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Description of key information

Not sensitising, Local Lymph Node Assay, OECD 429, Rattray 2004

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:
10 September 2003 to 16 September 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Young adults
- Weight at study initiation: 20.4 g to 23.3 g
- Housing: A maximum of 4 mice were housed per cage
- Diet: ad libitum
- Water: ad libitum, mains water
- Acclimation period: A minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 15 changes minimum
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial), 12 hours dark

IN-LIFE DATES: From: 10 September 2003 To: 16 September 2003
Vehicle:
dimethylformamide
Concentration:
1, 2.5 or 10 % w/v
No. of animals per dose:
Animals were tested in groups of four.
Details on study design:
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The results are expressed as disintegrations per minute (dpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test:control ratio for each concentration. The criterion for a positive response is that one or more concentrations of the test material should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. The assay is able to identify those materials that elicit responses in standard guinea pig test for skin sensitisation (Kimber et al 1994). Consequently, a test material which does not fulfil the above criterion is designated as unlikely to be a sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
A top dose of 10 % w/v of the test material was decided upon in order to maximise any potential sensitisation response without the risk of inducing a toxic response. Approximately 25 µL of 1 %, 2.5 % or 10 % w/v preparation of the test substance in DMF was applied using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using DMF alone. The procedure was repeated daily for 3 consecutive days.

Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing approximately 20 µCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining aurcular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a contained of PBS.

A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5 % w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4 °C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA.

The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to β-scintillation counting used a Packard Tri-Carb 2500TR Liquid Scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The application of hexylcinnamaldehyde at concentrations of 2.5 %, 5 % and 10 % w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at all three concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.
Key result
Parameter:
SI
Value:
0.74
Test group / Remarks:
1% w/v
Key result
Parameter:
SI
Value:
1.31
Test group / Remarks:
2.5% w/v
Key result
Parameter:
SI
Value:
1.11
Test group / Remarks:
10% w/v

Table 1: Results

Concentration (% w/v)

Number of lymph nodes assayed

Disintegrations per minute (dpm)

dpm per lymph node

Test control ratio

0 (vehicle only) Main test

8

3539

442

N/A

Test material

1

8

2598

325

0.74

2.5

8

4621

578

1.31

10

8

3908

489

1.11

0 (vehicle only) Positive control

8

2570

321

N/A

Hexylcinnamaldehyde

2.5

8

16088

2011

6.26

5

8

15659

1957

6.10

10

8

15611

1951

6.08

 

Table 2: Bodyweights

Dose

Animal number

Bodyweight (g)

Day 1

Day 6

0 % w/w (vehicle control)

1

21.9

22.7

2

20.9

21.1

3

20.4

20.9

4

21.5

23.2

1 % w/v test material

5

22.6

24.1

6

23.3

23.4

7

22.7

23.7

8

21.0

21.8

2.5 % w/v test material

9

23.2

23.5

10

21.1

21.0

11

20.4

22.9

12

19.5

20.6

10 % w/v test material

13

25.4

23.2

14

23.0

24.9

15

20.7

21.9

16

23.1

24.1

 

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
A top dose of 10 % w/v of the test material was decided upon in order to maximise any potential sensitisation response without the risk of inducing a toxic response. Under the conditions of the test, the isotope concentration was less than three-fold at all test concentrations and therefore, the test material is considered to be unlikely to be a skin sensitiser. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The skin sensitisation potential of the test material was determined in accordance with the standardised guidelines OECD 429 and EPA OPPTS 870.2600 using the mouse Local Lymph Node Assay. The test substance was applied as 1 %, 2.5 % or 10 % w/v preparation in dimethylformamide with a top dose of 10% w/v decided upon in order to maximise any potential sensitisation response without the risk of inducing a toxic response. The positive control was shown to have the capacity to cause skin sensitisation confirming the validity of the protocol used for this study. The isotope concentration induced by the test material was less than three-fold at all test concentrations and therefore the test material is considered to be unlikely to be a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitisation potential of the test material was determined in accordance with the standardised guidelines OECD 429 and EPA OPPTS 870.2600 using the mouse Local Lymph Node Assay. The test material was applied as 1 %, 2.5 % or 10 % w/v preparation in dimethylformamide with a top dose of 10% w/v decided upon in order to maximise any potential sensitisation response without the risk of inducing a toxic response. The positive control was shown to have the capacity to cause skin sensitisation confirming the validity of the protocol used for this study. The isotope concentration induced by the test material was less than three-fold at all test concentrations and therefore the test material is considered to be unlikely to be a skin sensitiser.

The study was performed in line with GLP and an accepted standardised guideline with a high standard of reporting. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.

The available data is considered to be complete and the conclusion, not sensitising, was taken forward for risk assessment.


Migrated from Short description of key information:
Not sensitising, female mouse, OECD 429, EPA OPPTS 870.2600, Rattray 2004

Justification for selection of skin sensitisation endpoint:
Only one study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material did not elicit a response in the Local Lymph Node Assay and therefore does not meet the criteria for classification as a skin sensitiser.