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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP and OECD guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(3-methyl-5-oxo-2-pyrazolin-1-yl)benzenesulphonic acid
EC Number:
201-901-3
EC Name:
4-(3-methyl-5-oxo-2-pyrazolin-1-yl)benzenesulphonic acid
Cas Number:
89-36-1
Molecular formula:
C10H10N2O4S
IUPAC Name:
4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzene-1-sulfonic acid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 25 g
- Housing:single caging
- Diet (e.g. ad libitum): pelleted standard diet (Harlan Laboratories GmbH, 33178 Borchen), ad libidum
- Water (e.g. ad libitum): tap water, (Gemeindewerke, 64380 Rossdorf), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness will be used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
5, 10, and 25%
No. of animals per dose:
4
Details on study design:
In order to study a possible allergenic potential of the test substance, three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter. After the lymph nodes have been excised, both ears of mice were punched at the apical area (Ø 8 mm corresponding to 0.5 cm2). For each animal the weight of both punches was determined.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations of the body weights were calculated.

The ANOVA (Dunnett-test) was conducted to assess whether the difference is statistically significant between ear weights of test item groups and the negative control (vehicle) group. Statistical significance was at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
Experiment performend in January 2009, 5, 10, and 25% alpha-Hexylcinnamaldehyde yielded a S.I. of 1.49, 4.17, and 4.90, respectively. The EC3 value calculated was 7.8%

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see table below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table below

Any other information on results incl. tables

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

24

---

---

---

---

---

BG II

24

---

---

---

---

0

1

4238

4214

8

526.8

1.00

5

2

19916

19892

8

2486.5

4.72

10

3

5846

5822

8

727.8

1.38

25

4

27964

27940

8

3492.5

6.63

BG=Background (1 ml 5% trichloroacetic acid) in duplicate 1=Control Group 2-4=Test Group S.I.=Stimulation Index

a)=The mean value was taken from the figures BG I and BG II b)=Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since the highest and the lowest test item concentration induced an S.I. greater than 3. The S.I. obtained at 10 % test item concentration was 1.38 and has to be considered an artefact.

Viability / Mortality

No deaths occurred during the study period.

 Clinical Signs

No symptoms of local toxicity at the ears of the animals and no signs of systemic toxicity were observed during the study period. From the third day of application onwards, the animals treated with the highest test item concentration showed discoloured urine.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

EarWeights

Group

 

Test Item Concentration % (w/v)

Ear Weight (punches, mg/animal)

Mean

SD

1

-

27.92

1.66

2

5

30.75

1.97

3

10

35.13

0.80

4

25

40.50

3.83

The measured ear weights of all animals treated were recorded at necropsy. A significant increase in ear weights was observed between the vehicle group and the groups treated with 10 and 25 % test item concentration (p<0.001).

Applicant's summary and conclusion

Interpretation of results:
other: sensitising (precausionary classification based on the findings in this study, under reserve of possible results of further clarifying studies)
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item was found to be a skin sensitizer under the conditions of this test.
Nevertheless, taking into account the dose dependent increase in ear weight, it cannot be excluded that the observed lymph node proliferation might be related to irritation effects. Clarification of the true nature of the the lymphoproliferative response of the test item may be obtained through further appropriate studies.

Executive summary:

The present study analyses the sensitising potential of the test item.

Three groups each of four female mice were treated daily with the test item at concentrations of 5, 10 and 25% (w/v) in dimethylsulfoxide by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (dimethylsulfoxide) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in ß-scintillation counter.

All treated animals survived the scheduled study period and no symptoms of local toxicity at the ears of the animals or signs of systemic toxicity were observed. From 24 hours after the second application onwards, the animals treated with the highest test item concentration showed discoloured urine.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 4.72, 1.38 and 6.63 were determined with the test item at concentrations of 5, 10 and 25% in dimethylsulfoxide.

The EC3 value could not be calculated, since only the highest and the lowest test item concentration induced an S.I. greater than 3. The S.I. obtained at 10% test item concentration was 1.38 and is most probably an artefact.

A significant increase in the ear punch biopsies was observed between the vehicle group and the groups treated with 10 and 25 % test item concentration (p<0.001). The increase in ear weights showed a clear dose dependency.

Even though a conventional dose response was not observed, the S.I. values obtained at test item concentrations of 5 and 25% were above 3 and the test item has therefore to be regarded as a skin sensitizer under the conditions of this test. However, there is some evidence for irritation effects (i.e. dose dependent increase in ear weight) as an alternative cause for lymph node proliferation in this study. Clarification of the true nature of the lymphoproliferative response of the test item may be obtained through further appropriate studies.