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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:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-N,N-dimethylpropanamide
EC Number:
609-066-0
Cas Number:
35123-06-9
Molecular formula:
C5H11NO2
IUPAC Name:
2-hydroxy-N,N-dimethylpropanamide
Details on test material:
- Name of test material (as cited in study report): Agnique AMD 3L
- Test substance No.: 11/0303-2
- Purity: 98.6% corr. area-% (separate report, Project No.: 11L00343)
- Batch: 0007864415
- Homogeneity: Given (vis.) (separate report, Project No.: 11L00343)
- Physical state/appearance: Liquid/yellowish, clear
- Storage Stability: Expiry date 6 Aug 2013. The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor
- Storage conditions: Ambient (RT), under light exclusion

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 146.4 - 183.2 g
- Housing: individually in Makrolon type M III cages (BECKER & CO., Castrop-Rauxel, FRG; floor area about 800 cm²) with dust free wood bedding. For enrichment, wooden gnawing blocks were offered (Typ NGM E-022, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria).
- Diet: ground Kliba maintenance diet mouse/rat "GLP", meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: Tap water ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 pm - 6.00 am dark; 6.00 am - 6.00 pm light)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the period of established stability. To prepare the dose solutions, the specific amount of test substance was weighed, topped up with drinking water in a graduated flask and intensely mixed by shaking until it was completely dissolved.

ADMINISTRATION VOLUME:
10 mL/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. Analytical verifications of the stability of the test substance in drinking water for a period of 7 days were carried out prior to the study in a similar batch (Project No.: 01Y0303/11Y027). Samples of the test substance preparations were sent to the analytical laboratory at the beginning of administration for verification of the concentrations. Given that the test substance is completely miscible with drinking water, solutions were considered to be homogenous without further analysis.
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.
Duration of treatment / exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning.
Frequency of treatment:
daily
Duration of test:
3 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 200, 500 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Mortality was checked twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20). The animals were examined at least once daily for clinical symptoms by cage-side examination for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-20). The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20. All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6). Dams were sacrificed on gestation day 20 under isoflurane anesthesia by cervical dislocation, in randomized order. After the dams had been sacrificed, they were necropsied and assessed for gross pathology. Uterus and ovaries were removed for subsequent examination.
Ovaries and uterine content:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
1) Live fetuses
2) 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)
- Conception rate and pre- and postimplantation losses were calculated.
Fetal examinations:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half were placed in Harrison’s fluid for fixation. The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded. The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.
Statistics:
- Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight: simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings: pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions.
- Proportions of fetuses with malformations, variations and/or unclassified observations in each litter: pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Indices:
- Conception rate (%): 100 x number of pregnant animals / number of fertilized animals
- Preimplantation loss (%), for each individual pregnant animal which underwent scheduled sacrifice: 100 x (number of corpora lutea - number of implantations) / number of corpora lutea
- Postimplantation loss (%), for each individual pregnant animal which underwent scheduled sacrifice: 100 x (number of implantations - number of live fetuses) / number of implantations

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
None of the dams died untimely and the clinical observations revealed no toxicologically relevant difference between the animals receiving the test substance and controls.
Terminal examinations of the dams:
- The mean gravid uterus weights of the animals of test group 1-3 (100, 200 and 500 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
- No test substance-related or spontaneous findings occurred at necropsy in any dam.
- Reproduction data: The conception rate reached 92% in the low- and mid-dose groups (100 and 200 mg/kg bw/d) and 100% in the control and the high-dose group (0 and 500 mg/kg bw/d). There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 100, 200 and 500 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

The following test substance-related, adverse effects were noted at the high dose level of 500 mg/kg bw/day:
- Reduced food consumption during treatment (10% below control)
- Reduced mean body weight gain during treatment (17% below control)
- Reduced corrected (net) body weight gain during treatment (34% below control) and net body (carcass) weight (5% below control)

No test substance-related adverse effects were observed in dams of the low (100 mg/kg bw/day) and mid (200 mg/kg bw/day) dose groups. In these groups the corrected body weight gain was slightly reduced (12% and 16% compared to controls). As this reduction was not associated with any considerable effect on gross or net weight at term it was not interpreted as adverse or toxicologically relevant.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
- The sex distribution of the fetuses in test groups 1-3 (100, 200 and 500 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
- Mean placental weights were statistically significantly reduced in the male fetuses, and as a consequence in all viable fetuses, of the high-dose group (500 mg/kg bw/day). This difference was considered to be related to the treatment.
- Mean fetal weights of test group 3 (500 mg/kg bw/d) were slightly but statistically significantly reduced (about 6% in comparison to the corresponding control value). This difference were considered to be related to the treatment. The extent of the fetal weight reduction was in the same order of magnitude as found for their mothers (5-6%). Since there were no effects on the degree of fetal maturity, the slight weight reduction is considered not to be an adverse, toxicologically relevant effect of the test substance.
External examination of the fetuses:
- Malformations: External malformations were recorded for two fetuses from two litters in the high-dose group (500 mg/kg bw/d). Both fetuses had more than one malformation affecting either the head or the abdomen. The total incidence of external malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.
- Variations: One external variation, i.e. limb hyperflexion, was recorded for one control fetus. This finding was considered to be spontaneous in nature.
- Unclassified observations: No unclassified external observations were recorded.
Soft tissue examination of the fetuses:
- Malformations: Soft tissue malformations were observed in one control and two high-dose litters (0 and 500 mg/kg bw/d). Each of the individual malformations can be found in the historical control data at a comparable frequency. Furthermore, the overall incidences of soft tissue malformations were comparable to those found in the historical control data.
- Variations: Two soft tissue variations, i.e. dilated renal pelvis and dilated ureter, were detected in all test groups including the controls. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups and therefore, considered not to be biologically relevant.
-Unclassified observations: No unclassified soft tissue observations were recorded.
Skeletal examination of the fetuses:
- Malformations: Skeletal malformations were noted in single fetuses of test groups 1, 2 and 3 (100, 200 and 500 mg/kg bw/d). These findings affected individual fetuses in one low dose litter and 2 mid- and high-dose litters each. Each of the individual malformations can be found in the historical control data at a comparable or higher frequency. Furthermore, the overall incidences of skeletal malformations were comparable to those found in the historical control data.
- 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 skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.
-Unclassified cartilage observations: Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the sternum. Although the incidence of bipartite processus xiphoideus was statistically significantly higher in the mid-dose group, it was well within the historical control range. Because of this finding the overall incidence for skeletal unclassified cartilage observations in the mid-dose group is statistically significantly higher, but within the historical control range. Thus, an association to the test substance and a toxicological relevance is not assumed.

An assessment of all fetal external, soft tissue and skeletal observations is given under 'Any other information on results incl. tables.'

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: developmental

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in all groups. When the different findings were combined, the following can be concluded:

Two high dose fetuses from different litters were multiple-malformed. Female fetus No. 82-05 had a gastroschisis, associated with a situs inversus, while the findings in male fetus No. 94-11 consisted of agnathia (comprising severely malformed skull bones), open eyes and astomia. 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 the high-dose group. Apart from gastroschisis all of them are present in the historical control data of the rat strain. Thus a relationship of these two cases of malformed fetuses to the treatment is not assumed.

Other malformations, such as diaphragmatic hernia, abnormal lung lobation, malpositioned and bipartite sternebrae, misshapen tuberositas deltoidea and misshapen basisphenoid, observed in all groups including controls are common for this rat strain and can be found in the historical control data at a comparable or higher frequency. An association of these findings to the treatment is also not assumed.

One external variation, two soft tissue variations and a broad range of skeletal variations occurred in all test groups including the controls. None of the incidences showed a relation to dosing and all variations can be found in the historical control data at a comparable or higher frequency.

A spontaneous origin is also assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3 (0, 100, 200 and 500 mg/kg bw/day). The distribution and type of these findings do not suggest any relation to treatment.

Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to the highest dose tested (500 mg/kg bw/day).

Applicant's summary and conclusion

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