1-Ethyl-3-methylimidazolium benzoate

Endpoint summary

Description of key information

In vivo:

In an in vivo dermal sensitization study (LLNA) according to OECD Guideline 429 (NOTOX, 2004), 1H-imidazolium, 3-ethyl-1-methyl-, ethyl sulfate (1:1) did not show skin sensitising properties.

In an in vivo dermal sensitization study (LLNA) similar to OECD Guideline 429 (Gildea et al., 2006), benzoic acid did not show skin sensitising properties.

In vitro:

In an integrated testing strategy (ITS), covering three key events in skin sensitisation, the following results for benzoic acid were obtained: Sensitising properties in an in chemico direct peptide reactivity assay (DPRA) similar to OECD Guideline 442C (Natsch et al., 2013), but no skin sensitising properties in an in vitro ARE-Nrf2 Luciferase Test method (KeratinoSens) similar to OECD Guideline 442D (Natsch et al., 2013), and an in vitro human Cell Line Activation assay (h-CLAT) similar to OECD Guideline 442E (Nukada et al., 2012).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004-10-25 to 2004-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

Deviations:

yes

Remarks:

-temperary fluctuations of relative humidity above level of 70 %. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 99/484
- Expiration date of the batch: 2005-06-10

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark.
- Solubility and stability of the test substance in the vehicle: Dimethyl formamide, at least 96 h

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: When required, the test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.

Species:

mouse

Strain:

CBA

Remarks:

inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: ~ 10 weeks
- Weight at study initiation: ± 20 % of the sex mean
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Housing: individually, Macrolon cages (type I)
- Diet : ad libitum, standard pelleted laboratory animal diet (code VRF 1, Altromin, Lage, Germany)
- Water: ad libitum, tap-water
- Acclimation period: at least 5 days
- Preparation of test formulations: the test substance formulations (wlw) were prepared within 4 hours prior to each treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3
- Humidity (%): 41 - 75
- Air changes (per hr): ~ 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Remarks:

Selected based on trial formulations performed at NOTOX.

Concentration:

25 %, 50 % in vehicle and 100 % (undiluted)

No. of animals per dose:

5 females

Details on study design:

RANGE FINDING TESTS:
- Concentrations: 50 %, 100 % (undiluted)
- No of animals: 1 per dose (total of 2)
- Age of animals: 5 - 14 weeks
- Duration of treatment: Each animal was treated with one concentration on three consecutive days.
- Procedure: Approximately 4 hours after the last exposure, the ear was cleaned of residual test substance with water and the irritation was assessed. Test system, procedure and techniques were identical to those used during days 1 to 3 of the main study unless where specified. Bodyweights were determined on day 3. No necropsy was performed after termination.

MAIN STUDY
INDUCTION
- Frequency of application: once daily for a total of 3 days (days 1, 2 and 3)
- Site of application: The dorsum surface of both ears
- Route of application: epidermal
- Volume applied: 25 µL/ear
- Concentrations: 25 %, 50 % in vehicle and 100 % (undiluted)

Injection of 3H-methyl thymidine (Amersham Pharmacia Biotech, NOTOX Substance 105624)
- Day of injection: 3 days after last treatment (day 6)
- Site of injection: tail vein
- Vehicle: PBS
- Volume injected: 0.25 mL
- Specific radioactivity: 20 µCi/injection

SACRIFICE
- Time schedule: 5 hours after injection of 3H-methyl thymidine
- Method: injection of phenobarbital

TISSUE PROCESSING AND MEASUREMENTS
- Lymph node processed: auricular lymph nodes
- Pooling of lymph nodes: yes
- Measurement of radioactivity: Packard scintillation counter (1900TR) (Counting time was to a statistical precision of ± 0.2 % or a maximum of 5 minutes whichever comes first)

OTHER
The test animals were checked twice daily for mortality/viability and at least once a day for toxicity signs. Bodyweight of the test animals was taken on day 1, prior to treatment and on day 6. On day 3 (3 - 4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded following the Draize numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

The SI values calculated for the substance concentrations 5,10 and 25 % were 1.0, 3.2, and 7.1 respectively. An EC3 value of 9.5 % was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20 %. The results of the 6 monthly HCA reliability checks of the recent years were 8.8, 5.5, 7.3 and 10.3 %.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

25 %

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

50 %

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

100 %

Parameter:

other: DPM/animal

Value:

411

Test group / Remarks:

25 %

Parameter:

other: DPM/animal

Value:

385

Test group / Remarks:

50 %

Parameter:

other: DPM/animal

Value:

243

Test group / Remarks:

100 %

Group	Induction	Mean DPM ± S.D.	SI ± S.D.
1	Vehicle Control (Dimethyl Formamide)	373 ± 48	1.0
2	25 % Test Substance	411 ± 188	1.1 ± 0.5
3	50 % Test Substance	385 ± 105	1.0 ± 0.3
4	100 % Test Substance	243 ± 137	0.7 ± 0.6

 

OBSERVATIONS (MAIN STUDY)

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study. The majority of the nodes of the experimental and control groups were considered normal in size (visual inspection). The nodes of two animals treated at 25 and 100 % were decreased in size. No other macroscopic abnormalities of the nodes were noted. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100 % were 411, 385, and 243 respectively. The mean DPM/animal value for the vehicle control group was 373 (NOTOX Project 418534). The SI values calculated for the substance concentrations 25, 50, and 100 % were 1.1,1.0, and 0.7 respectively. There was no indication that the test substance could elicit an SI > 3.

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015-02-04

GLP compliance:

not specified

Remarks:

scientific publication

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

in vitro/in chemico non-animal integrated testing strategy (ITS)

Details on the study design:

The principle of the test is to measure the reactivity of the test substance with lysine- or cysteine containing model hepta peptids. Purified peptides (> 90 %, HPLC) were prepared by SynPep Corporation (Dublin, CA, USA).
Peptide reactivity is based on the depletion of non-reacted peptide. The concentration of non-reacted peptide in the sample was compared to the non-reacted peptide concentration in the untreated control.
Preparation: 400 µL of a 1.25 mM peptide stock solution prepared in buffer and a 100 mM test chemical stock solution prepared in either acetonitrile or DMSO/acetonitrile were added to 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide or 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide. The final reaction, containing 0.5 mM of the peptide and 5 or 25 mM of the test chemical, representing 1:10 and 1:50 M ratios, was mixed and incubated in the dark for 24 h at 25 °C. Control samples and standards used for defining the calibration curve for each analysis were prepared without test chemical for each peptide and ranged from 0.0156 to 1.0 mM. All samples were prepared in triplicate. Following incubation, the peptide was quantified by reverse-phase HPLC (Waters 2695 Alliance) on a Zorbax SB-C18 column (3.5 µm, 100 x 2.1 mm) with UV detection at 220 nm (Waters 996 PDA detector) using an external standard linear calibration curve. The UV spectrum was collected from 210 to 400 nm to permit verification of the peptide peak identity.

Key result

Parameter:

other: Lysine [% peptide remaining]

Value:

31.9

Parameter:

other: Lysine [% peptide depleted]

Value:

68.1

Key result

Parameter:

other: Cysteine [% peptide remaining]

Value:

98.9

Parameter:

other: Cysteine [% peptide depleted]

Value:

1.1

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 442E (in vitro skin senssitisation: human Cell Line Activation, h-CLAT)

Version / remarks:

2016-07-26

GLP compliance:

not specified

Remarks:

scientific publication

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

in vitro/in chemico non-animal testing battery

Details on the study design:

In the h-CLAT assay, THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were used as surrogate for dermal dendritic cells. For dose finding, cytotoxicity tests were conducted and the concentration resulting in 75 % cell viability, termed CV75, was calculated based on the analysis of viable cells. THP-1 cells were treated with eight different concentrations, chosen based on dose finding cytotoxicity test, for 24 h. After removing the test substance, the expression of CD86 and CD54 on the cell surface was measured by flow cytometry. The relative fluorescence intensity (RFI) was used as an indicator of CD86 and CD54 expression. If the RFI of CD86 or CD54 was greater than 150 % or 200 % at any dose in at least two out of three experiments, the substance was judged as a sensitizer. Otherwise, it was considered a non-sensitizer (Ashikaga et al., 2006). From the dose-dependency curves of three experiments, the median concentration inducing 150 % of CD86 RFI and/or 200 % of CD54 RFI (EC150 or EC200) was calculated like EC3 value determination in the LLNA. The lower EC value was defined as minimal induction threshold (MIT) (Nukada et al., 2012).

Key result

Parameter:

other: CV75 [µM]

Value:

8 188.67

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2015-02-05

GLP compliance:

not specified

Remarks:

scientific publication

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

in vitro/in chemico non-animal testing battery

Details on the study design:

The standard operating procedure described (Natsch et al., 2011) and published online (ECVAM, 2014) was used to test additional substances in the KeratinoSens™ assay. Briefly, cells were grown for 24 h in 96-well plates. The medium was then replaced with medium containing the test substance and a final level of 1 % of the solvent DMSO. Each test substance was subsequently tested at 12 twofold dilutions (0.98 – 2000 µM). In each repetition, three parallel replicate plates were run for luciferase determination and a fourth parallel plate was prepared for cytotoxicity determination. Cells were incubated for 48 h with the test substances, and then luciferase activity and cytotoxicity (with the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) assay (Mosmann, 1983)) were determined. For each chemical the EC1.5, EC2 and EC3 values (concentration in µM for 1.5, 2 and 3- fold induction of the luciferase activity) were calculated along with IC50 values for the concentration yielding 50 % reduction in cellular viability. Substances were tested in at least two independent experiments. A substance is considered to have a sensitizing potential if an induction equal to or exceeding 1.5-fold compared to the vehicle control is observed at a concentration below 1000 µM and at which cells remain > 70 % viable. If the results of the two experiments were concordant, a prediction according to the prediction model was derived. Substances with discordant results or results close to the 1.5-fold threshold (borderline) were tested in additional independent experiments.

Key result

Parameter:

other: EC1.5 [µM]

Value:

2 000

Remarks on result:

no indication of skin sensitisation

Based on the OECD Guideline 442 D (adopted 2015-02-04) (OECD, Section 4, 2015), a KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:

1. the Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test)

2. the cellular viability is higher than (>) 70 % at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)

3. the EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)

4. there is an apparent overall dose-response for luciferase induction (or a biphasic response as mentioned under paragraph 33, OECD Guideline 442 D)

Interpretation of results:

GHS criteria not met

Reference 5

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010-07-22

GLP compliance:

not specified

Remarks:

scientific publication

Type of study:

mouse local lymph node assay (LLNA)

Key result

Parameter:

EC3

Value:

> 20

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No data on the skin sensitising properties of 1H-imidazolium, 3-ethyl-1-methyl-, benzoate are available. However, the test substance is an ionic liquid and dissolves in water to its ionic components benzoate and 3-ethyl-1 -methyl-Imidazolium. There are studies available which evaluate the skin sensitising properties of these single ionic components. Consequently, the skin sensitising properties of 1H-imidazolium, 3-ethyl-1-methyl-, benzoate were determined in an WoE-approach, taking into account the results of the single ionic components.

In an in vivo dermal sensitization study (LLNA) according to OECD Guideline 429 (NOTOX, 2004), ca.10 week old female CBA mice (5/dose) were tested at doses of 25 %, 50 %, (in dimethylformamide) and 100 % (undiluted) of 1H-imidazolium, 3-ethyl-1-methyl-, ethyl sulfate (1:1). Induction was performed by application of 25 µL test substance at the dorsum surface of both ears once daily for a total of 3 days. No mortality occurred and no symptoms of systemic toxicity were observed. To assess the lymph node swelling, 3H-methyl thymidine (20 µCi, 0.25 mL in PBS) was injected into the tail vein, 3 days after the last application. The mice were sacrificed 5 h after the injection. The results with the positive control substance hexyl cinnamic aldehyde (5, 10, and 25 %, in acetone:olive oil (4:1)) were valid. No abnormalities of the lymph nodes were observed. The calculated SI values for 25, 50 and 100 % were 1.1, 1.0, and 0.7 respectively. There was no indication that the test substance could elicit an SI > 3.

In a publication, the assessment capabilities of non-animal integrated testing strategies (ITS) for human skin sensitisation were compared with the benchmark test LLNA (Urbisch et al., 2015). Therefore published data inter alia of benzoic acid were evaluated. In an in vivo dermal sensitisation study (LLNA) similar to OECD Guideline 429 (Gildea et al. 2006), benzoic acid showed no skin sensitising properties.

This is supported by an in vitro test battery: Within the ITS, three key events for skin sensitisation were determined: 1.    Protein/peptide reactivity of the test substance, covered by the direct peptide reaction assay (DPRA). 2.    Keratinocyte activation, covered by the KeratinoSens, luciferase assay. 3.    Dendritic cell activation, covered by human Cell Line Activation assay (h-CLAT).

In an in chemico DPRA similar to OECD Guideline 442C (Natsch et al., 2013), benzoic acid showed direct peptide reactivity. In an in vitro KeratinoSens, luciferase assay similar to OECD Guideline 442 D (Natsch et al., 2013), benzoic acid did not induce keratinocyte activation. In an in vitro h-CLAT similar to OECD Guideline 442E (Nukada et al., 2012), benzoic acid did not induce immune-cell activation.

In addition to the experimental results OECD QSAR Toolbox v.3.2 data are available for benzoic acid. Neither the OASIS algorithms - Protein binding alerts, nor the OECD algorithms - Protein binding alerts did produce alerts for possible protein binding properties.

The evaluation of the WoE indicated that benzoic acid does not show skin sensitising properties, based on the result of the LLNA, supported by the results of the higher tier ITS assays (2. and 3.) and the result of the QSAR calculation.

Conclusion: both ionic components of H-imidazolium, 3-ethyl-1-methyl-, benzoate did not show skin sensitising properties. As a result of the read-across, H-imidazolium, 3-ethyl-1-methyl-, benzoate is not considered to have skin sensitising properties.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

As a result the substance is not considered to be classified as skin sensitising under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.