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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 2010-02-19 to 2010-03-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted GLP guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cis 2-Pentene Nitrile
- Physical state: colorless liquid
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 12 weeks old on the first day of treatment period
- Weight at study initiation: 22.8 +/- 1.1 g
- Housing: individually, in disposal crystal polystyrene cages (22.00 cm * 8.50 cm * 8.00 cm)
- Diet: SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water (filtered using a 0.22 micron filter) ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 30 to 70 %
- Air changes: approximately 12 cycles per hour of filtered, non-recycled air
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2010-03-03 To: 2010-03-15

Study design: in vivo (non-LLNA)

Induction
Concentration / amount:
Not applicable
Challenge
Concentration / amount:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Challenge controls:
Not applicable

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
Acetone, batch No. K38945413830 (Merck, Chelles, France) and olive oil, batch No. 1223873 (Fluka, Saint Quentin Fallavier, France).
Concentration:
0, 5, 10, 25, 50 and 100 %
No. of animals per dose:
4 females per dose for the main test
Details on study design:
RANGE FINDING TESTS:
To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was performed on a small number of animals, as follows:
- the test item was prepared at the concentrations of 10, 25, 50 and 100%
- for 3 consecutive days, the animals received applications of 25 µL of the dosage form preparations to the external surface of both ears (one concentration per ear),
- measurement of the ear thickness (using a micrometer) was performed each day before treatment and 72 hours after the last application.
- Compound solubility: The test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at the maximum tested concentration of 50%.
- Irritation: The test item was non-irritant in the preliminary test, whatever the concentration.


MAIN STUDY
ANIMAL ASSIGNMENT
- Allocation to group: hand procedure
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer when the Similation Indice for a dose group is >= 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION
The concentrations were expressed in % (v/v). The test item was prepared at the chosen concentrations in AOO by successive dilutions. The dosage form preparations were homogenized by vortex. The reference item was dissolved in AOO at the concentration of 25% (v/v). All dosage form preparations were made freshly on the morning of administration and any unused material was discarded that same day.

TREATMENT ADMINISTRATION:
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Simulation Indices (SI).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data

Results and discussion

Positive control results:
In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and a stimulation index exceeding the
threshold value of 3 (SI = 6.35) were noted. The study was therefore considered valid.
See Table 7.4.1/1

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
A significant lymphoproliferation (SI > 3) was noted at the concentration of 100% (SI= 4.43). In the absence of local irritation, the significant lymphoproliferative response observed was attributed to delayed contact hypersensitivity. The EC3 value for the test item Cis 2-Pentene Nitrile is equal to 79%. See Table 7.4.1/1
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 7.4.1/1

Any other information on results incl. tables

Table 7.4.1/1: Results of the main test

Treatment and Concentration

Cell count

Viability %

Amounts of cells (x106 cells)

Cellularity index

N° of nodes per group

Dpm per group

Dpm per node

SI

Increase ear thickness (% between day 1 and 6)

Irritation level

EC3 value

Viable

Dead

Vehicle

95

22

81.20

9.50

 

8

850.54

105.07

 

3.96

 

79%

Test item 5%

202

42

82.79

20.20

2.13

8

657.35

82.17

0.78

2.97

I

Test item 10%

100

21

82.64

10.00

1.05

8

756.29

94.54

0.90

3.00

I

Test item 25%

116

13

89.92

11.60

1.22

8

639.02

79.88

0.76

6.00

I

Test item 50%

124

36

77.50

12.40

1.31

8

903.73

112.97.

1.08

2.00

I

Test item 100%

267

28

90.51

26.70

3.75

6

2794.97

465.83

4.43

4.04

I

HCA 25%

668

77

89.66

66.80

7.03

8

5334.38

666.80

6.35

 

 

SI = simulation index = dpm of treated group / dpm of control group

dpm = disintegration per minute

I = non-irritant (increase in ear thickness < 10 %)

EC value = theoretical concentration resulting in a SI value of 3

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information weak sensitizer
Conclusions:
Under the test conditions, the test substance is classified as a skin sensitizer Category 1 (H317 May cause an allergic skin reaction) according to the CLP regulation (1272/2008) and Xi; R43 (May cause sensitisation by skin contact) according to the Directive 67/548/CEE.
Executive summary:

In a dermal sensitization study performed according to the OECD test guideline No. 429 and in compliance with Good Laboratory Practice, cis-pentene-2-nitrile was tested in female CBA/J mice using the Local Lymph Node Assay.

 

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

 

In the main test, twenty eight mice were allocated to seven groups:

· five treated groups of four animals receiving the test item Cis 2-Pentene Nitrile at the concentration of 5, 10, 25, 50 or 100% in a mixture acetone/olive oil (4/1; v/v) (vehicle),

· one negative control group of four animals receiving the vehicle,

· one positive control group of four animals receiving the reference item, α‑hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in the vehicle.

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Simulation Indices (SI).

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

  

The test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at the maximum tested concentration of 50%. Consequently, the concentrations selected for the preliminary test were 10, 25, 50 and 100%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (100%).

 

No mortality related to treatment and no clinical signs were observed during the study.

No cutaneous reactions and no notable increase in ear thickness were observed in the animals of the treated groups.

A significant lymphoproliferation was noted in the positive control group given HCA at 25%, the study was therefore considered valid.

A significant lymphoproliferation (SI > 3) was noted at the concentration of 100% (SI = 4.43).

In the absence of local irritation, the significant lymphoproliferative response observed was attributed to delayed contact hypersensitivity.

The EC3value for the test item Cis 2-Pentene Nitrile is equal to 79%.

 

Under the experimental conditions of this study, the test item Cis 2-Pentene Nitrile induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3 value obtained in this experiment, the test item Cis 2-Pentene Nitrile should be considered as a weak sensitizer.

 

Under the test conditions, the test substance is classified as a skin sensitizer Category 1 (H317 May cause an allergic skin reaction) according to the CLP regulation (1272/2008) and Xi; R43 (May cause sensitisation by skin contact) according to the Directive 67/548/CEE.