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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 11 OCT 2004 to 10 Nov 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 429)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands BV, Horst, The Netherlands
- Age at study initiation: 8-12 weeks
- Housing: Individually in Macrolon Type I cages with wire mesh top and granulated sooft wood bedding
- Diet: Pelleted standard diet (provided by Harlan Winkelmann GmbH, Borchen, Germany); ad libitum
- Water: Community tap water from Rossdorf, ad libitum
- Acclimation period: yes, but duration not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
0, 3.75, 7, and 15% (w/v)
No. of animals per dose:
5 females per dose group
2 females in the pre-test
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: DMSO was selected because in any other vehicle test item concentrations above 15% could not be obtained or due to the instability of the test item withinh the vehicle
- 15% (w/v) was the highest technically applicable concentration
- Irritation: no systemic toxicity after single application of test item concentrations up to 15% (w/v); irritating effects could not be assessed due to yellow staining at concentration of 15% (w/v), but no ear swelling was observed
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
1: exposure to at least one concentration of the test item resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index
2: data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression

TREATMENT PREPARATION AND ADMINISTRATION:
- individual preparation of weight volume dilutions using a magnetic stirrer as homogenizer
- test item preparations were made feshly before each dosing occasion
- 25 µl were spread over the entire dorsal surface of each ear lobe once daily for three consecutive days
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- calculations of mean values and standard deviations for body weight
- A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c; where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining Iymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the value of 3 an the local lymph node assay dose response Plot.
- The Dunnett-test (ANOVA) was conducted to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. Statistical significance was at the five per cent level (p < 0.05).

Results and discussion

Positive control results:
Stimulation indices of 1.96, 3.03 and 4.92 were determined with the positive control item at concentrations of 5%, 10% and 25% (w/v), respectively, in acetone:olive oil, 4:1 (v/v). An EC3 value was calculated (= 9.9% (w/v)).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Stimulation indices (SI) were all below 3. The following SI were calculated: 3.75% test item: 1.4 7% test item: 1.3 15% test item: 1.6
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM/lymph node Background of measurement: 0 - 3.4; mean: 1.7 (was taken for calculations) control group: 613.4 3.75% test item: 868.4 7% test item: 872.1 15% test item: 1026.9

Any other information on results incl. tables

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Calculation of the EC3 value was not performed because no test concentration produced a SI of 3 or higher.

Test item
concentration

Measurement
DPM

Calculation

Result

Calculation

DPM
per
lymph node
b)

Result

% (w/v)

Group

DPM-BG per animal
(2 lymph nodes)
a)

S.I. *

number
of
lymph nodes

S.1

-

BG I

3.4

 

---

 

 

--

 

BG II

0,0

---

---

---

-

--

--

 

CG 1_1

891.4

889,7

 

 

 

---

-

CG 1_2

980.9

979.2

---

--

 

 

 

CG 1_3

1392.9

1391.2

 

---

---

--

---

CG 1_4

1378.5

1376,8

 

-

-

 

 

CG 1_5

1679.0

1677.3

 

10

631.4

1.0

3.75

TG 2_1

2339.5

2337.8

1,9

 

 

 

3.75

TG 2_2

1481.9

1480.2

1,2

 

 

--

3.75

TG 2_3

1498.5

1496.8

1.2

 

 

--

3.75

TG 2_4

1743.1

1741.4

1.4

---

-

--

3.75

TG 2_5

1629.6

1627.9

1.3

10

868.4

1.4

7.0

TG 3_1

1140.0

1138.3

0.9

 

 

--

7.0

TG 3_2

1822.8

1821.1

1.4

 

---

 

7.0

TG 3_3

1665.0

1663.3

1.3

---

 

 

7.0

TG 3_4

2168.6

2166.9

1.7

---

---

--

7.0

TG 3_5

1483.4

1481.7

1.2

10

827.1

1.3

15.0

TG 41

2075.9

2074.2

1.6

 

 

--

15.0

TG 4_2

1870.6

1868.9

1.5

---

-

 

15.0

TG 4_3

2893.5

2891.8

2,3

 

 

15.0

TG 4_4

1936.7

1935.0

1.5

 

15.0

TG 4_5

1501.0

1499.3

1.2

10

1026.9

1.6

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.l. = Stimulation Index

* = mean net DPM value per lymph node of the mouse divided by the mean net DPMvalueof the vehiclecontrol group

a)        = The mean of the background value (BGI and BGH) was subtracted from the value.

b)        = DPM/node was determined by dividing the sum of the measured values from all lymph nodes within a
group by the number of lymph nodes taken from that group

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Tested in this study according to OECD test guideline no. 429 and GLP conditions the test item was not a skin sensitizer.
Executive summary:

In the study the test item suspended in DMSO was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay (LLNA) was performed using test item concentrationsof 3.75 %, 7.0 %, and 15.0 % (w/v).

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.4, 1.3, and 1.6 were determined with the test item at concentrations of 3.75 %, 7.0 %, and 15.0 % (w/v) in DMSO.

The test item was found to be not a skin sensitiser in this assay.