Registration Dossier

Administrative data

Description of key information

Weight of evidence. One LLNA (Local Lymph Node Assay) study (method similar to OECD 429) reports that the test item produced a significant increase in lymph node proliferation at concentrations equal or greater than 0.006%; three MEST (Mouse Ear Swelling Test) studies report the test item to cause an increase in ear thickness in various mice strains at concentrations from 0.006 to 0.05% test item. Based on the available information, the substance is deemed to be skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
See 'Principles of method if other than guideline'
Principles of method if other than guideline:
The method was a modification of the procedure developed by Kimber et al (1995).
Deviations:
- number of animals per treatment group is not specified.
- the period between exposure and extraction of the lymph nodes was 1 day instead of 2.
- the guideline states that pellets obtained from suspension of lymph node cells should be re-suspended in 1 ml TCA and transferred to scintillation vials containing 10 ml of scintillation fluid for 3H-counting; however, the experimental study used 5 ml of scintillation fluid.
- the substance used as positive control in the experimental study was not one of the recommended substances indicated in the guideline.
- results for each treatment group were not expressed as SI. Accordingly, classification could not rely on SI values (as indicated in the guideline).
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Aldrich Chemical Company (Milwaukee, WI)
- Purity: 99%
Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: hepatitis and Sendai virus free.
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 17-20 g
- Diet: Agway Rat and Mouse Ration (NIH 031), ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: animals were quarantined until 8 weeks of age.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light
Vehicle:
other: acetone
Concentration:
0.006%, 0.03%, 0.06% and 0.1% w/v
No. of animals per dose:
not specified
Details on study design:
PRE-SCREEN TESTS:
12.5 µl of the test material was applied to both sides of each ear pinna for four consecutive days (50 µl/mouse/day). 24 hours after the treatment, ear thickness was measured and compared with the measurements made prior to treatment. Percent ear swelling was calculated: [(mean post-treatment ear thickness/mean pre-treatment ear thickness)/100] - 100. The highest concentration producing a percent ear swelling not significantly different from the vehicle was considered the Maximal Non-Irritating Concentration (MNC). The lowest concentration of test article producing a percent ear swelling significantly greater than the vehicle was considered the Minimal Irritating Concentration (MIC). Initial concentrations tested were 0.001, 0.005, 0.01, 0.1, and up to 30% (w/v).
- Irritation: Animals experienced dose dependent irritant responses. The MNC was 0.06%, and the MIC was 0.1% (w/v).
- Systemic toxicity: Exposure to concentrations higher than 3% DCC resulted in significant toxicity. Animals exposed to 3% test item experienced significant weight
loss and presented with ataxia, hunched stance, and squinting eyes.

MAIN STUDY
Based on the results of the irritancy test, the highest dose chosen for testing was the MIC (0.1%), and the subsequent doses were MNC (0.06%), 0.5*MNC (0.03%) and 0.1*MNC (0.006%).

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: measurement of cell proliferation in the local lymph nodes.

TREATMENT PREPARATION AND ADMINISTRATION:
On days 1-3, 12.5 µl of test item (or vehicle) was applied on both the dorsal and the ventral surfaces of each ear. Animals were left untreated on day 4.

PROLIFERATION ASSAY
On day 5, mice were injected intravenously via tail vein with 0.2 ml (20 pCi) [3H]thymidine. Approximately 5 hours after injection, mice were euthanized with CO2 gas and cervical draining lymph nodes located at the bifurcation of the jugular veins were excised and adequatly treated: cells were washed twice in 10 ml of PBS and then precipitated in 3 ml of 5% trichloroacetic acid (TCA). Following overnight incubation at 4”C, the precipitates were pelleted, resuspended in 1 ml TCA, and transferred to 5 ml of scintillation solution. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine. Mean cpm for each group was calculated and compared to the vehicle control for significance and dose response comparation.
Positive control substance(s):
other: 1-Fluoro-2,4-dinitrobenzene
Statistics:
The data were first tested for homogeneity by the Bartlett’s Chi Square Test. If homogeneous, a one-way analysis of variance (ANOVA) was conducted. If the ANOVA showed significance at p < 0.05 or less, the Dunnett’s Testz was used to compare treatment groups with the appropriate control group. If data were non-homogeneous, a non-parametric analysis of variance and a Wilcoxon’s Rank Sum Test were used to compare treatment groups with the appropriate control groups. The Jonckheere’s Test was used to detect trend analysis and dose dependency.
Positive control results:
The positive control groups experienced a significant increase in lymph node cell proliferation as compared to control.
Key result
Parameter:
other: minimal concentration that produced lymph node proliferation
Value:
0.06
Test group / Remarks:
0.06% (w/v) was the first concentration of test item with p<0.01 (significant increase in lymph node proliferation)
Parameter:
SI
Test group / Remarks:
0.06% (w/v)
Remarks on result:
not determinable because of methodological limitations
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: a dose dependent increase in lymph node cell proliferation was detected. Significant increase observed at 0.06 and 0.1% (w/v).
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
EU criteria.
Conclusions:
The minimal concentration of the test item that produced a significant increase in lymph proliferation was found to be 0.06% w/v. The test item was considered a skin sensitizer.
Executive summary:

The skin sensitisation potential of the test item was evaluated in accordance with the LLNA method in female mice. A preliminary test was run with 11 concentrations of the test item (0.001 to 30% w/v) for determining the MNC (Maximal Non-Irritating Concentration) and the MIC (Minimal Irritating Concentration). Then, the main test was run with four concentrations: the highest one was the MIC (0.1% w/v), the other three were the MNC (0.06% w/v), half the MNC (0.03% w/v) and ten times below MNC (0.006% w/v). Mice were treated on both the dorsal and the ventral surfaces of each ear for three days and were left untreated on day 4. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine. The minimal concentration of the test item that produced a statistically significant increase in lymph proliferation was found to be 0.06% w/v. The test item was considered a skin sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Mouse Ear Swelling Test (MEST):
- The method followed the original procedure described by Gad et al (1986).
- Principle of test: to identify allergic responses using changes in the measurements of ear thickness exposed to the chemical
- Short description of test conditions:
Preliminary test (Irritancy assay): 12.5 µl of the test material was applied to both sides of each ear pinna for four consecutive days (50 µl/mouse/day). 24 hours after the treatment, ear thickness were measured and compared with the measurements made prior treatment. Percent ear swelling was calculated [(mean post-treatment ear thickness/mean pre-treatment ear thickness)/100] - 100. The highest concentration producing a percent ear swelling not significantly different from the vehicle was considered the Maximal Non-Irritating Concentration (MNC). The lowest concentration of test article producing a percent ear swelling significantly greater than the vehicle was considered the Minimal Irritating Concentration (MIC).
Main test (Contact Hypersensitivity Assay): 50 µl of the test material was applied on the shaved dorsal lumbar area of each mouse at different concentrations (< to the found MNC and the MNC). Mice were treated for three days and were left untreated for four days. On day 8, right ear thickness of each mouse was measured in duplicate (induction phase). After measurement, mice were challenged with 12.5 µl of vehicle or test article (MIC concentration) on the dorsal and ventral surfaces of the right ear pinna. The right ear then was measured in duplicate 24 and 48 hours (± 2 hr) after challenge and averaged (challenge phase). The percent ear swelling was calculated for each time point as described for the irritancy assay. Mean percent ear swelling for each dose group was compared to the mean percent ear swelling for the background control (BC, sensitization with acetone and challenge with MIC) for significance and dose response. The BC group was used as the control to account for swelling caused by irritation.
- Parameters analysed / observed: ear thickness measurements
GLP compliance:
not specified
Type of study:
mouse ear swelling test
Justification for non-LLNA method:
As stated in OECD Guideline 406, the MEST (Mouse Ear Swelling test) can be used as a first stage in the assessment of skin sensitisation potential. If a positive result is seen in the assay, a test substance may be designated as a potential sensitiser, and it may not be necessary to conduct further testing.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Aldrich Chemical Company (Milwaukee, WI)
- Purity: 99%

Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: hepatitis and Sendai virus free and quarantined until 8 weeks of age
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 17-20 g
- Diet ad libitum: Agway Rat and Mouse Ration (NIH 031)
- Water ad libitum: tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light
Route:
epicutaneous, open
Vehicle:
other: acetone
Concentration / amount:
0.003%, 0.006%, 0.03% and 0.06% %w/v
Day(s)/duration:
3 days
Adequacy of induction:
other: The highest concentration used is the MNC (Maximal Non-Irritating)
Route:
epicutaneous, open
Vehicle:
other: acetone
Concentration / amount:
0.1%
Day(s)/duration:
single-dose treatment, measurements at 24 and 48 hours
Adequacy of challenge:
other: Minimal Irritating Concentration (MIC)
No. of animals per dose:
16 females in control group (background group), positive control group, 0.006% and 0.03% groups
8 females in 0.003% and 0.06% groups
Details on study design:
RANGE FINDING TESTS: Preliminary test (Irritatncy study)
- Concentrations tested: 0.001%, 0.005%, 0.01%, 0.03%, 0.06%, 0.1%, 0.3%, 1%, 3%, 10% and 30% w/v.
- Exposure: 12.5 µl of each concentration applied to both sides of each ear pinna for four consecutive days.
- Twenty-four hours (± 2 hr) after the final treatment, ear thickness was measured and the percent ear swelling was calculated. MNC and MIC were determined.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 3 days
- Test groups: four groups
- Control group: two control groups, one vehicle control group and one positive control (treated with the sensitizing DNFB :1-Fluoro-2,4-dinitrobenzene)
- Site: shaved dorsal lumbar area
- Frequency of applications: daily
- Duration: 3 days exposure
- Concentrations: 0.003%, 0.006%, 0.03% and 0.06% w/v

B. CHALLENGE EXPOSURE
- No. of exposures: one single dose
- Day(s) of challenge: 1 day
- Exposure period: 2 days
- Test groups: four groups
- Control group: two control groups, one vehicle control group and one positive control (treated with the sensitizing DNFB :1 Fluoro-2,4-dinitrobenzene)
- Site: dorsal and ventral surfaces of the right ear pinna
- Concentrations: 0.06% w/v
- Evaluation (hr after challenge): 24h and 48h

Challenge controls:
Right ear thickness measurements
Positive control substance(s):
yes
Remarks:
1-Fluoro-2,4-dinitrobenzene (DNFB)
Positive control results:
The positive control group experienced a significant increase in percent ear swelling as compared to the vehicle control group at both the 24 and 48 hr time points.
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle control
No. with + reactions:
0
Total no. in group:
16
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
48
Group:
test group
Dose level:
0.006% (w/v)
Total no. in group:
5
Clinical observations:
changes in ear thickness
Remarks on result:
positive indication of skin sensitisation
Hours after challenge:
48
Group:
positive control
Dose level:
0.25% (v/v) 1-fluoro-2,4-dinitrobenzene
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

- Dose dependent contact hypersensitivity responses were observed for the test item.

- 0.006% (w/v) test item produced significant ear swelling 24 and 48h after application, compared to the background control group.

- No significant differences were seen in body weight changes between animals dosed with vehicle or test item at any dose.

- Acceptance of results: the positive control groups experienced a significant increase in percent ear swelling as compared to the background positive at both 24 and 48h time points.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
EU criteria.
Conclusions:
The concentration of 0.006% of the test substance was found to be sensitizing in the MEST.
Executive summary:

The MEST (Mouse Ear Swelling Test) was performed in Female B6C3F1 Mice. A preliminary test was run with 11 concentrations of the test item (0.001 to 30% w/v) for determining the MNC (Maximal Non-Irritating Concentration) and the MIC (Minimal Irritating Concentration). Then, the induction phase of the MEST was performed with 50 µl of the 0.003%, 0.006%, 0.03% and 0.6% w/v concentrations (being the highest dose tested the MNC) applied to the shaved dorsal lumbar area of each mouse for three consecutive days. Mice were left untreated for four days. On day 8, 12.5 µl of the MIC concentration (0.1% w/v) were applied on the dorsal and ventral surfaces of the right ear pinna of each mice and the ear thickness was measured on 24 and 48 hours. On the basis of this study, 0.006% w/v test substance concentration was found to produced significant ear swelling after 24 and 48 hours after application, so it was considered as sensitizing concentration.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Mouse Ear Swelling Test (MEST)
- Principle of test: to identify allergic responses using changes in the measurements of ear thickness exposed to the chemical

- Short description of test conditions:
Preliminary test (Irritancy assay): Prior to dosing on day 1, the ear thickness was measured with a dial thickness gauge (Ozaki MFG. Co.,Tokyo, Japan). Three mice per dose group received 12.5 µl of the test material, applied to both sides of each ear pinna. 24 hours after the treatment, ear thickness were measured and compared with the measurements made prior treatment. Statistical analysis was performed using the t-test. The highest concentration producing ear swelling not significantly different from the vehicle control was considered the maximum non-irritating concentration. The lowest concentration of the test article producing ear swelling significantly greater than the vehicle control was considered the minimum irritating concentration.

Main test (MEST): Three mice per group received 50 µl of the test material applied on the shaved dorsal lumbar area at different concentrations: the highest dose was the minimum irritating concentration, and three other lower concentrations including the maximum non-irritating concentration were selected. Mice were treated for three days and were left untreated for four days. On day 8, mice were challenged with 12.5 µl of the test article solution of maximum non-irritating concentration on the dorsal and ventral surfaces of the right ear pinna. The right ear then was measured on 24 after challenge. The increases in thickness were compared with control values. Statistical analysis was performed with the t-test.

- Parameters analysed / observed: ear thickness measurements
GLP compliance:
not specified
Type of study:
mouse ear swelling test
Justification for non-LLNA method:
As stated in OECD Guideline 406, the MEST (Mouse Ear Swelling test) can be used as a first stage in the assessment of skin sensitisation potential. If a positive result is seen in the assay, a test substance may be designated as a potential sensitiser, and it may not be necessary to conduct further testing.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity: 99%
- Source: Sigma-Aldrich Chemical (Wisconsin, USA)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test substance was dissolved in acetone.

Species:
mouse
Strain:
other: BALB/c, DBA/1, C57BL/6, CBA/J and C3H/HeN
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan (Hino, Japan)
- Sex: male
- Age at study initiation: 6 weeks old
- Weight at study initiation: not specified
- Diet: ad libitum, CRF rations
- Water: ad libitum, tap water
- Acclimation period: 1 week
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25ºC
- Humidity (%): 40-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Route:
epicutaneous, open
Vehicle:
other: acetone
Concentration / amount:
0.01, 0.03, 0.1 and 0.3 % w/v
Day(s)/duration:
3
Adequacy of induction:
other: the highest dose was the minimum irritating concentration
Route:
epicutaneous, open
Vehicle:
other: acetone
Concentration / amount:
0.1% w/v
Day(s)/duration:
single-dose treatment
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
three mice per dose group and per strain
Details on study design:
RANGE FINDING TESTS: Preliminary test (Irritancy study)
- Concentrations tested: 0.01%, 0.03%, 0.1%, 0.3%, 1% and 3% w/v.
- Exposure: 12.5 µl of each concentration applied to both sides of each ear pinna (single-dose)
- Twenty-four hours later, ear thickness was measured and compared with the measurements prior treatment.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 3 days
- Test groups: four groups
- Control group: three control groups, one vehicle control group and two positive control (one treated with the sensitizing DNCB :1-Chloro-2,4-dinitrobenzene, the other treated with OXA: 4-ethoxymethylene-2-phenyloxazolin-5-one )
- Site: shaved dorsal lumbar area
- Frequency of applications: daily
- Duration: 3 days exposure
- Concentrations: 0.01, 0.03, 0.1 and 0.3 % w/v

B. CHALLENGE EXPOSURE
- No. of exposures: one single dose
- Day(s) of challenge: 1 day
- Exposure period: 24 hours
- Test groups: four groups
- Control group: three control groups, one vehicle control group and two positive control (one treated with the sensitizing DNCB :1-Chloro-2,4-dinitrobenzene, the other treated with OXA: 4-ethoxymethylene-2-phenyloxazolin-5-one )
- Site: dorsal and ventral surfaces of the right ear pinna
- Concentrations: 0..01 % w/v
- Evaluation (hr after challenge): 24 hours
Challenge controls:
Right ear thickness measurements
Positive control substance(s):
yes
Remarks:
4-ethoxymethylene- 2-phenyloxazolin-5-one (OXA; 90% purity) and 1-Chloro-2,4-dinitrobenzene (DNCB; 99.0% purity)
Positive control results:
Both positive controls showed the expected sensitizing behaviour: DNCB and OXA caused ear swelling responses at concentrations above 0.1% w/v in BALB/c mice and above 0.03 % w/v in C3H/HeN mice (results with the other strains not shown).
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle control
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
24
Group:
test group
Dose level:
>= 0.03%
Clinical observations:
changes in ear thickness
Remarks on result:
positive indication of skin sensitisation
Remarks:
BALB/c mice
Key result
Hours after challenge:
24
Group:
test group
Dose level:
>= 0.03 %
Clinical observations:
changes in ear thickness
Remarks on result:
positive indication of skin sensitisation
Remarks:
C57BL/6 mice
Key result
Hours after challenge:
24
Group:
test group
Dose level:
>= 0.1 %
Clinical observations:
changes in ear thickness
Remarks on result:
positive indication of skin sensitisation
Remarks:
DBA/1 mice
Key result
Hours after challenge:
24
Group:
test group
Dose level:
> 0.3%
Clinical observations:
no changes in ear thickness
Remarks on result:
no indication of skin sensitisation
Remarks:
C3H/ HeN mice
Key result
Hours after challenge:
24
Group:
test group
Dose level:
> 0.3%
Clinical observations:
no changes in ear thickness
Remarks on result:
no indication of skin sensitisation
Remarks:
CBA/J mice
Hours after challenge:
24
Group:
positive control
Dose level:
1-Chloro-2,4-dinitrobenzene
Remarks on result:
positive indication of skin sensitisation

The test item induced significant responses from 0.03% in BALB/c mice, while no response was observed up to 0.3% in the C3H/HeN strain. Because the acetone treatment group challenged with 0.1% test item showed no response, it was considered that irritation did not affect the results.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
EU criteria.
Conclusions:
The test substance produced signifcant increases in ear thickness at concentrations equal to or greater than 0.03 % in BALB/c, DBA/1 and C57BL/6 mice strains . The test substance was considered to be skin sensitizing.
Executive summary:

The MEST ( Mouse Ear Swelling Test) was performed in five different mice strains: BALB/c, DBA/1, C57BL/6, C3H/HeN and CBA/J. A preliminary test was run with six concentrations of the test item (0.01 to 3 % w/v) for determining the maximum non-irritating concentration (0.1%) and the minimum irritating concentration (0.3%). Knowing these values, the induction phase of the MEST was performed with 50 µl of the test substance at 0.01, 0.03, 0.1 and 0.3 % w/v concentrations applied to the shaved dorsal lumbar area of each mouse for three consecutive days. Mice were left untreated for four days. On day 8, 12.5 µl of the minimum irritating concentration (0.1% w/v) were applied on the dorsal and ventral surfaces of the right ear pinna of each mice and the ear thickness was measured at 24 hours. BALB/c, DBA/1 and C57BL/6 mice strains exhibited positive responses at and above 0.03% concentration, but no positive reactions were observed in the C3H/HeN or CBA/J strains. The test substance was considered to be skin sensitizing.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to other study
Qualifier:
no guideline available
Principles of method if other than guideline:
- Method according to Flint et al. (2000, 2001); Mouse Ear Swelling Test (MEST).
- Principle of test: determination of ear swelling measuring edema and cellularity at the site of chemical challenge as a gauge of cutaneous sensitisation.
- Short description of test conditions: On Day 1 and 2, 0.025, 0.05, 0.1, and 0.25% concentrations of the test substance were applied to the back with a micropipette. On day 5, baseline pinnae thickness was measured. On day 6, the right pinnae were challenged with 0.2% of the test substance. Restraint was applied for 2 h prior to chemical application on day 1 or day 6. The thickness of the right and left ear pinnae was measured 24, 48, and 72 h after challenge using a digital micrometer.
- Parameters analysed / observed: baseline pinnae ear thickness.
GLP compliance:
not specified
Type of study:
mouse ear swelling test
Justification for non-LLNA method:
As stated in OECD Guideline 406, the MEST (Mouse Ear Swelling test) can be used as a first stage in the assessment of skin sensitisation potential. If a positive result is seen in the assay, a test substance may be designated as a potential sensitiser, and it may not be necessary to conduct further testing.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Aldrich, Milwaukee, WI
- Purity: 99%

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolved in acetone

Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: not specified
- Sex: young adult male
- Weight at study initiation: 20-25 g
- Housing: in groups of six in partitioned cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hours light - 12hours dark cycle
Route:
epicutaneous, open
Vehicle:
other: acetone
Concentration / amount:
0.025, 0.05, 0.1, and 0.25 %
Day(s)/duration:
two days
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, open
Vehicle:
other: acetone
Concentration / amount:
0.2 %
Day(s)/duration:
single-dose
Adequacy of challenge:
other: the concentrations of the sensitizing dose were selected based on a previous study by Hayes (1998).
No. of animals per dose:
6 animals per dose
Details on study design:
RANGE FINDING TESTS:
- The concentration of the sensitizing dose was selected based on previous studies by Hayes (1998); see 'Cross-reference'.
- Irritation: Animals experienced dose dependent irritant responses. The MNC (Maximal Non-Irritating Concentration) was 0.06%, and the MIC (Minimal Irritating Concentration) was 0.1% (w/v).
- Systemic toxicity: Exposure to concentrations higher than 3% DCC resulted in significant toxicity. Animals exposed to 3% test item experienced significant weight
loss and presented with ataxia, hunched stance, and squinting eyes.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: 4 days
- Test groups: four
- Control group: yes
- Site: on the back of each mouse
- Frequency of applications: 2 applications in 2 days
- Concentrations: 0.025, 0.05, 0.1, or 0.25%

B. CHALLENGE EXPOSURE
- No. of exposures: one
- Day(s) of challenge: one
- Exposure period: 3 days
- Test groups: four
- Control group: yes
- Site: right pinnae
- Concentrations: 0.2 %
- Evaluation (hr after challenge): 24, 48 and 72 hours
Challenge controls:
Baseline pinnae thickness was measured
Positive control substance(s):
no
Remarks:
The authors proposed the use of these three substances in this study because of their known sensitizing properties: DNFB (2,4 dinitrofluorbenzene), dicyclohexylcarbodiimide and OXA (oxazolone).
Group:
negative control
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
24
Group:
test group
Dose level:
>= 0.05 %
Clinical observations:
changes in ear pinnae thickness
Remarks on result:
positive indication of skin sensitisation
Key result
Hours after challenge:
48
Group:
test group
Dose level:
>= 0.05 %
Clinical observations:
changes in ear pinnae thickness
Remarks on result:
positive indication of skin sensitisation
Key result
Hours after challenge:
72
Group:
test group
Dose level:
>= 0.05 %
Clinical observations:
changes in ear pinnae thickness
Remarks on result:
positive indication of skin sensitisation
Group:
positive control
Dose level:
oxazolone
Remarks on result:
positive indication of skin sensitisation
Group:
positive control
Dose level:
2,4-dinitrofluorbenzene
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
EU criteria.
Conclusions:
The test substance produced increases in ear thickness at concentrations equal to or greater than 0.05 %. The test substance was considered sensitizing to the skin.
Executive summary:

In this study, the acute restraint modulation of the sensitizing dose of the test item. Male BALB/c mice were sensitized with the test item at different concentrations (0.025, 0.05, 0.1, and 0.25%) on their shaved backs at day 1 and 2 and left untreated until day 6, when challenged with a solution of 0.2 % test item applied on their right ear pinna and a solution of vehicle on the left ear pinna. The thickness of the right and left ear was measured at 24, 48 and 72 hours. At concentrations of 0.05 % or greater, a statistical increase in the ear thickness was observed at 24, 48 and 72 hours. Therefore, the substance was considered sensitizing to the skin.

Endpoint:
skin sensitisation, other
Type of information:
other: data from peer reviewed handbook.
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Principles of method if other than guideline:
Data from peer reviewed handbook, method not specified.
GLP compliance:
not specified
Type of study:
other: data from handbook: type of study not specified.
Key result
Parameter:
other: no quantitative result
Remarks on result:
other: susbtance defined as contact allergen
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
EU criteria.
Conclusions:
According to peer reviewed handbook data, the substance is considered a contact allergen.
Executive summary:

According to According to 'The Merck Index' (peer reviewed handbook), the substance is considered a contact allergen.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Weight of evidence approach. Four experimental studies with six different strains of mice are included. All data sources found the test substance to be a skin sensitizer. It is considered that the data set provided is sufficient to fulfill  the information requirements.

One of the studies performed an LLNA (Local Lymph Node Assay) on female B6C3F1 mice, showing that the test item at concentrations equal or greater than 0.006% produced a significant increase in lymph node proliferation, this being a consequence of the sensitization potential of the substance. The other three studies performed a MEST (Mouse Ear Swelling Test) based on increases in the ear thickness as a result of exposure and sensitization of the mice to the test substance. Depending on the strain of the mice, effects at different concentrations of the test substance were observed: in females B6C3F1 there were increases in ear thickness from 0.006% test item; in male BALB/c, DBA/1 and C57BL/6 at 0.03% or more; a second study on BALB/c males showed statistical changes in concentrations of 0.05% or more. The information contained in a peer-reviewed handbook is also provided, as a supporting study. In this handbook, the substance is defined as contact allergen. Based on the available information, the substance is sensitizing.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the substance is classified as Skin Sens. Category 1, H317, according to CLP Regulation (EC) No. 1272/2008.