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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:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented report of a guideline study conducted to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
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): Dimethyldiallylammoniumchlorid ca 60%
- Molecular formula (if other than submission substance): C8H16N.Cl
- Molecular weight (if other than submission substance): 161.68
- Smiles notation (if other than submission substance): C(=C)CN{+}(c)(c)(.Cl{-})CC=C
- InChl (if other than submission substance): 1/C8H16N.ClH/c1-5-7-9(3,4)8-6-2;/h5-6H,1-2,7-8H2,3-4H3;1H/q+1;/p-1
- Physical state: liquid (aqueous solution)
- Analytical purity:
- Composition of test material, percentage of components: 60% DADMAC in Water
- Lot/batch No.: R950 Partie 15
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, FRG
- Age at study initiation: 10 – 11 weeks
- Weight at study initiation: 16.7 g – 22.7 g
- Housing: Makrolon type I cages, Granulat Typ ¾ (staubfrei); SSNIFF
- Diet (e.g. ad libitum): ad libitum (Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland)
- Water (e.g. ad libitum): ad libitum (Tap water)
- Acclimation period: Acclimatization period (8 days before the first test-substance application)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 30 – 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
3%, 10% or 30% in propylene glycol
No. of animals per dose:
6
Details on study design:
Conduct of the study: 3 treatment groups, a vehicle control group and a concurrent untreated control group. Each group consisted of 6 mice.
Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages.
Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
Signs and symptoms: No detailed clinical examination of individual animals performed but obvious signs of systemic toxicity and/or local inflammation at application sites were noted in raw data.
Form of application: Epicutaneous.
Application volume: 25 µL per ear.
Site of application: Dorsal part of both ears
Frequency of application: 3 consecutive applications (day 0-2) to same application site. Animals of control group 0 remained untreated. Animals of control group 1 and test groups 2-4 were treated with vehicle or test-substance preparation.
Mortality: Twice each workday and once on Saturdays, Sundays and on public holidays.
Sacrifice: On study day 5 by CO2 inhalation.
Determination of ear weight: Immediately after death a circular piece of tissue (diameter 0.5 cm) was punched out of the apical part of each ear and weighed immediately after removal
Preparation of cell suspension and determination of cell count: Pooled lymph nodes of each animal were stored in phosphate buffered saline in an icewater bath until further preparation. A single cell suspension was prepared as soon as possible after dissection. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. Cell count was determined using a Casy®-Counter. Each suspension was measured in triplicate (3 dilutions of the standard suspension). The mean of the triplicate measurements was used for further evaluation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of cell count, lymph node weight and ear weight were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group. Cell count, lymph node weight, ear weight and body weight change were performed according to the Wilcoxon test.

Results and discussion

Positive control results:
No signs of systemic toxicity were noted. The test substance induced a statistically significant and biologically relevant auricular lymph node response when applied as 3%, 10% or 30% preparations in acetone to the ear of the mice. The ear weight indicated an irritant property of the test substance at these concentrations. However, only the irritation induced by high concentration (30%) was of a magnitude, which might have interfered with the lymph node response. The increased ear weights at the mid and low concentration (10% and 3%), the latter of which was not statistically different from the historical control data, indicate minimal irritation which is not considered to be of relevance for the lymph node response. Based on the results discussed above it is concluded that Alpha-Hexylcinnamaldehyde, techn. 85% has a sensitizing effect in the murine Local Lymph Node Assay under the test conditions chosen.

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
When applied as 3%, 10% and 30% test-substance preparations in propylene glycol, the test substance did not induce a biologically relevant (increase to 1.5 fold or above the vehicle control value = stimulation index (SI) = 1.5) or statistically significant response in the auricular lymph node cell counts. The statistical significance of the increase in lymph node weights observed after application of the 30% preparation was lost when compared to historical control data. In addition it was not reflected in a concomitant increase in cell counts and its magnitude is not considered to be of biological relevance.

Any other information on results incl. tables

No signs of systemic toxicity were observed. The expected body weight gain was generally observed in the course of the study. Increases in ear weights were not biologically relevant and not statistically significant at all concentrations.

CELL COUNT AND LYMPH NODE WEIGHT: TEST GROUP MEAN VALUES,
STANDARD DEVIATIONS AND STIMULATION INDICES

Test Group

Treatment

Cell Counts

Counts/Lymph Node Pair

Stimulation
Index (1)

Mean

S.D.

0

Untreated

8,385,000

924,208

1.13

1

vehicle propylene glycol

7,436,333

1,244,042

1.00

2

3% in propylene glycol

7,392,333

384,120

0.99

3

10% in propylene glycol

6,067,667

958,745

0.82

4

30% in propylene glycol

6,642,000

1,556,155

0.89

Test Group

Treatment

Lymph Node Weight

mg/Lymph Node pair

Stimulation
Index (1)

Mean

S.D.

0

Untreated

4.5

0.9

1.07

1

vehicle propylene glycol

4.2

1.1

1.00

2

3% in propylene glycol

4.5

0.6

1.06

3

10% in propylene glycol

4.4

0.5

1.04

4

30% in propylene glycol

4.9

1.8

1.16 #

EAR WEIGHT: TEST GROUP MEAN VALUES, STANDARD DEVIATIONS
AND STIMULATION INDICES

Test Group

Treatment

Ear Weight

mg/animal

Stimulation
Index (1)

Mean

S.D.

0

Untreated

21.6

0.8

0.99

1

vehicle propylene glycol

21.7

1.1

1.00

2

3% in propylene glycol

22.1

0.6

1.02

3

10% in propylene glycol

21.3

0.5

0.98

4

30% in propylene glycol

21.1

1.8

0.97

(1): test group x / test group 1 (vehicle control)

# = statistically significant for the value p=0.05

## = statistically significant for the value p=0.01

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Dimethyldiallylammonium chloride ca. 60% does not have a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions.