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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:
14-02-2006 to 28-02-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented study following a method equivalent to a recognised guideline under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
482-160-5
EC Name:
-
Cas Number:
130786-09-3
Molecular formula:
C12H13N
IUPAC Name:
(2Z)-2-phenylhex-2-enenitrile
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: In refrigerator (2-8°C) in original container under nitrogen
- Other: Colour not stated.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: approximately 8 weeks old
- Weight at study initiation: 16.7 - 22.8 grams
- Housing: Group housed with up to 5 mice per cage during acclimation and then housed individually in plastic shoebox-style cages for remainder of study
- Diet: Free access to rodent diet (certified, recognised supplier)
- Water: mains tap water ad libitum
- Acclimation period: at least 5 days (actual: 6 days)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (actual: 19.8 to 21.4°C)
- Humidity (%): 30 to 70 (actual: 31 – 58%)
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 22-02-2006 To: 28-10-2011

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
- Main test: 0% (vehicle control), 1%, 5%, 10%, 20%, and 40% v/v in Acetone : Olive Oil (4:1). Test concentrations were determined by the sponsor (not documented within the full study report). Applicant assessment indicates: the limitation of test item % v/v was potentially based on the results of other studies and/or to limit dermal irritation and/or systemic toxicity.
No. of animals per dose:
Main test: 5 mice per dose group: 1%, 5%, 10%, 20%, and 40% v/v and 7 per 0% (vehicle control) group.
5 per dose group in concurrent positive controls.

Details on study design:
RANGE FINDING TESTS:
Not applicable.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Following excision of the nodes. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer. Refer to: OECD TG Guideline 429 (2010).

TREATMENT PREPARATION AND ADMINISTRATION:
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected by the study sponsor. One group of seven animals was treated with vehicle. One groups of five animals were treated with the concurrent positive control (Isoeugenol) and investigative positive control (Hydroxy citronellal), respectively.
- Induction: The dorsal surface of both ears was topically treated (25 μL/ear) with the test item concentration, at approximately the same time on each day. The control animals were treated in the same way as the experimental animals, except that the vehicle or PC items were administered instead of the test item. The animals were rested on days 4 and 5 without treatment.
- Node excision: Each mouse was injected in the lateral tail vein with 0.25 mL containing 2 μCi of 125-Iodine- labelled IuDR and 1x10^-5 M FuDR in phosphate-buffered saline (PBS). Approximately 5 hours later, the mice were euthanized. The auricular lymph nodes were excised. The nodes were dissociated using the frosted ends of glass slides. The cell suspension was washed with Hanks' balanced salt solution with HEPES and then with PBS prior to being resuspended in 5% trichloroacetic acid (TCA) and refrigerated at approximately 4°C. The following day, the cells were centrifuged and resuspended in fresh 5% TCA. The radioactivity was measured using a gamma counter.
- Tissue processing and radioactivity measurements: See above. The results from each cell suspension counted on the gamma counter were recorded in counts per minute (CPM). The CPM values were converted to disintegrations per minute (DPM) by dividing by the gamma counter efficiency and multiplying by 100. After the
DPM values had been calculated, the mean “blank” DPM was subtracted from each mouse DPM to obtain corrected DPM values. The mean corrected DPM and standard
error of the mean (SE) were determined for each group. The stimulation index (SI) was then calculated by dividing the treatment-group mean DPM by the vehicle control group mean DPM. Any questionable data points were evaluated with a Q-test to identify statistical outliers.

Observations:
- Mortality/Viability: Daily.
- Bodyweights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
- Clinical Observations: Once daily.
- Irritation: Daily.
- Ear Thickness: Ear measurements were taken prior to dosing on Days 1 and 3 using a micrometer. Percentage increases in group mean ear thickness from Day 1 to Day 3 were calculated and, if a 10%-or-greater increase occurred, the lowest concentration of test item eliciting that response (minimal irritating concentration or MIC10) was identified. If the MIC10 was greater than the EC-3, confounding irritation is unlikely to have affected the LLNA. If the MIC10 was less than the EC-3, the concentration of the chemical that elicited the increase may have produced an SI value close to 3 because of the physiology surrounding the primary irritation reaction. Full details provided in the full study report.
Positive control substance(s):
other: 1. Isoeugenol and 2. Hydroxycitronellal
Statistics:
1. Any questionable data points were evaluated with a Q-test to identify statistical outliers.
2. A one-sample t test was performed to test whether the individual untransformed SI value for each dose level of each compound was greater than 3. The natural log-transformed DPM values for each compound were compared against vehicle with a Bartlett’s chi-square test for variance homogeneity. If the Bartlett’s chi-square results were found to be nonsignificant, a one-way analysis of variance (ANOVA) was performed using dose (concentration). If the ANOVA was statistically significant, a Dunnett’s t-test was performed.
3. If the Barlett’s chi-square term was found to be significant, nonparametric analyses were conducted, specifically a Kruskal–Wallis test (KW). If the nonparametric analyses were statistically significant, then a Jonckheere–Terpstra (Jonckheere’s) test was performed for dose-dependent trends.

The data from the concentrations tested were fit using a quadratic equation (a linear term and a square term of the concentration). If the quadratic term did not fit (p < 0.05), a simple regression model was used. The EC3 was derived from the appropriate regression equation.

All calculations were performed using MS Excel and SAS (v9.1): using GLM, FREQ, NPAR1WAY, and MEANS procedures.

Results and discussion

Positive control results:
The sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, isoeugenol. The positive control was tested at concentrations 5% in Acetone/Olive oil (4:1 v/v). The PC tested showed a Stimulation Index (SI) of 6.8 ± 1.5 (at 5% v/v) and met the criteria for a 'positive' result. An EC3 value of 1.7% was calculated using linear regression. Applicant assessment: The calculated EC3 value was found to be in the acceptable range of 0.5 and 3.3% from OECD TG 429 (2010).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
EC3
Remarks:
%
Value:
10.1
Remarks on result:
other: See table below
Parameter:
SI
Value:
1.6
Variability:
± 0.2
Test group / Remarks:
1% in acetone:olive oil (4:1)
Parameter:
SI
Value:
1.6
Variability:
± 0.4
Test group / Remarks:
5% in acetone:olive oil (4:1)
Parameter:
SI
Value:
2.7
Variability:
± 0.3
Test group / Remarks:
10% in acetone:olive oil (4:1)
Parameter:
SI
Value:
3.7
Variability:
± 1.0
Test group / Remarks:
20% in acetone:olive oil (4:1)
Parameter:
SI
Value:
10.8
Variability:
± 2.6
Test group / Remarks:
40% in acetone:olive oil (4:1)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: See tables.

DETAILS ON STIMULATION INDEX CALCULATION: See tables.

EC3 CALCULATION: The 20% and 40% concentrations of test item have SI values greater than 3 (SI = 3.7 and 10.8, respectively), but only the 40% value is statistically significant. The linear model is used to determine an EC3 of 10.1%. Since the quadratic equation did not fit the (p < 0.05) criterion.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

OTHER: None of the treatment or control groups had a mean increase in ear thickness of 10% or more from Day 1 to Day 3.

Any other information on results incl. tables

Table 1.0 - Results from the definitive test

Number

Treatment Group

DPM

Mean DPM

Mean SI

Standard Error of Mean

1

Vehicle control, Acetone/olive oil, 4:1 (AOO)

27.6

29.1

0.0

0.0

2

40.2

 

 

 

3

25.0

 

 

 

4

45.8

 

 

 

5

26.3

 

 

 

6

28.8

 

 

 

7

10.1

 

 

 

 

 

 

 

 

 

8

Test Item

1% v/v

31.9

47.7

1.6

0.2

9

49.8

 

 

 

10

37.4

 

 

 

11

64.7

 

 

 

12

54.9

 

 

 

13

Test Item

5% v/v

27.8

46.0

1.6

0.4

14

70.0

 

 

 

15

42.0

 

 

 

16

17.2

 

 

 

17

73.1

 

 

 

18

Test Item

10% v/v

49.3

79.3

2.7

0.3

19

87.7

 

 

 

20

81.6

 

 

 

21

73.1

 

 

 

22

104.7

 

 

 

23

Test Item

20% v/v

131.7

107.7

3.7

1.0

24

80.4

 

 

 

25

171.6

 

 

 

26

141.3

 

 

 

27

13.4

 

 

 

28

Test Item

40% v/v

146.1

315.0

10.8

2.6

29

299.5

 

 

 

30

198.2

 

 

 

31

343.8

 

 

 

32

587.5

 

 

 

44

Isoeugenol

5% v/v

335.9

196.7

6.8

1.5

45

123.9

 

 

 

46

120.6

 

 

 

47

260.9

 

 

 

48

142.3

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test item is considered to be sensitising to skin with EC3 of 10.1%.
Executive summary:

The study was performed under GLP according to a method equivalent or similar to OECD TG 429, EU Method B.42 and US EPA OPPTS 870.2600 guidelines to assess the skin sensitisation potential of the test material in the CBA/J strain mouse following topical application to the dorsal surface of the ear. For the definitive test 0% (vehicle control), 1%, 5%, 10%, 20%, and 40% v/v in Acetone : Olive Oil (4:1) was utilised for treatment on three consecutive days by direct application on the ears to treatment groups of five or seven females for the test item and vehicle control groups, respectively. The investigational positive control hydroxycitronellal, tested at 15% and 60% in AOO, and the positive control isoeugenol (known sensitizer), tested at 5% in AOO were included concurrently. Treatment-group mean ear thickness values, which were taken on Days 1 and 3, did not increase by 10% or more at any tested concentration of the test or control articles. No mortality occurred, and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights were within the range seen for vehicle control animals. All relevant validity criteria were met and the positive control (isoeugenol, EC3 1.7%) validated the conditions of the assay. Mean DPM/animal values for the experimental groups treated with test item concentrations 0% (vehicle control), 1%, 5%, 10%, 20%, and 40% v/v were: 29.1, 47.7, 46.0, 79.3, 107.7 and 315.0 DPM respectively. SI values calculated for the equivalent concentrations were 0.0, 1.6, 1.6, 2.7, 3.7, 10.8, respectively. These results show that the test item elicits an SI ≥ 3. The 20% and 40% concentrations of test item have SI values greater than 3 (SI = 3.7 and 10.8, respectively), but only the 40% value is statistically significant. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between 10 and 20%. The point estimate based on linear regression of the test item EC3 is 10.1%. Under the conditions of this study, the test item would be considered to be classified under Regulation (EC) No 1272/2008 as skin sensitizer category 1B.

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