Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented study according to international accepted guidelines and GLP compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Test item: 2,3-DICHLOROBENZOYL NITRILE
CAS No.: 77668-42-9
Batch No.: L36020N
Physical state: Yellow, crystalline powder
Colour: Light-yellow
Purity (GC): 96.48% (based on 2,3-DICHLOROBENZOYL NITRILE content)
Storage 15-30°C

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/Ca mice
Source: TOXI-COOP ZRT.
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Number of animals: 28 animals/main test (4 animals/treatment group)
Sex: Female, nulliparous, non pregnant
Age of animals: Young adult mice; 10-11 weeks old (at start of the main test)
Body weight range at starting: 15.7-21.7 g
Acclimatization time: 7 days
Animal health: Only healthy animals were used

Housing during acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
AOO (acetone:olive oil, 4:1) for positive control groups
Concentration:
The test item was administered at four different concentrations (2,5; 1; 0,5; 0,25 w/v%) according to the results of the dose range finding test.
No. of animals per dose:
4 animal/dose
Details on study design:
Selection of test item concentrations was based on the results of a formulation evaluation and also results of preliminary irritation/toxicity tests to find the best solvent and the maximum applicable concentration according to the relevant guidelines. Due to significant adverse effects (systemic toxicity and/or irritation) observed at high test concentrations “2,3-DICHLOROBENZOYL NITRILE” (CAS No. 77668-42-9) was examined in the main test at 2.5 %, 1 %, 0.5 % and 0.25 % (w/v) non-toxic, non-irritant concentrations as formulations in the selected vehicle of N,N-Dimethylformamide (DMF). Appropriate positive control (α-Hexylcinnamaldehyde [HCA] in 25 % concentration), furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.
The positive control item (25 % HCA in Acetone: Olive oil 4:1 (v/v) mixture, AOO) induced the appropriate stimulation over the control, thus confirming the validity of the assay.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) HCA solution (dissolved in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
Significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 10.4). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Based on the results EC3 value (dose calculated to induce a stimulation index of 3) of the test item was calculated. Dose-response relationship was evaluated by linear regression. All calculations were made by Microsoft Excel Software. (See Table1 fo the values.)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPN (DPM divided by the number of pooled lymph nodes). (See Table1 fo the values.)

Any other information on results incl. tables

 Test Group Name

Measured

DPM/group

Group * DPM 

DPN

(DPM/Node)

 Stimulation Index Values

Vehicle control for the positive control:

AOO

8955

8890.0

1111.3

1.0

Positive control:

25% HCA in AOO 

92586

92521.0

11565.1

10.4

 2,3-DICHLOROBENZOYL NITRILE

2,5% in DMF

77283

77218.0

9652.3

5.8

 2,3-DICHLOROBENZOYL NITRILE

1% in DMF

29287

29222.0

3652.8

2.2

 2,3-DICHLOROBENZOYL NITRILE

0,5 % in DMF

12180

12115.0

1514.4

0.9

 2,3-DICHLOROBENZOYL NITRILE

0,25% in DMF

9858

9793.0

1224.1

0.7

 Vehicle control :for the test item:

DMF

13336

13271.0

1658.9

1.0

 *Group DPM = measured DPM(group)- average DPM(background)

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
In conclusion, under the conditions of the present Local Lymph Node Assay, “2,3-DICHLOROBENZOYL NITRILE” (CAS No. 77668-42-9) tested at the maximum applicable (non-toxic, non-irritant) concentration of 2.5 % (w/v) and at concentrations of 1 %, 0.5 % and 0.25 % as formulations in an appropriate vehicle (DMF) was shown to have sensitization potential.
Based on the EC3 value calculated using the dose-response curve “2,3-DICHLOROBENZOYL NITRILE” (CAS No. 77668-42-9) was classified as a moderate sensitizer in this LLNA according to the published data.