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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 August 12 to October 06, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
guideline study with acceptable restrictions GLP Study performed according to the old version of the OECD test guideline No. 429 (2002), therefore ear thickness measurements were not included in the pre-screen test. The substance being irritating to the skin, this deviation may have an impact on the reliability of the study results. However no visible signs of irritation were observed at any of the concentration tested, therefore the study results are considered as reliable. The rational for the choice of the maximal dose tested (30%) is not very clear and not well reported (technical reason linked to the physical state of the substance - solid - expected since no higher dose was tested in the pre-test).

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted 24 April 2002
Deviations:
yes
Remarks:
tested only up to 30% (rational not clear), no ear thickness measurements, Age: 6-7 weeks old instead of 8-12 weeks old. Humidity: 30-95% instead of 30-70%, no duration of acclimatation period, single caging, use of hair dryer.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on July 19 to 22, 2004 / signed on January 06, 2005
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one
EC Number:
249-320-4
EC Name:
7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one
Cas Number:
28940-11-6
Molecular formula:
C10H10O3
IUPAC Name:
7-methyl-2H-1,5-benzodioxepin-3(4H)-one
Test material form:
solid
Details on test material:
- Storage condition of test material: At room temperature, protected from light, tightly sealed
Specific details on test material used for the study:
- Physical state: White solid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, Horst, The Netherlands.
- Age at study initiation: 6-7 weeks (beginning of acclimatization)
- Weight at study initiation: 19.0 ± 1.1 g (mean)
- Housing: Animals were individually housed in Makrolon Type I, with wire mesh top
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: no duration reported

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-95 %
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: August 24, 2005 To: September 27, 2005

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main test: 1, 10 and 30 % w/v in acetone/olive oil 4:1 (v/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Soluble at 30% in AOO 4:1.
- Irritation: No irritation effects were observed at the concentrations of 1, 10 and 30% after a single application.
- Systemic toxicity: 30% is the highest achievable concentration whilst avoiding systemic toxicity and excessive local irritation.
- Ear thickness measurements: not measured
- Erythema scores: not reported

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: pooled treatment group approach, using tritiated (3H)-methyl thymidine, according to the OECD 429 test guideline.
- Criteria used to consider a positive response: A test item is regarded as a sensitiser 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 stimulation index.
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.
The decision to select a stimulation index (S.I.) of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

TREATMENT PREPARATION AND ADMINISTRATION:
- All formulations were prepared freshly before each dosing. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
- Groups of four mice were treated with the test material at concentrations of 1, 10 and 30 % w/v in acetone/olive oil 4:1. The mice were treated by daily application of 25 μL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). A further group of four mice received the vehicle alone in the same manner. A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
Each animal was injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 20.4 μCi of 3H-methyl thymidine (3HTdR) on Day 6.
After five hours, all animals were killed by intraperitoneal injection of Na-thiopental and the draining (auricular) lymph node of each ear was excised. The nodes from the four mice were excised and pooled for each experimental group. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle mechanical disaggregation through gauze. LNC were washed two times with PBS. To precipitate out the radioactive material, the LNC were resuspended in 3 mL of 5 % Trichloroacetic acid (TCA) and incubated approximately 18 h incubation at approximately 4 °C. The precipitate was then resuspended in 1 mL of 5% TCA and transferred to 10 mL of scintillation fluid (Ultima gold) and thoroughly mixed. 3HTdR incorporation was measured bon a β-scintillation counter. Similarly background 3HTdR levels were also measured in two aliqots of 5% TCA.
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
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.
- 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 lymph 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 S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
α-hexylcinnamaldehyde at 25 % induced skin sensitisation (SI = 4.0)

In vivo (LLNA)

Results
Key result
Parameter:
SI
Value:
< 3
Test group / Remarks:
Stimulation index for 1, 10 and 30 % were 0.83, 0.83 and 1.25, respectively.
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
DPM / group for vehicle, 1, 10 and 30 % were 6293.56, 5225.82, 5253.89 and 7843.85, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 1, 10 and 30 % were 0.83, 0.83 and 1.25, respectively.

EC3 CALCULATION
The EC3 value could not be calculated wince all SI's are below 3.

CLINICAL OBSERVATIONS:
Mortality / Viability: No deaths occurred during the study period.
Clinical signs (local / systemic): No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals recorded was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Table 7.4.1/1: Results of skin sensitisation

Test item

Concentration %(w/v)

Group

Measurement

DPM

Calculation

 

 

Result

DPM-BGa

number of

lymph nodes

DPM per

lymph nodeb

S.I.

-

BG I

24.60

-

-

-

-

-

BG II

23.53

-

-

-

-

-

CG 1

6293.56

6269.5

8

783.7

 NA

1

TG 2

5225.82

5201.8

8

650.2

0.83

10

TG 3

5253.89

5229.8

8

653.7

0.83

30

TG 4

7843.85

7819.8

8

977.5

1.25

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

The EC3 Value could not be calculated, since all SI’s were below 3.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test material is not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/CaOlaHsd strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following a preliminary screening test in which no clinical signs of toxicity and no signs of local irritation were noted at the achievable concentrations of 0.5, 1, 10 and 30 % (w/v), the dose levels of 1, 10 and 30% (w/v) were selected for the main test.

 

The study comprised three groups, each comprising four female animals, were treated with 50 μl (25 μl per ear) of test material as a solution in acetone/olive oil 4:1 at concentrations of 1, 10 and 30 % (w/v) for 3 consecutive days. A further control group of four animals was treated with acetone/olive oil 4:1 alone. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per group and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

 

The SI obtained for 1, 10 and 30% v/v test item were 0.83, 0.83 and 1.25, respectively, which indicates that test item did not show the potential to induce skin sensitization. The EC3 value was therefore not calculated. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.  Bodyweight changes of the test animals between prior to the first application and prior to treatment with 3HTdR were comparable to those observed in the corresponding control group animals over the same period.

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 4.00, when tested at 25 % w/v. The test system was therefore considered to be valid.

 

Under the test conditions, test material is not classified as a skin sensitiser in the Local Lymph Node Assay according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.