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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:
From 16 december 2003 to 7 december 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted : 24th April 2002
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-1-phenyl-5-pyrazolone
EC Number:
201-891-0
EC Name:
3-methyl-1-phenyl-5-pyrazolone
Cas Number:
89-25-8
Molecular formula:
C10H10N2O
IUPAC Name:
5-methyl-2-phenyl-2,3-dihydro-1H-pyrazol-3-one
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch 62
- Expiration date of the lot/batch: september 2005
- Purity of test item : 98.5%
- Purity test date: 31 August 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:glass flask, at +4°C and protected from light and under nitrogen glass
- Stability under test conditions: known stability (data not shown)
- Solubility and stability of the test substance in the solvent/vehicle: known stability (data not shown)


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was ground to a fine powder, using a mortar and pestle
- Final dilution of a dissolved solid, stock liquid or gel: Dissolved at 25%, 10%, 5%, 2.5%, 1%, 0.5%, 0.25% and 0.1% in dimethylsulfoxyde (DMSO)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, France
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: not specified
- Age at study initiation: 9 weeks old
- Weight at study initiation: 20.6±1.3g
- Housing:The animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust is analysed by the supplier for composition and contaminant levels.
- Diet (e.g. ad libitum): All animals had free access to A04 C pelleted diet (SAFE,
France) and tap water (filtered using a 0.22 micron filter) contained in bottles. Each batch of food is analyzed by the supplier for composition and contaminant levels.
- Water (e.g. ad libitum): Bacteriological and chemical analyses of water, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines) are performed regularly by external laboratories. The results of these analyses are archived at CIT.
No contaminants are known to be present in the diet, drinking water or sawdust at levels which may be expected to interfere with or prejudice the outcome of the study.
- Acclimation period: at least 5 days before the beginning of the study.
- Indication of any skin lesions: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h
- IN-LIFE DATES: From: 27 February 2004 To: 8 March 2004 (First experiment)
From: 8 May 2004 To: 17 May 2004

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
The vehicle used in the study was chosen based on the results of solubility assay. Homogeneous solution was obtained at the maximum concentration of 25%.
Concentration:
First experiment : 25%, 10%, 5%, 2.5% and 1% of test substance in vehicle
Second experiment : 2.5%, 1%, 0.5%, 0.25% and 0.1% of test substance in vehicle
No. of animals per dose:
4 animals per dose group
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: known stability (data not shown) according to the solubility assay CIT/Study No. 26977
- Irritation: not specified
- Systemic toxicity: not specified
- Ear thickness measurements: done
- Erythema scores: done

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: animals were assigned to the treatment groups by hand procedure.
- Criteria used to consider a positive response: The study is considered valid if all the following criteria are met:
• the incorporation of 3H-TdR expressed as disintegration's/mn (dpm) in the vehicle control group should be at least 2 fold higher than that of the control blank,
• the SI for the positive controls should be higher than the threshold positive value of 3.
Stimulation Indices (SI) were calculated according to the following formula:
SI = dpm of treated group / dpm of control group
The same calculation was made for the positive control group.
The test item was considered as a skin sensitizer when the SI for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was prepared as a solution in the vehicle at the chosen concentrations.The test item dosage forms were prepared extemporaneously under nitrogen atmosphere and were stored protected from light and under nitrogen gas before use. They were used within the 4 hours following the preparation according to the known stability results.
On days 1, 2 and 3 of each experiment, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application
site. No rinsing was performed between each application. On day 6, animals received single intravenous injection of 250 µM of 0.9% NaCl containing 20 µCu of 3H-TdR. Five hours later, animals were sacrified.

PROLIFERATION ASSAY
Cell suspensions were washed with 15 mL of 0.9% NaCl and pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of
their viability by exclusion of Trypan blue.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical approach not specified

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was exceeded in the positive control group in the two experiments.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Remarks:
Second experiment
Test group / Remarks:
A039 tested at 2.5%
Remarks on result:
not determinable
Remarks:
The SI value did not reach the treshold of 3 to calculate the EC3.
Key result
Parameter:
SI
Remarks:
Second experiment
Value:
ca. 2.79 - < 3
Test group / Remarks:
A039 treated with 2.5% test item
Remarks on result:
other: SI value close to 3 and estimated by the authors as valuable for estimation of skin sensitization potency.
Key result
Parameter:
SI
Value:
1.67
Test group / Remarks:
Second Experiment : Test item at 0.1%
Key result
Parameter:
SI
Value:
0.72
Test group / Remarks:
Second Experiment : Test item at 0.25%
Key result
Parameter:
SI
Value:
2.77
Test group / Remarks:
Second Experiment : Test item at 0.5%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
viability = viable cells/viable cells + dead cells
cellularity index = amount of cells (x10E6 cells) in the treated group / amount of cells (x10E6 cells) in the vehicle group

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index = dpm of treated group / dpm of control group

EC3 CALCULATION
EC3 value = theorical concentration resulting in a SI value of 3

CLINICAL OBSERVATIONS:
No clinical signs and no mortality were observed.

BODY WEIGHTS
The body weight gain of the treated animals was similar to that of controls.

LOCAL IRRITATION
No cutaneous reactions and no noteworthy increases in ear thickness were observed during the
study. In the second experiment (performed at lowest concentrations than those of the
first experiment), slight or moderate increases in ear thickness were sometimes observed in the
treated groups, but without evidence of a dose-response relationship. Though no significant ear
oedema was observed in the vehicle control group in this experiment, reproducible moderate
increases in ear thickness are usually caused by DMSO itself. Therefore, these increases were
not attributed to an irritant effect of the test item but to an irritant effect of DMSO.

Any other information on results incl. tables

 Table 1 :First experiment study results

Groups

Treatment and concentrations

Cell count

Viability (%)

Amount of cells (x 106cells)

Cellular Index

Number of nodes per group

Dpmper group

Dpmper node

Stimulation Index

Increase in ear thickness (% between day 1 to day 6)

EC3 value

Viable

dead

1

DMSO 0

108

9

92.31

5.40

 

8

305.15

38.14

 

8.08

 

2

A039 1%

191

12

94.09

9.55

1.77

8

967.67

120.96

3.17

1.96

 

3

A039 2.5%

247

9

96.48

12.35

2.29

8

2790.97

348.87

9.15

6.86

 

4

A039 5%

287

17

94.41

14.35

2.66

8

4644.39

580.55

15.22

9.90

NA

5

A039 10%

325

24

93.12

16.25

3.01

8

3716.74

464.59

12.18

6.80

 

6

A039 25%

164

24

87.23

8.20

1.52

8

2993.69

374.21

9.81

3.92

 

7

HCA 25%

179

12

93.72

17.90

3.31

8

3495.46

436.93

11.45

 

 

NA = not applicable

Dpm= disintegration per minute

viability= viable cells/viable cells + dead cells

cellularityindex = amount of cells (x106 cells) in the treated group / amount of cells (x106 cells) in the vehicle group

Stimulation index =dpm of treated group /dpm of control group

EC3 value =theorical concentration resulting in a SI value of 3

 

Table 2 :Second experiment study results

Groups

Treatment and concentrations

Cell count

Viability (%)

Amount of cells (x 106cells)

Cellular Index

Number of nodes per group

Dpmper group

Dpmper node

Stimulation Index

Increase in ear thickness (% between day 1 to day 6)

EC3 value

Viable

dead

1

DMSO 0

185

17

91.58

9.25

 

8

605.75

75.72

 

6.93

 

2

A039 0.1%

158

17

90.29

7.90

0.85

8

1011.50

126.44

1.67

28.87

 

3

A039 0.25%

62

116

34.83

3.10

0.34

8

438.10

54.76

0.72

9.09

 

4

A039 0.5%

240

20

92.31

12.00

1.30

8

1676.20

209.53

2.77

8.33

NA

5

A039 1%

196

28

87.50

9.80

1.06

8

1027.09

128.39

1.70

13.86

 

6

A039 2.5%

178

42

80.91

8.90

0.96

8

1691.93

211.49

2.79

12.00

 

7

HCA 25%

107

67

61.49

10.70

1.16

8

3707.03

463.38

6.12

 

 

NA = not applicable

Dpm= disintegration per minute

viability= viable cells/viable cells + dead cells

cellularity index = amount of cells (x106 cells) in the treated group / amount of cells (x106 cells) in the vehicle group

Stimulation index =dpm of treated group /dpmof control group

EC3 value =theorical concentration resulting in a SI value of 3

 

 

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In the first experiment, lymphoproliferative responses were observed at all concentrations tested, which were attributed to delayed contact hypersensitivity in the absence of local irritation.
In the second experiment, the threshold positive value of 3 was approached at the concentration of 2.5%.

Under the conditions of this study, phenyl methyl pyrazolone induced delayed contact hypersensitivity. EC3 value could be estimated in these studies, phenyl methyl pyrazolone was considered to have sensitising potential. The discrepancy between the two experiments could be explained by a low incorporation of 3H-TdR in the vehicle control group (dpm/group = 305.15), leading to high SI values in the first experiment. Therefore, the skin sensitization potency of the test item should be based on the results of the second experiment, for which a more usual value of incorporation of 3H-TdR was observed in the vehicle control group (dpm/group = 605.75), but without EC3 calculation.

Under experimental conditions, the test item Phenylmethyl pyrazolone (A039) (batch No. 62) induces delayed contact hypersensitivity in the murine Local Lymph Node Assay.
According to current CLP criteria (1272/2008/CE consolidated), the test item Phenylmethyl pyrazolone (A039) should be considered as skin sensitizer category 1B.
Executive summary:

This GLP-compliant study was performed to evaluate the potential sensitization of phenylmethyl pyrazolone according to OECD Guideline 429 method (adopted the 24th April 2004) for Local Lymph Node Assay.

Materials and Method

This assessment was made through two independent experiments using 28 animals each.

In the first experiment, animals were separated in 7 groups (4 mice/group) consisting of:

5 treated groups receiving phenyl methyl pyrazolone at 1, 2.5, 5, 10 and 25% (w/v) in dimethylsulfoxide (DMSO). A negative control group receiving the vehicle (DMSO) alone and positive control group receiving alpha-hexylcinnamaldehyde (HCA) at 25% (v/v) in DMSO. A similar second experiment was conducted at the lower concentrations of 0.1, 0.25, 0.5, 1 and 2.5% (w/v) phenyl methyl pyrazolone.

On days 1, 2 and 3 of each experiment, a dose-volume of 25 μL of the control or registered item dosage was applied to the dorsal surface of both ears. On day 6, mice were sacrified by cervical dislocation. Aurical lymph node were excised to assessed lymphocyte proliferation determination through radioactive labelling.

Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.

Result

In the first experiment, lymphoproliferative responses were observed at all concentrations tested, which were attributed to delayed contact hypersensitivity. In the second experiment, the threshold positive value of 3 was approached at the concentration of 2.5%. Therefore, the skin sensitization potency of the test item should be based on the results of the second experiment, for which a more usual value of incorporation of 3H-TdR (radioactive labelling) was observed in the vehicle control group. No significant skin irritation were observed.

Conclusion

Under experimental conditions, the test item Phenylmethyl pyrazolone (A039) (batch No. 62) induces delayed contact hypersensitivity in the murine Local Lymph Node Assay.

According to the current CLP regulation criteria, the test item Phenylmethyl pyrazolone (A039) should be considered as skin sensitizer category 1B without EC3 calculation