minced and homogenized. Following DNA-staining, the homogenization-resistant sperm heads were enumerated. The daily sperm production was calculated. Sperm counts were conducted on the control and the high-dose males.

Splenic lymphocyte subpopulation analysis (Cohort 1A):
in 10 cohort 1A animals/sex/group. About half of the spleen was collected individually. A single cell suspension was prepared by dissociation of the spleen tissue, washing, erythrocyte lysing and filtering and resuspension at a density of 10 million cells/ml. For FACS analysis, cells were incubated with a specific antibody cocktail in the presence of Fc block. The antibody cocktail consisted of anti-CD45RA, CD161a, CD3, CD4 and CD8 antibodies in order to determine the frequency of CD4+ and CD8+ T lymphocytes (T-helper, T-cytotoxic cells), B lymphocytes and natural killer cells.

Reproductive performance (Cohort 1B):
- number of adult females with normal or abnormal estrous cycle and cycle duration
- number of females placed with males (and vice versa)
- number of females (not) mated and number of males (not) mated
- number of females (not) pregnant
- number of males that became sire
- number of females killed moribund or found dead
- number of females completing delivery
- number of females with liveborn pups
- number of females with (all) stillborn pups
- total and mean number of pups delivered (live + stillborn pups)
- total number of liveborn pups
- total number of stillborn pups
- total and mean number of live pups(/litter) at day n
- total number of culled pups
- total number of pups lost (dead, missing, cannibalized) (period)
- number and incidence of litters lost entirely (period)
- total and mean number of live male pups at day n
- total and mean number of implantation sites


-> F1-generation animals (Cohort 2A + 2B):

General clinical observations (Cohort 2A & 2B): daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity; in the afternoon for dead or moribund animals.

Detailed clinical observations (Cohort 2A): Once weekly. Between postnatal days 63–75 arena testing was part of the Functional Observational Battery tests.

Body weight (Cohort 2A & 2B): recorded weekly; Additionally, body weights were recorded on the day of attainment of vaginal patency or balano-preputial separation. Body weights of male and female animals in cohort 2A were recorded on the day of auditory startle testing. Terminal body weight was determined for all Cohort 2A and 2B animals prior to necropsy.

Food consumption (Cohort 2A): topped up when necessary and refreshed weekly. Food consumption was measured per cage over the same periods as the body weights were measured. The results are expressed in g per animal per day.

Intake of test item (Cohort 2A): per kg body weight per day; calculated from the nominal dietary concentration, the feed consumption and the body weight at the end of the pertaining period.

Sexual maturation (Cohort 2A):
The following landmarks for sexual maturation were recorded:
- males: preputial separation from postnatal day 39 onwards
- females: vaginal opening from postnatal day 31 onwards
Daily checks were discontinued after 95% of the animals reached sexual maturation. The day the animals were scored positive for these landmarks, the body weight was recorded.

Auditory startle response (Cohort 2A):
On postnatal day 24 (+/- 1 day) in all cohort 2A animals. On the day of testing, at least 30 minutes prior to the start of an experiment, the selected animals were transferred to the examination room. Each of the selected animals was put into an animal holder which was placed in an auditory startle chamber. Subsequently, the animals were acclimatized for 10 minutes. Groups of up to four animals were tested simultaneously. Dose groups were evenly distributed with respect to auditory startle chamber and time as much as possible.
The auditory startle test was performed using a computer-controlled system (Coulbourn Instruments, Animal Acoustic Startle System). Animals were acclimatized in the auditory startle chambers for 10 minutes in the presence of 61.5 +/- 0.5 dBA background noise before the auditory startle test. This background noise remained present throughout the entire testing session. Immediately following the acclimation period, animals received a series of 150, 85±1 dBA, 10 milliseconds bursts of white noise with 2 ms rise/fall time and with a 20 seconds intertrial interval. Recorded auditory startle responses were expressed as gram-force (gf). Auditory startle responses were analyzed using Generalized Estimating Equations (GEE). In addition, the latency (ms) to the maximum auditory startle amplitude was analyzed using Generalized Estimating Equations (GEE).

Functional Observational Battery and spontaneous motor activity (Cohort 2A): In all Cohort 2A animals approximately between postnatal days 63-75.
FOB: At least one hour prior to the start of the observations, the animals were placed individually in macrolon cages in a waiting area in the examination room. First, measurements were carried out in the cage. The rat's posture, palpebral closure and the possible presence of clonic and tonic convulsions were recorded. Then the rat was removed from the cage and the ease of removal and handling was rated. Palpebral closure and any lacrimation or salivation were also rated, and the presence or absence of piloerection and vocalizations was recorded. In addition, other signs, such as changes in skin and fur, exophthalmus, crustiness around the eyes, bite marks on the tail or paws, missing toe nails or emaciation (shallow stomach, protruding spinal vertebrae) were recorded. The rat then was placed in an open arena and observed for 3 minutes. Rears (both supported and unsupported) were counted. At the same time, gait characteristics were recorded and ranked, the ease with which the rat locomotes was ranked, and arousal was assessed and recorded. Further, the occurrence of clonic and/or tonic convulsions, stereotypies and bizarre behaviour were recorded. At the end of the observation period, the number of faecal boluses and urine pools were recorded. Following this observation period, reflex testing was conducted. Reflex testing consists of recording the rat's responses to the approach of a pencil, a touch of a pencil to the rump, a click stimulus, tail pinch, and the constriction of the pupil to light. Aerial righting was rated next. Forelimb and hindlimb gripstrength were measured. Three valid determinations (from a maximum of five attempts) were taken for each gripstrength measure. The rectal temperature was taken with the rat restrained by hand. Finally, the hindlimb feet was painted lightly and landing foot splay was measured.
Parameters assessed:
- Autonomic (lacrimation, salivation, pupil response to light, palpebral closure, piloerection , defecation , urination)
- Neuromuscular (gait, mobility, forelimb and hindlimb gripstrength, landing foot splay, righting reflex)
- Sensorimotor (response to tail pinch, click, touch and approach of a visual object)
- Convulsive (clonic and tonic movements)
- Excitability (ease of removal, handling reactivity, arousal, vocalizations, Activity rearing, posture
- Physiological (body temperature)
Motor activity measurements: Changes in spontaneous motor activity were assessed using an automated quantitative microprocessor-based video image analysis system. Rats were placed individually in open roofed cages on the insides and equipped with a video camera suspended above the test cage. The position of the rat was continuously monitored throughout the test session. Spontaneous motor activity was expressed as the total distance run in a 30 minute test period. In addition, habituation of activity was evaluated. Each session was divided into 5 time blocks of 6 minutes each. Squads of up to eight animals could be monitored
simultaneously. Dose groups were evenly distributed over motor activity test cages and over time as good as possible. Motor activity testing of a squad was conducted immediately after functional observations for that squad was finished.
Postmortem examinations (parental animals):
-> F0-generation animals:

Gross necropsy: all adult animals fasted overnight (water was freely available); At scheduled necropsy, sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anesthesia and
examined grossly for pathological changes.
- Male animals were sacrificed after the mating period.
- Female F0 animals were sacrificed shortly after weaning of the F1-pups All females that did not mate with their assigned male and all non-pregnant females were sacrificed at least one week after the end of the gestation period.

Organ weights - Organs assessed:
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Pituitary gland
Prostate
Seminal vesicles with coagulation glands
Spleen
Testis
Thymus
Thyroid (including parathyroid)
Uterus with cervix

Histopathology:
Organs preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; or Bouin’s fixative (testes). Tissues for microscopic examination were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS.
Organs assessed:
Adrenal glands
Bone marrow (of sternum)
Brain
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs
Lymph node axillary + mesenteric
Mammary gland (♂and ♀)
Ovaries
Oviducts
Optic nerve
Pancreas
Peyer’s patches
Pituitary gland
Prostate
Rectum
Sciatic nerve
Seminal vesicles with coagulation glands
Skeletal muscle (tigh)
Spinal cord (cervical, mid-thoracic and lumbar)
Spleen
Stomach
Testis
Thymus
Thyroid (including parathyroid)
Trachea
Urinary bladder
Uterus with cervix
Vagina
Vas deferens
Postmortem examinations (offspring):
-> F1-generation animals (pups):

Neonatal pup pathology:
Grossly malformed pups were sacrificed and examined. Necropsy was performed on stillborn pups and pups dying or sacrificed in a moribund state during the study.

Culled pup pathology:
Pups were sacrificed at culling by decapitation in random order.

Pup pathology non-selected pups at weaning:
At weaning on PND 21 (or shortly thereafter), pups not selected for any of the cohorts were sacrificed by exsanguination via heart puncture under CO2/O2 anaesthesia in random order.
Following organs were weighed:
Brain
Spleen
Thymus

Hormone determinations: T4 and TSH
Culled pups (PND4)-> Blood was collected per litter for evaluation of T4 in serum.
Non-selected pups at weaning (PND 21 or shortly thereafter) -> Blood was sampled for serum hormone determinations (evaluation of T4 and TSH).


-> F1-generation animals (Cohort 1A + Cohort 1B):

Gross necropsy (Cohort 1A & 1B): all adult animals fasted overnight (water was freely available); At scheduled necropsy, sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anesthesia and examined grossly for pathological changes.

Organ weights (Cohort 1A) - Organs assessed:
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Lymph node axillary
Lymph node mesenteric
Ovaries
Pituitary gland
Prostate
Seminal vesicles with coagulation glands
Spleen
Testis
Thymus
Thyroid (including parathyroid)
Uterus with cervix

Organ weights (Cohort 1B) - Organs assessed:
Epididymides
Ovaries
Pituitary gland
Prostate
Seminal vesicles with coagulation glands
Testis
Uterus with cervix

Histopathology (Cohort 1A):
Organs preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; or Bouin’s fixative (testes). Tissues for microscopic examination were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS.
Organs assessed:
Adrenal glands
Bone marrow (of sternum)
Brain
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs
Lymph node axillary + mesenteric
Mammary gland (♂and ♀)
Ovaries
Oviducts
Optic nerve
Pancreas
Peyer’s patches
Pituitary gland
Prostate
Rectum
Sciatic nerve
Seminal vesicles with coagulation glands
Skeletal muscle (tigh)
Spinal cord (cervical, mid-thoracic and lumbar)
Spleen
Stomach
Testis
Thymus
Thyroid (including parathyroid)
Trachea
Urinary bladder
Uterus with cervix
Vagina
Vas deferens

DOFC (Cohort 1A): In 10 female Cohort 1A animals per dosing group a differential ovarian follicle count (DOFC) was conducted.


-> F1-generation animals (Cohort 2A + Cohort 2B):

Gross necropsy (Cohort 2A & 2B): All surviving animals Cohort 2A (between postnatal days 75-90) and 2B (postnatal day 22) animals; sacrificed by perfusion fixation under pentobarbital anaesthesia (2 ml/kg body weight of a 60 mg/ml pentobarbital solution by IP injection). Animals of Cohort 2A were sacrificed after overnight fasting, whereas the pups of Cohort 2B were not fasted before sacrifice. At sacrifice, the animals were examined grossly for pathological changes.

Organ weights (Cohort 2A + Cohort 2B) - Organs assessed:
Brain

Histopathology (Cohort 2A):
The musculus gastrocnemius of the right hind leg was samples and preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The left hind leg was preserved in
its entirety for subsequent isolation of nerves. Tissues for microscopic examination were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin, except for sections of the sciatic nerve, the spinal cord, the spinal ganglia, the tibial nerve, the trigeminal ganglia and the dorsal and ventral root fibers. These were embedded in epoxin resin, sectioned and stained with toluidine blue. Tissues assessed:
Brains (analysed on eight levels)
Eyes with retina and optic nerve
Musculus gastrocnemius (of right hind leg)
Nose (nasal cavity) - olfactory epithelium (nose level III)
Pituitary gland
Sciatic nerve, proximal section
Spinal cord, cervical part (C1-C6)
Spinal cord, thoracic part (Th5-Th8)
Spinal cord, lumbar part (L1-L4)
Spinal ganglia, cervical part (3x)
Spinal ganglia, lumbar part (3x)
Tibial nerve (knee) proximal section
Tibial nerve (lower leg) distal section
Trigeminal ganglia (with part of nerve)
Root fibers, dorsal (C1-C6 & L1-L4)
Root fibers, ventral (C1-C6 & L1-L4)

Histopathology (Cohort 2B): Tissues assessed:
Brains (analysed on eight levels)

Brain measurements (Cohort 2A & 2B): The length and width of the brain; in all animals.

Morphometry (Cohort 2A):
Thickness measurements of major brain layers (Level 4: neocortex (frontal and parietal cortices), striatum, corpus callosum, Level 5: hippocampus gyrus, Level 7: cerebellum) were performed. Measurements were
carried out bilaterally in the left and right halves of the brain with the exception of the corpus callosum and the cerebellum. Selection of the planes and measurements was performed according to the best practices, Bolon et al, Toxicological Pathology 34, 296-313 (2006).
Statistics:
see below
Reproductive indices:
F0-generation animals + F1-generation animals Cohort 1B (calculated per group):
. Pre-coïtal time (period) = time between the start of mating and successful copulation
. Duration of gestation = time between gestation day 0 and day of delivery (=gestation days)
. Male mating index = no. of males mated*100/no. of males placed with females
. Female mating index = no. of females mated*100/no. of females placed with males
. Male fertility index = no. of males that became sire*100/no. of males placed with females
. Female fertility index = no. of females pregnant*100/no. of females placed with males
. Female fecundity index = no. of females pregnant*100/no. of females mated
. Gestation index = no. of females with a viable litter *100/ no. of females pregnant
Offspring viability indices:
F0-generation animals + F1-generation animals Cohort 1B (calculated on a litter basis):
. Number of lost implantations = no. of implant sites – no. of liveborn pups
. Post-implantation loss = no. of implant sites – no. of liveborn *100 /no. of implant sites
. Live birth index = no. of liveborn pups *100/no. of newborn pups
. Stillborn index = no. of stillborn pups *100/no. of newborn pups
. Viability index 0 - 4 = no. of live pups on day 4 *100/no. of liveborn pups
. Viability index 4 - n = no. of pup live pups on day n *100/no. of live pups post cull day 4
. Sex ratio (day n) = no. of live male pups on day n *100/no. of live pups on day n

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Daily observations did not show any treatment-related clinical signs in male animals during the study neither in females during the premating period, the gestation period and lactation period.
Weekly detailed clinical observations did not show any treatment-related effect of the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights: Significant decrease in high dose group (% of ctrl):
Males day 90 93
Females day 70 premating 91
Females day 21 gestation 87
Females day 21 lactation 95
Males Terminal BW 93
Females Terminal BW 92

Mean body weight change: Significant changes in high dose group (% of ctrl):
Females gestation 0-7 90
Females gestation 7-14 85
Females gestation 14-21 76
Females lactation 0-4 101
Females lactation 4-7 158
Females lactation 7-14 169
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: statistically significantly lower in high-dose group between premating days 0-7, 21-42 and 49-70, the difference reached a maximum of 10% between days 35-42.
Females:
Premating: statistically significantly lower in high-dose group during almost the entire premating period, the difference reached a maximum of 19% between days 0-7.
Gestation: statistically significantly lower in high-dose group (approximately 85%) during almost the entire gestation period.
Lactation: statistically significantly lower in high-dose group (approximately 90%) during almost the entire lactation period.


Mean food consumption: Significant decrease in high dose group (% of ctrl):
Females gestation 0-7 87
Females gestation 7-14 86
Females gestation 14-21 84
Females lactation 0-4 86
Females lactation 4-7 90
Females lactation 7-14 89
Females lactation 14-21 92

Mean food consumption: Significant decrease in mid dose group (% of ctrl):
Females lactation 0-4 94
Females lactation 4-7 93
Females lactation 7-14 91
Females lactation 14-21 91
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Red blood cell and coagulation parameters: no statistically significant effects observed
White blood cell parameters: no statistically significant effects observed (males)
Females:
- increase in WBC (144% of ctrl in low dose)
- increase in Neutrophils (158% of ctrl in high dose 158% of ctrl in low dose)
- increase in Eosinophils (186% of ctrl in low dose)
Since no dose-relationship could be established, and these differences did not occur in the F1 generation, these effects were considered chance findings and not related to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The increased GGT activity in animals of the high-dose group is higher than historical control values and is considered treatment-related:
- GGT statistically significantly increased in high dose (males: 414% of ctrl; females: 1183% of ctrl)

Other statistically significant changes were observed only in one sex and/or only in one generation and/or were inconsistent over the generations:
- Bilirubin statistically significantly decreased in high dose males (63% of ctrl).
- Chloride + Sodium statistically significantly decreased in high dose (96% of ctrl) and low dose (97% of ctrl) males.
- Inorganic phosphate statistically significantly increased in high dose (117% of ctrl) females.

Hormone determinations: concentrations of TSH and T4 hormones in sera of male and female animals were not statistically significantly affected by test item.

Mean (+/-SD) T4 males: 483.1 (+/- 93.6); 501.9 (+/-134.5); 556.3 (+/-182.5); 518.8 (+/-189.7) ng/ml in control, low, mid and high dose group.
Mean (+/-SD) T4 females: 379.6 (+/-62.4) ; 356.0 (+/-99) ; 493.8 (+/-237.5) ; 390.9 (+/-59.9) ng/ml in control, low, mid and high dose group.
Mean (+/-SD) TSH males: 2158.1 (+/-616.6) ; 2440.6 (+/- 272.8); 2195.9 (+/- 542.2) 2470.8 (+/-599.9) pg/ml in control, low, mid and high dose group.
Mean (+/-SD) TSH females: 3401.2 (+/-1460.1); 3303.0 (+/-832); 3408.6 (+/-668.5); 3724.0 (+/-836.9) pg/ml in control, low, mid and high dose group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Volume and density: no effects were observed
Semi-quantitative observations: no effects were observed
Microscopic observations: Except for the score for crystals, that was statistically significantly increased in the female
animals of the low- and high-dose groups, no statistically significant effects were observed on any of the microscopic observations of the urinary sediment of animals. Since there was no dose-relationship for the score of crystals, the increased scores were considered chance findings not related to treatment.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased liver and thyroid weight as observed in the high-dose group were considered to be related to treatment. The effects on the liver and thyroid weights are considered treatment-related but the absolute and relative weights of the liver and thyroid as observed in male and female animals of the high-dose group of the F0-generation and of Cohort 1A are at the upper end or slightly above (relative weight of thyroid of females of the F0-generation) the historical control ranges. In addition, the effects on liver- and thyroid weights as induced by the test substance are considered as adaptive in rats and of less toxicological relevance.

Liver:
Males:
Mean weights absolute: significant increase in high dose (112% of ctrl) and mid dose (107% ctrl)
Mean weights relative: significant increase in high dose (121% of ctrl) and mid dose (107% ctrl) and low dose (104% ctrl)
Females:
Mean weights absolute: significant increase in high dose (112% of ctrl)
Mean weights relative: significant increase in high dose (121% of ctrl)

Thyroid:
Males:
Mean weights absolute: significant increase in high dose (122% of ctrl)
Mean weights relative: significant increase in high dose (132% of ctrl)
Females:
Mean weights relative: significant increase in high dose (126% of ctrl)

All other findings observed on organ weight were considered of no toxicological relevance and not related to treatment. Other statistically significant findings on organ weights were related to the lower terminal body weight, were inconsistent between the F0- and F1-generation, were observed in only one generation and/or were not dose-related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy no treatment related macroscopic changes were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Thyroid gland:
Statistically significantly increased incidence of activated appearance of the thyroid gland; characterised by loss of colloid from the follicles and hypertrophy and hyperplasia of follicular epithelial cells:
Males: 18/28 high-dose; 14/28 mid-dose.
Females: 17/28 high-dose
This effect is considered adaptive in rats.

The statistically significantly decreased incidence of inflammatory infiltrates in the liver of the high dose males was merely caused by a relatively high incidence in the control animals and not related to treatment, since this is a common background finding.

The statistically significantly decreased incidence of luminal dilatation of the uterus of high dose females was considered a chance finding (variation of normal cyclic changes).

The other organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No effects were observed on any of the estrus cycle related parameters between the control and the treatment groups.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
- Epididymal sperm motility: Except for a statistically significant increased amplitude of lateral head displacement (ALH) of the epidydimal sperm cells of males of the mid- and high-dose groups, no effects were observed on any of the epididymal sperm motility parameters. The observed difference in ALH was mainly due to a low value for ALH in the control group and this parameter was not affected in the F1 cohort. For these reasons, the difference in ALH was considered of no toxicological relevance.
- Epididymal sperm count: No statistically significant differences observed
- Epididymal sperm morphology: No statistically significant differences observed
- Testicular sperm count: No statistically significant differences observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Lower mean number of implantation sites, and consequently, the lower number of pups delivered in female animals of the high-dose group of the F0-generation were at the lower end of historical control ranges and the effects were considered to be related to treatment and adverse.

- Implantation sites: 12.3; 12.0; 11.6; 10.7 in control, low, mid and high dose group. (HC range: 9.8-12.7)
- Pups delivered: 11.4; 11.3; 10.6; 9.6* in control, low, mid and high dose group. (HC range: 9.3-12.0)
- Live pups/litter day 0: 11.4; 11.3; 10.3; 9.6* in control, low, mid and high dose group. (HC range: 9.3-12.0)
*statistically significantly different from control

No other treatment-related effects were observed on fertility and reproductive performance of male and female animals of the F0-generation.
Male and female mating indices and the pre-coital time were comparable among the groups.
The number of males that became sire, the male and female fertility indices and the female fecundity index were comparable among the groups.
No pregnant females died during gestation, and all the females delivered live born pups, the gestation index was 100% for all groups.
The duration of gestation (gestation days) was comparable among the groups.
The live birth- and stillborn indices were comparable among the groups.
No effects were observed on the number of lost implantations and on post-implantation loss.

Female/Male mating index: 100%; 100%, 100%, 96.4% in control, low, mid and high dose group.
Female/Male fertility index: 100%; 100%, 100%, 92.9% in control, low, mid and high dose group.
Female feucundity index: 100%; 100%, 100%, 96.3% in control, low, mid and high dose group.
Gestation index: 100%; 100%, 100%, 100%in control, low, mid and high dose group.
Precoital time: 2.4; 3.0; 2.4; 2.8 in control, low, mid and high dose group.
Gestation days: 22.3; 22.3; 22.3; 22.2 in control, low, mid and high dose group.
Livebirth index: 100%, 100%, 97.7%, 100% in control, low, mid and high dose group.
Post-implantation loss: 7.2%; 5.6%, 11.1%, 10.0% in control, low, mid and high dose group.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
General Toxicity
Effect level:
2 100 ppm (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
Remarks on result:
other: Overall mean dose:
Dose descriptor:
NOAEL
Remarks:
Fertility and reproductive performance
Effect level:
2 100 ppm (nominal)
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: Overall mean dose:

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
1 female (high-dose group) showed piloerection and a hunched posture, at an age of 31 days (day 5 of the F1-generation). Further daily observations did not show any treatment-related clinical signs in male animals during the study neither in females during the premating, gestation and lactation period.
In addition, the results of the weekly detailed clinical observations did not show any treatment-related effect of the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
1 Female animal of the high-dose group was sacrificed in a moribund condition, showing piloerection and a hunched posture at an age of 31 days, day 5 of the F1-generation). Otherwise, no mortalities were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights: Significant decrease in high dose group (% of ctrl):
Males day 84 94
Females day 70 premating 94
Females day 21 gestation 91
Females day 21 lactation 98
Males Terminal BW 93
Females Terminal BW 96

Mean body weight change: Significant changes in high dose group (% of ctrl):
Females gestation 0-7 91
Females gestation 7-14 93
Females gestation 14-21 88
Females lactation 0-4 145
Females lactation 4-7 84
Females lactation 7-14 210
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption: Significant decrease in high dose group (% of ctrl):
Females gestation 0-7 88
Females gestation 7-14 88
Females gestation 14-21 90
Females lactation 0-4 90
Females lactation 4-7 93
Females lactation 7-14 91
Females lactation 14-21 91
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Absolute organ weights:
No statistically significant effects were observed on the absolute weights of the reproductive organs of male and female animals.

Relative organ weights:
No statistically significant effects were observed on the relative weights of the reproductive organs of male and female animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related macroscopic changes observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Based on the results of the microscopic examination of the reproductive organs of the F0- generation animals, the reproductive organs of the animals of Cohort 1B were not examined microscopically.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Lower mean number of implantation sites, and consequently, the lower number of pups delivered in female animals of the high-dose group of the F1-generation were lower than (or at the lower end of) historical control ranges and the effects were considered to be related to treatment and adverse.

- Implantation sites: 10.7; 11.6; 11.1; 9.3 in control, low, mid and high dose group. (HC range: 9.8-12.7)
- Pups delivered: 10.3; 10.8; 10.2; 9.3* in control, low, mid and high dose group. (HC range: 9.3-12.0)
- Live pups/litter day 0: 10.2; 10.8; 9.9; 9.3* in control, low, mid and high dose group. (HC range: 9.3-12.0)
*statistically significantly different from control

No other treatment-related effects were observed on fertility and reproductive performance of male and female animals of the F1-generation.
The male and female mating indices and the pre-coital time were comparable among the groups.
The number of males that became sire, the male and female fertility indices and the female fecundity index were comparable among the groups.
The duration of gestation (gestation days) was comparable among the groups.
The live birth- and stillborn indices were comparable among the groups.
No effects were observed on the number of lost implantations and on post-implantation loss.

Female/Male mating index: 96%; 100%, 100%, 100% in control, low, mid and high dose group.
Female/Male fertility index: 96%; 96%, 96%, 100% in control, low, mid and high dose group.
Female feucundity index: 100%; 96%, 96%, 100% in control, low, mid and high dose group.
Gestation index: 100%; 100%, 95.8%, 100% in control, low, mid and high dose group.
Precoital time: 2.9; 2.4; 2.6; 2.3 in control, low, mid and high dose group.
Gestation days: 22.3; 22.1; 22.1; 22.1 in control, low, mid and high dose group.
Livebirth index: 100%, 100%, 98.7%, 100% in control, low, mid and high dose group.
Post-implantation loss: 5.2%; 7.3%, 9.5%, 3.8% in control, low, mid and high dose group.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
-> F1-generation animals (pups):

Pup observations: no treatment-related clinical observations

-> F1-generation animals (Cohort 1A):

No treatment-related clinical signs in Cohort 1A animals. In addition, the results of the weekly detailed clinical observations did not show any dose-related effect of the test item.

-> F1-generation animals (Cohort 2A):

No treatment-related clinical signs in Cohort 2A animals. In addition, the results of the weekly detailed clinical observations did not show any dose-related effect of the test item.

-> F1-generation animals (Cohort 2B):

There were no treatment-related clinical observations in the pups of cohort 2B.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
-> F1-generation animals (Cohort 1A):
1 Male animal of the high-dose group was sacrificed in a moribund condition (unsteady locomotion, body flattened an hindlimb paresis) at an age of 42 days, day 13 of the F1-generation).

-> F1-generation animals (Cohort 2A):
No mortalities observed.

-> F1-generation animals (Cohort 2B):
No mortalities observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
-> F1-generation animals (pups):

Pup weights were comparable in all groups at birth. On postnatal days 14 and 21, mean weights of the male pups and female pups of the high-dose group were statistically significantly lower than the corresponding weights of pups of the control group. In addition, on postnatal day 7, 14 and 21 the combined weight of male and female pups of the high-dose group were also statistically significantly lower than the corresponding weights of pups of the control group

Mean pup weights: Significant decrease in high dose group (% of ctrl):
Males day 21 90
Females day 21 90
Total day 21 90
Males Terminal BW 89
Females Terminal BW 91
Total Terminal BW 91

-> F1-generation animals (Cohort 1A):

Males: Mean body weight of the high-dose group were lower than the body weights of the control animals, reaching the level of statistical significance on days 7, 14, 28, 35, 49 and 56, whereas, in this group no statistical significant differences were observed on body weight changes.
Females: mean body weights in the high-dose group were lower than of the control animals, reaching statistical significance on days 0, 7, 35, 42, 49, 56 and 62. Some incidental statistically significant effects were observed on body weight changes in the low- (between days 14-21), mid- (between days 7-14) and high- (between days 7-14 and 14-21) dose groups, respectively.

Mean body weights: Significant decrease in high dose group (% of ctrl):
Males day 62 93
Females day 62 94
Males Terminal BW 93
Females Terminal BW 92

-> F1-generation animals (Cohort 2A):

Males: Mean body weights of the high-dose group were lower than of the control animals, reaching the level of statistical significance on days 0, 7 and 21. Body weight changes did not differ among the control and treatment groups.
Females: no statistically significant differences were observed on body weights and body weight changes among the control and treatment groups.

Mean body weights (% of ctrl):
Males day 47 91
Females day 47 91
Males Terminal BW 90
Females Terminal BW 91
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
-> F1-generation animals (Cohort 1A):

Males: comparable to control animals (maximally -6%)
Females: high-dose group; statistically significantly lower (down to -15%) than in the corresponding control animals between days 35-56.

-> F1-generation animals (Cohort 2A):

No statistically significant effects were observed.

Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
-> F1-generation animals (Cohort 1A):

Red blood cell and coagulation parameters: no statistically significant effects observed (females)
Males:
- increase in MCHC (102% of ctrl in low dose)
White blood cell parameters: no statistically significant effects observed

The statistically significant effects on hematology parameters were observed only in one sex and only in the low-dose group and only in one generation. For that reason, the observed changes on hematology parameters were considered as fortuitous findings and not to be related to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
-> F1-generation animals (pups):

Hormone determinations -culled pups:
No statistically significant differences in T4 hormone concentrations were observed among the various groups.
Mean (+/-SD) T4: 9.6 (+/-2.9); 8.7 (+/-2.3); 8.4 (+/-2.4); 7.2 (+/-1.4) ng/ml in control, low, mid and high dose group.

Hormone determinations - PND 21 pups:
Except for a statistically significant higher concentration of TSH in female PN day 21 pups (as compared to control PN day 21 female pups), no differences were observed on T4- and TSH-hormone concentrations in serum of F1-generation pups sacrificed on postnatal day 21. As the difference in TSH hormone was only observed in the mid-dose group, and not in high-dose animals, the difference was considered a chance finding and considered not to be related to treatment.

Mean (+/-SD) T4 males: 94.5 (+/-45); 69.1 (+/-18); 113.5 (+/-39.3); 78.9 (+/-36.2) ng/ml in control, low, mid and high dose group.
Mean (+/-SD) T4 females: 85.1 (+/-34.2); 115.6 (+/-73.5); 111.7 (+/-47.4); 102.9 (+/-84.4) ng/ml in control, low, mid and high dose group.
Mean (+/-SD) TSH males: 10357.1 (+/-3886.5); 7761.8 (+/-2754.1); 9051.1 (+/-2166.4); 7865.5 (+/-3488.4) pg/ml in control, low, mid and high dose group.
Mean (+/-SD) TSH females: 7897.6 (+/-3530.1); 9318,9 (+/-3536.8); 12621.7 (+/-2670.4)*; 7504.4 (+/-3307.5) pg/ml in control, low, mid and high dose group.
*p<0.01

-> F1-generation animals (Cohort 1A):

The increased GGT activity in animals of the high-dose group is higher than historical control values and is considered treatment-related:
- GGT statistically significantly increased in high dose (males 2.39; females 4.21 vs. 0 in ctrl)

Other statistically significant changes were observed only in one sex and/or only in one generation and/or were inconsistent over the generations:
- Chloride + Sodium statistically significantly increaed in high dose (109-110% of ctrl) and low dose (109% of ctrl) males.
- Kalium statistically significantly increased in high dose (109% of ctrl) and low dose (108% of ctrl) males.
- Albumin statistically significantly decreased in low dose (94% of ctrl) males.
- Albumin/Globulin ratio statistically significantly decreased in high dose (94% of ctrl) and low dose (90% of ctrl) males.
- Bile acids statistically significantly decreased in high dose (51% of ctrl) females.
- Cholesterol statistically significantly increased in high dose (124% of ctrl) females.
- Urea statistically significantly increased in high dose (118% of ctrl) females.

Hormone determinations: concentrations of TSH and T4 hormones in sera of male and female animals were not statistically significantly affected by test item.

Mean (+/-SD) T4 males: 820.1 (+/-212.2); 830.9 (+/-179.2); 838.8 (+/-238.6); 706.6 (+/-248.9) ng/ml in control, low, mid and high dose group.
Mean (+/-SD) T4 females: 702.8 (+/-95.6); 646.5 (+/-177.2); 714.6 (+/-226.5); 723.8 (+/-128) ng/ml in control, low, mid and high dose group.
Mean (+/-SD) TSH males: 1962.6 (+/-442.4); 2153.1 (+/-303.6); 2421.5 (+/-485); 1904.6 (+/-398.6) pg/ml in control, low, mid and high dose group.
Mean (+/-SD) TSH females: 3202.0 (+/-978.7); 3018.2 (+/-2437.5); 2529.9 (+/-713.5); 2873.9 (+/-1072.2) pg/ml in control, low, mid and high dose group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
-> F1-generation animals (Cohort 1A):

Volume and density:
Males: no effects observed.
Females: mid-dose group; the volume of urine was statistically significantly lower than in control animals.
Since no effect on the volume was observed in the high-dose group, the difference in the mid-dose group was considered as a chance finding. The density of the urine did not show any differences among the various groups.
Semi-quantitative observations: no effects observed.
Microscopic observations: no effects observed.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
-> F1-generation animals (pups):

Preputial separation: The timing in the male pups of the high-dose group was statistically significantly delayed as compared to the corresponding control group. No effect was observed on the mean body weight at the day of preputial separation
- Preputial separation (n): 55, 55, 55, 54 in control, low, mid and high dose group.
- Day of Preputial separation: 43.0; 43.3; 42.9; 46.6** in control, low, mid and high dose group.
** = p < 0.01

Vaginal opening: The timing of vaginal opening in the female pups of the high-dose group was statistically significantly delayed as compared to the corresponding control group. No effect was observed on the mean body weight
at the day of vaginal opening
- Vaginal opening (n): 55, 55, 54, 55 in control, low, mid and high dose group.
- Day of Vaginal opening: 31.4; 31.4; 31.9; 33.9** in control, low, mid and high dose group.
** = p < 0.01
The onset of preputial separation as well as vaginal opening was delayed in male and female pups of the high-dose group. These effects were related to the (statistically significant) decreased body weights of the pups in this group. Achievement of developmental parameters as preputial separation and vaginal opening is more related to body weight and to a lesser extend to age of the pups since at the time/day the pups were scored positive for these parameters, body weights were comparable.


-> F1-generation animals (Cohort 1A):

Estrus cycle:
Vaginal patency in the pups of the high-dose group was slightly, but statistically significantly delayed as compared to control pups. The day of first estrus stage was also delayed. The difference between the mean day of vaginal opening and the mean day of first EST stage was comparable between the control and the high-dose animals. Effects were considered to be related to the lower body weights of the pups. In the control-, low-, mid- and high-dose groups, 2, 1, 1 and 4 animals, respectively, did not show an EST stage within 5 days after vaginal opening.
Estrus cycle evaluation for 3 weeks before sacrifice did not show any statistically significant difference between the control and treatment groups in mean estrus cycle length and normality of the cycle in the female animals of the high-dose group.
- Vaginal patency: 31.3; 31.3; 32.1; 33.9** in control, low, mid and high dose group.
- First EST: 32.8; 32.6; 32.6; 35.5** in control, low, mid and high dose group.
** = p < 0.01
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
-> F1-generation animals (pups):

In male pups, no differences were observed in the absolute- and corrected anogenital distances as measured on postnatal day 4. In female pups of the high-dose group, both the absolute- and corrected anogenital distances were slightly, but statistically significantly higher. However, the differences were small and not observed in F2 animals. In addition, the values for the anogenital distance as observed in the high-dose group were within the historical control range. In conclusion, the differences observed were on anogenital distance were considered to be incidental.

- Mean AGD males (mm): 5.986; 6.051; 6.126; 6.184 in control, low, mid and high dose group. (HC range 5.508-6.780)
- Mean AGD females (mm): 3.399; 3.576; 3.473; 3.641* in control, low, mid and high dose group. (HC range 3.140-4.350)
- Mean AGD corrected males: 2.668; 2.665; 2.703; 2.706 in control, low, mid and high dose group. (HC range 2.475-3.020)
- Mean AGD corrected females: 1.535; 1.598; 1.553; 1.651* in control, low, mid and high dose group. (HC range 1.410-1.749)
* = p < 0.05
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
-> F1-generation animals (pups):

On post-natal days 13 and 21, there were no effects on nipple retention in male pups.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
-> F1-generation animals (pups):

Absolute pup organ weights:
- Spleen: In the high-dose group, the combined absolute weight of the spleen of male and female pups was statistically significantly decreased (83% of control). The absolute weights of the spleen of the male- (78% of control) and female- (87% of control) pups were also decreased but these differences did not reach the level of statistical significance as compared to the corresponding controls.
- Thymus: the combined absolute weight of the thymus of male and female pups together (89% of controls) and of male pups (83% of controls) was decreased, but these differences did not reach the level of statistical significance.

Relative pup organ weights:
Brain: In high-dose group, the relative brain weight was statistically significantly increased, mainly due to the lower terminal pup weights. No statistically significant effects were observed on the relative weights of the spleen and thymus.

The decreased absolute weights of the spleen and thymus and the increased relative brain weight as observed in pups of the high-dose group were considered as related to the lower weight of the pups and of no direct toxicological relevance.

-> F1-generation animals (Cohort 1A):

Increased liver and thyroid weight as observed in the high-dose group were considered to be related to treatment. The effects on the liver and thyroid weights are considered treatment-related but the absolute and relative weights of the liver and thyroid as observed in male and female animals of the high-dose group of the F0-generation and of Cohort 1A are at the upper end or slightly above (relative weight of thyroid of females of the F0-generation) the historical control ranges. In addition, the effects on liver- and thyroid weights as induced by the test substance are considered as adaptive in rats and of less toxicological relevance.

Liver:
Males:
Mean weights absolute: significant increase in high dose (114% of ctrl) and mid dose (112% ctrl)
Mean weights relative: significant increase in high dose (123% of ctrl) and mid dose (110% ctrl) and low dose (107% ctrl)
Females:
Mean weights absolute: significant increase in high dose (121% of ctrl)
Mean weights relative: significant increase in high dose (131% of ctrl)

Thyroid:
Males:
Mean weights relative: significant increase in high dose (124% of ctrl)

All other findings observed on organ weight were considered of no toxicological relevance and not related to treatment. Other statistically significant findings on organ weights were related to the lower terminal body weight, were inconsistent between the F0- and F1-generation, were observed in only one generation and/or were not dose-related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
-> F1-generation animals (pups):
Macroscopic examination of stillborn pups and pups that died on postnatal day 0 did not reveal any effect.
Macroscopic observations of selected pups (10/sex/group) at necropsy on postnatal day 21 did not reveal any treatment-related effects.

-> F1-generation animals (Cohort 1A):
No treatment related macroscopic changes were observed.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
-> F1-generation animals (Cohort 1A):

Thyroid gland:
Statistically significantly increased incidence of activated appearance of the thyroid gland; characterised by loss of colloid from the follicles and hypertrophy and hyperplasia of follicular epithelial cells:
Males: 16/20 high-dose
Females: 13/20 high-dose
This effect is considered adaptive in rats.

The statistically significantly decreased incidence of mineralisation in the kidneys of the high dose females was merely caused by a relatively high incidence in the control animals and not related to treatment, since this is a common background finding.

The other organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Differential ovarian follicle count: No treatment-related effects were observed

Splenic lymphocyte subpopulation analysis:
Males: no effects on any of the splenic lymphocyte subpopulation parameters.
Females: statistically significant increased number of B-lymphocytes in the spleen of female animals of the low-dose group (and not in the mid- and high-dose groups). No other effects were observed on any of the splenic lymphocyte subpopulation parameters.
Other effects:
no effects observed
Description (incidence and severity):
-> F1-generation animals (pups):

No litters were lost entirely. The viability indices of post-natal days 0-4 and 4-21 were comparable among the groups.
The sex ratios on post-natal day 0 and 21 were comparable among the groups.
- Pups dead, missing, cannibalized: 2, 1, 8, 1 in control, low, mid and high dose group.
- Viability index d0-4: 99%, 100%, 97%, 100% in control, low, mid and high dose group.
- Viability index d4-21: 100%, 100%, 100%, 100% in control, low, mid and high dose group.
- Sex ratio day 0 (males): 55.7%, 50.2%, 47.4%, 53.6% in control, low, mid and high dose group.
- Sex ratio day 21 (males): 53.9%, 50.4%, 48.2%, 53.1% in control, low, mid and high dose group.

-> F1-generation animals (Cohort 1A):

Sperm analysis:
- Epididymal sperm motility: no statistically significant differences observed.
- Epididymal sperm count: no statistically significant differences observed.
- Epididymal sperm morphology: no statistically significant differences observed.
- Testicular sperm count: no statistically significant differences observed.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
-> F1-generation animals (Cohort 2A):

FOB and spontaneous motor activity:
No neurotoxic effects were observed from detailed clinical observations, functional observations and motor activity assessment when treated with the test item during the study period.

Auditory startle response:
Results of the auditory startle response examinations did not indicate any neurotoxic potential of the test item in rats.

Brain measurements:
Animals were sacrificed at an age of 74-78 days. No statistically significant effects were observed on terminal body weights and on the absolute and relative brain weights. In addition, brain length and brain width measurements did not reveal any difference between the various groups.

Brain morphometry:
- Left and right hippocampus gyrus: Changes in thickness
In males, enlargement observed, differences were not statistically significant.
In females, statistically significant reductions were observed in the low- and mid-dose groups but not in the high-dose group.
Males:
Mean thickness - Hippocampus gyrus left (% of ctrl): 116%, 111%, 121% in low, mid and high dose group.
Mean thickness - Hippocampus gyrus right (% of ctrl): 114%, 110%, 115% in low, mid and high dose group.
Females:
Mean thickness - Hippocampus gyrus left (% of ctrl): 90%*, 89%*, 94% in low, mid and high dose group.
Mean thickness - Hippocampus gyrus right (% of ctrl): 91%, 90%*, 98% in low, mid and high dose group.
* = p < 0.05

- Frontal cerebral cortex: Enlargement in females
Males:
Mean thickness - Frontal cerebral cortex left (% of ctrl): 102% in high dose group.
Mean thickness - Frontal cerebral cortex right (% of ctrl): 97% in high dose group.
Females:
Mean thickness - Frontal cerebral cortex left (% of ctrl): 104%* in high dose group.
Mean thickness - Frontal cerebral cortex right (% of ctrl): 102% in high dose group.
* = p < 0.05
Other parameter assessed in brain morphomentry did not show any test substance related changes.
The differences observed were only small and there was a large variance between individual animals, which increase the chance
for fortuitous findings. No differences occurred in all the neurobehavioural tests, and no changes were observed by histopathology. These results indicate that the effects on the thickness of the brain layers observed were chance findings which were not related to treatment.

Macroscopic observations:
No treatment related macroscopic changes were observed.

Microscopic observations:
No treatment related histopathological changes observed. Changes were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

-> F1-generation animals (Cohort 2B):

Brain measurements:
Animals were sacrificed at an age of 22 days. No statistically significant effects were observed on the absolute brain weight, whereas the relative weight of the brains of the male and female pups of the high-dose group were statistically significantly higher than of the corresponding control pups (116 and 114%, respectively). The increased relative brain weights were considered to be related to the lower pup weights and are of no toxicological relevance.
In male animals, no statistically significant effects were observed on the length and width measurements. In female animals of the low- and mid-dose groups, slight, but statistically significant effects on the length (in the low- and mid-dose groups) and width (in the mid-dose group) measurements were observed. Since no effects on these parameters were observed in the high-dose group and no findings were observed by histopathological examinations, the former differences were considered to be incidental.

Macroscopic observations:
No treatment related macroscopic changes were observed.

Microscopic observations:
Microscopic examination of the brain at 8 different levels did not reveal treatment related histopathological changes.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
General and sexual development
Generation:
F1
Effect level:
2 100 ppm (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Overall mean dose:
Dose descriptor:
NOAEL
Remarks:
Neuro(developmental) toxicity
Generation:
other: Cohort 2 A/B
Effect level:
7 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: no effects on neuro(developmental) parameters
Remarks on result:
other: Overall mean dose:

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
Pup observations: no treatment-related clinical observations
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see "other effects".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
On postnatal days 4, 7, 14 and 21 the mean weights of the male pups, of the female pups and the combined weight of male and female pups of the high-dose group were statistically significantly lower than the corresponding weight of pups of the control group.

Mean pup weights: Significant decrease in high dose group (% of ctrl):
Males day 21 92
Females day 21 91
Total day 21 91
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hormone determinations -culled pups/ PND 21 pups:
No treatment-related effects on T4 concentrations in culled PND 4 pups and and on T4- and TSH-hormone concentrations in PND 21 pups.

Culled pups:
Mean (+/-SD) T4: 5.1 (+/-2.5); 4.6 (+/-1.7); 5.0 (+/-4.6); 3.4 (+/-1.6) ng/ml in control, low, mid and high dose group.

PND 21 pups:
Mean (+/-SD) T4 males: 69.4 (+/-31.6); 76.6 (+/-58); 93.7 (+/-71.8); 83.3 (+/-41.1) ng/ml in control, low, mid and high dose group.
Mean (+/-SD) T4 females: 81.8 (+/-33.5); 69.3 (+/-21.6); 106.0 (+/-77.7); 102.2 (+/-99.4) ng/ml in control, low, mid and high dose group.
Mean (+/-SD) TSH males: 10956.6 (+/-3627.2); 10655.8 (+/-4215.1); 9486.2 (+/-3827); 9537.7 (+/-4085.8) pg/ml in control, low, mid and high dose group.
Mean (+/-SD) TSH females: 10789.8 (+/-2957.1); 10271.1 (+/-2812.8); 12032.0 (+/-3484); 11005.5 (+/-4106.4) pg/ml in control, low, mid and high dose group.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Males: Mid and high-dose group; absolute, but not for body weight corrected, anogenital distances was were slightly, but statistically significantly lower than in the control group. The values for the anogenital distance were within the historical control range.
Females: no effect on anogenital distance was observed.

- Mean AGD males (mm): 6.681; 6.557; 6.481*; 6.469* in control, low, mid and high dose group. (HC range 5.508-6.780)
- Mean AGD females (mm): 3.879; 3.853; 3.851; 3.818 in control, low, mid and high dose group. (HC range 3.140-4.350)
- Mean AGD corrected males: 2.924; 2.903; 2.850; 2.903 in control, low, mid and high dose group. (HC range 2.475-3.020)
- Mean AGD corrected females: 1.716; 1.731; 1.716; 1.735 in control, low, mid and high dose group. (HC range 1.410-1.749)
* = p < 0.05
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
On post-natal days 13 and 21, there were no effects on nipple retention in male pups.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations of dead pups:
No treatment-related effects observed.

Macroscopic observations of selected pups at necropsy on PN 21:
No treatment-related effects observed.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No litters were lost entirely. The viability indices of post-natal days 0-4 and 4-21 were comparable among the groups.
The sex ratios on post-natal day 0 and 21 were comparable among the groups.

- Pups dead, missing, cannibalized: 1, 6, 5, 2 in control, low, mid and high dose group.
- Viability index d0-4: 100%, 98%, 98%, 99% in control, low, mid and high dose group.
- Viability index d4-21: 100%, 99%, 100%, 100% in control, low, mid and high dose group.
- Sex ratio day 0 (males): 47.3%, 54.4%, 48.9%, 55.6% in control, low, mid and high dose group.
- Sex ratio day 21 (males): 47.1%, 53.2%, 49.8%, 55.6% in control, low, mid and high dose group.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Dose descriptor:
NOAEL
Remarks:
General and sexual development
Generation:
F2 (cohort 1B)
Effect level:
2 100 ppm (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
7 000 ppm
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Results of diet analysis

- The test substance was considered to be homogeneously distributed in the diets;

- The test substance was considered to be stable under the experimental conditions (storage in the animal room for 7 days and after storage in a refrigerator (2-10°C) for at least 5 weeks);

- The concentration of the test substance in the diets was considered close to the intended concentration for all batches of diets and all dose levels, except for the mid-dose of the diet prepared during the lactation phase of the F0-generation). The concentration of this diet deviated >-10% (-15% at first analysis and -12% at re-analysis) from the intended concentration.

Mean test substance intake

Generation/Cohort Sex/Phase Group
(anticipated dose of the test item (mg/kg body weight/day))
Low-dose Mid-dose High-dose
50 135 450
Mean overall calculated test item intake
(mg/kg body weight/day)
F0 Males  46 127 425
F0 Females premating 56 155 498
F0 Females gestation 46 128 396
F0 Females lactation 67 177 629
F1-Cohort 1A Males 57 159 574
F1-Cohort 1A Females 60 172 578
F1-Cohort 1B Males  51 144 502
F1-Cohort 1B Females premating 59 168 626
F1-Cohort 1B Females gestation 46 131 414
F1-Cohort 1B Females lactation 66 187 620
         
F1-Cohort 2A Males 64 181 635
F1-Cohort 2A Females 65 184 636
 Overall mean*  Males  55  153  534
 Overall mean*  Females  58  163  550

*Overall mean is sum of means of each period per sex as described in this table and divided by the number of periods (4 periods for male animals and 8 periods for female animals).

Applicant's summary and conclusion

Executive summary:

The objective of this study was to provide data on the possible effects of Octocrylene on reproductive performance of Wistar rats and the development of pups consequent to daily oral administration of various dietary concentrations to male and female rats during a premating period of 10 weeks and during mating (max. 2 weeks), gestation and lactation until postnatal day 21. At weaning, pups were distributed to different cohorts and were exposed to the same dose levels of the test substance as their parents during their growth into adulthood. Cohorts 1A and 1B of this study assessed reproductive performance and Cohorts 2A and 2B focused on neurodevelopmental endpoints. Animals of Cohort 1B were used for breeding a second generation.

General observations

The test substance was considered homogenously distributed in the diets and was stable under the experimental conditions. Except for one batch of diets of the mid-dose used during the lactation period of the F0-generation (12-15% lower than intended), the concentration of the test substance in the diet was considered close to the intended concentrations.

In total, two animals (one high-dose male animal of Cohort 1A and one high-female of Cohort 1B) were sacrificed in a moribund condition. The macro- and micro-observations in these animals were not considered to be related to treatment. Furthermore, no treatment-related (detailed) clinical signs were observed during the study.

Body weights of the male and female animals of the high-dose group were lower than the corresponding control animals throughout almost the entire study (down to -7% in F0 males and down to -11% (premating), -13% (gestation) and -11% (lactation) in F0 females; down to -8% and -14% in F1 -cohort 1A males and females, respectively)

. In general, food consumption of the male and female animals of the high-dose group was slightly but statistically significantly lower than of the control animals. The observed effects on body weight and food consumption in the high-dose group were considered to be related to treatment.

Except for the increase in gamma glutamyl transferase activity (GGT) in male and female animals of the high-dose group of both the F0- and Cohort 1A F1-generation, no treatment-related effects were observed on hematology, clinical chemistry and urinalysis in F0- generation and Cohort 1A animals.

The decreased terminal body weights (~5 to 10% below controls) and the increased relative liver (males ~20%, females ~30%)) and thyroid weight (males ~25 to 30%, females F0 ~25%) as observed in male and female animals of the high-dose group of the F0- and Cohort 1A F1-generation were considered to be related to treatment. The decreased terminal body weights were considered to be adverse, whereas the increased liver and thyroid weights were considered to be adaptive changes in rats. All other findings observed on organ weight were considered of no toxicological relevance and not related to treatment. No effects were observed on the weight of the reproductive organs of Cohort 1B F1-generation animals.

At necropsy of F0-generation and Cohort 1A F1-generation animals, no treatment-related macroscopic changes were observed. In F0-generation animals of the mid- and high-dose group and in Cohort 1A animals of the high-dose group an increased incidence of activated appearance of the thyroid gland in comparison with the controls was observed. It was characterised by loss of colloid from the follicles and hypertrophy and hyperplasia of follicular epithelial cells. These findings were considered to be related to treatment but to be adaptive changes in rats. Other organs and tissues did not reveal treatment related histopathological changes.

Fertility and reproductive parameters

No treatment-related effects were observed on estrus cycle related parameters in female animals of the F0-generation and in animals of Cohort 1A of the F1-generation. No treatment-related effects were observed on epididymal and testicular sperm parameters in male animals of the F0-generation and in animals of Cohort 1A of the F1-generation.

The lower mean number of implantation sites, and consequently, a lower number of pups delivered in female animals of the high-dose group of the F0-generation (implantation sites: 10.7 versus 12.3 in controls; pups: 9.6 versus 11.4 in controls) and of Cohort 1B of the F1- generation (implantation sites: 9.3 versus 10.7 in controls; pups: 9.3 versus 10.3 in controls) was considered to be related to treatment and adverse.

No treatment-related effects were observed on fertility and reproductive performance of male and female animals of the F0-generation and of Cohort 1B of the F1-generation.

No effects were observed on TSH and T4 analysis in animals of the F0-generation and in adult F1-generation animals of Cohort 1A. No effects were observed on T4 concentrations in serum of F1- and F2-generation pups culled on PN day 4 and, in addition, no treatment-related effects were observed on T4- and TSH concentrations in serum of F1- and F2-generation pups sacrificed on PN day 21.

General and sexual developmental parameters

The mean number of live pups on postnatal day 0 was lower in the high-dose group of the F0- generation and in the high-dose group of Cohort 1B of the F1-generation. This finding was considered to be related to the lower number of implantation sites and the lower number of pups delivered as observed in the animals of these groups. The effects were considered to be related to treatment. No other relevant effects were observed on implantation loss, stillborn pups, dead, missing and/or cannibalized pups, litter loss, pup viability indices and sex ratio. No treatment-related effects were observed on clinical signs of pups nor on macroscopic observations at sacrifice and of dead pups in F1-generation pups and Cohort 1B F2-generation pups.

Overall, in the high-dose group, the body weight of F1-generation pups and of Cohort 1B F2-generation pups was lower than of the corresponding control pups (~10% on postnatal day 21), which is considered due to relatively high compound intake. This finding was considered to be related to treatment.

Preputial separation (control: 43.0 days, high dose 46.4 days), vaginal opening (control: 31.4 days, high dose: 33.9 days), and first estrus stage occurred later in Cohort 1A-generation offspring. However, these differences were not considered as delayed sexual development but as a consequence of delayed general development (lower pup weights).

No direct effects were observed on organ weights of F1-generation pups.

No effects were observed on nipple retention in male F1-generation pups and Cohort 1B F2-generation pups.

No treatment-related effects were observed on the development of the ovarian follicles from primordial small follicles into corpora lutea.

No treatment-related effects were observed on splenic lymphocyte subpopulation analysis in Cohort 1A F1-generation animals.

Neuro(developmental) parameters

Functional observation battery (FOB) and spontaneous motor activity analysis did not reveal any effect of the test substance in animals of Cohort 2A of the F1-generation. The results of the auditory startle response did not indicate any neurotoxic potential of the test substance in animals of Cohort 2A of the F1-generation. No treatment-related adverse effects were observed on brain weight, brain length and brain

width in animals of Cohort 2A and Cohort 2B, respectively. Brain morphometric analysis of the thicknesses of 10 major regions of the brain did not reveal any compound-related adverse effect. Macroscopic observations at sacrifice of animals of Cohort 2A and Cohort 2B did not reveal any treatment-related abnormalities. Mircoscopic observations of brains and neuronal tissues of animals of Cohort 2A and brain of animals of Cohort 2B revealed no treatment-related abnormalities.

Conclusion:

- Based on the effects on body weights the NOAEL for parental effects was placed at the mid-dose concentration of 2100 mg test substance per kg diet (mean test substance intake 153 mg/kg bw/d in males and 163 mg/kg bw/d in females).

- Based on the lower number of implantation sites and the lower number of pups delivered, the NOAEL for fertility and reproductive performance was placed at the mid-dose concentration of 2100 mg test substance per kg diet (mean test substance intake 153 mg/kg bw/d in males and 163 mg/kg bw/d in females). The differences in reproductive parameters occurred at a maternally toxic dose level.

- Based on the effects on pup body weights the NOAEL for general and sexual development was placed at the mid-dose concentration of 2100 mg test substance per kg diet (mean test substance intake 153 mg/kg bw/d in males and 163 mg/kg bw/d in females).

- There were no effects of the test item on neuro(developmental) parameters. The NOAEL for neuro(developmental) parameters was placed at the high-dose concentration of 7000 mg test substance per kg diet (mean test substance intake 534 mg/kg bw/d in males and 550 mg/kg bw/d in females).