Soybean oil, epoxidized, reaction products with methanol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun-Aug 2021

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:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Kaiser-Friedrich-Straße 7 55116 Mainz

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Soybean oil, epoxidized, reaction products with methanol

EC Number:

287-837-7

EC Name:

Soybean oil, epoxidized, reaction products with methanol

Cas Number:

85586-35-2

Molecular formula:

C H4 O . Unspecified

IUPAC Name:

1-({13-[(1-{2,3-bis[(9,13-dihydroxy-10,12-dimethoxyoctadecanoyl)oxy]propoxy}-10-methoxy-1-oxooctadecan-9-yl)oxy]-9-hydroxy-10,12-dimethoxyoctadecanoyl}oxy)-3-[(9-hydroxy-10-methoxyoctadecanoyl)oxy]propan-2-yl 9,13-dihydroxy-10,12-dimethoxyoctadecanoate; 1-[(9,13-dihydroxy-10,12-dimethoxyoctadecanoyl)oxy]-3-[(9-hydroxy-10-methoxyoctadecanoyl)oxy]propan-2-yl 9,13-dihydroxy-10,12-dimethoxyoctadecanoate; 3-({8-[3-(3-hydroxy-2-methoxyoctyl)oxiran-2-yl]octanoyl}oxy)-2-[(8-{3-[(3-pentyloxiran-2-yl)methyl]oxiran-2-yl}octanoyl)oxy]propyl 9-hydroxy-10-methoxyoctadecanoate; 3-[(8-{3-[3-({1-[3-({8-[3-(3-hydroxy-2-methoxyoctyl)oxiran-2-yl]octanoyl}oxy)-2-[(8-{3-[(3-pentyloxiran-2-yl)methyl]oxiran-2-yl}octanoyl)oxy]propoxy]-10-methoxy-1-oxooctadecan-9-yl}oxy)-2-methoxyoctyl]oxiran-2-yl}octanoyl)oxy]-2-[(8-{3-[(3-pentyloxiran-2-yl)methyl]oxiran-2-yl}octanoyl)oxy]propyl 9-hydroxy-10-methoxyoctadecanoate

Details on test material:

Name of test material (as cited in study report): Soybean oil, epoxidized, reaction products with methanol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch identification: 0021987711
- BASF compound no.: 12/0114-2
- Expiry date: 05 Dec 2021
- The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- The stability of the test substance in corn oil at room temperature over a period of 7 days had been verified prior to the start of the study in the same batch.
- Homogeneity: Given (The homogeneous distribution of the test substance in corn oil was demonstrated)
- Purity: 100 % UVCB
- Content: 99.85 g/100 g

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The specific amount of test substance was weighed in a calibrated beaker, topped up with corn oil and intensely mixed with the magnetic stirrer
- Preparations were produced at intervals which guarantee stability
- Preparations were kept homogeneous with a magnetic stirrer during applications

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- in the vehicle: corn oil

OTHER SPECIFICS
- Physical state/Appearance: liquid, viscous / yellowish, clear

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany, Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males: about 77 - 90 days old/ Females: about 70 - 76 days old
- Weight at study initiation: male 389g (mean), female 215g (mean)
- Housing:
• Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany during pretreatment (up to 5 animals per sex and cage), during premating (2 animals per sex and cage), during mating and postmating (2 animals per cage (males only))
• Polycarbonate cages type III females during mating, gestation, lactation and after weaning and all animals during functional observational battery (FOB) (1 animal per cage with following exceptions: during overnight mating:1 male/1 female per cage and during rearing up to PND 13: 1 dam with her litter)
• For motor activity (MA) measurements the animals were housed individually in
polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany, with
wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material
• Pregnant females were provided with nesting material (2 SAFE® compact nesting large) toward the end of gestation.
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Granovit
AG, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water (from water bottles); ad libitum
- Acclimation period: approximately 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15 times per hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 01 Jun 2021 To: Males: 20 Jul 2021/ Females: 20 Aug 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.

PREPARATION OF DOSING SOLUTIONS:
- Dose volume: 4 mL/kg body weight
- The calculation of the administration volume was based on the most recent individual body weight

VEHICLE
- Control animals: Received the vehicle alone at 4 mL/kg body weight
- Vehicle: Corn oil

Details on mating procedure:

- M/F ratio per cage: 1M / 1F (overnight; same dose group)
- placing the female in the cage of the male mating partner from about 16.00 h until 6.30 - 9.00 h
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear (sperm identification); denoted " gestation day (GD) 0"
- Further matings after two unsuccessful attempts: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the test substance preparations:
- Analyses were carried out at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany
- At beginning, twice during gestation and once during lactation: 3 samples (from top, middle and bottom of the vessel) were taken from the lowest and highest conc. for homogeneity analyses and as a concentration control
- One sample from the mid concentration was taken for concentration control
- Reserve samples were stored at the Lab. Mechanistic Tox. frozen (at -20 °C)

Food analyses:
- The food was assayed for chemical and for microbiological contaminants

Drinking water analyses:
- The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities of Frankenthal and the Environmental Analytics Water/Steam Monitoring of BASF SE.

Bedding and Enrichment analyses:
- The bedding and the enrichment is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals) by the producer.

Duration of treatment / exposure:

After the acclimatization period, the test substance was administered to the parental animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice (Males: 28 days and Females: 59 days). The animals of the control group were treated with the vehicle (corn oil), in the same way.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

low-dose level

Doses / concentrations 2

Dose / conc.:

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

Remarks:

mid-dose level

Doses / concentrations 3

Dose / conc.:

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

Remarks:

high-dose level

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a test study in male and female Wistar rats, oral administration by gavage (Project No.01R0114/12R186) no relevant adverse signs of systemic toxicity at the highest tested dose of 1000 mg/kg bw/d, were seen. Therefore, it can be concluded that dose levels up to 1000 mg/kg/day can be chosen for a subsequent reproductive toxicity study.

- Fasting period before blood sampling for clinical biochemistry: about 16 to 20 hours

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least daily for any signs of morbidity, pertinent
behavioral changes and signs of overt toxicity before the administration as well as within 2
hours and between 2 and 5 hours after the administration.
The parturition and lactation behavior of the dams was generally evaluated in combination with the daily clinical inspection of the dams. Only particular findings (e.g., inability to deliver) were documented on an individual dam basis.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration (day 0) and at weekly intervals during the administration period.
- examined parameters: Abnormal behavior in “handling”, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos (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 until sacrifice;
The body weight change of the animals was calculated from these results.
• During the mating period the parental 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) and on PND 1, 4, 7, 10
and 13.
Females without positive evidence of sperm, without litter and females after weaning (PND 13)
were weighed weekly.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Once a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental
animals).
• Food consumption of the females with evidence of sperm was determined on GD 0 - 7,
7 - 14 and 14 - 20.
• Food consumption of the females which gave birth to a litter was determined on
PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
- Food consumption was not determined in females without positive evidence of sperm during
the mating and the gestation period and in females without litter during the lactation period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption will be monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
In the morning blood was taken from the retro-bulbar venous plexus from 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
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. Clotting tests (Prothrombin time (Hepato Quick’s test) (HQT)) were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
In the morning blood was taken from the retro-bulbar venous plexus from 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
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters checked in table [No.2] were examined. An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the
clinicochemical parameters

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:

- Functional observational battery:
A functional observational battery (FOB) was performed in first surviving 5 male and selected
surviving 5 female animals with litter per group at the end of the administration period starting
at about 10.00 h (Males: study day 23 / Females: study day 57). . The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

- Home cage observations
The animals were observed in their closed home cages (for a short period: about 10-30
seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these
examinations in order not to influence the behavior of the animals. Attention was paid to:
Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings

- Open field observations
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The
following parameters were examined: Behavior on removal from cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypy, Gait, Activity/arousal level, Feces (appearance/ consistency) within 2 minutes, Urine (amount/color) within 2 minutes, Rearing within 2 minutes, Other findings

- Sensory motoric tests/Reflexes
The animals were removed from the open field and subjected to following sensory motor or
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 (Startle 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

- Motor activity measurement
The measurement of motor activity (MA) was measured at the end of the administration period
in first surviving 5 male and selected surviving 5 female animals with litter per group (Males: study day 23 / Females: study day 57). The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in
new clean polycarbonate cages with a small amount of bedding for the duration of the
measurement. Eighteen beams were allocated per cage. The number of beam interrupts were
counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were
placed in the cages was selected at random. On account of the time needed to place the
animals in the cages, the starting time was "staggered" for each animal. The measurement
period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food
or water was offered to the animals during these measurements and the measurement room
was darkened after the transfer of the last animal.

- Thyroid Hormones
Blood samples were taken from all surplus pups pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling.
All generated serum samples were frozen at -80°C until measurement. Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). (For technical reasons the values of PND 13 pups (males nos 201-240, females nos 301-340) were listed in the mean and individual tables under test groups 10, 11, 12 and 13 instead of 0, 1, 2 and 3).
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Oestrous cyclicity (parental animals):

- Before allocation - Stock females:
For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization.

- Females allocated to groups:
For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.

- Before despatch to necropsy:
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Litter observations:

- Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

- 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 Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. Pups which died accidentally or had to be sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated.

- Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 13 after birth.

- Pup body weight data
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters.

- Anogenital distance
Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.

- Anogenital index
The anogenital index was calculated.

- Nipple/areola anlagen
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen was counted.

Postmortem examinations (parental animals):

The male and female animals were sacrificed by decapitation under isoflurane anesthesia 28 and 59 days, respectively, after the beginning of the administration, and examined. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
Animal No. 138 died intercurrently and was necropsied and assessed by gross pathology as soon as possible after its death.

GROSS PATHOLOGY: Yes

- Organ weights

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Epididymides
3. Ovaries
4. Prostate (ventral and dorsolateral part together, fixed)
5. Seminal vesicles with coagulating glands (fixed)
6. Testes
7. Thyroid glands (with parathyroid glands) (fixed)
8. 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):
1. Adrenal glands (fixed)
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus (fixed)
All paired organs were weighed together (left and right).

- Organ/tissue fixation

The following organs or tissues of all parental animals were fixed in in 4% neutral buffered
formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Esophagus
12. Epididymides (modified Davidson’s solution)
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic, and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus
48. Vagina

The testes, epididymides, eyes with optic nerve and ovaries of animals that died/were
sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.
The uteri of all cohabited female F0 parental animals were examined for the presence and
number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus
horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able
to identify early resorptions or implantations (SALEWSKI E (1964)). Then the uteri were rinsed
carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the
uteri were transferred to the Pathology Laboratory for further processing.

HISTOPATHOLOGY: Yes (see table 3)

Fixation was followed by histotechnical processing, examination by light microscopy, and
assessment of findings according to the table 3.
Animals that die or are sacrificed in a moribund state were processed histotechnically and
assessed like control animals.
The organs were trimmed according to the “Revised guides for organ sampling and trimming
in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
Special attention was given to the stages of spermatogenesis and histopathology of interstitial
testicular cell structure. Special attention was given to the synchrony of the morphology of the
estrous cycle in ovaries, uterus, cervix, and vagina.
Whenever the diagnosis „no abnormalities detected” was used in the ovary, it implies that all
the different stages of functional bodies (especially corpora lutea) were present and normal.

Postmortem examinations (offspring):

- Pup necropsy observations
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 (see 3.9.). After sacrifice, the pups were examined externally and eviscerated,
and the organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was 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 with CO2. After sacrifice, all pups were
examined externally and eviscerated, and their organs were assessed macroscopically.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated
and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic
evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case
basis, depending on the type of finding noted.

Statistics:

Different statistical tests were used. For details please refer to table [No.4].

Reproductive indices:

Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs
- mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods")

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected as well as the gestational status were recorded for F0 females.
- mating, fertility and gestation indices, live birth index, postimplantation loss were calculated for F1 litters (for formulas see "Any other information on materials and methods")

Offspring viability indices:

Viability index, survival index, Sex ratio, Anogenital index (for formulas see "Any other information on materials and methods").

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All males and females of the high-dose (1000 mg/kg bw/d), three out of ten males and four out of ten females of the mid-dose (300 mg/kg bw/d) and four out of ten females of the low-dose (100 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) in
different frequencies and continuance during the treatment period. Furthermore, all males and females of the high-dose (1000 mg/kg bw/d) ploughed nose-first into bedding on several days with showing salivation. These findings were considered as treatment-related.

Female animal no. 138 of test group 3 (1000 mg/kg bw/d) showed blood in bedding, pale skin and piloerection on gestation days 23 – 24, semiclosed eyelids and additionally hypothermia on gestation day 24 and was found dead on gestation day 25 not being able to deliver. Female animal no. 136 of the same test group as well showed blood in bedding and piloerection on
gestation day 23 but then was able to deliver. In female animal no. 134 of test group 3 a mass was palpable through skin from lactation day 17 – 20. These findings were considered being incidental and spontaneous by nature and not treatment-related.

No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the test groups (1 - 3; 100, 300 and 1000 mg/kg bw/d) during the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal (no. 138) was found dead on gestation day 25 being unable to deliver after showing blood in bedding, pale skin and piloerection on gestation days 23 - 24 and additionally semiclosed eyelids and hypothermia on gestation day 24.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of male animals of test group 3 (1000 mg/kg bw/d) were significantly reduced from study day 7 onwards (max. -7.9% in comparison to the control group); see table 5.

Mean body weights of all male parental animals of test groups 1 and 2 (100 and 300 mg/kg bw/d) and all female parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study.

Mean body weight change of the high-dose parental males was significantly below the concurrent control values during the entire administration period (day 7 to 27). This finding was considered to be treatment-related and adverse (see table 6).
Mean body weight change of the parental males in test group 2 was significantly reduced at the start of the administration period (day 7). Since the mean body weights in this test group were only slightly reduced and only at the start of treatment this was considered to be possibly
treatment-related but not adverse (see table 6).
During the lactation period parental females of test group 2 showed a significantly increased body weight gain for the overall interval (PND 0 to 13) this was considered to be incidental as no dose response was given.

Mean body weight change of all test substance-treated females and of the mid- and low-dose males was comparable to the concurrent control values during the entire study.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. At the end of the administration, in dams of test group 3 (1000 mg/kg bw/d) absolute and relative eosinophil counts were decreased (absolute not statistically significantly). Additionally, absolute neutrophil cell and monocyte counts were among these individuals were increased although not statistically significantly. However, all mentioned values were within historical control ranges (dams, relative eosinophils 1.2-2.8 %), absolute eosinophils 0.08-0.19 Giga/L, absolute neutrophils 2.37-3.89 Giga/L, absolute monocytes 0.18-0.32 Giga/L). Therefore, these changes were regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed. At the end of the administration period, in dams of test group 3 (1000 mg/kg bw/d) total bile acid levels (TBA) were significantly increased and globulin values were significantly decreased. TBA levels were above the historical control range, globulin values within their range (dams, total bile acids 13.8-44.9 μmol/L; globulins 22.10-28.42 g/L). Total bile acid levels were the only relevantly changed clinical pathology parameter. Therefore, this alteration was regarded as maybe treatment related but non-adverse whereas globulin level decreases within the historical control range were evaluated as incidental and not treatment related.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The histologically detected minimal follicular hypertrophy/hyperplasia in the thyroid glands in 2 out of 10 female animals in test groups 1 and 3 was not accompanied by dose-dependent alteration of thyroid hormone values or organ weight changes. Therefore, this finding was regarded as incidental and without any relation to treatment.

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.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Detailed clinical observations:
In female animal no. 134 of test group 3 (1000 mg/kg bw/d) a mass was palpable through skin on study day 56 (lactation day 17). This finding was considered being incidental and spontaneous by nature and not treatment-related. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the other male and female animals in any of the groups.

- Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

- Open field observations:
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

- Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups. One out of five examined female animals of dose group 1 showed vocalizations always when touched. Since this was not related to dose, it was assessed as spontaneous.

- Quantitative Parameters:
No test substance-related impaired parameters were observed in male and female animals of all test groups.

- Motor activity measurement:
No treatment-related, adverse changes on motor activity data (summation of all intervals) was observed in the male and female animals of all test substance-treated groups in comparison to the concurrent control values.

- Thyroid hormones:
In parental males (test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d) and in male and female pups at PND13 (test groups 11, 12 and 13; 100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was similar: 3.9 / 3.9 / 3.9 and 3.9 days in test groups 0 - 3, respectively. One female in test group 1 (100 mg/kg bw/d) showed a long diestrous. This was considered to be incidental and spontaneous in nature.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Male reproduction data:
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (0 - 3). Fertility was proven for all F0 parental males of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) and 9 F0 parental males of test group 0 (0 mg/kg bw/d) within the scheduled mating interval for F1 litter. Thus, the male fertility index was 90% in test group 0 and 100% for all other test groups.

- Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups (0 - 3). The mean duration until sperm was detected (GD 0) varied between 2.0 and 2.6 days without any relation to dosing. One female animal (No. 103) in test group 0 (0 mg/kg bw/d) was not pregnant. All other female animals delivered pups or had implants in utero. For animal No. 138 which was found dead, unable to deliver a number of 8 pups in uteri was documented in the electronic raw data. The fertility index was 90% in test group 0 and 100% for all other test groups (1 - 3). The mean duration of gestation values varied between 22.2 (control), 22.1 (test group 1), 22.2 (test group 2) and 22.7 (test group 3). The gestation index was 100% in the control and test groups 1 - 2 and 90% in test group 3. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.9 / 14.0 / 14.4 and 12.8 implants/dam in test groups 0 - 3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.2 / 13.1 / 14.2 and 11.8 pups/dam in test groups 0 - 3, respectively). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 97.3% / 99.2% / 99.3% and 98.1% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
Thus, the test substance did not adversely affect reproduction and delivery of the F0 generation parental females.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Description (incidence and severity):

The viability index indicating pup survival during lactation (PND 0 - 4) varied between 100% /100% / 99.2% and 100% in test groups 0 - 3, respectively. The pups surviving index indicating pup survival during lactation (PND 4 - 13) was 100% in all test groups. Thus, the test substance did not influence pup survival in any of the treated groups (100, 300 and 1000 mg/kg bw/d).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study. Mean body weight gain of the male pups and subsequently male and female pups combined of test group 3 (1000 mg/kg bw/d) were significantly decreased from PND 7 to 13. The overall body weight gain (PND 1 to 13) of the male pups was, caused by this temporary change, also significantly decreased. As this weight change was only minor, temporary and inconsistent it was assessed as incidental. One male runt was seen in test group 1 (100 mg/kg bw/d) and one female runt was seen in test group 2 (300 mg/kg bw/d).

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A few pups showed spontaneous findings at gross necropsy, fluid-filled or empty stomach and dilated renal pelvis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few pups showed spontaneous findings at gross necropsy, fluid-filled or empty stomach and dilated renal pelvis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- Pup number and status at delivery
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

- Sex ratio
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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 5: Summary Body Weights - BW / Body Weights [g]; Sex: Male; Phase: In-life

		Test Group 0/M	Test Group 1/M	Test Group 2/M	Test Group 3/M
		0 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
day 0	Mean	402.4 n	396.1	400.6	396.0
	S.d.	20.0	13.4	18.0	15.8
	N	10	10	10	10
	Deviation Vs Control [%]		-1.6	-0.4	-1.6
day 7	Mean	420.3 n	411.2	408.2	400.1 *
	S.d.	18.1	11.2	19.2	16.1
	N	10	10	10	10
	Deviation Vs Control [%]		-2.2	-2.9	-4.8
day 13	Mean	427.1 n	417.6	413.9	403.7 *
	S.d.	18.0	13.5	20.6	15.6
	N	10	10	10	10
	Deviation Vs Control [%]		-2.2	-3.1	-5.5
day 21	Mean	436.0 n	423.9	423.8	407.1 **
	S.d.	18.5	13.5	21.4	13.4
	N	10	10	10	10
	Deviation Vs Control [%]		-2.8	-2.8	-6.6
day 27	Mean	445.4 n	430.8	431.8	410.4 **
	S.d.	19.4	15.0	22.5	13.6
	N	10	10	10	10
	Deviation Vs Control [%]		-3.3	-3.1	-7.9
Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics; n=DUNNETT

 

Table 6: Summary Changes Body Weights - BW / Body Weights [g]; Sex: Male; Phase: In-life

		Test Group 0/M	Test Group 1/M	Test Group 2/M	Test Group 3/M
		0 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
d 0 -> 7	Mean	17.9 n	15.2	7.6 *	4.1 **
	S.d.	9.6	11.6	3.1	2.8
	N	10	10	10	10
d 7 -> 13	Mean	6.8 n	6.3	5.7	3.6 *
	S.d.	2.8	2.9	2.9	2.4
	N	10	10	10	10
d 13 -> 21	Mean	8.9 n	6.3	10.0	3.4 *
	S.d.	4.0	2.8	5.6	4.6
	N	10	10	10	10
d 21 -> 27	Mean	9.5 n	6.9	7.9	3.4 **
	S.d.	2.0	3.5	3.2	3.2
	N	10	10	10	10
d 0 -> 27	Mean	43.1 n	34.7	31.1	14.4 **
	S.d.	12.4	13.7	10.0	5.9
	N	10	10	10	10
Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
d = day;  n=DUNNETT

Applicant's summary and conclusion

Conclusions:

Based on the results of the present study, the NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.

Executive summary:

METHODS
The test substance was administered daily as an emulsified preparation to groups of 10 male and
10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body
weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed
daily with the vehicle only (corn oil).
The duration of treatment covered a 28 days in-life period in the males (including premating,
mating [mating pairs were from the same test group] and postmating period) and a 2-weeks
premating and mating period, the entire gestation and approximately 3 weeks of lactation
period in the females.

OBSERVATIONS
After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation
pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm
were detected in the vaginal smear. F0 animals were examined for their reproductive
performance including determination of the number of implantation sites and the calculation of
postimplantation loss for all F0 females.
A detailed clinical observation (DCO) was performed in all animals before the start of the
administration period and, as a rule, thereafter at weekly intervals.
Food consumption of the F0 parents was determined once weekly during premating. In dams
food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10 and
10 - 13.
Body weights of F0 parents were determined once a week, in males throughout the study and
in females during premating. During gestation and lactation period, F0 females were weighed
on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 1, 4, 7, 10 and 13.
Estrous cycle data were evaluated for F0 generation females over a two weeks period prior to
mating until evidence of mating occurred. Moreover, the estrous stage of each female was
determined on the day of scheduled sacrifice.
The pups were sexed and examined for macroscopically evident changes on PND 0. They
were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 13, all
pups were sacrificed with CO2 under isoflurane anesthesia (except the selected pups for blood
sampling) and examined macroscopically for external and visceral findings.
Anogenital distance (defined as the distance from the anus [center of the anal opening] to the
base of the genital tubercle) measurements were conducted in a blind randomized fashion,
using a measuring ocular on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on
PND 13. The number of nipple/areola anlagen were counted.
Blood samples 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 for hormone
measurement.
Clinico-chemical and hematological examinations were performed in 5 animals per sex and
group towards the end of the administration period.
Blood samples from all dams at PND 14 and all males at termination were taken by puncturing
the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement.
At the end of the administration period a functional observational battery was performed and
motor activity was measured in 5 parental males and females per group.
All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were
assessed by gross pathology. Weights of selected organs were recorded and a
histopathological examination was performed.

Analysis
The various analyses:
• Demonstrated the stability of the test substance preparations in corn oil for a period of 7
days at room temperature.
• Confirmed the homogeneous distribution of the test substance in corn oil
• Confirmed the overall accuracy of the prepared test substance concentrations

Effects
The following test substance-related relevant effects/findings were noted:

1000 mg/kg bw/d:
F0 PARENTAL ANIMALS
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE
• Significantly decreased body weights in males from study day 7 onwards
• Significantly decreased body weight changes in males for the entire study period
CLINICAL PATHOLOGY/ PATHOLOGY
• No test substance-related, adverse findings were noted.
F1 PUPS
CLINICAL EXAMINATIONS/ GROSS FINDINGS
• No test substance-related, adverse findings were noted

300 mg/kg bw/d
F0 PARENTAL ANIMALS
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/PATHOLOGY
• No test substance-related, adverse findings were noted.
F1 PUPS
CLINICAL EXAMINATIONS/ GROSS FINDINGS
• No test substance-related, adverse findings were noted

100 mg/kg bw/d
F0 PARENTAL ANIMALS
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/PATHOLOGY
• No test substance-related, adverse findings were noted.
F1 PUPS
CLINICAL EXAMINATIONS/ GROSS FINDINGS
• No test substance-related, adverse findings were noted

CONCLUSION
In conclusion, under the conditions of the present OECD 422 combined repeated dose toxicity
study with the reproductive/developmental screening test in Wistar rats after oral administration
of the test substance by gavage, the NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.