3-methylpyrazole

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-05-31 - 2011-07-12

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Dose Range Finding Animals
Species and strain: CRL: NMRI BR mice
Source: TOXI-COOP ZRT.
H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during the test: Good conventional
Justification of strain: The aim of the preliminary screen was to assess the ability of the test item causing irritation and/or systemic toxicity (not the sensitizing effect), CRL: NMRI BR mice were used in the pre-screen test for a better visibility of erythema and oedema.
Number of animals: 6 animals (2 animals/group);
Sex: Female, nulliparous, non pregnant
Age of animals: Young adult mice, 8-9 weeks old

Main Test Animals
Species and strain: CBA/Ca mice
Source: TOXI-COOP ZRT.
H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during
the study: Good conventional
Justification of strain: On the basis of comparative investigations in other laboratories, mice of the CBA/Ca strain were found to exhibit a more marked response than other strains. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals/treatment group; 24 animals/main test
Sex: Female, nulliparous, non pregnant
Age of animals: Young adult mice, 9-12 weeks old (at start of the experiment),
Body weight range at starting: 16.6-22.0 g; The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
Acclimatization time: 7 and 21 days

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

2.5, 5, 10, 25 %

No. of animals per dose:

4

Details on study design:

Dose Range Finding Test
The maximum dose selection was performed according to the OECD 429 guideline. The preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main test except there was no assessment of lymph node proliferation and fewer animals were used.
The preliminary test was performed with test item concentrations of 100 % (the undiluted test item itself), 50 % and 25 % in AOO. The applicability of the undiluted test item was acceptable. The 50 % and 25 % concentration of test item in the vehicle (AOO) was a real solution and applicability of these formulations on the ears of animals was also acceptable. Groups of 2 CRL: NMRI BR mice were treated with the test item (100 %) and its formulations (50 % and 25 %). All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to the first treatment (Day 1) and prior to termination (Day 6, where applicable). Both ears of each mouse were observed for erythema and scored using Table 2. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
Mortality and signs of systemic toxicity were observed in the 100 % and 50 % dose groups. The observed effect in the 100 % dose group was decreased activity on Day 1 after the first treatment. Both animals were found to be dead on the next day (Day 2).
In the 50 % dose group the first symptoms were observed on Day 2 after the second treatment. The observed effects were piloerection (1/2 animals), decreased activity (1/2 animals), incoordination (1/2 animals) and intermittent twitch of the head (2/2 animals) on Day 2. On Day 3 piloerection (2/2 animals) and decreased activity (1/2 animals) were observed prior to the third treatment. Incoordination, decreased activity and piloerection were observed on both animals approximately 2 hours after the third treatment. Approximately 4 hours after the treatment incoordination (1/2 animals), decreased activity (2/2 animals, 1/2 animals was completely inactive), piloerection (2/2 animals), low respiration rate (1/2 animals), tremor (1/2 animals) and tympany (1/2 animals) were observed in this dose group. On Day 4 one animal was found dead.
The other one was euthanized, because the observed effects (tympany, piloerection, tremor, incoordination, decreased activity, hunched back posture) were considered to exceed an acceptable level.
In the 25 % dose group piloerection (2/2 animals) was observed on Day 2 (after the treatment) and on Day 3 (1/2 animals prior the treatment, 2/2 animals after the treatment). Both animals were normal on the subsequent days (Day 4, 5 and 6). No treatment related effect on the body weights was observed in this treatment group. No significant signs of irritation (indicated by an erythema score > 3 and/or an increased ear thickness 25 %) were observed in this dose group. Tabulated results of the above preliminary test are given in Table 4-7 of Appendix I.
According to the preliminary results, test item concentration of 25 % was selected to be the highest test concentration in the main test. Although the observed effect (piloerection) was considered not to be unacceptable, ensure that the test will not be invalid because of an unexpected adverse effect four test concentrations were examined in the main test.

Main Test Design
The acetone:olive oil 4:1 mixture (AOO) was the negative control and used as vehicle for the test item and also for the PC substance. Concentration of 25 % of the PC substance was tested as positive control groups. The test item was administered in four concentrations according to the results of the dose range finding test.


In the main assay 24 female CBA/Ca mice were allocated to six groups of four animals each:

- four groups received 3-Methylpyrazol at four different concentrations of 25 %, 10 %, 5 % or 2.5 %,

- the negative control group received the vehicle (AOO),

- the positive control groups received a-Hexylcinnamaldehyde (HCA) at concentration of 25 %.

Each substance was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the obtained values were used to calculate stimulation indices (SI).

Proliferation Assay
Mortality (1/4 animals) and significant symptoms of systemic toxicity was observed in the 25 % test item treated group. The observed effect were considered to be unacceptable, animals in this dose group was euthanized on Day 4. Hence lymph nodes in this dose group were not processed. No significant signs of systemic toxicity or irritation were observed in the other treatment groups, and no technically failed treatment was observed during the test. All animals were processed in the 10 %, 5 % and 2.5 % dose groups.

Injection of 3H-methyl-thymidine
On day 6, the animals were intravenously injected via the tail vein with 250 Ll of sterile PBS containing 20 LCi of 3H-methyl-thymidine using a hypodermic needle with 1 ml sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
6.5.2 Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were anesthetized by asphyxiation with Isofluran CP® and sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 ml) of PBS to keep the nodes wet before processing.
Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (lNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 ml). lNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 ml supernatant above each pellet. The pellets were gently agitated before making up to 10 ml with PBS and resuspending the lNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
Determination of Incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % trichloracetic acid (TCA) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8 °C overnight (approx. 18 hrs), each precipitate was removed and the pellets were resuspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were stored at 2-8 °C until measurement. Before measurement of incorporated radioactivity each precipitate was stirred and than transferred to a suitable sized scintillation vial with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a P-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample.
The P-counter expresses the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA.
Instrument used for the measurement:
Name: Packard Tri-Carb 2300 TR Liquid Scintillation Analyzer
Serial Number: 406 117

Positive control substance(s):

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

Positive control results:

The positive control group animals were treated with 25 % (w/v) HCA solution (dissolved in AOO) concurrent to the test item groups.
No mortality, cutaneous reactions or signs of toxicity were observed. Significant lymphoproliferative response (SI > 3) was noted for HCA with a stimulation index value of 7.4. The results of the positive control demonstrated the appropriate performance of the test in accordance with the OECD Guideline 429 and confirmed the validity of the assay.

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

10 %

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

5 %

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

2.5 %

Test Group	Measured	Group*	DPN	Stimulation
Name	DPM/group	DPM	(DPM/Node)	Index Values
Negative (vehicle) control:	5088	5038.5	629.8	1.0
AOO
Positive control	37158	37108.5	4638.6	7.4
25 %HCA in AOO
3-Methylpyrazol	-	-	-	-
25 %in AOO
3-Methylpyrazol	8887	8837.5	1104.7	1.8
10 %in AOO
3-Methylpyrazol	9676	9626.5	1203.3	1.9
5 %in AOO
3-Methylpyrazol	8151	8101.5	1012.7	1.6
2.5 %in AOO

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of the present assay 3-Methylpyrazol, tested at the maximum feasible concentration of 10 % and concentrations of 5 % and 2.5 % as formulations in a suitable vehicle (AOO), was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of 3-Methylpyrazol following dermal exposure in the Local Lymph Node Assay.

The maximum dose selection was performed according to the OECD 429 guideline[1]. The test item was a liquid hence the maximum concentration of 100 % (the undiluted test item) could be tested. A preliminary solubility test was performed to select suitable vehicle for the further test concentrations. Solubility of the test item in Acetone: Olive oil 4:1 (v/v) mixture (AOO) was examined and found to be acceptable. Preliminary irritation/toxicity test was performed with test item concentrations of 100 % (the undiluted test item itself), 50 % and 25 % in AOO. The applicability of the undiluted test item and the formulations in AOO (50 % and 25 %) was acceptable.

In this preliminary test mortality and significant signs of systemic toxicity were observed in the 100 % and 50 % dose groups. The observed effect (piloerection) in the 25 % test item treated group was considered not to be unacceptable hence test item concentration of 25% was selected to be the highest test concentration in the main test.

In the main assay 24 female CBA/Ca mice were allocated to six groups of four animals each:

- four groups received 3-Methylpyrazol at four different concentrations of 25 %, 10 %, 5 % or 2.5 %,

- the negative control group received the vehicle (AOO),

- the positive control groups received α-Hexylcinnamaldehyde (HCA) at concentration of 25 %.

Each substance was applied on the external surface of each ear (25 μL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the obtained values were used to calculate stimulation indices (SI).

No sign of irritation was observed in any of the treatment groups.

Mortality and signs of systemic toxicity were observed in the 25 % test item treated group. The symptoms observed on Day 2 (after the second treatment) were piloerection and decreased activity (4/4 animals). Same effects were observed on Day 3 (4/4 animals) with less intensity before the third treatment, but the symptoms were more severe after the treatment on this day. One animal was found dead on Day 4. Piloerection (3/3 animals), severe decreased activity (3/3 animals), increased respiration rate (3/3 animals), convulsions (2/3 animals), tremor (1/3 animals) and trepidancy (2/3 animals) were observed on the other animals in this dose group on Day 4. The symptoms observed were considered to be unacceptable hence animals (3/3) in this dose group were humanely killed on the same day.

In the 10 % test item treated group piloerection (1/4 animals) was observed on Day 2 (after the second treatment), but the animal was normal on the subsequent day prior to the treatment. After the third treatment on Day 3 slight piloerection (4/4 animals) was observed. All animals were normal on the next day (Day 4) and remained symptom free till the terminal killing (on Day 6).

No clinical sign of systemic toxicity was observed in the other treatment groups (including control groups).

Stimulation index values of the test item were 1.8, 1.9 and 1.6 at the evaluated treatment concentrations of 10 %, 5 % and 2.5 %, respectively. No dose-related response was observed.

α-Hexylcinnamaldehyde, dissolved in AOO at concentration of 25 % (w/v) was used as positive control to demonstrate the appropriate performance of the assay [1]. A significant lymphoproliferative response (SI ≥ 3) was noted for the positive control chemical with a stimulation index value of 7.4. The positive control results obtained confirmed the validity of the assay.

In conclusion, under the conditions of the present assay 3-Methylpyrazol, tested at the maximum feasible concentration of 10 % and concentrations of 5 % and 2.5 % as formulations in a suitable vehicle (AOO), was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The available study is GLP-compliant and has Klimisch score 1.

Short description of key information:

3-Methylpyrazol, tested at the maximum feasible concentration of 10 % and concentrations of 5 % and 2.5 % as formulations in a suitable vehicle (AOO), was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.

Justification for selection of skin sensitisation endpoint:

Only one study available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

3 -Methylpyrazole has not to be classified as skin sensitiser.