Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March 2010 - ................
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

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
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): 1,2-DICHLOROETHANE
- Physical state: colourless liquid
- Analytical purity: 99.95%
- Impurities (identity and concentrations): organic impurities: 0.0473%
- Purity test date: 17 February 2010
- Lot/batch No.: R1104 du 17/02/2010
- Expiration date of the lot/batch: 17 February 2015
- Storage conditions of test material: at room temperature and protected from light, moisture and heat

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: 9 weeks old for the main test and 11 weeks old for the preliminary test
- Weight at study initiation: 21.2 +/- 0.9 g
- Housing: the animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm).
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): tap water (filtered usingi a 0.22 µm filter)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h

IN-LIFE DATES: From: 07 April 2010 To: 23 April 2010

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100%.
No. of animals per dose:
4 females per dose.
Details on study design:
PRELIMINARY TEST:
- Concentrations: 10, 25, 50 and 100%
- Irritation: no irritation whatever the concentration applied

MAIN STUDY
- Criteria used to consider a positive response: Stimulation Index > 3

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

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

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Group 2: test item 25%: 0.46 Group 3: test item 50%: 0.86 Group 4: test item 100%: 2.16 Group 5: HCA 25%: 6.65
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM per group: Group 1: vehicle: 1150.47 Group 2: test item 25%: 526.98 Group 3: test item 50%: 990.73 Group 4: test item 100%: 2484.29 Group 5: HCA 25%: 7650.27 DPM per node: Group 1: vehicle: 143.81 Group 2: test item 25%: 65.87 Group 3: test item 50%: 123.84 Group 4: test item 100%: 310.54 Group 5: HCA 25%: 956.28

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions of this study, the test item, 1,2-DICHLOROETHANE, (batch No. R1104 du 17/02/2010), did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

The potential of the test item, 1,2-DICHLOROETHANE, to induce delayed contact hypersensitivity, was evaluated using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel.

 

Methods

 

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 female CBA/J mice were allocated to five groups:

·          three treated groups of four animals receiving the test item at the concentration of 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, a-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 Stimulation 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.

 

Results

 

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%).

 

Neither mortality nor 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.

No notable lymphoproliferation was noted at any tested concentration.

 

The results are presented in the following table:

 

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

25

non-irritant

0.46

Test item

50

non-irritant

0.86

Test item

100

non-irritant

2.16

HCA

25

-

6.65

 

Conclusion

Under the experimental conditions of this study, the test item, 1,2-DICHLOROETHANE, (batch No. R1104 du 17/02/2010), did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.