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Toxicological information

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 2003-04-2 until 2003-05-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Remarks:
RCC Ltd
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: White solid
- Storage condition of test material: In the original container at room temperature (20 °C ± 3 °C), away from direct sunlight.
- Stability under test conditions: Stable under storage conditions

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15.3 g - 21.1 g
- Housing: groups of four in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard Kliba 3433, ad libitum
- Water: Community tap water, ad libitum
- Acclimation period: Under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 2, 2003 To: May 28, 2003

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5 %, 5 % and 10 % (for Groups 2-4); 2.5 %, 5 % and 10 % (for Groups 6-8: in acetone:olive oil, 4:1, w/v); Groups 1 and 5 were treated with the vehicle (control groups)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- The highest non-irritant and technically applicable test item concentration was determined in a non-GLP pretest in two mice with concentrations of 1 %, 5%, 10% and 25 % (w/v). The test item in the main study was assayed at three consecutive concentrations. The top dose (10%) was the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.

MAIN STUDY
- Name of test method: LLNA
- Criteria used to consider a positive response: regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls.

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle (acetone:olive oil, 4:1 (v/v)) was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
- Topical application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 2.5 %, 5 % and 10 % (for Groups 2-4); 2.5 %, 5 % and 10 % (for Groups 6-8) (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. Two control groups of mice were treated with an equivalent volume of the relevant vehicle alone. A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
- Administration of 3H-Methyl Thymidine: Five days after the first topical application, all mice were administered with 250 µl of 78.4µCi/ml 3HTdR (equal to 19.6 µCi 3HTdR) (for Groups 1-4) and 85.0 µCi/ml 3HTdR (equal to 21.3 µCi 3HTdR) (for Groups 5-8) by intravenous injection via a tail vein.
- Determination of incorporated 3HTDR: Approximately five hours after treatment with 3HTdR all mice were euthanized. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of “Ultima Gold” scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a P-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
- Interpretation of Data: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (Stimulation Index) (S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
1. First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the SI.
2. Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:
SI of 2.5, 3.7 and 9.7 were determined with the positive control at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: In this study, SI of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). (See Table 1)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 1

Any other information on results incl. tables

- Viability / Mortality: No deaths occurred during the study period.

- Clinical signs: No test item-related clinical signs were observed. On the third application day, a slight ear erythema was observed at both dosing sites in all mice of Group 7 (5 %) and Group 8 (10 %). On the second application day, a slight ear swelling was observed at both dosing sites in all mice of Group 8 (10 %). These signs persisted for the remainder of the in-life phase of the study.

- Body weights: The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

- LLNA: A dose related increase (> 26 folds) in SI was observed. An exact EC3 value could not be calculated because for such calculation, data immediately above and below SI value of 3 is required (Table 1).

 

Table 1: Results of individual data

Test item concentration % (w/v)

 

Measurement DPM

Calculation

SI

DPM-BG (a)

No. of lymph nodes

DPM per lymph node (b)

--

BG I

3

--

--

--

--

--

BG II

1

--

--

--

--

--

CG 5 (c)

5497

5495

8

687

--

2.5

TG2

121387

121385

8

15173

22.1*

5

TG3

172678

172676

8

21585

31.4*

10

TG4

165859

165857

8

20732

30.2*

2.5

TG5

146953

146951

8

18369

26.7

5

TG6

190092

190090

8

23761

34.6

10

TG8

212233

212231

8

26529

38.6

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

CG = Control Group

TG = 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

(c) = The date of vehicle control Group 5 was used to calculate S.I. The date of vehicle control Group 1 was not reported due to measurement problems occurred during evaluation

* The data was just as reference, because the S.I. value was calculated based on dpm/LN of vehicle control Group 5.

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
In this study STIMULATION INDICES of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). The test item was therefore found to be a strong sensitizer.
Executive summary:

In order to study a possible contact allergenic potential of the test substance, a GLP-compliant local lymph node assay according to OECD guideline 429 was performed. Six groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 5 % and 10 % (for Groups 2-4); 2.5 %, 5 % and 10 % (for Groups 6-8), (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. Two control groups of each four mice were treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labeled 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 ^H-methyl thymidine measured in a β-scintillation counter. No test item-related clinical signs were observed in any animals of the control groups (Groups 1 and 5), Group 2 (2.5 %), Group 3 (5 %), Group 4 (10 %) or Group 6 (2.5 %). On the third application day, a slight ear erythema was observed at both dosing sites in all mice of Group 7 (5 %) and Group 8 (10 %). On the second application day, a slight ear swelling was observed at both dosing sites in all mice of Group 8 (10 %). These signs persisted for the remainder of the in-life phase of the study. All treated animals survived the scheduled study period. In this study STIMULATION INDICES of 26.7, 34.6 and 38.6 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.). As a result, the test item was found to be a strong sensitizer.