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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:
2009-11-24 through 2009-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study and GLP conform

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
according to §19b Abs. 1 Chemikaliengesetz (issued 30th march 2009)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

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., 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 wks
- Weight at study initiation: 19 - 22 g
- Housing: single caging, Makrolon Type II cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: =< 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50, and 100 %
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: not specified (solution or dispersion)
- Irritation: no signs of irritation or systemic toxicity at 50 and 100 % ( 1 h and 24 +-4 h p.a.)
- Lymph node proliferation response: not determined in the pre-test

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
Stimulation index (SI) is ≥ 3
Response increases in dose-related manner
Criteria for irritation potential: The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and
excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
Note: Ear weight or ear thickness were not determined.


TREATMENT PREPARATION AND ADMINISTRATION:
Days 1-3: 25 µL test suspension over an area of ~8 mm in diameter, 1x on day 1, 2, and 3
Days 4 and 5: no treatment
Day 6: Injection of 250 μL of 3H-methyl thymidine solution into all test and control mice via the tail vein. Five hours later, the animals were killed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Calculation of mean values and standard deviations

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
- test concentration 25%: SI = 3.6 - 50% : SI = 7.23 - 100%: SI = 7.83 The EC3 value could not be calculated, since all Stimulation indices are above 3. But it can be estimated that EC3 is in the range of 20 %. Based on test results, no conclusion can be drawn about the sensitisation potential at lower concentrations.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Table 1 below

Any other information on results incl. tables

Table 1: Calculation and Results of Individual Data

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM minus - BGa)

number of lymph nodes

mean DPM per lymph nodeb)

S.I.

---

BG I

14

---

---

---

---

---

BG II

15

---

---

---

---

---

1

1014

1000

8

124.9

 

25

2

3608

3594

8

449.2

3.60

50

3

7243

7229

8

903.6

7.23

100

4

7843

7829

8

978.6

7.83

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.

Irritation potential:

24 hours after the second and 1 hour after the third application swelling and redness of the ears of all four animals of the highest concentration (100%) was observed. These local signs of irritation resolved until the day of preparation (day 6).

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item n/i-C13-C15 aldehyde was found to be a skin sensitiser in a valid LLNA assay according to OECD TG 429 and conform to GLP.
Executive summary:

n/i-C13 -C15 -Aldehyde was examined for contact allergenic potential in a Local Lymph node Assay (LLNA) that was conducted under GLP conditions and in accordance with OECD TG 429. Three groups each of four female mice were treated daily with the test item at concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4+1) by topical application of 25 µL 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 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 a scintillation counter.

All treated animals survived the scheduled study period and no systemic signs of toxicity were observed. 24 hours after the second and 1 hour after the third application swelling and redness of the ears of all four animals of the highest concentration (100%) was observed. These local signs of irritation resolved until the day of preparation (day 6). The incorporation of thymidine was increased in all treated groups. Stimulation Indices of 3.60, 7.23, and 7.83 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4+1). The EC3 value could not be calculated, since all obtained SI´s were above 3. But it can be estimated that the EC3 values is in the range of 20%.

Based on the test results n/i-C13 -C15 -Aldehyde is considered a skin sensitizer in the LLNA. The study is considered to be valid and useful for assessment (Harlan, 2009).