Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-04-04 - 2011-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on Jan 22, 2001
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1-Ethyl-3-methylimidazolium ethylsulphate (Lab test substance number: 06/0104-3)
- Physical state: liquid/yellowish clear
- Analytical purity: 99.5 g /100 g
- Lot/batch No.: 06/0104-3
- Stability under test conditions: the stability of the test substance under storage conditions over the test period was guaranteed.
- Storage condition of test material: room temperature, under N2, protected against humidity
- Other: the test substance was homogeneous under the testing conditions

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-12 weeks
- Housing: Makrolon cages type M III, one animal per cage
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water supplied from water bottles; ad libitum
- Acclimation period: 7 days

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

IN-LIFE DATES: From: 22 March 2011 To: 13 April 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: drinking 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 were weighed, topped up with drinking water in a calibrated beaker and intensely mixed with a magnetic stirrer until it is completely dissolved. A magnetic stirrer was also used to keep the preparations homogeneous during treatment of the animals.

VEHICLE
- Concentration in vehicle: 0, 1000, 3000 and 10000 mg/100 mL water, respectively in the 0, 100, 300 and 1000 mg/kg bw dose groups
- Amount of vehicle (if gavage): 10 mL of the aqueous preparations were applied per kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water at room temperature over a period of 7 days had been verified prior to the start of the study in a similar batch. Given that the test substance is completely miscible with water, solutions were considered to be homogenous without further analysis.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
The animals arrived on the same day (gestation day [GD] 0) at the experimental laboratory. The following day was designated as “GD 1”.
Duration of treatment / exposure:
from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approx. the same time in the morning
Frequency of treatment:
once a day
Duration of test:
On GD 20, all surviving dams were sacrificed and examined macroscopically.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
analytical conc.
No. of animals per sex per dose:
25 animals per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on available toxicological information for the substance

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occur, the animals were examined several times daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily; a check for mortality was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights was recorded on GD 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption will be recorded for GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
On GD 20, the dams were sacrificed under isoflurane anesthesia by cervical dislocation (in randomized order according to a randomization plan) 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 and distribution of implantation sites (classified as: live fetuses, dead implantations [early resorptions, late resorptions, and dead fetuses]). After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters were conducted by operators unaware of treatment group in order to minimize bias.
Ovaries and uterine content:
The following examinations will be carried out:
- Number of corpora lutea
- Number of implantations (differentiated according to live and dead fetuses and early or late resorptions)
- Early resorptions according to SALEWSKI in animals that do not appear to be pregnant and animals with single-horn pregnancy
- Site of implantations in the uterus
Fetal examinations:
After the fetuses have been removed from the uterus, the following examinations or weight determinations were carried out:

- Weight of each fetus
- Sex
- Viability of the fetuses and condition of placentae, umbilical cords, fetal membranes, and fluids
- Weight of the placentas
- Gross-pathological examination of the fetuses after dissection from the uterus (including abnormalities of the fetal membranes, placentas, amniotic fluid and umbilical cord); then all fetuses will be sacrificed by subcutaneous injection of pentobarbital (e.g. Narcoren®, dose: 0.1 ml/fetus).
- About half of the fetuses of each dam was skinned, fixed in ethyl alcohol and, after fixation, stained according to a modified method (KIMMEL and TRAMMELL ) to show the skeleton and the cartilage. After the skeletons/cartilage have been examined, these fetuses were archived individually.
- The other half of the fetuses of each dam was fixed in Harrison’s fluid. After fixation, the soft tissues of these fetuses were examined according to a modified microdissection method (BARROW and TAYLOR ). After the examination, these fetuses were discarded.
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses will be carried out:
- Food consumption, body weight, body weight change, corrected body weight gain, weight of the unopened uterus, weight of the placentas and fetuses, corpora lutea, implantations, pre and postimplantation losses, resorptions and live fetuses: DUNNETT’s test
- Number of pregnant animals at the end of the study, mortality rate (of the dams) and number of litters with fetal findings: FISHER's exact test
- Proportion of fetuses with findings per litter: WILCOXON test.
Indices:
Conception rate, preimplantation loss, postimplantation loss
Historical control data:
Historical control data were available

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: Temporary salivation occurred in the high dose group from GD7 onwards immediately after each administration, but was no longer present after cessation of treatment. This salivation was not considered to be a sign of systemic toxicity.

Details on maternal toxic effects:
I Clinical examinations of the dams
- Mortality: there were no substance-related or spontaneous mortalities in any of the groups.
- Clinical symptoms: salivation occurred from GD 7 onwards in the high dose group, and persisted in the respective females for a few minutes immediately after each administration. After cessation of treatment on GD 19, salivation was no longer observed in these rats. This temporary salivation is considered to be treatment-related. It was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.
No clinical symptoms were noted in the low-and the mid dose group (100 and 300 mg/kg bw/d).
- Food consumption: any differences between control rats and test substance-treated animals did not show a relation to dosing and were considered to be without biological relevance.
- Body weight: the mean body weights and the average body weight gain of the low-, mid- and high-dose rats (100, 300 and 1000 mg/kg bw/d) were substantially similar to the concurrent control values. The observable differences between the test substance-treated groups and the controls are without any biological relevance.
- Corrected (net) body weight gain: the corrected body weight gain revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.

II Terminal examinations of the dams
- Uterus weight: the mean gravid uterus weights of the animals of all test groups (100, 300 or 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance. Considering the fluctuations in the mean number of live fetuses/dam, they reflect the normal degree of variation for rats of the strain used in this study.
- Necropsy findings: No test substance-related or spontaneous findings occurred at necropsy in any dam.
- Reproduction data: the conception rate reached 92% in the mid dose group (300 mg/kg bw/d), 96% in the low and the high dose group (100 and 1000 mg/kg bw/d) and 100% in the control group (0 mg/kg bw/d). With these rates, a sufficient number of pregnant females were 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 or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Sex distribution of the fetuses: the sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.
- Weight of the placentae: the mean placental weights were comparable between the dosed groups (100, 300 and 1000 mg/kg bw/d) and the corresponding control. Differences observed in comparison to the control were neither statistically significant nor biologically relevant.
- Weight of the fetuses: the mean fetal weights were not influenced by the test substance and did not show any biologically relevant differences between the test substance-treated groups and the control. The observable differences between the groups reflect the usual fluctuation for this parameter. All values are well within the historical control range.
- Fetal external malformations: only one fetus with two external malformations (i.e. mandibular micrognathia in combination with aglossia) was observed in the control group (0 mg/kg bw/d). These findings were considered to be spontaneous in nature.
- Fetal external variations: no external variations were observed.
- Fetal external unclassified observations: one unclassified external observation, i.e. blood coagulum around placenta, was recorded for 3 littermates of the low and the mid dose group and was assessed as spontaneous in nature.
- Fetal soft tissue malformations: no soft tissue malformations were observed.
- Fetal soft tissue variations: 2 soft tissue variations, i.e. uni- or bilateral dilation of renal pelvis and/or ureter, were detected. These findings observed in 2 to 3 fetuses of 2 to 3 litters in test groups 0 and 1 (0 and 100 mg/kg bw/d) showed no dose-response relationship. The observable differences between the groups reflect the usual fluctuation for this parameter and were clearly within the range of the historical control data.
- Fetal soft tissue unclassified observations: no unclassified soft tissue observations were recorded.
- Fetal skeletal malformations: skeletal malformations were noted in fetuses of test groups 0, 1 and 2 (0, 100 and 300 mg/kg bw/d). Although some of these findings are not in the historical control data, each of them affected individual fetuses and neither statistically significant differences between the test groups nor a dose-response relationship were observed. The incidences of skeletal malformations were comparable to the historical control data.
- 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. The overall incidences of skeletal variations were comparable to the historical control data.
- Fetal skeletal unclassified cartilage observations: some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all groups including the controls. The observed unclassified cartilage findings were related to the ribs and the sternum and did not show any relation to dosing. The percentages of affected fetuses per litter are nearly within the overall historical control range (mean value 47.2%; range per study: 22.1 – 64.5%) and do not show any relation to dosing. Thus, a toxicological relevance for these findings is not assumed.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of 1-Ethyl-3-methylimidazolium ethylsulphate to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) elicited that the test compound did not caused substance-related, adverse effects/findings up to the highest dose group tested (1000 mg/kg bw/d).
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity is 1000 mg/kg body weight/day. No adverse maternal and fetal findings of toxicological relevance were evident at any dose.
Executive summary:

In a prenatal developmental toxicity study the test compound 1-Ethyl-3-methylimidazolium ethylsulphate was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance. No test substance-related effects on the dams concerning mortality, food consumption, body weight, body weight gain, gestational parameters, uterine and placental weights, as well as necropsy observations up to and including a dose of 1000 mg/kg bw/day, had been determined. Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights as well as no direct and specific effect on fetal morphological structures in all dose groups.