(1-hydroxyethane-1,1-diyl)bis(phosphonic acid), compound with 2-aminoethanol (1:[?])

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-04 to 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(1-hydroxyethane-1,1-diyl)bis(phosphonic acid), compound with 2-aminoethanol (1:?)

EC Number:

814-283-0

Cas Number:

42220-47-3

Molecular formula:

C4 H13 N1 O7 P2

IUPAC Name:

(1-hydroxyethane-1,1-diyl)bis(phosphonic acid), compound with 2-aminoethanol (1:?)

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0013537161
- Expiration date of the lot/batch: 30 Dec 2017

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Females nulliparous and non-pregnant: yes
- Age at supply: about 11-12 weeks (males animals); about 10 weeks (female animals)
- Weight at study initiation: (P) Males: 392 +/- 11 g; Females: 213 +/- 10 g
- Housing during pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III
- Housing during pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeiflenberg, Germany
- No of animals per cage: 1; exceptions: during pre-treatment: 5 animals per sex and cage; during mating: 1 male/1 female per cage; during rearing up to PND 13: 1 dam with her litter.
- Bedding: Dust-free wooden bedding
- Diet and water: ad libitum
- Type of diet: Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland
- Acclimation period: 28 days prior to the beginning of administration period

REASON FOR SELECTION:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA. This Wistar rat strain (Crl:WI(Han)) is selected because extensive historical control data is available for these rats.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 /12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a suspension. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, ultrapure water was filled up to the desired volume and intensely mixed with a magnetic stirrer. The test substance preparations were produced weekly, at least.

VEHICLE
- Justification for use and choice of vehicle: recommended by the guideline

Details on mating procedure:

- Premating period: 14 days
- Mating period: until there is evidence of copulation or for maximum 14 days
- M/F ratio per cage: 1 male/1 female
- Length of cohabitation: female was placed into the cage of the male at 16.00 h and seperated from the male between 06.30 and 09.00 h in the following morning.
- Proof of pregnancy: sperm in vaginal smear referred to as (gestation) day 0 of pregnancy
- Pairs: each female was mated with a predetermined male.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany under the responsibility of the Study Director of this test facility. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in ultrapure water over a period of 7 days at room temperature was proven. Concentration control analyses of test-substance preparations were performed at the beginning and during lactation in all concentrations. A homogeneity control analysis was not performed, because the test item was administered as a solution in ultrapure water. Of each sample, one additional reserve sample was retained. The samples collected at the beginning of the administration period and during lactation were analyzed in the Analytical Laboratory.

Duration of treatment / exposure:

females: at least 63 days
males: at least 29 days

Frequency of treatment:

Daily, on a 7 days per week basis

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

vehicle control (ultrapure water)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Adjusted due to the water content of 49.9% of the test substance

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Adjusted due to the water content of 49.9% of the test substance

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

Adjusted due to the water content of 49.9% of the test substance.
Dose level of the high dose group was reduced due to signs of severe toxicity at days around delivery to 1000 mg/kg bw/d.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale: based on dose-range finding study
- Rationale for animal assignment: not random

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Parameters observed: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily from Mondays to Fridays and once daily on weekends and public holidays
- Parameters observed: Abnormal behavior in handling; Fur; Skin; Posture; Salivation; Respiration; Activity/arousal level; Tremors; Convulsions; Abnormal movements; Gait abnormalities; Lacrimation; Palpebral closure; Exophthalmus (Protruding eyeball); Assessment of the feces excreted during the examination (appearance, consistency); Assessment of the urine excreted during the examination; Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning)
- During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0), PND 4. PND 7 PND 10 and PND 13.
- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume, body weight data of these individuals were only reported in the individual tables).
- Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of the administration volume, body weight data of these individuals were only reported in the individual tables).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption was determined once a week for the male and female parental animals.
- Food consumption was determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
- Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
- Food consumption of the females which gave birth to a litter was determined for PND 1-4, 4-7, 7-10 and 10-13.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily by visual inspection of the water bottles for any changes in volume

HAEMATOLOGY: Yes
- Time schedule: Prenatal day14
- Anaesthetic used for blood collection: Yes, under isoflurane anesthesia
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males per group at termination and the first 5 females with litters (in order of delivery) per group at PND 14.
- The following parameters were observed: leukocytes; erythrocytes; haemoglobin; haematocrit; mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC); platelets; differential blood count; reticulocytes; preparation of blood smears (only evaluated blood smears were archived); prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule: Prenatal day 14
- Anaesthetic used for blood collection: Yes, under isoflurane anesthesia
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males per group at termination and the first 5 females with litters (in order of delivery) per group at PND 14.
- The following parameters were observed: alanine aminotransferase; aspartate aminotransferase; alkaline phosphatase; serum y-glutamyl transferase; sodium; potassium ; chloride; inorg. phosphate; calcium; urea; creatinine; glucose; total bilirubin; total protein; albumin; globulins; triglycerides; cholesterol; bile acids.

THYROID HORMONES: Yes
- Blood samples for T3, T4 and TSH measurement were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia.
- If not sufficient serum could be sampled from PND 4 pups, samples were pooled per sex and litter. If not at least 8 pools per sex were sufficient for the hormone measurements, samples were pooled regardless of sex per litter.
- Additionally, blood samples for the above mentioned hormones were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia from all dams at PND 14 and all adult males at termination. The adults were fastened before the blood sampling.
- Blood samples from the PND 13 pups and the adult males were assessed for serum levels for T4 and TSH.
All generated serum samples were frozen at -80° at least until finalization of the report.

BEHAVIOUR (FUNCTIONAL FINDINGS): Yes
FUNCTIONAL OBSERVATIONAL BATTERY
- The functional observational battery (FOB) was carried out once, towards the end of the administration period, in the first 5 surviving parental males and the first 5 surviving parental females with litter per group (in order of delivery).
- The examinations were generally started in the morning at about 10:00 h. The FOB was carried out in a randomized sequence. The animals were not h transferred to new cages before the test, nor were food or drinking water withdrawn. The FOB was started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable.
- Home cage observation: besides other abnormalities, posture; tremors ; convulsions; abnormal movements; gait were observed.
- Open field observation: besides other abnormalities, behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (appearance/consistency), urine excreted within 2 minutes (amount/color), rearings within 2 minutes and other findings were observed.
- Sensory motor tests/reflex tests: the animals were removed from the open field and were subjected to the sensory motor and reflex tests; reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (startte response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test were performed.

MEASUREMENT OF MOTOR ACTIVITY
- The measurement of motor activity (MA) was carried out once, towards the end of the administration period in the first 5 surviving parental males per group and the first 5 surviving parental females with litter per group (in order of delivery).
- For this purpose, the animals were placed in clean polycarbonate cages with a small amount of bedding for the duration of the measurement. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. Eighteen beams will be allocated per cage.
- The number of beam interrupts were counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out from 14.00 h onwards.
- On account of the measuring variant "staggered", the starting time varied by the time which was needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and ended exactly 1 hour later.
- The animals were given no food or water during the measurements. During measurement the pups were placed in a different room, separated from the dams. After the transfer of the last animal in each case, the room of measurement was darkened. The program required a file name for the measured data to be stored. This name consists of the reference number and a serial number

Oestrous cyclicity (parental animals):

For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all F0 female animals.

Sperm parameters (parental animals):

- Parameters examined in male parental generation: testes (histologically), weight of testes and of epididymides.

Litter observations:

Pup status and litter size after birth: the status (sex, liveborn or stillborn) and number of all pups delivered from the parents were determined as soon as possible after birth. At the same time, the pups were examined for gross-morphological changes.
- Pup viability/mortality: in general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on weekends and public holidays. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.
- Clinical signs: all live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.
- Nipple/areola anlagen: all surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen were counted.
- Body weights: the pups were weighed on the day after birth (PND 1) as well as on PNDs 4, 7 and 13. The body weight determined on PND 1 was also used to determine runts. Those pups whose body weight was ≥ 25% below the mean body weight of the control group (separately according to male and female pups) were defined as runts.
- Anogenital distance: anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth.

THYROID HORMONES: Yes
- Blood samples for T3, T4 and TSH measurement were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia.
- If not sufficient serum could be sampled from PND 4 pups, samples were pooled per sex and litter. If not at least 8 pools per sex were sufficient for the hormone measurements, samples were pooled regardless of sex per litter.
- Additionally, blood samples for the above mentioned hormones were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia from all dams at PND 14 and all adult males at termination. The adults were fastened before the blood sampling.
- Blood samples from the PND 13 pups and the adult males were assessed for serum levels for T4 and TSH.
All generated serum samples were frozen at -80° at least until finalization of the report.

Postmortem examinations (parental animals):

SACRIFICE
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including the cervical and thoracic

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals; Epididymides; Ovaries; Prostate; Seminal vesicles with coagulating glands; Testes; Thyroid glands; Uterus (with cervix)
- The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands; Brain; Heart; Kidneys; Liver; Spleen; Thymus
- The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson's solution: All gross lesions; Adrenal glands; Aorta; Bone marrow (femur); Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Eyes with optic nerve; Esophagus; Extraorbital lacrimal glands; Epididymides (modified Davidson's solution); Femur with knee joint; Heart; Ileum; Jejunum (with Peyer's patches); Kidneys; Larynx; Liver; Lungs; Lymph nodes (axillary and mesenteric); Mammary gland (male and female); Nose (nasal cavity); Ovaries (modified Davidson's solution); Oviducts; Pancreas; Parathyroid glands; Pharynx; Pituitary gland; Prostate gland; Rectum; Salivary glands (mandibular and sublingual); Sciatic nerve; Seminal vesicles; Skeletal muscle; Spinal cord (cervical, thoracic and lumbar cord); Spleen; Sternum with marrow; Stomach (forestomach and glandular stomach); Testes (modified Davidson's solution); Thymus; Thyroid glands;Trachea ;Urinary bladder; Uterus; Vagina.

Postmortem examinations (offspring):

- On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs will be assessed macroscopically.
- On PND 13, one selected male and one female pup per litter were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing.
- The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs will be assessed macroscopically.
- Pups that die or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death, paying particular attention to potential pericardial blood vessel effects.
- Pups showing clinical symptoms or gross-morphological findings may have been further examined using appropriate methods. Organs/tissues with gross-morphological findings may have been preserved in a suitable manner for potential histopathological examination. All pups without any notable findings were discarded after their macroscopic evaluation.

Statistics:

Statistics of clinical examinations:
- Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
- Food consumption (parental animals), body weight and body weight change (parental animals): DUNNETT test (two-sided)

Statistics of clinical pathology
- Means, medians and standard deviations were calculated
- Clinical pathology parameters: KRUSKAL-WALLIS and WILCOXON

Reproductive indices:

- Male and female mating indices
- Male and female fertility indices
- Gestation index
- Anogetical index: anogetical distance [mm] / cubic root of pup weight [g]

Offspring viability indices:

- viability Index
- survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Except gestation and lactation period:
Salivation within 2 hours after administration was observed in test group 3 (2000/1000 mg/kg bw/d) in two male animals during premating, in 8 male and one female animal during mating phase as well as in 7 male animals during post-mating phase. No test substance-related, adverse findings were observed in male and female animals of test groups 1-3 (200, 600, and 2000/1000 mg/kg bw/d).

Gestation:
Salivation shortly after treatment (<2 hours after treatment) was observed from slight to moderate in 6 animals of test groups 3 (2000/1000 mg/kg bw/d) during gestation. In test group 3 (2000/1000 mg/kg bw/d) female no. 132 showed tremors and semiclosed eyelid on GD 21, animal no. 136 showed tremors, hypothermia, poor general condition, reduced nutritional condition and semiclosed eyelid between GD 20 and 21, female animal no. 138 showed poor general condition and semiclosed eyelid between GD 22 and 23. Animal no. 139 showed semiclosed eyelid on GD 22 and tremors on GD’s 22 and 23 before all pups were stillborn on GD 23. In test group 2 (600 mg/kg bw/d) one female animal (No. 121) showed hypothermia and poor general state on GD 22, one female animal (No. 126) showed only poor general state on GD’s 22 and 23. With the exception of salivation, the effects were considered to be related to the test substance and assessed as adverse.

Lactation:
Slight salivation shortly after treatment (<2 hours after treatment) was observed in three female animals of test groups 3 (2000/1000 mg/kg bw/d) during lactation. In test group 3 female no. 135 showed paleness, poor general condition, reduced nutritional condition on PND 0, female animal no. 139 showed tremors after treatment (<5 hours after treatment) PND 0. In test group 2 (600 mg/kg bw/d) one female animal (No. 126) showed poor general condition on PND 0 and one female animal (No. 127) showed insufficient maternal care und pups not properly nursed on the day of birth (PND 0). With the exception of salivation, the effects were considered to be related to the test substance and assessed as adverse.

Detailed clinical observations:
In the detailed clinical observations on study days 0, 7, 14, 21 and 28 in parental males and females and additionally, on study days 35, 42, 49 and 56 in parental female animals no adverse findings were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Three female animals of test group 3 (2000/1000 mg/kg bw/d) were found dead during gestation. Another female of this test group was found dead during lactation. One female animal of test group 2 (600 mg/kg bw/d) was sacrificed in a moribund state after showing hypothermia and severe poor general condition.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test substance-related changes in mean body weights were observed for male and female animals of test groups 1-3 (200, 600 and 2000/1000 mg/kg bw/d) when compared to the control group, except for male animals of test group 3 on post-mating day 6, body weight was significantly decreased (-4.5%). The latter finding was assessed as treatment-related and but based on in its small degree of decrease as non-adverse.
The body weight change of the high-dose male animals (2000 mg/kg bw/d) was significantly decreased during premating days 0-7 (-58% below control) and, consequently, if calculated for the entire premating period (days 0-13; -44%). These findings were assessed as treatment-related and without an correlating effect on the body weight itself as non-adverse. The body weight change of test group 2 (600 mg/mg bw/d) was significantly decreased during gestation days 7-14 (-19% below control). This isolated finding without dosedependency was assessed as incidental and not related to treatment.

The body weight change of the high-dose female animals (2000/1000 mg/kg bw/d) was significantly decreased during lactation days 0-4 (-135% below control) and over the entire lactation period (days 0-13; -62%). These findings were assessed as treatment-related and
adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was significantly decreased in males of test group 2 (600 mg/kg bw/d; -6.9%) and test group 3 (2000/1000 mg/kg bw/d; -12.0%) on study day 7 during premating as well as in males of test group 3 between study days 0-13 (-9.6%). These finding were assessed as treatment-related but without any correlating effect of body weight as nonadverse. In female animals of test group 2 (600 mg/kg bw/d) food consumption was significantly decreased (-9.5%) between gestation day 7 and 14. This isolated finding without dosedependency was assessed as incidental and not related to treatment. In female animals of test group 3 (2000/1000 mg/kg bw/d) food consumption was significantly decreased during the entire lactation period (day 1 to 13 -25%). The effects were considered to be treatment-related and adverse.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed. In rats of both sexes of test group 3 (2000 mg/kg bw/d; females reduced to 1000 mg/kg bw/d beginning at study day 37) and additionally in females of test group 2 (600 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly reduced. The values were slightly below the historical control ranges (HQT, males 34.7-40.9 sec; females 29.9-32.9 sec). The cause for this reduced prothrombin time was most probably a higher biosynthesis of coagulation factors by the liver due to a liver cell enzyme induction. This was the only relevantly changed liver parameter and therefore, this alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). In males of test group 3 (2000 mg/kg bw/d) absolute eosinophil cell counts were significantly increased. This change was not dose-dependent and the mean was only marginally above the historical control range (males absolute eosinophils 0.06-0.14 Giga/L). Therefore, this change was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes, adverse among clinical chemistry parameters were observed.
In males of test group 3 (2000 mg/kg bw/d) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were significantly increased. The ALT mean was marginally above the historical control range and the AST mean within this range (males ALT, 0.55-0.92 μkat (L; AST 1.31-2.28 μkat/L). An ALT increase was regarded as adverse not before a two-fold increase (Hall et al., 2012). Therefore, the ALT and AST activity increases were regarded as treatment-related, but not adverse. In females of test group 2 (600 mg/kg bw/d) chloride and sodium values and additionally in females of test group 3 (2000 mg/kg bw/d, reduced to 1000 mg/kg bw/d beginning at study day 37) chloride levels were significantly increased. Chloride levels were within the historical control range (females chloride 90.5-96.8 mmol/L) and sodium values in females of test group 2 were not dose-dependently changed. Therefore, the electrolyte value change in females of test groups 2 and 3 were regarded as incidental and not treatment-related.

Thyroid Hormones:
In parental males (test groups 1, 2 and 3; 200, 600 and 2000 mg/kg bw/d) and in male and female pups at PND13 (test groups 11, 12 and 13; 200, 600 and 2000 mg/kg bw/d, the latter dose reduced to 1000 mg/kg bw/d beginning at study day 37), no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery:
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a doseresponse relationship or occurred in single animals only, these observations were considered as incidental. In touch response the results showed a dose-dependent increase of incidence of no reaction and decrease of incidence of orientation to the stimulus. Both findings represent normal reactions in the test of touch response. Therefore, these findings were assessed as incidental and not related to treatment. Rearing was significantly decreased in female animals of test groups 1 (200 mg/kg bw/d; N = 8) and 3 (2000/1000 mg/kg bw/d (N = 7) in comparison to current control (N = 11). These numbers were within the historical control range (mean 10.0, minimum 5.6 and 13.2 maximum ). Therefore, the changes were assessed as being spontaneous in nature and not related to treatment.

Motor Activity:
Regarding the overall motor activity, no test substance-related deviations were noted for male and female animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Degeneration of the zona glomerulosa and fasciculata of the adrenal cortex occurred in female animals that died or the one animal that was sacrificed moribund. This finding was regarded to be treatment-related. In the fore- and glandular stomach, erosion/ulcer, sero-cellular crusts, inflammatory cell infiltrates and edema were observed, mainly in test group 3 males (2000 mg/kg bw/d) and females (1000/2000 mg/kg bw/d). In the glandular stomach of males and females of the same test group a glandular atrophy was observed, characterized by a (multi)focal loss of glandular epithelial cells, which were then replaced by an acellular fibroid tissue. These findings were regarded to be treatment-related. In the heart of females of test group 2 and 3 (600 and 1000/2000 mg/kg bw/d) a minimal to severe necrosis/fibrosis of the myocardial fibers was observed. This finding was regarded to be treatment-related. The special stains performed on the heart were negative.

In the kidneys of males of test group 3 (2000 mg/kg bw/d) a slightly higher incidence of basophilic tubules was observed and regarded to be treatment-related. In the liver of females of test group 2 and 3 (600 and 1000/2000 mg/kg bw/d) single animals showed a diffuse or centrilobular hypertrophy and a minimal to slight increase in fatty change (demonstrated via positive ORO and Sudan black stain) in the periportal area. A treatmentrelated effect is assumed.

In the mesenteric lymph nodes of test group 2 and 3 (600 and 1000/2000 mg/kg bw/d) males and females, an increase of foamy macrophages was observed. Additionally, in males of all test groups and females of test group 2 and 3 (600 and 1000/2000 mg/kg bw/d) an increase of small macrophage aggregates in the parenchyma of the mesenteric lymph nodes was seen. These findings were regarded to be treatment-related. No special stains (fat stains) were performed on the mesenteric lymph nodes, as no native tissue was left after processing.

Special stains (ORO and Sudan black) performed on Peyer’s plaques were negative. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) ranged from 3.9 to 4.0 days.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Parental Males:
The male mating index calculated after the mating period for F1 litter was 100% in all test groups. Fertility was proven for all F0 parental males within the scheduled mating interval to produce F1 litter. Thus, the male fertility index was 100% in all test groups 0-3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Parental Females:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 3.4 days for test group 0, 2.8 days for test group 1, 4.9 days for test groups 2 and 2.9 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. All sperm positive showed implants. Thus, the female fertility index was 100% in all test groups 0-3.

The mean duration of gestation was comparable in all test groups, i.e. 22.2 days (test group 0 and 1), 22.3 days (test group 2) and 22.6 days (test group 3). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data The gestation index was 100% in test group 0 and 1, 80% in test group 2, and 50% in test group°3. The decreased indices of test group 2 and 3 are below the historical control range (87.5-100%). These findings were assessed as treatment-related and adverse. The gestation index was 100% in test group 0 and 1, 80% in test group 2, and 50% in test group 3. The decreased indices of test group 2 and 3 are below the historical control range (87.5-100%). These findings were assessed as treatment-related and adverse.

Live birth indices
The rate of liveborn pups was 100% in test groups 0 and 1 (0 and 200 mg/kg bw/d), 86.5% in test group 2 (600 mg/kg bw/d) and 58.3% in test group 3 (1000 mg/bw/d). The decreased indices of test group 2 and 3 are below the historical control range (91.7 -100%). These findings were assessed as treatment-related and adverse.

Postimplantation loss
The postimplantation loss was 10.2% in test group 0 (control), 9.6% in test group 1 (200°mg/kg bw/d), 4.4% in test group 2 (600 mg/kg bw/d) and 8.7% in test group 3 (1000°mg/kg bw/d).
The incidences of postimplantation loss in all test groups showed no dose-dependent effects and were within the historical control range (0-18.12%, see PART III, Supplement) and assessed as incidental and not treatment-related.
Implantation sites of deceased or moribund animals were included in this calculation since it was not possible to decide if the pups died before maternal death / moribund state or thereafter.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: myocard and adrenal glands

Organ:

adrenal glands

heart

Treatment related:

yes

Dose response relationship:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The numbers of delivered F1 pups per dam was evenly distributed about the test groups 0 (116 pups), 1 (117 pups) and 2 (104 pups). The decreased number of delivered pups in test group 3 (72 pups) was caused by the reduced number of dams delivering pups. In all test groups the mean numbers of delivered pups per dam were comparable (test group 0 11.6, test group 1 11.7, test group 2 11.6 and test group 3 10.3) and within the historical control
range (9.3-13.9). The stillborn pups were (significantly) observed in test group 2 (600 mg/kg bw/d; 13.5%) and in test group 3 (2000/1000 mg/kg bw/d; 41.7%). Both values are outside of the historical control range (0.0-8.3%, see PART III, Supplement). The stillborn pups were found in 4 litters of test group 2 (44.4%) and in 3 litters of test group 3 (42.9%). Thereby, in one litter of test group 2 (11.1%) and two litters of test group 3 (28.6%) all pups were stillborn. These findings were assessed as treatment-related and adverse.

The surviving F1 pups of test groups 0-3 did not show adverse clinical signs up to scheduled sacrifice on PND 4, resp. PND 13.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The viability index indicating pup mortality during PND 0-4 varied between 100% in test group 0 (control) and test group 1 (200 mg/kg bw/d), 95.9% in test group 2 (600 mg/kg bw/d) and 95.0% in test group 3 (1000 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (89.4-100% days). The survival index indicating pup mortality on PND 4 – 13 was 100% in all test groups without showing any relation to the treatment.Please also refer to attached diagram.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights of all pups in all test groups were comparable to the control group. One male and 1 female runt was seen in the control group on PND 1. Two male and 2 female runt were seen in test group 1 (200 mg/kg bw/d) as well as in test group 2 (600 mg/kg bw/d) on PND 1. Respectively one male runt was seen in test group 3 (1000 mg/kg bw/d) on PND 1. All values were within the range of the biological variation inherent in the strain of rats used for this study. This includes also the statistically significantly decreased body weight change of male lactation pups between day 7 and 13.

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:

no effects observed

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed. In rats of both sexes of test group 3 (2000 mg/kg bw/d; females reduced to 1000 mg/kg bw/d beginning at study day 37) and additionally in females of test group 2 (600 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly reduced. The values were slightly below the historical control ranges (HQT, males 34.7-40.9 sec; females 29.9-32.9 sec). The cause for this reduced prothrombin time was most probably a higher biosynthesis of coagulation factors by the liver due to a liver cell enzyme induction. This was the only relevantly changed liver parameter and therefore, this alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
In males of test group 3 (2000 mg/kg bw/d) absolute eosinophil cell counts were significantly increased. This change was not dose-dependent and the mean was only marginally above the historical control range (males absolute eosinophils 0.06-0.14 Giga/L). Therefore, this change was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes, adverse among clinical chemistry parameters were observed. In males of test group 3 (2000 mg/kg bw/d) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were significantly increased. The ALT mean was marginally above the historical control range and the AST mean within this range (males ALT, 0.55-0.92 μkat (L; AST 1.31-2.28 μkat/L). An ALT increase was regarded as adverse not before a two-fold increase (Hall et al., 2012). Therefore, the ALT and AST activity increases were regarded as treatment-related, but not adverse. In females of test group 2 (600 mg/kg bw/d) chloride and sodium values and additionally in females of test group 3 (2000 mg/kg bw/d, reduced to 1000 mg/kg bw/d beginning at study day 37) chloride levels were significantly increased. Chloride levels were within the historical control range (females chloride 90.5-96.8 mmol/L) and sodium values in females of test group 2 were not dose-dependently changed. Therefore, the electrolyte value change in females of test groups 2 and 3 were regarded as incidental and not treatment-related.

Thyroid hormones:
In male and female pups at PND13 (test groups 11, 12 and 13; 200, 600 and 2000 mg/kg bw/d, the latter dose reduced to 1000 mg/kg bw/d beginning at study day 37), no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Two male and two female pups of test group 2 (600 mg/kg bw/d) showed an empty stomach. One female pup of test group 1 (200 mg/kg bw/d) showed situs inversus. One male pup of test group 3 (1000 mg/kg bw/) showed a green discolored liver lobe and small left parts of liver.
These finding occurred without any relation to dosing and/or, with the exception of the discolored liver lobe, can be found in the historical control data at comparable or even higher incidences. The discoloration of the liver lobe in one pup was assessed as incidental and not related to treatment as the small part of the liver observed in the same pup (no. 140-04).

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Anogenital distance/anogenital index
Anogenital distance and anogenital distance index of male and female pups was not influenced in all test groups.

Nipple/areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

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

Dose response relationship:

yes

Any other information on results incl. tables

Reproductive parameter indices, in percent

Test group (mg/kg bw/d)	0 (0)	01 (200)	02 (600)	03 (2000/1000)
P0 Males
Mating Index	100	100	100	100
Fertility Index	100	100	100	100
P0 Females
Mating Index	100	100	100	100
Fertility Index	100	100	100	100
Gestation Index	100	100	80	50
Live birth Index	100	100	86.5	58.3
F1 Pups
Viability Index	100	100	95.9	95.0

 

Reproduction report_females 

			Test Group 0/ F 0 mg/kg bw/d		Test Group 1/ F 200 mg/kg bw/d		Test Group 2/ F 600 mg/kg bw/d	Test Group 3/ F 2000/1000 mg/kg bw/d
No. of females at start	N		10		10		10	10
No. of females mated	N		10		10		10	10
Without evidence of mating	N		0		0		0	0
- Pregnant	N		0		0		0	0
- Not pregnant	N		0		0		0	0
Females with defined Day 0 pc	N		10		10		10	10
Pregnant	N		10		10		10	10
- found dead	N		0		0		0	4
- sacrificed moribund	N		0		0		1	0
- sacrificed scheduled	N		10		10		9	6
Not pregnant	N		0		0		0	0
Pregnant, not delivering	N		0		0		1	3
Delivering	N		10		10		9	7
-- With liveborn pups	N		10		10		8	5
	%		100.0		100.0		88.9	71.4
-- With all pups stillborn	N		0		0		1	2
	%		0.0		0.0		11.1	28.6

Delivery report

No. of females at start	N	Test Group 0/ F 0 mg/kg bw/d 10		Test Group 1/ F 200 mg/kg bw/d 10		Test Group 2/ F 600 mg/kg bw/d 10	Test Group 3/ F 2000/1000 mg/kg bw/d 10
No. of females mated	N	10	f-	10		10	10
	%	100.0		100.0		100.0	100.0
Pregnant	N	10	f-	10		10	10
	%	100.0		100.0		100.0	100.0
Without delivery	N	0		0		1	3
- Pregnant	N	0		0		1	3
- Not pregnant	N	0		0		0	0
-- Delivering	N	10	f-	10		9	7
	%	100.0		100.0		90.0	70.0
-- With liveborn pups	N	10	f-	10		8	5 *
Gestation Index	%	100.0		100.0		80.0	50.0
Gestation days	Mean	22.2	n	22.2		22.3	22.6
	S.d.	0.4		0.4		0.7	0.5
	N	10		10		9	7
-- With stillborn pups	N	0	f+	0		4 *	3
	%	0.0		0.0		44.4	42.9
-- With all pups stillborn	N	0	f+	0		1	2
	%	0.0		0.0		11.1	28.6

Litter report-pup status

		Test Group 0/ F		Test Group 1/ F	Test Group 2/ F	Test Group 3/ F
		0 mg/kg bw/d		200 mg/kg bw/d	600 mg/kg bw/d	2000/1000 mg/kg bw/d
Pups delivered	N	116		117	104	72
	Mean	11.6	x-	11.7	11.6	10.3
	S.d.	2.0		2.4	1.6	2.0
	N	10		10	9	7
Postimplantation Loss	Mean%	10.2	x+	9.6	4.4	8.7
	S.d.	10.6		10.2	5.7	7.2
	N	10		10	9	7
Pups liveborn	N	116		117	90	42
	%	100.0		100.0	86.5	58.3
	Mean	11.6	x-	11.7	10.0	6.0 *
	S.d.	2.0		2.4	4.0	4.9
	N	10		10	9	7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test

(one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX

		Test Group 0/ F		Test Group 1/ F	Test Group 2/ F	Test Group 3/ F
		0 mg/kg bw/d		200 mg/kg bw/d	600 mg/kg bw/d	2000/1000 mg/kg bw/d
Pups stillborn	N	0		0	14	30
	%	0.0		0.0	13.5	41.7
	Mean	0.0	x+	0.0	1.6	4.3
	S.d.	0.0		0.0	3.6	5.6
	N	10		10	9	7
Perinatal Loss	Mean%	0.0	x+	0.0	13.9	37.7
	S.d.	0.0		0.0	32.6	48.5
	N	10		10	9	7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test

(one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX

 

 

Litters with liveborn pups	N	Test Group 0/ F 0 mg/kg bw/d 10	Test Group 1/ F 200 mg/kg bw/d 10	Test Group 2/ F 600 mg/kg bw/d 8	Test Group 3/ F 2000/1000 mg/kg bw/d 5
Pups delivered	N	116	117	104	72
Day 0	Mean	11.6 x-	11.7	11.2	8.4
	S.d.	2.0	2.4	1.6	3.2
	N	10	10	8	5
Day 4	Mean	11.6 x-	11.7	10.8	8.2
	S.d.	2.0	2.4	1.6	3.6
	N	10	10	8	5
Day 7	Mean	8.0 x-	8.0	8.0	7.0
	S.d.	0.0	0.0	0.0	2.2
	N	10	10	8	5
Day 13	Mean	8.0 x-	8.0	8.0	7.0
	S.d.	0.0	0.0	0.0	2.2
		10	10	8	5

 Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-) statistics

x=WILCOX

 

Dose Group	Animal number	Sing Type	Sing	Modifier	First Day	Last Day	Duration (Days)
Test Group 2/F 600mg/kg bw/d	121 SM	dead	sacrificed moribund	Descriptions: -	22	22	1
Test Group 3/ F 2000/1000 mg/kg bw/d	132 DIED	dead	Found dead	Descriptions: -	21	21	1
	136 DIED	dead	Found dead	Descriptions: -	21	21	1
	138 DIED	dead	Found dead	Descriptions: unable to deliver	24	24	1

SM=Sacrificed moribund; DIED= Died Intercurrently