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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 - 30 Jul 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study followed the standard guideline of reference (OECD 429), which describes a procedure designed to evaluate this endpoint, but with an important protocol deviation. Cellular proliferation was not determined by incorporated radioactivity, thus no disintegrations per minute (DPM). Instead, lymph node cell count was used as direct measurement of cell proliferation and a lower cut-off value of 1.4 times increase of stimulation index was used. The results were reviewed for reliability and assessed as valid, and the study was conducted under GLP condition.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2002
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2010
Deviations:
yes
Remarks:
Proliferation assessed on Day 4 instead Day 6 by lymph node cell count instead of incorporated radioactivity and the analysis was not part of the GLP study, no Draize scoring, pos. control was run in an independent experiment
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
adopted in 2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid
EC Number:
700-093-4
Cas Number:
176969-34-9
Molecular formula:
C6H6F2N2O2
IUPAC Name:
3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederland, AD Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 8 weeks
- Weight at study initiation: 24 - 30 g
- Housing: During the acclimatisation period up to 8 mice were housed together in conventional Makrolon type III cages. During the study period the animals were housed individually in type II cages. Low-dust wood shaving was used as bedding (Lignocel BK 8-15, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany)
- Diet: Provimi Kliba SA 3883 (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
3, 10 and 30% (w/w)
No. of animals per dose:
6 animals
Details on study design:
MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Lymph node weight, cell count, ear swelling and ear weight were determined for the control and treatment animals.
- Criteria used to consider a positive response: Stimulation Index of 1.4 for the cell count and increase of 2 x 10E-2 mm for ear swelling (about 10% of the control values).

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of the test item formulation were applied epicutaneously onto the dorsal part of both ears of the animals. The treatment was repeated on three consecutive days. One day after the last application (study Day 4) the animals were sacrificed and the lymphatic organs (auricular lymph nodes) were isolated. Lymph nodes were weighed and the cell counts were determined. In addition, ear thickness was determined prior to the first treatment and before sacrifice. The thickness of both auricles of the animals was measured using a spring-loaded micrometer. At study termination, the ear weights of the sacrificed animals were measured using an 8 mm ear punch of each ear.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the vehicle control group by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogenous (p<0.05), a nonparametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5%. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffe's method, which according to Sachs can be used for both equal and unequal sample sizes. In this method of statistical processing of measurements a large number of comparisons is made, and as a result of the multiple tests the overall probability of error is considerably greater than the p values suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of these problems the biological and lexicological relevance is also taken into consideration in the evaluation of statistical significance. For this reason, in the case of indices only the standard deviations between groups and difference analysis of the mean values were used in the evaluation of the biological relevance.

Results and discussion

Positive control results:
The test methodology was checked for reliability in a test on female NMRI mice using alpha hexyl cinnamic aldehyde formulated in different vehicles (PEG 400, DAE 433, DMF, MEK, acetone/olive oil (4:1) and Cremophor EL/ physiological saline solution 2% v/v) at concentrations of 3%, 10% and 30%. The results for the positive control were valid, but not included in the study report of the present study.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control
Remarks on result:
other: SI = Cell count of treated group / Cell count of control group
Key result
Parameter:
SI
Value:
0.96
Test group / Remarks:
3% formulation
Remarks on result:
other: SI = Cell count of treated group / Cell count of control group
Key result
Parameter:
SI
Value:
0.69
Test group / Remarks:
10% formulation
Remarks on result:
other: SI = Cell count of treated group / Cell count of control group
Key result
Parameter:
SI
Value:
0.66
Test group / Remarks:
30% formulation
Remarks on result:
other: SI = Cell count of treated group / Cell count of control group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
There was no increase in the stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test item. The "positive level" which is 1.4 for the cell count index was never reached or exceeded in any dose group. The "positive level" of ear swelling, which is 2 x 10E-2 mm increase, i.e. about 10% of the control values, has not been reached or exceeded in any dose group. No substance specific effects were determined for ear weights either.

CLINICAL OBSERVATIONS:
No clinical signs of toxicity were reported for any animal.

BODY WEIGHTS
Body weights of the animals were not affected by any treatment.

Any other information on results incl. tables

Table 1: Direct LLNA

Dose (%) Weight index ± SD (mean ± SD in %) Cell count index ± SD (mean ± SD in %)
DMF 1.00 ± 12.60 1.00 ± 14.88
3 1.00 ± 28.76 0.96 ± 38.62
10 0.84 ± 21.02 0.69 ± 30.15
30 0.95 ± 24.94 0.66 ± 29.73
DMF: Dimethylformamide, vehicle control

Table 2: Ear swelling

Dose (%) Day 1 (mean ± SD in %) Day 4 (mean ± SD in %) Index Day 4
DMF 17.33 ± 2.84 17.58 ± 2.93 1.00
3 17.58 ± 3.80 18.00 ± 3.35 1.02
10 17.75 ± 2.55 17.92 ± 2.87 1.02
30 17.58 ± 2.93 17.75 ± 2.55 1.01
DMF: Dimethylformamide, vehicle control

Table 3: Ear weight

Dose (%) Day 4 (mean ± SD in %) Index Day 4
DMF 11.91 ± 5.07 1.00
3 11.64 ± 6.56 0.98
10 11.98 ± 4.82 1.01
30 11.84 ± 4.33 0.99
DMF: Dimethylformamide, vehicle control

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Conclusions:
The substance was tested for skin sensitisation in the LLNA test with mice according to OECD guideline 429. No skin sensitisation could be evidenced.