1H-Imidazole, hydrobromide (1:1)

Endpoint summary

Description of key information

Skin sensitisation: not sensitising (OECD 429; GLP compliant)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation, other

Remarks:

in vivo (LLNA)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2004-12-01 to 2004-12-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read-across from imidazole hydrobromide to imidazole hydroiodide is justified since both substances only differ in the respective counterion iodide or bromide. Both anions iodide and bromide are quite similar concerning toxicological behaviour and thus do not influence the toxicological results received.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002-04-24

Deviations:

yes

Remarks:

justification for vehicle is missing

GLP compliance:

yes (incl. QA statement)

Remarks:

signed May 2002

Type of study:

mouse local lymph node assay (LLNA)

Justification for non-LLNA method:

not applicable

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (20 °C ± 5 °C), protected from light
- Stability of test item: stable under storage conditions

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst/ The Netherlands
- Females nulliparous and non-pregnant: yes
- Age: 8 - 12 weeks (beginning of acclimatization)
- Weight: 16 g - 24 g
- Housing: individual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG)
- Diet (ad libitum): pelleted standard Kliba 3433 (batch no. 42/04 mouse maintenance diet (Provimi Kliba AG)
- Water (ad libitum): coomunity tap water from Füllinsdorf (Switzerland)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 30 - 70 %
- Air changes: 10 - 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: ethanol/water (7/3, v/v)

Concentration:

10 % (w/v), 25 % (w/v), and 50 % (w/v) of the test item

No. of animals per dose:

4 female mice

Details on study design:

TREATMENT PREPARATION.
- test item was placed into a volumetric flask on a tared Mettler balance and the vehicle was quantitatively added.
- weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer.
- test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears.
- homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.

PRE-SCREEN TESTS:
In a non-GLP solubility pre-test, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v) and ethanol/water (7/3, v/v). A suitable vehicle (ethanol/water (7/3, v/v)) was
selected and used in the main test.

In a non-GLP conform pre-test in two female mice, test item concentrations of 5 %, 10 %, 25 % and 50 % (w/v) were tested on one ear each. After 24 hours, no irritation effects were observed at these concentrations after a single application. 50 % (w/v) was the highest technically applicable concentration in the chosen vehicle.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay

Each test group of mice was treated by topical application to the dorsal surface of each ear lobe with different test item concentrations of 10 %, 25 % and 50 % (w/v) in the vehicle. The application volume, 25 μL, was spread over the entire dorsal surface (diameter: approx. 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Five days after the first topical application, all mice were administered with 250 μL of 78.42 μCi/mL ³HTdR (equal to 19.6 μCi ³HTdR) by intravenous injection via a tail vein.

Approximately five hours after treatment with ³HTdR all mice were euthanized.

The draining lymph nodes were excised and pooled for each experimental group (8 nodes/group). Single cell suspensions (phosphate buffered saline) of pooled lymph
node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing twice with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approx. 4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of 'lrga-Safe Plus' scintillation liquid and thoroughly mixed.

The level of ³HTdR incorporation was then measured on a β-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations/minute (DPM).

- Calculation of stimulation index:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations/minute/lymph node (DPM/node) and as the ratio of 3HTdR
incorporated into lymph node cells of test group relative to that recorded for control group (stimulation index) (S.I.). Before DPM/node values were determined, mean
scintillation-background DPM was subtracted from test and control raw data.
Stimulation index is calculated according to the following formula:
Stimulation index (S.I.) = (dpm/LN of treated group)/(dpm/LN of control group)

- Criteria used to consider a positive response:
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
1) exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
2) the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS:
- mortality/viability: twice daily from acclimatization start to the termination of in-life phase.
- body weights: on the test day 1 (prior to the first application) and on the test day 6.
- clinical signs (locval/systemic): daily from acclimatization start to the termination of in-life phase. Particular attention was paid to the treatment sites.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Positive control results:

The stimulation index for the three concentrations of alpha-hexylcinnamaldehyde were as follows:
5 % (w/v): 2.7*
10 % (w/v): 3.4*
25 % (w/v): 12.4
* this value was used in calculation of EC3.
A clear dose-response relationship was observed.
A EC3 value of 7.1 % (w/v) was calculated.

Observations:
- viability/mortality: no deaths occurred during the study period.
- clinical signs: no clinical signs were observed in any animals of the control group or 5 % concentration group. On the second application day, a slight ear erythema was observed at both dosing sites in all mice of 10 % concentration group and 25 % concentration group (with the exception of one animal), persisting for the remainder of the in-Iife phase of the study or persisting for a total of three days (in two mice, on a left or a right ear), separately. On the second application day, a slight ear swelling was observed at both dosing sites in all mice of 25 % concentration group, persisting for the remainder of the in-life phase of the study or persisting for a total of three days (in one animal, on the left ear), separately.
- body weights: body weight of the animals was within the range commonly recorded for animals of the strain and age (17 g - 25 g).

Key result

Parameter:

SI

Value:

2.9

Test group / Remarks:

10 % (w/v) test item

Key result

Parameter:

SI

Value:

2.4

Test group / Remarks:

25 % (w/v) test item

Key result

Parameter:

SI

Value:

2.7

Test group / Remarks:

50 % (w/v) test item

Cellular proliferation data / Observations:

No dose-response relationship was observed.

EC3 CALCULATION
Calculation of the EC3 value was not performed because no test concentrations produced a stimulation index of 3 or higher.

CLINICAL OBSERVATIONS:
- viability/mortality: no deaths occurred during the study period.
- clinical signs: no clinical signs of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals was within the range commonly recorded for animals of the strain and age.

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not a skin sensitiser.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation

The substance has no skin sensitisation potential based on a reliable local lymph node assay (OECD 429).

Justification for classification or non-classification

Skin sensitisation

The substance has no skin sensitisation potential based on a local lymph node assay (OECD 429). Thus, the substance does not require classification as skin sensitiser according to Regulation (EC) No 1272/2008 and subsequent adaptations.