Soybean oil, epoxidized, reaction products with methanol

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Soybean oil, epoxidized, reaction products with methanol

EC Number:

287-837-7

EC Name:

Soybean oil, epoxidized, reaction products with methanol

Cas Number:

85586-35-2

Molecular formula:

C H4 O . Unspecified

IUPAC Name:

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

Details on test material:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch 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.

Test animals

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

Administration / exposure

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).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Remarks:

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

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

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

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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.

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

1

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)

2

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:

Fetal abnormalities

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.

Overall developmental toxicity

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

Any other information on results incl. tables

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])

 

Applicant's summary and conclusion

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).