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Administrative data

Description of key information

Skin sensitisation (LLNA): not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jan - 11 Feb 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted Jul 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Type of study:
mouse local lymph node assay (LLNA)
Justification for non-LLNA method:
Test was done before LLNA as first-choice method for in-vivo testing was set into force.
Species:
mouse
Strain:
other: CBA/CA (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Bicester, UK
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 16 - 22 g
- Housing: the animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: 2014 Teklad Global Rodent diet (Harlan Teklad, Bicester, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
dimethylformamide
Concentration:
5, 10 and 25% w/w
No. of animals per dose:
5 females
Details on study design:
RANGE FINDING TESTS: in the preliminary screening test, 1 mouse was treated by daily application of 25 µL of the test substance at the maximum attainable concentration of 25% w/w in dimethylformamide, to the dorsal surface of each ear for 3 consecutive days (Day 1 - 3). The mouse was observed twice daily on Day 1 - 3 and once daily on Day 4 - 6. The body weight was recorded on Day 1 (prior to dosing) and on Day 6. No signs of systemic toxicity were reported and no adverse effects on body weight were noted.
- Compound solubility: the solubility of the test substance was assessed by mixing the test substance to a dilution of 25 and 50% with acetone/olive oil (4:1), dimethyl formamide, butanone, dimethyl sulphoxide, acetone, ethanol/distilled water (7:3) and 1% pluronic L92 in distilled water, respectively. The most suitable vehicle was used in the range-finding and main study.
- Irritation: there were no signs of irritation in the treated skin area.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: if the results indicate a SI ≥ 3, the test substance is considered to be a skin sensitiser

TREATMENT PREPARATION AND ADMINISTRATION:
A formulation of the test substance in dimethyl formamide was prepared within 2 hours before application. 25 µL was applied with a micropipette to the dorsal surface of both ears of the mice for 3 consecutive days. On Day 6, each animal was injected via the tail vein with 0.25 mL phosphate buffered saline containing 20 µCi of ³H-methyl thymidine (ARC UK Ltd., UK). After approximately 5 hours, the mice were sacrificed and the draining lymph nodes of the ears were excised. 1 mL PBS was added to the nodes of each animal and the nodes were processed per animal. A single cell suspension of lymph node cells was prepared in PBS by gentle mechanical disaggregation through a stainless steel gauze (diameter 200 µm). Lymph node cells were washed twice with an excess of PBS and the DNA precipitated with 5% trichloroacetic acid (TCA) at 4 ºC for approximately 18 hours. The precipitate was recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL Optiphase Trisafe as the scintillation fluid. The number of Disintegrations Per Minute (DPM) was measured using a Beckman LS6500 scintillation system (Beckman Instruments Inc., Fullerton, USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one-way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non-homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Positive control results:
The Stimulation Index for the positive control group used in the study was 6.46.

In addition, reliability checks are regularly performed by the testing laboratory in order to check the sensitivity of the test system and the reliability of the experimental techniques applied. The historical results were given in the study report. In November 2009 a study was performed (project number 0039/1116), in which groups of 5 female CBA mice were exposed to 15% alpha-hexylcinnamaldehyde in dimethyl formamide or the vehicle alone. The SI value for the positive control group was 5.16, showing the validity of the study.
Key result
Parameter:
SI
Value:
>= 1.13 - <= 1.45
Test group / Remarks:
Test group 5, 10 and 25%
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 350 - <= 471
Test group / Remarks:
Test group 5, 10 and 25%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index for the 5, 10 and 25% groups was 1.13, 1.45 and 1.34, respectively (see Table 1 and 2 under 'Ant other information on results incl. tables'). The mean DPM/animal values for the 5, 10 and 25% groups were 350, 389 and 471, respectively. The mean DPM/animal value for the control group was 352.

CLINICAL OBSERVATIONS:
No clinical signs were observed.

BODY WEIGHTS
The body weights and body weight gains were comparable between the control and the treatment groups.

Table 1: Radioactivity measurements, individual results

Dose group (%)

Animal

DPM/animal

Vehicle control

1

1306.85

Vehicle control

2

1425.22

Vehicle control

3

1781.19

Vehicle control

4

764.77

Vehicle control

5

812.59

5

1

1672.19

5

2

1102.57

5

3

1107.50

5

4

1489.06

5

5

1539.24

10

1

2257.42

10

2

1759.47

10

3

1833.76

10

4

1620.48

10

5

1358.38

25

1

1976.34

25

2

1442.97

25

3

1526.26

25

4

1809.78

25

5

1425.67

Positive control

1

9207.36

Positive control

2

12528.48

Positive control

3

6388.06

Positive control

4

6475.71

Positive control

5

4719.74

 

Table 2: Disintegrations per minute (DPM) and stimulation index (SI)

Dose group (%)

DPM (mean ± SD)

SI

Vehicle control

1218.12 ± 429.47

1.0

5

1382.11 ± 261.64

1.13

10

1765.90 ± 329.19

1.45

25

1636.20 ± 244.73

1.34

Positive control

7863.87 ± 3064.07

6.46

Interpretation of results:
other: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising potential of Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene was evaluated in a local lymph node assay (LLNA) performed according to OECD guideline 429 and under GLP conditions (Sanders, 2010). 25 µl of a 5, 10 and 25% w/w suspension of test substance in dimethylformamide was applied to the dorsal surface of both ears of 5 CBA mice/dose for 3 consecutive days. On Day 6, each animal was injected via the tail vein with 0.25 mL phosphate buffered saline containing 20 µCi of ³H-methyl thymidine. After approximately 5 hours, the mice were sacrificed and the draining lymph nodes of the ears were excised. The nodes of each animal were processed individually. PBS was added to the nodes and DNA was precipitated with 5% TCA at 4 °C for 18 hours. The number of disintegrations per minute (DPM) was determined by beta-scintillation count. The positive control group (hexyl cinnamic aldehyde) was treated concomitantly and shown to be valid. The mean DPM/animal values for the control, 5, 10 and 25% group were 352, 350, 389 and 471, respectively. The stimulation index (SI) calculated for the 5, 10 and 25% group was 1.13, 1.54 and 1.34, respectively. The SI was lower than 3 at all dose levels; therefore the test substance is considered to be not skin sensitising. 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.