Soybean oil, epoxidized, reaction products with methanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the results of a combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity
Screening Test in Wistar Rats (Oral Administration; gavage) according to OECD 422, the NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and female Wistar rats (the highest dose tested).

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Kaiser-Friedrich-Straße 7 55116 Mainz

Limit test:

no

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

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

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

Dose / conc.:

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

Remarks:

low-dose level

Dose / conc.:

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

Remarks:

mid-dose level

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

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").

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: 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.

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

Critical effects observed:

no

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Critical effects observed:

no

Reproductive effects observed:

no

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

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test:

In an OECD 422 study, CAS 85586-35-2 was administered daily 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, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity. 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 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 females. Parental females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or 13. The stability of these preparations was demonstrated over a period of 7 days under ambient conditions. Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. Regarding clinical examinations, in females no test substance related, adverse signs of (systemic) toxicity were observed up to limit dose of 1000 mg/ kg bw/d. All males and females of the high-dose (1000 mg/kg bw/d) and two out of ten males of the middose (300 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is most likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding. In male body weight development, a slight overall downward trend of the high dose group may indicate the beginning of systemic toxicity. This reduction was small (about 8% body weight below control values) and only a borderline case; however, as toxicity cannot be excluded, it is considered as adverse. Neither water nor food consumption were adversely affected in any of the tested dose groups. Regarding clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, target organs were the kidneys and the liver in males of test group 3. As the increase of the mean relative (+9.4%) and absolute (+19.0%) kidney weight as well as of the mean relative liver weight (+15.0%) was not accompanied by histopathological changes, these findings were regarded as possibly treatment-related, but not adverse. 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. In conclusion, the oral administration of CAS 85586-35-2 by gavage to female Wistar rats resulted in no adverse signs of systemic toxicity up to limit dose of 1000 mg/kg bw/d. In male rats the NOAEL for general, systemic toxicity of the test substance is 300 mg/kg bw/d based on the changes to body weight parameters at 1000 mg/kg bw/day. 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. (BASF 2022)

Effects on developmental toxicity

Description of key information

Based on the results of a Prenatal Developmental Toxicity Study in Wistar Rats (Oral Administration; gavage) according to OECD 414 (GLP compliant) a NOAEL of  1000 mg/kg bw/day for maternal toxixity and 300 mg/kg bw/day for praenatal developmental toxicity was obtained.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Sep 2021(Study initiation date) - 19 Jul 2022 (Experimental completion date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: 0021987711
- Purity: 99.85 g/100 g
- Storage stability: Expiry date: 05 Jun 2022
- Physical state/appearance: liquid, viscous / yellowish, transparent

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle under test conditions and during storage: The stability of the test substance in corn oil over a period of 7 days at room temperature was demonstrated before the start of the study. The homogeneous distribution of the test substance in the vehicle (corn oil) was demonstrated.

Species:

rat

Strain:

Wistar

Remarks:

(Crl:WI(Han))

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: supplied by Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 170.3 – 229.8 g
- Fasting period before study: not specified
- Housing: housed individually
- Diet: mouse and rat maintenance diet “GLP”, meal, supplied by Granovit AG, Kaiseraugst, Switzerland; ad libitum (The food used in the study was assayed for chemical and for microbiological contaminants.)
- Water: potable tap water in water bottles; ad libitum (The drinking water was regularly assayed for chemical contaminants as well as for bacteria by a contract laboratory.
- Acclimation period: The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h

IN-LIFE DATES: not specified

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

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

PREPARATION OF DOSING SOLUTIONS: The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with corn oil in a calibrated beaker and intensely mixed with a magnetic stirrer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Corn oil as vehicle, assumed as 100 g/100 g, delivered by the sponsor

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results of the analyses of the test substance preparations confirmed the correctness of the prepared concentrations. The measured concentrations of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.
The stability of the test substance in corn oil over a period of 7 days at room temperature was demonstrated before the start of the study. The homogeneous distribution of the test substance in the vehicle (corn oil) was demonstrated.

Details on mating procedure:

- The animals were paired by the breeder (“time-mated”)
- Proof of pregnancy: the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0

Duration of treatment / exposure:

from implantation to one day prior to the expected day of parturition (GD 6-19)

Frequency of treatment:

daily

Duration of test:

From the acclimatization phase to necropsy (GD 20).

Dose / conc.:

100 mg/kg bw/day

Remarks:

A standard dose volume of 4 mL/kg body weight was used for each test group.

Dose / conc.:

300 mg/kg bw/day

Remarks:

A standard dose volume of 4 mL/kg body weight was used for each test group.

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

A standard dose volume of 4 mL/kg body weight was used for each test group.

No. of animals per sex per dose:

25/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: At the request of the sponsor, the following dose levels were chosen for the present prenatal developmental toxicity study in Wistar rats:
100 mg/kg body weight/day: as low-dose level
300 mg/kg body weight/day: as mid-dose level
1000 mg/kg body weight/day: as high-dose level
The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified
- Time of day for (rat) dam blood sampling: on GD 20, blood samples were obtained in a randomized order from all females by retrobulbar venous puncture; notfurther specified

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and between 2 and 5 hours after administration.

- Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.

Corrected (net) body weight gain: Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
- The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order.
- Organs examined: After the dams had been sacrificed, they were necropsied and assessed for gross pathology (except thyroids), special attention being given to the reproductive organs. Thyroid glands (including parathyroid glands) were sampled.

OTHER:

Clinical pathology: Blood samples were taken from all females by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood and serum examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer. The following parameters were measured in all pregnant females:
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units.

Thyroid hormones: The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T3 and T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
- Parameters: Total triiodothyronine (T3), Total thyroxine (T4) and Thyroid stimulating hormone (TSH)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: Yes
- Serum: Yes
- Volume collected: not specified

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
- Anogenital distance of all live rodent pups: Yes: The anogenital distance (defined as the distance from the center of the anal opening to the base of the genital tubercle) measurements were conducted, using a measuring ocular, on all liveborn fetuses.

Statistics:

Statistical analyses were performed according to tables 1- 3 in section "Any other information on materials and methods incl. tables".

Indices:

The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals / number of fertilized animals) x 100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of corpora lutea – number of implantations / number of corpora lutea) x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of implantations – number of live fetuses / number of implantations) x 100

The anogenital index was calculated according to the following formula:
anogenital index = anogenital distance [mm] / cubic root of fetal weight [g]

Historical control data:

Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Nearly all females (24 out of 25) of the high-dose group (1000 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred in the respective animals shortly after treatment (i.e. within 0-2 h) and was observed during GD 9-19. This finding is considered to be treatment-related, most likely as a local irritation of the upper digestive tract or as a result of the bad taste of the test substance/vehicle preparation. No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 100, 300 or 1000 mg/kg bw/d).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

The corrected body weight gain of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of the dams in test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid hormones:
In dams of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) no treatment-related alterations of T3, T4 and TSH levels were observed.

TSH mean value in dams of test group 3 (1000 mg/kg bw/d) was 37% higher compared to study controls but attained no statistical significance and the values were within the historical control ranges (dams, TSH 4.92-8.69 μg/L). Therefore, this change was regarded as incidental and not treatment related.

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

All mean absolute and relative thyroid gland weights did not show significant differences when compared to the control group 0.

The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings were not observed. There occurred a dilated renal pelvis in high-dose female No. 84 (right kidney). As this was a spontaneous finding in one single animal, it was assessed as incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All 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:

no effects observed

Description (incidence and severity):

All 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.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The conception rate was 92% in test group 1 (100 mg/kg bw/d), 96% in test group 3 (1000 mg/kg bw/d) and 100% in test groups 0 and 2 (0 and 300 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed up to and including the highest tested dose.

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean fetal weights of test group 3 (1000 mg/kg bw/d) were slightly but statistically significantly reduced (about 8% below control, both sexes combined). The mean fetal weights of test groups 1 and 2 (100 and 30 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were recorded in one low-dose fetus: male fetus No. 50-03 (100 mg/kg bw/d) had anasarca and a cleft palate associated with multiple skeletal malformations. Since there was no relation to dose, it was assessed as not treatment-related and not adverse. The overall incidences of external malformations were comparable to those found in the historical control data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in one fetus, each, of the control and test group 3 and in two fetuses of the low-dose group. Male low-dose fetus No. 50-03 (100 mg/kg bw/d) had multiple skeletal malformations affecting the skull, sternum, fore-and hindlimbs, pelvic girdle and the vertebral column compared with additional external malformations. All findings are assessed as not treatment-related since they occurred in single fetuses without a relation to dose. The total incidences of skeletal malformations did not differ significantly from control and were comparable to the historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus in test group 2 (300 mg/kg bw/d) had multiple soft tissue malformations. However, these findings affecting the heart/great vessels (persistent truncus arteriosus, right-sided aortic arch, malpositioned subclavian origin, membranous ventricular septum defect) were isolated events in one single fetus and no cluster of any of these individual malformations were seen in the other offspring of this or the other treated groups. Thus, it is assessed as not treatment-related. The total incidence of soft tissue malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Weight of the placentae:
The mean placental weights of the low-, mid- and high-dose groups (100, 300 and 1000 mg/kg bw/d) were comparable to the corresponding control group.

Fetal external variations: One external variation (limb hyperflexion) was recorded in one fetus, each, of test groups 0 and 1 (0 and 100 mg/kg bw/d). The incidence of this single finding was not statistically significantly different from control and there is no dose response relationship visible. Thus, it was considered as not treatment-related.

Fetal external unclassified observations: One external unclassified observation, i.e. placentae fused, occurred in one mid-dose fetus (300 mg/kg bw/d). This finding can be found in the historical control data at a comparable incidence. Thus, it is considered as not treatment-related.

Fetal soft tissue variations: Four soft tissue variations were detected: dilated carotid in one fetus of test group 1, enlarged atrial chamber of heart in one control fetus, dilated renal pelvis in all test groups and dilated ureter in test group 1. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. Therefore, they are assessed as not treatment-related.

Fetal skeletal variations: For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.
The mean values of ‘misshapen sacral vertebra’ were not related to dose and were clearly inside the historical control ranges. Therefore, this finding is assessed as not treatment-related. The mean values of ‘Incomplete ossification of parietal; unchanged cartilage’, ‘Incomplete ossification of sacral arch; cartilage present’, and ‘Unilateral ossification of sternebra; unchanged cartilage’ were clearly inside the historical control ranges and therefore assessed as not treatment-related.
Some variations, affecting some elements of the skull, vertebral column, sternebrae and ribs, were, at the top dose (1000 mg/kg bw/d), present at incidences above the historical control range. These findings are considered to be treatment-related.
The findings ‘Incomplete ossification of skull; unchanged cartilage’ and ‘Supernumerary thoracic vertebra’ were statistically significantly increased also in test group 2 and outside the historical control range. These findings might be associated with the treatment, but since these are the only two increased skeletal variants in this group, only slightly exceeding the historical background incidence and only of mild adversity, they are evaluated as of no toxicological relevance.

For more detailed information please refer to table 4 in section "Any other information on results incl. tables".


Fetal skeletal unclassified cartilage observations: Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing.

Details on embryotoxic / teratogenic effects:

External, soft tissue and skeletal malformations were noted in one control, two low-dose, one mid-dose and one high-dose fetuses (0, 100, 300 and 1000 mg/kg bw/d). Three fetuses carried more than one malformation. Female control fetus No. 17-03 had a shortened scapula and a shortened humerus. For male low-dose fetus No. 50-03 (100 mg/kg bw/d) anasarca and a cleft palate combined with multiple skeletal malformations affecting the skull, sternum, fore-and hindlimbs, pelvic girdle and the vertebral column were recorded. Furthermore, female mid-dose fetus No. 61-10 (300 mg/kg bw/d) had multiple malformations of heart/great vessels, such as persistent truncus arteriosus, right-sided aortic arch, malpositioned subclavian origin, membranous ventricular septum defect. Further malformations, split scapula and cleft sternum, were observed unrelated to dose in individual fetuses. All these findings were single cases without relation to treatment, no ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern, nor do they influence the overall rate of malformations in this study. There is no evidence for any association of these scattered findings with the treatment.

One external variation, four soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. None of the total incidences showed a relation to dosing. The majority of individual variations are equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency.
The findings ‘Incomplete ossification of skull; unchanged cartilage’ and ‘Supernumerary thoracic vertebra’ were statistically significantly increased in test group 2 and outside the historical control range. These findings might be associated with the treatment, but since these are the only two increased skeletal variants in this group, only slightly exceeding the historical background incidence and only of mild adversity, they are evaluated as of no toxicological relevance.

However, at the top dose (1000 mg/kg bw/d) skeletal variations affecting elements of the skull, vertebral column, sternebrae and ribs, were present at incidences above the historical control range. Variations in contrast to malformations are less serious and usually transient or repairable (Carney & Kimmel, 2007). Anyhow, it has to be noted that almost all of these variations appear at high background incidences in the population of the used rat breed and are usually of relatively low toxicological concern.
Specifically, the affected fetuses/litter incidences of ‘Incomplete ossification of interparietal (unchanged cartilage)’, ‘Incomplete ossification of skull (unchanged cartilage)’, ‘Incomplete ossification of temporal’, ‘Incomplete ossification of thoracic centrum (unchanged cartilage)’ and ‘Unossified sternebra (unchanged cartilage)’, represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases.

Unclassified soft tissue observations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for the unclassified external and skeletal cartilage observations which were observed in several fetuses of all test groups (0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment.

Dose descriptor:

LOAEL

Remarks:

prenatal developmental toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: increased incidences of skeletal variations at the high-dose level (1000 mg/kg bw/d)

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to and including the highest tested dose

Remarks on result:

other:

Abnormalities:

effects observed, treatment-related

Localisation:

other: skeletal variations at the top dose (1000 mg/kg bw/d), affecting elements of the skull, vertebral column, sternebrae and ribs

Description (incidence and severity):

These findings (LOAEL 1000 mg/kg bw/day) represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases which may qualify them to be of relatively low toxicological concern.

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Table 4: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter).

Finding	Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d	HCD Mean % (range)
Incomplete ossification of parietal; unchanged cartilage	3.9	6.1	8.2	10.2*	9.1 (1.9 - 19.8)
Incomplete ossification of interparietal; unchanged cartilage	17.4	19.7	23.7	32.4**	19.9 (11.8 - 31.8)
Incomplete ossification of skull; unchanged cartilage	7.7	8.1	20.7**	37.5**	9.1 (2.1 - 17.1)
Incomplete ossification of temporal	1.0	0.7	1.2	7.5**	0.7 (0.0 - 2.1)
Incomplete ossification of thoracic centrum; unchanged cartilage	0.0	0.7	2.6	11.2**	0.5 (0.0 - 2.8)
Supernumerary thoracic vertebra	10.3	6.9	16.2*	19.4*	4.8 (2.4 - 9.4)
Misshapen sacral vertebra	5.1	7.3	10.3*	8.0	4.5 (0.7 - 11.2)
Incomplete ossification of sacral arch; cartilage present	0.0	2.0*	2.2*	3.5*	0.9 (0.0 - 3.8)
Unossified sternebra; unchanged cartilage	9.1	6.3	10.2	32.2**	3.6 (0.0 - 10.1)
Misshapen sternebra; unchanged cartilage	28.6	30.0	33.2	52.0**	23.7 (11.4 - 32.2)
Unilateral ossification of sternebra; unchanged cartilage	0.0	0.7	0.0	2.0*	0.9 (0.0 - 3.7)
Wavy rib	3.4	3.8	2.9	18.6**	3.9 (0.7 - 13.1)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

** = p ≤ 0.01 (Wilcoxon-test [one-sided])

 

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused no evidence of maternal toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the highest tested dose of 1000 mg/kg bw/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg bw/d based on the increased incidences of skeletal variations indicating a generalized delay at the high-dose level (1000 mg/kg bw/d).

Executive summary:

In a GLP compliant prenatal developmental toxicity study (according to OECD TG 414), the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle. Regarding clinical examinations, nearly all females (24 out of 25) of the high-dose group (1000 mg/kg bw/d) showed transient salivation within 2 hours after treatment. This finding is considered to be treatment-related, most likely as a local irritation of the upper digestive tract or as a result of the bad taste of the test substance/vehicle preparation. However, it was not assessed as sign of systemic toxicity. At 300 and 100 mg/kg bw/d, no treatment-related, adverse effects were observed. Concerning thyroid hormone measurement, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, no treatment related findings in thyroid glands were noted. All 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. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose.

The findings ‘Incomplete ossification of skull; unchanged cartilage’ and ‘Supernumerary thoracic vertebra’ were statistically significantly increased in test group 2 and outside the historical control range. This finding might be associated with the treatment, but since these are the only two increased skeletal variants in this group, only slightly exceeds the historical background incidence and is only of mild adversity, it is evaluated as of no toxicological
relevance.

However, at the top dose (1000 mg/kg bw/d) skeletal variations affecting elements of the skull, vertebral column, sternebrae and ribs, were present at incidences above the historical control range. Variations in contrast to malformations are less serious and usually transient or repairable (Carney & Kimmel, 2007). Anyhow, it has to be noted that almost all of these variations appear at high background incidences in the population of the used rat breed and are usually of relatively low toxicological concern.

Specifically, the affected fetuses/litter incidences of ‘Incomplete ossification of interparietal (unchanged cartilage)’, ‘Incomplete ossification of skull (unchanged cartilage)’, ‘Incomplete ossification of temporal’, ‘Incomplete ossification of thoracic centrum (unchanged cartilage)’ and ‘Unossified sternebra (unchanged cartilage)’, represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases. The ossification of the bony structures occurs at the end of gestation and the fetal ossification status is highly influenced by many factors, such as difference in mating time of just a few hours, time of cesarean section or by stress due to maternal toxicity (Carney & Kimmel, 2007).

According to the evaluation of Carney and Kimmel (2007), the fetal skeletal ossification is tightly linked to the overall rate of fetal growth with subsequent catch-up postnatally and a delay in skeletal ossification is mechanistically independent from malformation. Hence, delayed ossification does not seem to have general predictive value for teratogenesis.

A generalized delay is characterized by reduced ossification of bones that normally exhibit rapid ossification during the last few days of gestation (e.g. phalanges, sternebrae, calvarium and the cervical, thoracic, sacral and caudal vertebral centra) (Carney & Kimmel, 2007). The presence of normal cartilage, which is the case in the pups in this study, provides additional evidence of a simple delay.

This assessment is supported by the fact, that the mean fetal body weights in test group 3 (1000 mg/kg bw/d) were significantly reduced (about 8% below the concurrent control value) which again indicates a delay in overall development going along with the delay in ossification. The reduced fetal body weight in the high dose group can be a result of fetal toxicity or the higher mean number of pups per litter in the high dose group (11.8) compared to the control group (10.9). Hence, the reduced fetal weight and consequently the delay in overall development could also be consequence of the higher number of pups per litter in the high dose group that have to be supplied by the dams.

Despite of this possibility, the above-mentioned findings in the high-dose group (1000 mg/kg bw/d) were assessed as a minor delay or disturbance of ossification indicating a generalized delay that can be considered treatment-related. Consequently, the lowest observed adverse effect level (LOAEL) for prenatal developmental toxicity is 1000 mg/kg bw/d.

In conclusion, under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused no evidence of maternal toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the highest tested dose of 1000 mg/kg bw/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg bw/d based on the increased incidences of skeletal variations indicating a generalized delay at the high-dose level (1000 mg/kg bw/d).

 

 

 

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP compliant prenatal developmental toxicity study (according to OECD TG 414), the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle. Regarding clinical examinations, nearly all females (24 out of 25) of the high-dose group (1000 mg/kg bw/d) showed transient salivation within 2 hours after treatment. This finding is considered to be treatment-related, most likely as a local irritation of the upper digestive tract or as a result of the bad taste of the test substance/vehicle preparation. However, it was not assessed as sign of systemic toxicity. At 300 and 100 mg/kg bw/d, no treatment-related, adverse effects were observed. Concerning thyroid hormone measurement, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, no treatment related findings in thyroid glands were noted. All 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. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose.

The findings ‘Incomplete ossification of skull; unchanged cartilage’ and ‘Supernumerary thoracic vertebra’ were statistically significantly increased in test group 2 and outside the historical control range. This finding might be associated with the treatment, but since these are the only two increased skeletal variants in this group, only slightly exceeds the historical background incidence and is only of mild adversity, it is evaluated as of no toxicological
relevance.

However, at the top dose (1000 mg/kg bw/d) skeletal variations affecting elements of the skull, vertebral column, sternebrae and ribs, were present at incidences above the historical control range. Variations in contrast to malformations are less serious and usually transient or repairable (Carney & Kimmel, 2007). Anyhow, it has to be noted that almost all of these variations appear at high background incidences in the population of the used rat breed and are usually of relatively low toxicological concern.

Specifically, the affected fetuses/litter incidences of ‘Incomplete ossification of interparietal (unchanged cartilage)’, ‘Incomplete ossification of skull (unchanged cartilage)’, ‘Incomplete ossification of temporal’, ‘Incomplete ossification of thoracic centrum (unchanged cartilage)’ and ‘Unossified sternebra (unchanged cartilage)’, represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases. The ossification of the bony structures occurs at the end of gestation and the fetal ossification status is highly influenced by many factors, such as difference in mating time of just a few hours, time of cesarean section or by stress due to maternal toxicity (Carney & Kimmel, 2007).

According to the evaluation of Carney and Kimmel (2007), the fetal skeletal ossification is tightly linked to the overall rate of fetal growth with subsequent catch-up postnatally and a delay in skeletal ossification is mechanistically independent from malformation. Hence, delayed ossification does not seem to have general predictive value for teratogenesis.

A generalized delay is characterized by reduced ossification of bones that normally exhibit rapid ossification during the last few days of gestation (e.g. phalanges, sternebrae, calvarium and the cervical, thoracic, sacral and caudal vertebral centra) (Carney & Kimmel, 2007). The presence of normal cartilage, which is the case in the pups in this study, provides additional evidence of a simple delay.

This assessment is supported by the fact, that the mean fetal body weights in test group 3 (1000 mg/kg bw/d) were significantly reduced (about 8% below the concurrent control value) which again indicates a delay in overall development going along with the delay in ossification. The reduced fetal body weight in the high dose group can be a result of fetal toxicity or the higher mean number of pups per litter in the high dose group (11.8) compared to the control group (10.9). Hence, the reduced fetal weight and consequently the delay in overall development could also be consequence of the higher number of pups per litter in the high dose group that have to be supplied by the dams.

Despite of this possibility, the above-mentioned findings in the high-dose group (1000 mg/kg bw/d) were assessed as a minor delay or disturbance of ossification indicating a generalized delay that can be considered treatment-related. Consequently, the lowest observed adverse effect level (LOAEL) for prenatal developmental toxicity is 1000 mg/kg bw/d.

In conclusion, under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused no evidence of maternal toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the highest tested dose of 1000 mg/kg bw/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg bw/d based on the increased incidences of skeletal variations indicating a generalized delay at the high-dose level (1000 mg/kg bw/d).

 

 

 

 

Justification for classification or non-classification

Based on the available data, the test substance may induce a generalized delay at the limit dose. However, according to the evaluation of Carney and Kimmel (2007), the fetal skeletal ossification is tightly linked to the overall rate of fetal growth with subsequent catch-up postnatally and a delay in skeletal ossification is mechanistically independent from malformation. Consequently, the substance does not need to be classified according to Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 as amended for the fifteenth time in Regulation (EC) No. 2020/1182.