Registration Dossier

Administrative data

Description of key information

The test item is considered not to be a skin sensitiser.

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:
4 June 2003 to 10 June 2003
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:
OECD guideline reference 429 (2002): Skin sensitisation: Local Lymph Node Assay
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
United States Environmental Protection Agency, Health Effects Test Guidelines, OPPTS 870.2600 (1998): Skin Sensitisation.
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Species: Mouse
Strain: CBA/Ca/Ola/Hsd
Source: Harlan Interfauna UK Limited, Blackthome, Bicester, Oxon, UK
Sex: Female
Number used: 4 per group
Specification: Young adults

Accommodation and husbandry
A maximum of 4 mice was housed per cage, in cages suitable for animals of this strain and weight range.
The animal room was designed to give the environmental conditions shown as follows.
Temperature: 22 ± 3 °C
Relative humidity: 30-70%
Air: A minimum of 15 changes per hour
Light cycle: Artificial, giving 12 hours light, 12 hours dark

Both temperature and relative humidity were recorded daily. The recorded values were within the specified ranges.
Diet (RM1), supplied by Special Diet Services Limited, Witham, Essex, UK, and mains water, supplied by an automatic system, were available ad libitum.
Each batch of diet is routinely analysed for composition and for contaminants. Water is also periodically analysed for contaminants. No contaminants were found in the diet or water at levels considered likely to interfere with the purpose or outcome of the study.

Acclimatisation
The animals were housed under the experimental conditions for at least 5 days, prior to the start of dosing.
Vehicle:
propylene glycol
Concentration:
Approximately 25 μl of a 5, 10 or 30% w/v preparation of the test substance in propylene glycol was applied
No. of animals per dose:
Four female mice per dose.
Details on study design:
Justification for test system selection
The CBA/Ca mouse is the species and strain of choice because previous examination of strain difference in lymphocyte proliferation responses to skin sensitisers showed this strain of mouse to exhibit the most vigorous response (Kimber et al 1994).

Dose preparations
All dose preparations were used within 24 hours of preparation.

Test method for the evaluation of C.I. Disperse Red 82
Groups of four female mice were used for this study. Approximately 25 μl of a 5, 10 or 30% w/v preparation of the test substance in propylene glycol was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using propylene glycol 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 μl Ci 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 auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container 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 using a Packard Tri-Carb 2500TR Liquid Scintillation Counter.

Clinical observations
Animals were checked at least once daily for signs of systemic toxicity.

Positive control study
The sensitisation potential of hexylcinnamaldehyde was assessed using the method described above.
Approximately 25 μl of a 2.5%, 5% or 10% w/v preparation of hexylcinnamaldehyde in acetone was applied, and a vehicle control group was similarly treated using acetone.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The results are expressed as a counts 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 substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.
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:
0.78
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
0.85
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.08
Test group / Remarks:
30%
Cellular proliferation data / Observations:
Application of C.l Disperse Red 82 in propylene glycol did not result in an increase in isotope incorporation which was greater than 3-fold at concentrations up to and including 30% w/v. Consequently, the test substance was considered unlikely to be a skin sensitiser.

SKIN SENSITISATION POTENTIAL OF C.I. DISPERSE RED 82

Concentration of test substance

(% w/v)

Number of lymph nodes assayed

Disintegrations per minute

(dpm)

dpm per lymph node

(x10-2)

Test control ratio

0 (vehicle only)

8

3538

4.42

N/A

5% w/v

8

2765

3.46

0.78

10% w/v

8

3018

3.77

0.85

30% w/v

8

3809

4.76

1.08

N/A – not applicable

 

SENSITISATION POTENTIAL OF THE POSITIVE CONTROL SUBSTANCE (HEXYLCINNAMALDEHYDE)

Current CTL Study No.: GM7744

Concentration of hexylcinnamaldehyde

(% w/v)

Number of lymph nodes assayed

Disintegrations per minute

(dpm)

dpm per lymph node

(x10-2)

Test control ratio

0 (vehicle only)

8

2570

3.21

N/A

2.5

8

16088

20.11

6.26

5

8

15659

19.57

6.10

10

8

15611

19.51

6.08

N/A – not applicable

 

BODYWEIGHTS (g)

GROUP: 1            COMPOUND: Y01015/040

DOSE: 0% w/w

ANIMAL NUMBER

DAY

1

DAY

6

FEMALES

17

18.8

19.9

18

16.1

17.6

19

16.7

19.1

20

17.1

18.8

 

GROUP: 2            COMPOUND: Y12512/001

DOSE: 5% w/w

ANIMAL NUMBER

DAY

1

DAY

6

FEMALES

21

17.2

18.5

22

16.7

18.6

23

18.2

20.0

24

16.1

17.4

 

GROUP: 3            COMPOUND: Y12512/001

DOSE: 10% w/w

ANIMAL NUMBER

DAY

1

DAY

6

FEMALES

25

15.8

17.8

26

16.7

18.7

27

16.2

17.1

28

18.6

20.5

 

GROUP: 4            COMPOUND: Y12512/001

DOSE: 30% w/w

ANIMAL NUMBER

DAY

1

DAY

6

FEMALES

29

17.7

18.8

30

16.8

18.5

31

17.7

19.4

32

17.0

18.4

 

Interpretation of results:
GHS criteria not met
Conclusions:
C.l. Disperse Red 82 was shown not to have the capacity to cause skin sensitisation when applied as a preparation in propylene glycol at a concentrations up to and including 30% w/v.
Executive summary:

Study design

A sample of C.I. Disperse Red 82 was assessed for its skin sensitisation potential using the mouse Local Lymph Node Assay. The assay determines the level of T lymphocyte proliferation in the lymph nodes draining the site of chemical application, by measuring the amount of radiolabelled thymidine incorporated into the dividing cells. The test substance was applied as 5, 10 or 30% w/v preparations in propylene glycol.

 

Results

C.I. Disperse Red 82 was shown not to have the capacity to cause skin sensitisation when applied as a preparation in propylene glycol at a concentrations up to and including 30% w/v.

In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as 2.5%, 5% or 10% w/v preparations in acetone, confirming the validity of the protocol used for this study.

 

Conclusion

C.l. Disperse Red 82 was shown not to have the capacity to cause skin sensitisation when applied as a preparation in propylene glycol at a concentrations up to and including 30% w/v..

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

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 429 (local lymph node assay) in compliance with GLP. The assay determines the level of T lymphocyte proliferation in the lymph nodes draining the site of chemical application, by measuring the amount of radiolabelled thymidine incorporated into the dividing cells. The test substance was applied as 5, 10 or 30% w/v preparations in propylene glycol. C.I. Disperse Red 82 was shown not to have the capacity to cause skin sensitisation when applied as a preparation in propylene glycol at a concentrations up to and including 30% w/v. In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as 2.5%, 5% or 10% w/v preparations in acetone, confirming the validity of the protocol used for this study. Under the study conditions, the test substance was considered to be not sensitizing to mouse skin.

 

In an earlier study with the same batch of the test substance, the skin sensitisation potential of the test substance was tested according to OECD Guideline 406 and EU Method B.6 (Guinea pig maximization test), in compliance with GLP. Albino guinea-pigs were exposed to the test substance at a concentration of 15% (in vaseline). Several steps were conducted: a pre-test (intradermal injections - epidermal application) and a main test. The pre-test was carried out to identify a maximally tolerated concentration of the test substance suitable for the induction phase of the main study. A concentration of 5% in paraffin oil was selected for intradermal induction, concentrations of 25% and 15% in vaseline was selected for dermal induction and challenge, respectively. However, as the test item stained the skin deep violet, no clear reading could be achieved in the pre-test for intradermal induction. Also after the induction phase, evaluation of the skin for irritating effects was not possible due to the dark violet staining. Approximately 22 h prior to the epidermal induction, the test sites of the animals were pretreated with a 10% SLS solution in paraffinum perliquidum. Two weeks after the epidermal induction application the challenge was completed by epidermal application of the test substance at 15% in vaseline under occlusive dressing (clipped, shaved skin). The animals of the control group were induced with bi-distilled water and FCA/physiological saline, pretreated with 10% SLS and challenged similarly to those of the test group. For evaluation of the skin after dermal challenge, the discoloured skin was depilated with Veet Cream 21 hours after removal of the dressing. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 h after removal of the dressing. No erythematous or oedematous reaction was observed in the control animals Positive reactions were observed in 74% and 100% of the animals at the 24 and 48 h readings, respectively, when treated with the test substance at 15% in vaseline.

The Guinea Pig Maximisation Test (GPMT) was disregarded as unreliable in this evaluation based on the clear negative results of the Local Lymph Node Assay (LLNA). This is based on the fact that

- the LLNA with the same batch (ca 93% active ingredient) did not lead to any evidence for sensitising effects at concentrations of 5, 10 or 30%.

- due to the deep violet discolouration of the skin it could not clearly evaluated that the correct test substance concentrations were selected

- the use of SLS causes often false positive results in the GPMT

- the substance treated areas were depilated with Veet Cream to remove the red staining of the test substance which prevented erythema evaluation - this cream is also able to cause sensitising effects

- with 100% positive reaction in the treatment group, the LLNA, which was validated based on the GPMT should show also a positive result if the substance had skin sensitising properties.

 

 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The results of the above studies do not trigger a classification for skin sensitisation according to CLP (EC 1272/2008) criteria.