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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-05-2020 - 03-02-2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 Jun 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxyethyl acrylate
EC Number:
212-454-9
EC Name:
2-hydroxyethyl acrylate
Cas Number:
818-61-1
Molecular formula:
C5H8O3
IUPAC Name:
2-hydroxyethyl acrylate
Test material form:
liquid

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Species and strain
Sexually mature, virgin New Zealand White rabbits (Crl:KBL(NZW)) were supplied at 15-18 weeks of age by Charles River Laboratories, Research Models and Services, Germany GmbH (Breeder: Charles River Laboratories, France). Only animals free from clinical signs of disease were used for the investigations.

Animal identification
The breeder produced unique identification of the rabbits by ear tattoo.

Reason for species selection
The strain was selected since historical control data is available from the test facility for New Zealand White rabbits. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

HOUSING AND DIET
During the acclimatization and study period, the rabbits were housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH, Hohenpeißenberg, Germany (floor space 4264 mm², internal height 450 mm).

For enrichment, wooden gnawing blocks were added (Typ SAFE® gnawing block), supplied by
J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany).

The animals were accommodated in fully air-conditioned rooms in which central air conditioning maintained a range of temperature of 17-21°C and a range of relative humidity of 45-65%. The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study.

The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h).

Before the study started, the animal room was completely disinfected using a disinfector (Geschko MLT 17 hydrogen peroxide gas generator (PEA; Germany)). The walls and the floor were cleaned once a week with water containing an appropriate disinfectant.

The food used was pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland. Food and drinking water (potable tap water in water bottles) were available ad libitum throughout the study (from the day of arrival to the day of necropsy).

Based on the pregnant animals the body weight on GD 0 varied between 3251 – 4432 g.

IN-LIFE DATES: From: 29 May 2020 To: 14 Jul 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water over a period of a maximum of 7 days at room temperature had been verified prior to the start of the study with the same batch.

Samples of the test substance preparations were sent once (at the beginning of administration) to the analytical laboratory for verification of the concentrations.

All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology. All reserve samples and further samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C) Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to original samples after agreement by the Study Director.

The results of the analyses of the test substance preparations in drinking water confirmed the correctness of the prepared concentrations. The measured concentrations of the samples of treatment groups corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.
Details on mating procedure:
After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination.

A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.

During the acclimatization period the animals were assigned to the different test groups according to a randomization plan (NIJENHUIS and WILF) and on the basis of their body weights.

The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.
Duration of treatment / exposure:
The test substance was administered to the animal orally by gavage from implantation to one day prior to the expected day of parturition (GD 6-28) always at approximately the same time of day. The animals of the control group were treated in the same way with the vehicle (drinking water). The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
On GD 29 blood samples were obtained in randomized order from all surviving animals from the ear veins. After blood sampling, the females were euthanized and examined. The fetuses were removed from the uterus and investigated.

Dose selection rationale
The high dose was selected based on signs of toxicity noted at dose levels of 90 and 180 mg/kg bw/d in a previously conducted maternal toxicity range-finding study (BASF project 20R0173/16R191, non-GLP) which preceded this definitive prenatal developmental toxicity study.
In this maternal toxicity range‐finding study, 5 presumed pregnant NZW rabbits were adminis- tered the test substance via oral gavage from gestational day (GD) 6 through GD 28, at doses of 90 and 180 mg/kg bw/d.
The 180 mg/kg bw/d dose caused in the pregnant rabbits an immediate cessation of food consumption after the first dosing. One animal was found dead and all remaining animals were killed for humane reasons after the second dosing.
The 90 mg/kg bw/d dose caused a significant reduction of food consumption (up to 61% below control) during the first two treatment weeks (GD 6-19). During the last treatment week recovery was noted, however, mean food consumption stayed about 14 % below control during the treatment period. In addition, clinical signs like poor general condition, high-stepping gait, blood in bedding and no or reduced defecation were observed in individual rabbits. One animal aborted.
Clinical pathology revealed no relevant findings.
At macroscopic pathology discolorations and foci were detected in the stomach of 3 rabbits at 180 mg/kg bw/d and one rabbit at 90 mg/kg bw/d. Histopathologically erosions/ulcers were detected in all 180 mg/kg bw/d rabbits and one 90 mg/kg bw/d rabbit. At 180 mg/kg bw/d these were accompanied by signs of minimal or mild to moderate hemorrhages and inflammation.
Thus, the selected high dose for the present definitive study (60 mg/kg bw/d) represented a dose which was supposed to cause a moderate reduction of food consumption and/or body weight/body weight gain in pregnant female rabbits. Considering the specific characteristic of rabbit gastrointestinal physiology and the consequences of their disruption for the maintenance of pregnancy in rabbits this procedure meets the principles of guidelines OECD 414 (adopted 2018) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in compliance with EU Directive 2010/63/EU on animal protection.

Examinations

Maternal examinations:
Mortality
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).


Clinical symptoms
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.
During the administration period (GD 6-28) all animals were checked daily for any abnormal clinical signs before the administration as well as within 5 hours after the administration.


Food consumption
The consumption of food was recorded daily during GD 0-29.


Body weight data
All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.


Corrected (net) body weight gain
The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).


Clinical Pathology
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.


The following examinations were carried out in all animals per test group:

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):

Parameters and methods:

Parameter Unit Method
Leukocyte count (WBC) giga/L cytochemistry coupled with flow cytometry
Erythrocyte count (RBC) tera/L flow cytometric laserlight scattering
Hemoglobin (HGB) mmol/L cyanmethemoglobin method; according to ICSH
Hematocrit (HCT) L/L calculation: MCV x erythrocytes
Mean corpuscular volume
(MCV) fL RBC/PLT method; mean of RBC volume distribution curve (histogram)
Mean corpuscular
hemoglobin (MCH) fmol calculation: hemoglobin/erythrocytes
Mean corpuscular
hemoglobin concentration
(MCHC) mmol/L calculation: hemoglobin/hematocrit
Platelet count (PLT) giga/L flow cytometric laserlight scattering
Differential blood count % & giga/L cytochemistry coupled with flow cytometry
Reticulocytes (RETA) giga/L cytochemistry coupled with flow cytometry


Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters
Parameters and methods:
See table below in any other information


Necropsy
On GD29 all surviving animals were sacrificed by an intravenous injection of pentobarbital (dose: 2ml/animal) in a randomized sequence. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
All animals which died intercurrently (animal No. 31) or were sacrificed in a moribund state (including animals with abortion; animal No. 91) were necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights were determined in all does sacrificed on schedule:
1. Adrenal glands
2. Kidneys
3. Liver
4. Spleen
All paired organs were weighed together (left and right).

The carcass weights (GROSSE-System) were transferred to the ACOPAT-System to calculate the relative organ weights. Organ weights of animals, that were not pregnant, were excluded from the calculation of mean values.

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Duodenum
4. Kidneys
5. Liver
6. Spleen
7. Stomach (forestomach and glandular stomach)

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the following table:

Organs Test groups
0 1 2 3
1. Stomach (forestomach and glandular stomach) A1 A1 A1 A1
2. Duodenum A1 A1 A1 A1
A = hematoxylin and eosin (H&E) stain 1 = all animals/test group

A correlation between gross lesions and histopathological findings was attempted.

Ovaries and uterine content:
Cesarean section

On GD 29, the surviving does were euthanized in randomized order by intravenous injection of pentobarbital (Narcoren®; dose 2 mL/animal). After exsanguination, the animals were necropsied and assessed by gross pathology.

The uteri and the ovaries were removed and the following data were recorded:

- Weight of the unopened uterus

- Number of corpora lutea

- Number and distribution of implantation sites classified as:

• Live fetuses

• Dead implantations:

a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)

b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)

c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the does (except of gross pathology including organ sampling and weights) and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
Blood sampling:
In the morning blood was taken from the ear vein from not-fasted animals without anesthesia. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.


Additional blood samples for non-GLP research purposes

Additional samples during venipuncture were collected for external research projects beyond the scope of this study and without GLP status. The results of the study were not influenced by this procedure because blood sampling occurred just prior to sacrifice/at necropsy. The last aliquot was taken for these projects, thus blood sampling for the regulatory investigations was not affected. Thus, the sampling procedures did not affect the outcome and compliance of the GLP study. The data from these research projects do not affect the outcome, assessment and compliance of this GLP study.
From each animal 1 mL of blood was collected with EDTA as anticoagulant (10 µL of a 10% solution). The samples were centrifuged. The plasma was separated. The blood was sampled and prepared in original Eppendorf tubes. The preparation of the samples was done under cooling. All samples were stored covered with a N2 atmosphere and then stored at –80°C for research.
Fetal examinations:
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

Examinations of the fetuses after dissection from the uterus
At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded.
Thereafter, the fetuses were sacrificed by an intraperitoneal injection of pentobarbital (Narcoren®; dose: 0.2 mL/fetus; one part Narcoren® diluted with one part physiological saline).

Soft tissue examination of the fetuses
After the fetuses had been sacrificed, the abdomen and thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure.
The sex of the fetuses was determined by examination of the gonads in situ.

After these examinations, the heads of approximately one half of the fetuses per doe (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN’s solution and were, after fixation, processed and evaluated according to WILSON’s method (WILSON and WARKANY). About 10 transverse sections were prepared per head. After the examination the heads were discarded.

All fetuses (including those without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the intact fetuses were removed from the fixative and a transversal incision was made into the frontal/parietal head bones. The two halves of the calvarium were cautiously bent outward and the brain was thoroughly examined. Subsequently, these fetuses were placed back into the fixative for further fixation.

Skeletal examination of the fetuses
After fixation in ethyl alcohol, the skeletons (with and without skulls) were stained according to a modified method of KIMMEL and TRAMMELL. The stained skeletons were placed on an illuminated plate, investigated and archived individually.

Evaluation criteria for assessing the fetuses
Classification and assessment of fetal findings is a matter of ongoing discussion (see e.g. BELTRAME and GIAVINI, CHAHOUD, SOLECKI). Despite considerable efforts to harmonize the nomenclature used to describe observations of fetal morphology, the terms still vary considerably between laboratories, investigators and textbooks in the fields of teratology and developmental toxicity.
In the present study the internationally harmonized glossary of WISE et al. (1997) and the updated version MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI:

Malformation
A permanent structural change that is likely to adversely affect survival or health.

Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.

All fetal findings were listed in tables according to these classifications.
Statistics:
see any other information below
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

Historical control data:
see attached file

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No defecation was observed in three control females, one low-dose, two mid-dose and six high-dose females. Reduced defecation was observed in eight control, four low-dose, four mid- dose and seven high-dose females (0, 6, 20 and 60 mg/kg bw/d). Incidence and distribution of these findings do not indicate a relationship to the test substance.

There were no further clinical findings in the other does in this study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One low-dose female (No. 31 - 6 mg/kg bw/d) was found dead before treatment on GD 26, after showing no or reduced defecation during several days before. The cause of this preterminal death remains unknown, because of the lacking dose-response it is, however, considered not to be related to the test substance.

One high-dose female (No. 91 - 60 mg/kg bw/d) was sacrificed after abortion ahead of schedule (GD 25). Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, this was considered to be a spontaneous incident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant difference was observed for the mean body weights (BW) of the high- dose females (60 mg/kg bw/d) when compared to the control group.

Collectively, the mean body weight gain during the treatment period was comparable between all test groups (6, 20 and 60 mg/kg bw/d) and the control group.

Notably, there were episodes of significant differences between control and high-dose group such as GD 19-21 where the high-dose does lost less weight (-3.9 g vs. -55.4 g in control), or GD 23-25 where they gained more weight than the control does (47.2 g vs. 6.8 g in control). These episodes compensated for a longer time period (GD 6-19) where the high-dose rabbit gained consistently (but not statistically significantly) less body weight than the controls.

Corrected (net) body weight gain
Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were not significantly different between all test groups including controls (0, 6, 20 or 60 mg/kg bw/d).

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In comparison to the control group the mean food consumption of the does in test group 3 (60 mg/kg bw/d) was reduced from GD 12 onwards, attaining statistical significance on GD 12-14 and GD 15-18 (up to 40% below control). Afterwards it recovered, being comparable to or even exceeding the control values (attaining statistical significance on GD 25-27). Overall, the high- dose does consumed about 9% less food than the concurrent control does during the treatment period (GD 6-28).

The food consumption of the low- and mid-dose rabbits (6 and 20 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes of hematological parameters were observed.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.

At gestation day 29, in does of test group 3 (60 mg/kg bw/d) cholesterol values were significantly increased, but the mean was within the historical control range (cholesterol 0.18-0.53 mmol/L). Therefore, this change was regarded as incidental and not treatment related.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute weights
All mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative kidney weights were statistically significantly increased in test group 3 :

Relative weights
Test group (mg/kg bw/d) 1 (6) 2 (20) 3 (60)
Kidneys 96% 104% 110%**
*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Since only the relative kidney weights in test group 3 were significantly increased and since relative kidney weights lay within the range of historical control values, weight changes were regarded as incidental and not treatment related.


Uterus weights
The mean gravid uterus weight of the rabbits of test groups 1-3 (6, 20 or 60 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.
Gross pathological findings:
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: 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
Details on results:
Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.

The following females were excluded from the above-mentioned calculations:

Test group 0 (0 mg/kg bw/d):
• females Nos. 14, 18, 22, 23 - not pregnant

Test group 1 (6 mg/kg bw/d):
• females Nos. 43, 48 - not pregnant
• female No. 31 - died intercurrently

Test group 2 (20 mg/kg bw/d):
• females Nos. 53, 60, 65, 75 - not pregnant

Test group 3 (60 mg/kg bw/d):
• females Nos. 77, 80, 88 - not pregnant
• female No. 91 - sacrificed after abortion


Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, non-treatment-related
Early or late resorptions:
effects observed, non-treatment-related
Dead fetuses:
effects observed, non-treatment-related
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Female rabbits were placed into the study in four cohorts. Each dose group was represented in each cohort. The conception rate was 84% in the control and mid-dose groups (0 and 20 mg/kg bw/d), 88% in the high-dose group (60 mg/kg bw/d) and 92% in the low-dose group (6 mg/kg bw/d). A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study (according to the test guidelines).

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for pre- and post-implantation losses, the number of resorptions and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

In high-dose doe No. 98 (60 mg/kg bw/d) 2 early resorptions were recorded after staining the apparently non-pregnant uterus at C-section (i.e. pregnant by stain), resulting in a 100% post- implantation loss for this animal. This isolated finding did not exert a significant effect on the group mean and is regarded as incidental and not treatment related.

Two dead fetuses were found at C-section of high-dose doe No. 93 which is a rare finding but may occur spontaneously in this rabbit strain. As there was no evidence whatsoever for any potential developmental toxicity of the test item in this study, this is considered to be an incidental finding.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 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 (6, 20 and 60 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal external malformations
One external malformation was recorded for two fetuses, each, in test groups 0 and 1 (0 and 6 mg/kg bw/d) as listed in the table below. In one control fetus it was associated with an additional visceral malformation. However, this finding was an isolated event in single fetuses, thus, it is considered to be incidental. No statistically significant differences of overall incidences were noted between the groups

Individual fetal external malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 3-02 M umbilical hernia
21-09 F a) umbilical hernia
1 (6 mg/kg bw/d) 30-06 F umbilical hernia
35-02 M umbilical hernia
2 (20 mg/kg bw/d) none
3 (60 mg/kg bw/d) none
a) fetus with additional visceral malformations

Total external malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N(%) 2 (1.1) 2 (1.2) 0.0 0.0
Litter
incidence N(%) 2 (9.5) 2 (9.1) 0.0 0.0
Affected
fetuses/litter Mean% 1.3 1.1 0.0 0.0



Fetal external variations
One external variation (paw hyperflexion) was recorded in two control fetuses, one mid-dose fetus and two high-dose fetuses. This finding can be found in the historical control data at a comparable incidence, thus it is considered to be incidental.

Total external variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 2 (1.1) 0.0 1 (0.5) 2 (1.2)
Litter
incidence N (%) 2 (9.5) 0.0 1 (4.8) 2 (10)
Affected
fetuses/litter Mean% 1.1 0.0 0.4 1.3


Fetal external unclassified observations
One unclassified observation, i.e. placentae necrobiotic, was recorded in one fetus of test group 3 (60 mg/kg bw/d). This finding is considered not to be related to treatment.

Total external unclassified observations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 0.0 0.0 0.0 1 (0.6)
Litter
incidence N (%) 0.0 0.0 0.0 1 (5.0)
Affected
fetuses/litter Mean% 0.0 0.0 0.0 0.6

Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were detected in single fetuses of all test groups including the control (0, 6, 20 and 60 mg/kg bw/d), as shown in the table below. Two fetuses had associated soft tissue malformations. All findings were considered to be spontaneous in origin and not treatment-related.

No statistically significant differences between the groups were noted. The overall incidences were well within the historical control range of the test facility.

Individual fetal skeletal malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 19-04 M fused thoracic arch, fused rib
1 (6 mg/kg bw/d) 41-01 M additional vertebral arch and corresponding rib, intercostal rib
41-03 M a) cleft sternum
2 (20 mg/kg bw/d) 73-10 F absent lumbar vertebra
3 (60 mg/kg bw/d) 82-07 M severely malformed vertebral column and/or ribs
87-07 M a) severely malformed vertebral column and/or ribs
a) fetus with additional soft tissue malformations


Total skeletal malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 1 (0.5) 2 (1.2) 1 (0.5) 2 (1.2)
Litter
incidence N (%) 1 (4.8) 1 (4.5) 1 (4.8) 2 (10)
Affected
fetuses/litter Mean% 0.4 4.5 0.5 1.1


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 dosing. The overall incidences of skeletal variations were comparable to the historical control data.

Total fetal skeletal variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 175 (93) 143 (88) 165 (87) 161 (93)
Litter
incidence N (%) 21 (100) 22 (100) 21 (100) 20 (100)
Affected
fetuses/litter Mean% 93.5 86.4 86.2 92.7


For a better overview, all skeletal variations with statistically significant differences between the control and the treated groups were compiled in the table below. All incidences were expressed on a fetus per litter basis.

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

Finding Test group 0 Test group 1 Test group 2 Test group 3 HCD
Mean % (range)

Misshapen
sacral vertebra 2.1 2.3 5.3 4.8* 5.0 (1.9 - 8.6)

Incomplete
ossification
of talus;
cartilage present 2.6 2.9 2.3 6.7* 2.3 (0.0 - 4.5)

Unossified talus;
cartilage present 0.5 1.2 3.1 5.8* 1.3(0.0 - 2.6)

HCD = Historical control data
* = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])


The findings ‘incomplete ossification of talus’ and ‘unossified talus’ (with present cartilage, respectively) were statistically significantly increased and outside the historical control range in test group 3 (60 mg/kg bw/d). These findings may represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases.

The increased incidence of ‘misshapen sacral vertebra’ in test group 3 was not related to dose and clearly inside the historical control range. Thus, this finding is considered not to be associated with treatment.


Fetal skeletal unclassified cartilage observations
Some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they were assessed as not treatment-related.

Total unclassified cartilage observations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 20 (11) 12 (7.4) 30 (16) 13 (7.5)
Litter
incidence N (%) 11 (52) 8 (36) 12 (57) 5 (25)
Affected
fetuses/litter Mean% 10.7 6.0 14.3 8.5
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue malformations occurred in the test groups 0, 1 or 3 (0, 6 and 60 mg/kg bw/d), as listed in the table below. Three fetuses in different test groups had additional external or skeletal malformations.

The distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. The total incidence of soft tissue malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

Individual fetal soft tissue malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 15-10 F small thymus
21-09 F a) small spleen
24-05 M, 24-06 F, 24-08 M small spleen
1 (6 mg/kg bw/d) 39-04 F small spleen
41-03 M b) cor triloculare
2 (20 mg/kg bw/d) none
3 (60 mg/kg bw/d) 87-07 M b), 87-08 F small spleen
97-05 F small spleen
a) fetus with additional external malformations
b) fetus with additional skeletal malformations

Total soft tissue malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 5 (2.6) 2 (1.2) 0.0 3 (1.7)
Litter
incidence N (%) 3 (14) 2 (9.1) 0.0 2 (10)
Affected
fetuses/litter Mean% 2.0 2.8 0.0 1.8


Fetal soft tissue variations
The examinations of the soft tissues revealed malpositioned carotid branches and an absent lung lobe (Lobus inferior medialis) in all test groups including the control (0, 6, 20 and 60 mg/kg bw/d). Other variations, such as cystic dilatation of the brain and supernumerary branch from aortic arch (test group 2, respectively), dilated cerebral ventricle (test groups 1 and 2), malpositioned carotid origin (test group 0), dilated aortic arch and narrowed pulmonary trunk (test group 1, respectively) and dilated renal pelvis (test group 3) occurred in individual fetuses of the different test groups.

No statistically significant or toxicologically relevant differences between the groups were noted, and the overall incidences were within the historical control range.

Total soft tissue variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 4 (2.1) 7 (4.3) 7 (3.7) 7 (4.0)
Litter
incidence N (%) 4 (19) 6 (27) 6 (29) 4 (20)
Affected
fetuses/litter Mean% 1.7 5.2 4.3 4.1

Fetal soft tissue unclassified observations


Three unclassified soft tissue observations were recorded. A blood coagulum around urinary bladder was seen in two control, one low-dose, two mid-dose and one high-dose fetuses. This finding can be found in the historical control data at a comparable incidence, therefore, it was neither assessed as treatment-related nor as adverse. Furthermore, a discolored spleen was seen in three fetuses of the same high-dose litter, and an infarct of liver was recorded in one fetus of test group 3. These findings are considered not to be treatment-related.

Total soft tissue unclassified observations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 2 (1.1) 1 (0.6) 2 (1.1) 5 (2.9)
Litter
incidence N (%) 1 (4.8) 1 (4.5) 2 (9.5) 3 (15)
Affected
fetuses/litter Mean% 1.6 0.5 1.0 2.5

Description (incidence and severity):
The mean placental weights in test groups 1, 2 and 3 were not influenced by the test substance and were comparable to the control value.
Details on embryotoxic / teratogenic effects:
Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in all test groups (0, 6, 20 or 60 mg/kg bw/d). The distribution of total malformations about the groups was not related to dose.

Six fetuses had more than one malformation. Male control fetus No. 19-04 had a fused thoracic arch and a fused rib, while female control fetus No. 21-09 showed an umbilical hernia and a small spleen. Furthermore, for male low-dose fetus No. 41-03 (6 mg/kg bw/d) a cor triloculare combined with a cleft sternum was recorded. Male low-dose fetus No. 41-01 had malformations affecting the rib cage, i.e. additional vertebral arch and corresponding rib and an intercostal rib. Male high-dose fetus No. 87-07 (60 mg/kg bw/d) showed a small spleen and a severely malformed vertebral column and/or ribs. Lastly, male high-dose fetus No. 82-07 had also a severely malformed vertebral column and/or ribs. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups.

The findings ‘umbilical hernia’ and ‘small spleen’ which were seen in the multiple malformed fetuses, occurred also in further individual fetuses of test groups 0, 1 and 3. Other malformations, such as small thymus (test group 0) and absent lumbar vertebra (test group 2) were scattered observations in individual fetuses. They all were not dose-related and most of them can be found in the historical control data at comparable frequency. An association of these findings to the treatment is not assumed.

Total fetal malformations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 7 (3.7) 5 (3.1) 1 (0.5) 4 (2.3)
Litter
incidence N (%) 5 (24) 4 (18) 1 (4.8) 3 (15)
Affected
fetuses/litter Mean% 3.2 6.2 0.5 2.3



A spontaneous origin is assumed for the external variations, soft tissue variations and the broad range of skeletal variations which were noted in fetuses of all test groups including controls.

If all different types of variations are summarized, none of the incidences showed a relation to dosing and can be found in the historical control data at comparable frequency.

The only exception: statistically significantly increased findings ‘incomplete ossification of talus’ and ‘unossified talus’ represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases. As these non or incomplete ossifications were solely found in this single location, and no other signs of a delay of skeletal development were noted, a spontaneous origin of these findings is assumed. In addition, such minor developmental delays are considered not to be adverse events.

Total fetal variations
Test group 0 Test group 1 Test group 2 Test group 3
Litter N 21 22 21 20
Fetuses N 189 163 189 173
Fetal
incidence N (%) 175 (93) 143 (88) 165 (87) 161 (93)
Litter
incidence N (%) 21 (100) 22 (100) 21 (100) 20 (100)
Affected
fetuses/litter Mean% 93.5 86.4 86.2 92.7


A spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups. The distribution and type of these findings do not suggest any relation to treatment.

Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (60 mg/kg bw/d).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of Hydroxyethyl acrylate to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused beginning evidence of maternal toxicity at a dose of 60 mg/kg bw/d, such as slightly but consistently reduced food consumption as well as reduced body weight/weight gain during the first two treatment weeks.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is
20 mg/kg bw/d.

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is
60 mg/kg bw/d, the highest dose tested.

The test substance is not teratogenic in rabbits at the tested dose levels.
Executive summary:

In a prenatal developmental toxicity study, Hydroxyethyl acrylate was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28).

Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle.

Clinical examinations revealed no toxicologically relevant difference between the animals receiving 6 or 20 mg/kg bw/d Hydroxyethyl acrylate and the controls.

At the high-dose level of 60 mg/kg bw/d slightly, but consistently lower food consumption as well as lower body weight gain during the first two treatment weeks may represent beginning signs of maternal toxicity.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 60 mg/kg bw/d.

Regarding pathology, 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.

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and postimplantation losses, the numbers of resorptions and viable fetuses. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development.