2-(2-aminoethoxy)ethanol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2020 - May 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

Deviations:

no

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:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Commission Regulation (EC) No 440/2008, 30 May 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-aminoethoxy)ethanol

EC Number:

213-195-4

EC Name:

2-(2-aminoethoxy)ethanol

Cas Number:

929-06-6

Molecular formula:

C4H11NO2

IUPAC Name:

2-(2-aminoethoxy)ethan-1-ol

Test material form:

liquid

Specific details on test material used for the study:

- CAS No.: 929-06-6
- Batch No.: 22057368E0
- Purity: 99.4 area % corrected with the water content
- Identity: confirmed
- Homogeneity: given
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.
- Expiry date: 06 Apr 2021
- appearance: liquid / colorless, clear
- Storage conditions: Room temperature/under N2


Test substance preparations:
- Stability analysis: The stability of the test substance in deionized water at room temperature over a period of 7 days was demonstrated prior to the start of the study.
- Homogeneity analyses of the test substance preparations: Given that the test substance is completely miscible with deionized water, solutions are considered to be homogenous without further analysis.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH.
- Age at study supply: 14-17 weeks of age.
- Age at study initiation: 15 - 18 weeks of age.
- Weight at study initiation: 3757 - 3814 g.
- Housing: 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 Söhne GmbH + Co KG, Rosenberg, Germany).
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 7 days
- Only animals free from clinical signs of disease were used for the investigations.
- Identification: ear tattoo.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21°C.
- Humidity (%): 45-65%.
- Air changes (per hr): 15 times per hour.
- Photoperiod (hrs dark / hrs light): 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).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

TEST SUBSTANCE PREPARATION AND PREPARATION FREQUENCY:
- 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 preparation, the specific amount of the test substance was weighed, topped up with deionized water in a calibrated beaker and intensely mixed with a magnetic stirrer until it was completely dissolved.
- Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

EXPERIMENTAL EXPOSURE:
- 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.
- Based on the pregnant animals the body weight on GD 0 varied between 3342 – 4300 g.
- 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 (deionized 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.
- On GD 29, the females were euthanized in randomized order and examined. The fetuses were removed from the uterus and investigated.

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 in deionized 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.

Duration of treatment / exposure:

23 days

Frequency of treatment:

daily

Duration of test:

Complete duration of the test (all cohorts): 43 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

25

Details on study design:

- Dose selection rationale: In a previous test study , the test substance was tested orally by gavage at dose levels of 1000, 600, 300 and 0 mg/kg bw/d in each three non- pregnant female New Zealand White rabbits. At 1000 mg/kg bw/d, one female was found dead on study day 4 and clinical findings (reduced/no feces) were observed. Mean food consumption was statistically significantly reduced. High-dose females consumed remarkably less food than controls (95% below control during study days 3-4). For animal welfare reasons, this dose group was terminated. At 600 mg/kg bw/d, one female was found dead on study day 12. Females showed also reduced/no defecation and a reduction in mean food consumption (around about half of the controls, range of 61-116 g) throughout the study. Body weight was decreased in the beginning of the study but recovered in the remaining two females from study day 12 onwards. At 300 mg/kg bw/d, no clinical signs were observed. Mean food consumption was reduced in the beginning of the study (first week: up to 43% below control during study days 0-1) but recovered towards the end of the study to almost control level. Body weight was almost comparable to controls. At necropsy, erosions and ulcerations were observed in the stomach of several treated females. Ulcerations were seen in three high-dose and two mid-dose females. At 300 mg/kg bw/d, one female showed an erosion. Based on the above-mentioned findings, dose levels of 300 (as half-lethal dose) and 100 mg/kg bw/d were used in the following maternal toxicity study in pregnant rabbits. At 300 mg/kg bw/d, food consumption was reduced during GD6-28 (11%) with a maximum of 27% below control during GD15-16. Correspondingly, body weight change was decreased during GD 14-16 (17g versus 68g in controls). At 100 mg/kg bw/d, food consumption and body weight were not affected. At necropsy, foci in the glandular stomach
were seen in three out of five high-dose and one low-dose female. Based on the above-mentioned findings at 300 mg/kg bw/d and at request of the sponsor, the following doses were chosen for the present prenatal developmental toxicity study in New Zealand White rabbits: 20, 70, 200 mg/kg bw/day.
- Exposure roue rationale: The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- 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.

Examinations

Maternal examinations:

CLINICAL EXAMINATIONS OF THE DOES
- 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: Furthermore, 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).
- Blood samples for non-GLP research purposes: Blood samples 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 sampling procedures did not affect the outcome and compliance of this GLP study. The data from these research projects did not affect the outcome, assessment and compliance of this GLP study.


TERMINAL EXAMINATIONS OF THE DOES
- 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.
These data were used to calculate conception rate and pre- and postimplantation losses for each individual pregnant animal which underwent scheduled sacrifice according to the following formulas:

Conception rate (%) = number of pregnant animals x 100 / number of fertilized animals

Preimplantation loss (%) = (number of corpora lutea – number of implantations) x 100 / number of corpora lutea

Postimplantation loss (%) = (number of implantations – number of live fetuses) x 100 / number of implantations


PATHOLOGY
- Necropsy: On GD29 all surviving animals were sacrificed by an intravenous injection of pentobarbital (e.g. Narcoren®; 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.
- Organ/tissue fixation: The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Stomach
No further examinations or procedures were performed in the study.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.data
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.

Fetal examinations:

EXAMINATIONS OF THE FETUSES
- 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 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. and the updated version MAKRIS et al. 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.

Statistics:

See "Any other information on materials and methods incl. tables"

Historical control data:

Yes.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One high-dose doe (No. 78 - 200 mg/kg bw/d) was sacrificed moribund before treatment on GD 21 after showing several clinical findings on the respective day of
sacrifice or the days before: reduced defecation (GD 17-18), no defecation (GD 20-21), blood in bedding (GD 20-21), hypothermia (GD 21) and a poor general state (GD 21). Furthermore, one control doe (No. 5) had blood in bedding before/after treatment on GD 24.
In total, reduced defecation was observed in seven control, nine low-dose, five mid dose and twelve high-dose females (0, 20, 70 and 200 mg/kg bw/d). No defecation was observed in two control females and in one female, each, of the low-dose and high-dose groups. 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 high-dose female (No. 78 - 200 mg/kg bw/d) was sacrificed moribund on GD 21 (before treatment) for animal welfare reasons. The animal showed numerous clinical findings such as no/reduced defecation, blood in bedding, hypothermia and poor general state. Since only one single animal in the high-dose group was affected and the above-mentioned findings can be also seen in control animals, this was assessed as not related to treatment and as incidental.
There were no further substance-related or spontaneous mortalities in any female of all test groups (0, 20, 70 or 200 mg/kg bw/d).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and the average body weight gain of the low-, mid- and high-dose groups (20, 70 and 200 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
The statistically significantly increased body weight gain value in test group 1 on GD 21-23 is assessed as incidental.

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 comparable among all test groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption of the low-, mid- and high-dose rabbits (20, 70 and 200 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.
The statistically significantly reduced food consumption value in test group 1 on GD 7-8 is assessed as incidental since there was no relation to dose.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Uterus weights
- The mean gravid uterus weight of the rabbits of test groups 1-3 (20, 70 or 200 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.

Weight of the placentae
- 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.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopically, in 2/25 high-dose animals, a focus in the stomach was observed. There was a minimally increased incidence compared to the control group, where none of the animals showed a focus. Therefore, a relation to treatment can not be excluded.
A histopathologic examination of the stomach was not performed.
All other findings occurred only individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the values calculated for pre- and post implantation losses.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the number of resorptions.

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the number of viable fetuses.

One dead fetus was found at cesarean section of high-dose doe No. 80 (200 mg/kg bw/d) which is a rare finding but occurs also spontaneously in this rabbit strain. Therefore, this is considered to be an incidental finding.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Female rabbits were placed into the study in five cohorts. Each dose group was represented in each cohort. The conception rate was 76% in the control, 84% in the mid-dose group (70 mg/kg bw/d) and 92% in the low- and high-dose groups (20 and 200 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.

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.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No treatment-related adverse effects observed up to the highest tested dose.

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:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (20, 70 and 200 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

Changes in litter size and weights:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two external malformations were recorded (see "Any other informations on results incl. tables"): an umbilical hernia occurred in one fetus of test group 1, which had additionally a malrotated limb, and in one fetus of test group 3. However, these findings were isolated events in single fetuses and can be found in the historical control data at comparable incidences (Supplement, HCD, fetal external malformations, umbilical hernia: affected fetuses per litter: range of 0.0 – 1.3%). Thus, they are considered to be incidental. No statistically significant differences of overall incidences were noted between the groups.

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, 20, 70 and 200 mg/kg bw/d), as shown in "Any other informations on results incl. tables". One fetus (No. 96-01) had associated soft tissue malformations. No statistically significant differences between the groups were noted (see "Any other informations on results incl. tables"). The overall incidences were well within the historical control range of the test facility.
Two of the high-dose findings, such as absent cervical center, absent lumbar vertebra, were within the range of the historical control data. The other two were isolated events in single fetuses. Therefore, they were not assessed as treatment related. Findings in test group 2 were without relation to dose and, therefore, assessed as not treatment-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations occurred in test groups 0, 2 and 3 (0, 70 and 200 mg/kg bw/d), as listed in "Any other informations on results incl. tables". One female fetus in test group 3 (No. 96-01) had additional skeletal malformations. Furthermore, for one male fetus of test group 3 (No. 99-11) multiple external malformations, such as diaphragmatic hernia, small spleen, small pancreas, absent subclavian, malpositioned carotid branch and absent lung lobes, were recorded.
Most of the findings, such as aortic arch atresia, absent subclavian, heart: membranous ventricular septum defect, small spleen, can be found in the historical control data at comparable incidences. All other findings in the high dose group were isolated events in single fetuses. Therefore, they were assessed to be not related to treatment.
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.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal external variations:
- No external variations were recorded.

Fetal external unclassified observations:
- One unclassified external observation (placentae necrobiotic) was recorded in one control fetus. This finding is considered to be incidental (see "Any other informations on results incl. tables").

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, 20, 70 and 200 mg/kg bw/d). Other variations, such as cystic dilatation of the brain and narrowed aortic arch (test group 2, respectively), dilated aorta (test groups 1 and 3), dilated pulmonary trunk (test groups 1 and 2), narrowed pulmonary trunk (test group 3) and dilated aortic arch (test group 0) occurred in individual fetuses of the different test groups.
- The overall incidences in test groups 2 and 3 (see "Any other informations on results incl. tables", affected fetuses per litter: mean% 5.9 and 9.1, respectively) were outside the historical control range (Supplement, total soft tissue variations HCD: mean% 3.1 (0.4-5.6)). However, for the overall and also for the individual variations, no statistically significant differences between the groups were noted. No ontogenetic pattern is recognizable. Therefore, this was assessed as not relevant and not related to treatment.

Fetal soft tissue unclassified observations:
- Two unclassified soft tissue observations were recorded. A blood coagulum around urinary bladder was seen in eight control, five low-dose, 14 mid-dose and seven high-dose fetuses. This finding can be found in the historical control data at a comparable incidence. Therefore, it is neither assessed as treatment-related nor as adverse. Furthermore, a fluid-filled abdomen was seen in one control fetus.

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 without a relation to dosing (see "Any other informations on results incl. tables"). The overall incidences of skeletal variations were comparable to the historical control data.
- All skeletal variations with statistically significant differences between the control and any treated groups were compiled in the table below (see "Any other informations on results incl. tables"). All incidences were expressed on a fetus per litter basis.
- Concerning the statistically significant finding, no dose dependency was observed and all values were clearly inside the historical control range. Thus, an association to the test substance and a toxicological relevance is not assumed.

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 (see "Any other informations on results incl. tables"). The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to the dose.
- The incidence of ‘cartilaginous part of ribs not connected with sternum’ was statistically significantly increased in the mid-dose group (test groups 1-3: mean% 11.8 / 14.0* / 10.0 affected fetuses per litter versus 4.8% in control [* = p ≤ 0.05]). However, since this was not related to dose, it was assessed as incidental.
- The overall incidence of unclassified cartilage observations was statistically significantly increased in test group 1 and 2 (20 and 70 mg/kg bw/d). However, both values were well within the historical control range (HCD: mean% 11.3 [3.1-30.5]) and there was no relation to dose. Therefore, it is assessed as not treatment-related.

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, 20, 70 or 200 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 low-dose fetus No. 42-08 (20 mg/kg bw/d) had an umbilical hernia and a malrotated limb, while female mid-dose fetus No. 70-01 (70 mg/kg bw/d) showed a severely malformed vertebral column and/or ribs (i.e. thoracic arches fused with ribs, fused thoracic arch cartilages, unilateral, dumbbell, incomplete or bipartite ossification of thoracic centrum). Furthermore, for female mid-dose fetus No. 72-06 a small pancreas, an absent subclavian and a small spleen were recorded. Male high-dose fetus No. 81-09 (200 mg/kg bw/d) had an absent cervical center and a small thoracic arch. Female high dose fetus No. 96-01 showed a membranous ventricular septum defect at the heart combined with severely fused sternebrae (bony plate). Lastly, male high-dose fetus No. 99-11 had multiple visceral malformations, such as diaphragmatic hernia, small spleen, small pancreas, absent subclavian, malpositioned carotid branch and absent lung lobes. 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’, ‘absent subclavian’ and ‘small spleen’ which were seen in the multiple malformed fetuses, occurred also in further individual fetuses of test group 3. Other malformations, such as enlarged lens, cardiomegaly, small interparietal and malpositioned and bipartite sternebra (test group 0, respectively), right-sided aortic arch, asplenia and absent lumbar vertebra (test group 3, respectively), aortic arch atresia (test groups 0 and 3) and absent gallbladder (test groups 0 and 2), cleft sternum (test group 1) and fused rib (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.
- The total incidences of malformations are summarized in "Any other informations on results incl. tables".
- External variations did not occur in any fetus in this study. A spontaneous origin is assumed for the 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 (see "Any other informations on results incl. tables") and can be found in the historical control data at comparable frequency.
- 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 (0, 20, 70 and 200 mg/kg bw/d). 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 (200 mg/kg bw/d).

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed up to the highest tested dose.

Fetal abnormalities

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related adverse effects observed up to the highest tested dose.

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Clinical examinations
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. 3, 13, 15, 20, 22, 24 - not pregnant
• Test group 1 (20 mg/kg bw/d): females Nos. 37, 38 - not pregnant
• Test group 2 (70 mg/kg bw/d): females Nos. 62, 71, 74, 75 - not pregnant
• Test group 3 (200 mg/kg bw/d): female No. 97- not pregnant, female No. 78 - sacrificed moribund
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).

 

Tab. 3: Individual fetal external malformations

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (20 mg/kg bw/d)	42-08 M	umbilical hernia, malrotated limb
2 (70 mg/kg bw/d)	none
3 (200 mg/kg bw/d)	89-06 F	umbilical hernia

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

 

Tab. 4: Total external malformations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 211
Fetal incidence	N (%)	0.0	1 (0.5)	0.0	1 (0.5)
Litter incidence	N (%)	0.0	1 (4.3)	0.0	1 (4.3)
Affected fetuses/litter	Mean%	0.0	0.4	0.0	0.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 5: Total external unclassified observations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	1 (0.5)	0.0	0.0	0.0
Litter incidence	N (%)	1 (5.3)	0.0	0.0	0.0
Affected fetuses/litter	Mean%	0.5	0.0	0.0	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 6: Individual fetal soft tissue malformations

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	5-07 M	aortic arch atresia
	9-03 F, 9-05 F, 9-08 M	absent gallbladder
	16-11 M	cardiomegaly
	23-02 F	enlarged lens
1 (20 mg/kg bw/d)	none
2 (70 mg/kg bw/d)	72-04 F	absent gallbladder
	72-06 F	small pancreas, absent subclavian, small spleen
	72-12 F	small spleen
3 (200 mg/kg bw/d)	77-03 M	right-sided aortic arch
	80-13 D	aortic arch atresia
	81-10 F	asplenia
	93-02 F	small spleen
	96-01 F a)	heart: membranous ventricular septum defect
	99-07 F	absent subclavian
	99-11 M	multiple visceral malformations

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female; D = dead. a) fetus with additional skeletal malformations.

 

Tab. 7: Total soft tissue malformations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	6 (3.1)	0.0	3 (1.5)	7 (3.3)
Litter incidence	N (%)	4 (21)	0.0	1 (4.8)	6 (26)
Affected fetuses/litter	Mean%	3.2	0.0	1.1	3.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 8: Total soft tissue variations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	6 (3.1)	7 (3.2)	8 (3.9)	11 (5.2)
Litter incidence	N (%)	6 (32)	5 (22)	6 (29)	10 (43)
Affected fetuses/litter	Mean%	3.1	3.6	5.9	9.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

Tab. 9: Total soft tissue unclassified observations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	9 (4.7)	5 (2.3)	14 (6.8)	7 (3.3)
Litter incidence	N (%)	4 (21)	2 (8.7)	4 (19)	6 (26)
Affected fetuses/litter	Mean%	4.1	2.0	6.6	3.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 10: Individual fetal soft skeletal malformations

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	11-04 M 12-01 M	malpositioned and bipartite sternebra small interparietal
1 (20 mg/kg bw/d)	33-06 F	cleft sternum
2 (70 mg/kg bw/d)	60-03 F	fused rib
	70-01 F	severely malformed vertebral column and/or ribs
3 (200 mg/kg bw/d)	81-09 M 81-10 F 96-01 F a)	absent cervical center, small thoracic arch absent lumbar vertebra sternebrae severely fused (bony plate)

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female; D = dead

1. a) fetus with additional soft tissue malformations.

 

Tab. 11: Total skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	2 (1.0)	1 (0.5)	2 (1.0)	3 (1.4)
Litter incidence	N (%)	2 (11)	1 (4.3)	2 (9.5)	2 (8.7)
Affected fetuses/litter	Mean%	0.9	0.5	0.9	1.3

 mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 12: Total fetal skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	174 (91)	193 (89)	189 (92)	197 (93)
Litter incidence	N (%)	19 (100)	23 (100)	21 (100)	23 (100)
Affected fetuses/litter	Mean%	92.0	89.3	92.3	92.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 13: 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 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d	HCD Mean % (range)
Incomplete ossification of sternebra; unchanged cartilage	34.6	40.6	34.3	48.5*	36.6 (13.3 - 49.5)

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

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

 

Tab. 14: Total unclassified cartilage observations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	20 (10)	39 (18)	47 (23)	34 (16)
Litter incidence	N (%)	10 (53)	17 (74)	16 (76)	15 (65)
Affected fetuses/litter	Mean%	9.6	17.9*	22.1*	14.8

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

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

 

Tab. 15: Total fetal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	8 (4.2)	2 (0.9)	5 (2.4)	9 (4.2)
Litter incidence	N (%)	6 (32)	2 (8.7)	3 (14)	7 (30)
Affected fetuses/litter	Mean%	4.1	0.9	2.0	4.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 16: Total fetal variations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	175 (91)	193 (89)	189 (92)	197 (93)
Litter incidence	N (%)	19 (100)	23 (100)	21 (100)	23 (100)
Affected fetuses/litter	Mean%	92.5	89.3	92.3	92.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Applicant's summary and conclusion

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) at doses as high as 200 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 200 mg/kg bw/d.

Executive summary:

The test substance was tested for its prenatal developmental toxicity in New Zealand White rabbits. The test substance was administered as an aqueous solution to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 20, 70 and 200 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The vehicle control group, consisting of 25 females, was dosed with the vehicle (deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 19-23 females per group had implantation sites.

Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, all surviving females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

Analytics: The stability of the test substance in deionized water over a period of 7 days at room temperature was demonstrated. The correctness of the prepared concentrations was shown.

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

• Test group 3 (200 mg/kg bw/d): No test substance-related adverse effects on does, gestational parameters or fetuses.


• Test group 2 (70 mg/kg bw/d): No test substance-related adverse effects on does, gestational parameters or fetuses.


• Test group 1 (20 mg/kg bw/d): No test substance-related adverse effects on does, gestational parameters or fetuses.

Conclusion: Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) at doses as high as 200 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 200 mg/kg bw/d.