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Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-10-25 to 2012-11-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according guideline (OECD 429) under GLP

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
DMI pure
IUPAC Name:
DMI pure
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): DMI pure or test item
- Description: Pale yellow liquid
- Analytical purity: 99.6%
- Lot/batch No.: AN2012-0131
- Expiration date of the lot/batch: 2014-04-05
- Storage condition of test material: Approximately 4 °C in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
2.5, 5, 10, 25 or 50% (v/v)
No. of animals per dose:
5
Details on study design:
Preliminary Screening Test

- Compound solubility: Homogeneity in the vehicle (dimethylformamide) obtained to visually acceptable levels.
- Irritation: tested at 50% (v/v) with 1 animal respectively, daily application of 0.025 mL to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on days 1, 2 and 3 and once daily on days 4, 5 and 6. Local skin irritation was scored daily according to the scale below:
No erythema (score 0)
Very slight erythema (barely perceptible) (score 1)
Well-defined erythema (score 2)
Moderate to severe erythema (score 3)
Severe erythema (beef redness) to eschar formation preventing grading of erythema (score 4)

Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on day 1 (prior to dosing) and on day 6. The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre dose on day 1, post dose on day 3 and on day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods days 1 to 3 and days 1 to 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Main Test

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of five mice were treated with the test item at concentrations of 50%, 25%, 10%, 5% or 2.5% (v/v) in dimethyl formamide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of five mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:80 µCi/mL, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations:

Clinical Observations: All animals were observed twice daily on days 1, 2 and 3 and on a daily basis on days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Bodyweights: The bodyweight of each mouse was recorded on day 1 (prior to dosing) and day 6 (prior to termination).

Terminal Procedures:

Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by beta-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, 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. Probability values (p) are presented as follows:

P<0.001 ***
P<0.01 **
P<0.05 *
P>=0.05 (not significant)

Interpretation of Results:

The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".
The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is: EC3 = c + [[(3-d)/(b-d)] x (a-c)]

Results and discussion

Positive control results:
A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals No. 429 and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.

Test Item: α-Hexylcinnamaldehyde, tech., 85%
Project number: 41202697
Study dates: 16 May 2012 to 22 May 2012

Methods: A group of five animals was treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in dimethyl formamide at a concentration of 15% (v/v). A further control group of five animals was treated with dimethyl formamide alone.

Results: The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

Concentration: 15% ( v/v) in dimethyl formamide
Stimulation Index: 5.74
Result: positive

Conclusion:α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: See other information on results incl. tables.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See other information on results incl. tables.

Any other information on results incl. tables

Individual Disintegrations per Minute and Stimulation Indices:

Concentration
(% v/v) in
dimethyl formamide

Animal Number

dpm/
Animal
a

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle

1-1

1532.18

1578.34
(±66.33)

na

na

1-2

1693.99

1-3

1555.72

1-4

1570.48

1-5

1539.34

2.5

2-1

1110.90

2697.36
(±1090.64)

1.71

negative

2-2

3628.44

2-3

3519.32

2-4

2028.96

2-5

3199.17

5

3-1

6732.02

6575.66
(±1641.35)

4.17

positive

3-2

6781.32

3-3

4884.87

3-4

5373.53

3-5

9106.57

 10

4-1

17826.37

12906.74***
(±3206.16)

8.18

positive

4-2

10132.52

4-3

14312.02

4-4

10445.72

4-5

11817.09

25

5-1

8934.68

11441.51***
(±5721.95)

7.25

positive

5-2

20117.12

5-3

13594.03

5-4

5027.27

5-5

9534.47

 50

6-1

8243.93

5638.66
(±3346.30)

3.57

positive

6-2

2926.30

6-3

10194.16

6-4

3414.53

6-5

3414.40

dpm=Disintegrations per minute

a=     Total number of lymph nodes per animal is 2

b=     Stimulation Index of 3.0 or greater indicates a positive result

na=     Not applicable

*** =   Significantly different from control group p<0.001

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%v/v) in
dimethyl formamide

Stimulation Index

Result

2.5

1.71

negative

5

4.17

positive

10

8.18

positive

25

7.25

positive

50

3.57

positive

Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test:

Concentration
(% 
v/v) in
dimethylformamide

Animal Number

Bodyweight (g)

Clinical Observations

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

50

S-1

22

21

0

0

0

0

0

0

0

0

0

0=     No signs of systemic toxicity

Local Skin Irritation – Preliminary Screening Test:

Concentration
(% 
v/v) in
dimethylformamide

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

50

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test:

Concentration
(% 
v/v) in
dimethyl

formamide

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

50

S-1

0.220

0.230

0.240

0.235

0.225

0.230

overall mean (mm)

0.225

0.238

0.228

overall mean
ear thickness change (%)

na

5.556

1.111

na = not applicable

Individual Clinical Observations and Mortality Data:

Concentration
(% v/v) in
dimethylformamide

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Vehicle

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

1-5

0

0

0

0

0

0

0

0

0

2.5

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

2-5

0

0

0

0

0

0

0

0

0

5

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

3-5

0

0

0

0

0

0

0

0

0

10

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0

4-5

0

0

0

0

0

0

0

0

0

25

5-1

0

0

0

0

0

0

0

0

0

5-2

0

0

0

0

0

0

0

0

0

5-3

0

0

0

0

0

0

0

0

0

5-4

0

0

0

0

0

0

0

0

0

5-5

0

0

0

0

0

0

0

0

0

50

6-1

0

0

0

0

0

0

0

0

0

6-2

0

0

0

0

0

0

0

0

0

6-3

0

0

0

0

0

0

0

0

0

6-4

0

0

0

0

0

0

0

0

0

6-5

0

0

0

0

0

0

0

0

0

0=No signs of systemic toxicity

Individual Bodyweights and Bodyweight Changes:

Concentration
(% v/v) in
dimethyl formamide

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

17

18

1

1-2

19

20

1

1-3

18

22

4

1-4

20

21

1

1-5

19

20

1

2.5

2-1

18

20

2

2-2

20

21

1

2-3

19

21

2

2-4

19

19

0

2-5

19

20

1

5

3-1

17

19

2

3-2

20

20

0

3-3

18

19

1

3-4

18

20

2

3-5

19

20

1

10

4-1

18

19

1

4-2

20

21

1

4-3

19

21

2

4-4

17

18

1

4-5

17

17

0

25

5-1

21

21

0

5-2

19

21

2

5-3

20

21

1

5-4

20

21

1

5-5

21

22

1

50

6-1

19

21

2

6-2

20

19

-1

6-3

19

20

1

6-4

18

19

1

6-5

19

20

1

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information sub-category 1B Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a sensitiser under the conditions of the test.


Executive summary:

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the following: - OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) - Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008 Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% (v/v), this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Five groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in dimethyl formamide at concentrations of 50%, 25%, 10%, 5% or 2.5% (v/v). A further group of five animals was treated with dimethyl formamide alone. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%v/v) in
dimethyl formamide

Stimulation Index

Result

2.5

1.71

negative

5

4.17

positive

10

8.18

positive

25

7.25

positive

50

3.57

positive

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 3.81%.