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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
The stability of the test item in the vehicle did not meet the acceptance criteria defined in the RCC-SOP (± 10% of the mean value of the respective homogeneity sample). The test item was not stable in the vehicle at room temperature for seven days. The 7-day stabilities were found to be between 51% and 92% of the respective nominal formulation concentrations. Therefore, the dose levels were re-calculated using the respective analytical results assuming that at least this minimum dose had been daily administered. The results obtained in this study are considered to correspond to this re-calculated doses. The study is considered to be valid.
Deviations:
yes
Remarks:
see remarks
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(1,3-benzodioxol-5-ylamino)ethanol hydrochloride
EC Number:
303-085-5
EC Name:
2-(1,3-benzodioxol-5-ylamino)ethanol hydrochloride
Cas Number:
94158-14-2
Molecular formula:
C9H11NO3.ClH
IUPAC Name:
2-(1,3-benzodioxol-5-ylamino)ethanol hydrochloride
Test material form:
solid: particulate/powder
Details on test material:
beige, cryst. powder

Test animals

Species:
rat
Strain:
other: HanBrl: WIST (SPF Quality)
Details on test animals or test system and environmental conditions:
Acclimatization: Seven days (minimum) prior to pairing under test conditions with an evaluation ol the health status.
Age at pairing: 10 weeks
Conditions: Animals were housed under standard Iabaratory conditions: air-conditioned with 10-15 air changes per hour; the environment monitared continuously with recordings of temperature (target range 22 ± 5°C) and relative humidity (target range 30- 70%), 12 hours artificial fluorescent light I 12 hours dark with background music played at a centrally defined low volume for at least 8 hours du ring the light period.
Accommodation: Animals were housed individually in Makroion cages (type-3) with wire mesh tops and standardized granulated softwood bedding.
Diet: Pelleted standard Kliba-Nafag 3433 rat/mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland) was available ad libitum (Batch Nos. 42/04, 53/04).
Water: Community tap water from Füllinsdorf in botltes was available ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Bi-distilled water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampies for determination of concentration, homogeneity and stability (7 days) of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentration and homogeneity were taken during the last week of the administration period. On each occasion three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. Stability samples were taken from the middle only. The samples were frozen (-25°C to -15°C) pending analysis. Sampies were sent on dry ice to Dr. D. Flade, RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 ltingen I Switzerland. Analyses were performed using a method provided by the Sponsor (HPLC). The test item was used as analytical standard.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually, if:
a) the daily vaginal smear was sperm positive. Or
b) a copulation plug was observed.
The day of mating was designated day 0 post coitum. Male rats of the same source and strain were used for mating only. These male rats were in the possession of RCC, and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored.
Frequency of treatment:
once daily in the morning from day 6 through to day 20 post coitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
control group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
group 2: corresponds to an achieved minimal dose Ievel of 26 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
group 3: corresponds to an achieved minimal dose Ievel of 152 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
group 4: corresponds to an achieved minimal dose Ievel of 688 mg/kg bw/day
reduced dose level (starting level: 1000 mg/kg bw/day (nominal)) from day 10 to end
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
corresponds to an achieved minimal dose Ievel of 702 mg/kg bw/day
starting level, reduced to 750 mg/kg bw/day (nominal)) from day 10 to end due to an occurrence of overt toxicity and mortalities
No. of animals per sex per dose:
22 females
Control animals:
yes

Examinations

Maternal examinations:
All animals were checked at least twice daily for any mortalities. Any female sacrificed or found dead during the study was subjected to macroscopic examination with ernphasis on the uterus and its contents. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. All animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health.Food consumption was recorded on 3-day intervals: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum. Body weights were recorded daily from day 0 until day 21 post coitum.
Ovaries and uterine content:
Post mortem examination, including gross macroscopic examination of all internal organs, with emphasis on the uterus, uterine contents, positin of fetuses in the uterus and number of corpora lutea, was performed and the data recorded. The uteri (and contents) of all Iernaies with live fetuses were weighed at necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
Fetal examinations:
Fetuses were. removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, killed by a subcutaneous injection of sodium pentobarbital (Vetanarcol®) and allocated to one of the following procedures: 1) Microdissection technique (sectioning/dissection technique). At least one half of the fetuses from each litter were fixed in Bouin's fixalive (one fetus per container). They were examined by a
combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination the tissue was preserved in a solution of glycerine/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded. The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The Skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually in plastic bags. lf no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites. When considered appropriate, macroscopic changes in the dams were photographed and samples of tissue fixed in neutral phosphate buffered 4% formaldehyde solution for possible microscopic examination. Fetuses with abnormalities were photographed, when considered appropriate.
Statistics:
The following statistical methods were used to analyse body weights, food consumption, reproduction and skeletal examination data:
- Means and standard deviations of various data were calculated and included in the report.
- lf the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
- The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Tests were performed on the 5% significance Ievel.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Any of the following signs indicating overt toxicity were noted before the deaths: Occurrence of ruffled fur, hypoactivity, hunched posture, lacrimation, ventral recumbency, or hardened abdomen. ln all animals from group 4, occurrence of slight sedation was noted from the beginning to end of the dosing period. After the daily applications animals pushed their heads through the bedding which was considered to be indicative of discomfort. ln all cages, yellowish discoloration of the bedding was observed. ln group 3, pushing of heads through the bedding was also noted, but only during the secend half of the treatment period. On days 18-21 p.c., animal no. 61 presented ruffled for and animal no. 57 alopecia at the chest wall. Neither clinical signs nor behavioral changes were noted in any animal from group 2.
Mortality:
mortality observed, treatment-related
Description (incidence):
ln group 4, the following animals died or were sacrificed in moribund condition: Nos. 69 (day 13 p.c.), 72 (day 20 p.c.), 73 (day 11 p.c.), 74 (day 14 p.c.), 77, (day 15 p.c.), 79 (day 16 p.c.), 83 (day 19 p.c.), and 88 (day 15 p.c.).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During treatment period, the body weight development of animals in group 4 was markedly depressed. The mean weight gain (from day 6 p.c. to day 21 p.c.) was only 2%, compared to 44% in the control group. The corrected body weight gain (corrected for uterus weight) was decreased in group 3 (gain of 6%) and in group 4 (loss of 16%), compared to the control group value (gain of 11 %). Body weight development and the corrected niean body weight gain of group 2 animals was not influenced by treatment with the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During treatment period, overall food consumption was markedly reduced in group 4 (-56%, only dams with live fetuses considered) and slightly reduced in group 3 (-10%), compared to the control group's values.
The food consumption of group 2 animals was similar to that of the control group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At necropsy, all dams from group 4 (including the prematurely deceased animals) presented severe signs of test item-related effects, of which those on the stomach were most striking: Stomach wall having multiple foci with or without perforations, sometimes with bloody contents in the stomach. ln addition, enlarged adrenals, small or enlarged spleens, grayish kidneys with or without multiple foci were seen. Some of the prematurely deceased animals showed also signs of putrefaction. There was one dam (no. 61) presenting test item-related findings in group 3: Enlarged adrenals, liver with spots, small spleen, bloody contents in the stomach and the stomach wall with multiple foci. There were no test item-related findings in group 2.

Maternal developmental toxicity

Details on maternal toxic effects:
Only dams with live fetuses were used for calculation of reproduction parameters. Of 22 animals mated in each group, 22, 21, and 21 dams were pregnant in dose groups 1, 2, and 3, respectively. ln group 4, only 13 dams of which 12 were pregnant survived to scheduled necropsy dates. Of these, 9 had live fetuses. Dam nos. 68, 78, and 81 had total resorptions. ln group 4, the post Implantation loss was increased, due to fetal resorptions (8.5% vs. 1% in the control group). Therefore, the number of fetuses pertotal Implantation sites was lower (83%) than in the control group (93%). All relevant reproduction parameters (post Implantation loss, embryonie/fetal resorptions, number of fetuses per dam) calculated for group 2 and group 3 dams were similar to those of the control group animals.

Effect levels (maternal animals)

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

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Markedly lower fetal body weights were measured in group 4. Statistically significantly mean weights were calculated for male and for female fetuses both on an individual basis (2.3 g vs. 4.7 g in the control group) and on a litter basis (2.2 g vs. 4.8 g in the control group). ln group 3, mean fetal weights were also lower when compared with the values of the control group. On a litter basis, the mean value was 4.6 g compared to 4.8 g in the control group. On an individual basis, the value was 4.6 g compared to 4.7 g in the control group (statistically significant). Individual weights of the fetuses from litter no. 61 were between 2.4 g and 3.1 g. The fetal body weights in group 2 were not influenced by treatment with the test item.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
At all dose Ievels, the treatment had no influence on the sex ratio values.
External malformations:
no effects observed
Description (incidence and severity):
External exarnination at Caesarean necropsy did not reveal any findings which were related to the treatment with the test item. ln group 2, occurrence of thread-like tail (fetus no. 212 from litter no. 28) and shortened
lower jaw (fetus no. 431 from litter no. 36) was noted. ln the absence of dose-relationship these isolated occurrences were considered to be incidental.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
SKELETAL EXAMINATION OF FETUSES (ABNORMALITIES AND VARIANTS).
During skeletal examination abnormal findings were noted in:
2 of 132 examined fetuses (2 of 22 litters) of group 1
3 of 113 examined fetuses (3 of 20 litters) of group 2
7 of 128 examined fetuses (4 of 21 litters) of group 3
31 of 46 examined fetuses (8 of 9 litters) of group 4.
ln group 4, increased incidences of a multitude of test item-related findings occurred, which, ln its entirety, were considered to reflect mal-development (retardations and/or disturbances) of the fetuses: Uni- and /or bilateral fused zygomatic processes of jugal and maxilla; bipartite occipital and supraoccipital bones; bent scapula, humerus and radius; bipartite sternebra bones; unossified or absent thoracic and/or lumbar vertebral bodies; bipartite thoracic and lumbar vertebral bodies; dumbbell-shaped thoracic, lumbar, and sacral vertebral bodies; extra 15th and 16th ribs, fused ribs; pelvic girdle displacements. In group 3, one fetus (no. 855, litter 65) showed the following findings which were considered to be possibly related to the treatment with the test item: Fusion of cervical vertebral bodies 1 and 2 with the exoccipital bone and occurrence of a bilateral bone connection between the basisphenoid bone and the tympanic rings. There were no findings in group 2 which were considered to be related to the treatment with the test item. One fetus (no. 212, litter 28, group 2) had a shortened vertebral column (thread-like tail was seen at external examination). As isolated individual finding which occurred in the low dose only it was considered incidental. Other findings in groups 2, 3, and 4 were of isolated incidental occurrence and/or common findings known to occur in this strain of rat and covered by the range of historical reference data.

SKELETAL EXAMINATION OF FETUSES (STAGE OF DEVELOPMENT)
ln group 4, markedly increased incidences of non-ossified and incompletely ossified bones concerning the cranium, vertebra, ribs, sternebra, as weil as extremities, both on an individual and litter based evaluation, occurred. ln addition, increased occurrence of supernumerary ribs was noted. ln group 3, numbers of non-ossified cervical vertebral bodies 3 and 4 were significantly increased on an individual basis. There were no findings in group 2 which were considered to be related to the treatment with the test item.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Du ring visceral examination of fixed fetuses findings were noted in:
63 of 139 examined fetuses (22 of 221itters) of group 1
70 of 123 examined fetuses (18 of20 litters) of group 2
68 of 135 examined fetuses (21 of 21 litters) of group 3
49 of 51 examined fetuses ( 9 of 9 litters) of group 4.
Compared to the control group, distinctly increased incidences of the following test item-related findings occurred in group 4: Haemorrhages (subcutaneous, intrathoracic or intraabdominal), dilated lateral brain ventricles, thymus cranial displacement and/or elongation, thymus reduced in size, thyroid enlargement/discoloration, trachea narrowed, heart abnormalities (misshapen, enlarged, ventricular chamber enlarged, atrium distended), aorta and pulmonary trunk abnormalities (displacement, dilation or narrowness), lungs not inflated and abnormal lobulation, thinning of tendinusou/muscular region of the diaphragm and diaphragm hernia, bilateral azygous vein, cranial testis displacement, absence of innominate artery.
ln group 3, the following findings were observed:
One fetus in group 3 (number 713, litter 64) had multiple malformations of the heart (bulbous and malrotated, interventricular septal defect), and accompanying/additional findings in aortic arch and pulmonary trunk (displacements, enlargements) and lungs (not inflated, abnormal lobulation). As these signs were also observed in fetuses of group 4, it cannot be excluded that they were test item-related. There were no other findings which were considered to be related to the treatment with the test item. The occurrence of locally thinned tendinous region of the diaphragm was found in 8 fetuses (5 litters). This condition was also found in similar incidence in the control group (4 fetuses in 4 litters) and was therefore considered to be not related to the treatment with the test item.
ln group 2, the following findings were observed:
There were no findings which were considered to be related to the treatment with the test item. One fetus in group 2 (no. 431, litter 36) had agnathia, microglossia, misshapen palate, and an interrupted nasopharynx. As this type of multiple finding was of isolated occurrence in the low dose group only and it is known to occur commonly at low incidence in this strain of rat it was considered to be incidental. The tendinous region of the diaphragm was locally thinned in 10 fetuses (7 litters). This condition was also found in the control group (4 fetuses in 4 litters) and in dose group 3 (8 fetuses in 5 litters). As there was no increase in incidence
proportional to dose, as the numbers of affected fetuses were low and the numbers of affected litters were similar, this finding was considered to be unrelated to the treatment with the test item.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
During cartilage examination findings were noted in:
0 of 132 examined fetuses (0 of 22 litters) of group 1
0 of 113 examined fetuses (0 of 20 litters) of group 2
5 of 128 examined fetuses (4 of 21 litters) of group 3
16 of 46 examined fetuses (5 of 9 litters) of group 4.
ln group 4, increased incidences of the following findings which were considered related to the treatment with the test item were noted: Dumbbell-shaped or split cartilage of cervical vertebra; fused cartilage of cervical vertebra; dumbbell-shaped cartilage of thoracic vertebra; short, off-set, fused, or branched costal cartilage; uni- or bilaterally costal cartilage connected to sternum; split cartilage of sternebra; cervical and thoracic ribs fused; incidences of Ieng as weil as interrupted costal cartilage 11 were increased on an individual and on a litter basis; increased incidences of supernumerary costal cartilages. ln group 3, the following findings which were considered related to the treatment with the test item were noted: Absent, dumbbell-shaped or split cartilage of cervical vertebra; off-set, fused, or branched costal cartilage; costal cartilage connected to sternum. There were no findings in group 2 which were considered to be related to the treatment with the test item. lncidences of interrupted costal cartilages 11 in groups 2 and 3 were within the range of the historical reference data and therefore considered to reflect normal variability.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
26 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
visceral malformations

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
152 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Applicant's summary and conclusion

Conclusions:
In this prenatal developmental toxicity study in rats, severe effects were noted on reproduction and on the development of the fetuses at the high dose group (702/688 mg/kg body weight). At this dose Ievel, the maximum tolerated dose Ievel was clearly exceeded as it led to pronounced maternal toxicity and several cases of death. The effects at this dose Ievel were therefore considered to be due to the high maternal toxicity. Similar, but less pronounced maternal and embryo-fetal toxicity were noted at the mid dose Ievel (152 mg/kg body weight). No test-item related effects were observed at the low dose Ievel (26 mg/kg body weight). Therefore, a No-Observable-Adverse-Effect Level (NOAEL) for maternal and for embryo-fetal effects was established at the low dose Ievel of 26 mg/kg body weight.
Executive summary:

The purpese of this study was to assess the effects of Hydroxyethyl-3,4-methylenedioxyaniline HCl on pregnant females and on embryonie and fetal development when administered orally, by gavage once daily to mated female rats from day 6 (implantation) through to day 20 post coitum (one day prior to scheduled Caesarean section, inclusive). Each group consisted of 22 mated female rats. Hydroxyethyl-3,4-methylene-dioxyaniline HCl was administered once daily at nominal dose levels of:

Group 1: 0 mg/kg body weightlday (vehicle control)

Group 2: 50 mg/kg body weightlday

Group 3: 250 mg/kg body weight/day

Group 4: 1000/750 mg/kg body weight/day *

* ln agreement with the Sponsor, the high dose (1 000 mg/kg body weight) was reduced to 750 mg/kg body weight/day, effective as on November 03, 2004, due to occurrence of overt toxicity and mortalities at the 1000 mg/kg/day dose level.

These dose levels of this study had been selected based on a former teratology study with the test item in rats (REPROTOX GmbH, Order no. 681, 1981) where doses of 0, 500 and 1000 mg/kg body weight were applied, suspended in a different vehicle (gum arabic). As no toxic effects had been observed in that study, an additional range-finding study was not performed. In this study with water as vehicle, overt toxic effects occurred at the 1000 mg/kg body weight (nominal) dose level and still after decrease to 750 mg/kg body weight. It is considered that the cause for this different outcome at the same dose level was the difference in the vehicles. Due to occurrence of overt toxicity and mortalities at the 1000 mg/kg/day dose level, the highdose was reduced to 750 mg/kg body weight/day, effective as of November 3, 2004 (corresponding to individual treatment days between 9 and 15 post coitum, respectively). Using a conservative approach by taking into account the stability data of the analytical examinations, minimal dose levels of 26, 152, and 702/688 mg/kg body weight were achieved in dose groups 2, 3, and 4, respectively. A standard dose volume of 10 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bi-distilled water). All females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section. Examinalien of dams and fetuses was performed in accordance with international recommendations. Treatment of dams at 702/688 mg/kg body weight caused occurrence of overt toxicity, poor general development and even death. The maximum tolerated dose level was clearly exceeded. At necropsy severe gastric effects were noted in all animals of the high dose group and in addition in one animal treated at 152 mg/kg body weight. in the high dose group, the majority of all severe effects on reproduction and fetal weights and of findings at visceral, skeletal and cartilage examinations is therefore considered to be consequent to the high matemal toxicity leading to mal-development of fetuses during the gestational period. At the 152 mg/kg body weight dose level, decreased food consumption of dams was noted. Fetal weights were found to be decreased. In addition, some of the visceral, skeletal or cartilage effects which occurred in the high dose group occurred in increased incidences at this dose level. At the 26 mg/kg body weight dose level, no findings were noted which were considered related to treatment with the test item.