2-[N-(2-cyanoethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]anilino]ethyl acetate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From May 13, 2002 to May 21, 2002

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:

yes

Remarks:

male mice instead of female mice were used; groupe housing instead of single housing; no body weight measurement

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan, Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Sex: male
- Age at study initiation: young adults
- Weight at study initiation:
- Housing: 4/cage
- Diet (e.g. ad libitum): RM1 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 d
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Air changes (per hr): minimum 15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: The main study was initiated on 13 May 2002. The experimental phase started on 14 May 2002 and was completed on 21 May 2002.
For the positive control study, the experimental phase started on 18 September 2001 and was completed on 25 September 2001.

Vehicle:

other: acetone

Concentration:

3%, 10% and 50% w/v

No. of animals per dose:

groups of 4 males

Details on study design:

Groups of four male mice were used for this study. Approximately 25 µL of a 3%, 10% or 30% w/v preparation of the test substance in acetone was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all animals were injected, via the tail, with approximately 250 µL of PBS containing about 20 µCi of a 2.0Ci/mmol specific activity 3H-methyl thymidine (for beta-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation Counter). After 5 d, the animals were sacrificed. 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.

The results are expressed as a counts per minute (cpm) value per lymph node for each group. The stimulation index for each test group is then calculated by dividing the cpm value per lymph node by the equivalent value for the control (vehicle only) group.
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. The assay is able to identify those materials that elicit responses in standard guinea pig tests for skin sensitisation (Kimber et al 1994). Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a sensitiser.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

The application of hexylcinnamaldehyde at concentrations of 1, 3 and 10% w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at all three concentrations.

The validity of the protocol was confirmed.

Key result

Parameter:

SI

Value:

0.83

Test group / Remarks:

3% of test substance

Key result

Parameter:

SI

Value:

1.19

Test group / Remarks:

10% of test substance

Key result

Parameter:

SI

Value:

1.06

Test group / Remarks:

30% of test substance

Cellular proliferation data / Observations:

The application of the test substance at concentrations of 3%, 10% and 30% w/v in acetone resulted in an increase in isotope incorporation which was less than 3-fold at all three concentrations.

Concentration of test substance (% w/v)	Number of lymph nodes assayed	Counts per minute (cpm)	cpm per lymph node (x10^-2)	Test control ratio
0 (vehicle only)	8	1092	1.37	N/A
3	8	913	1.14	0.83
10	8	1303	1.63	1.19
30	8	1160	1.45	1.06

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was considered to be not sensitizing to mouse skin (LLNA).

Executive summary:

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. Male CBA/Ca mice were exposed to the test substance at concentrations of 0, 3, 10 and 30% in acetone. Isotope (3H-methyl thymidine) incorporation was measured in lymph nodes. The negative (vehicle alone) and positive (hexylcinnamaldehyde at concentrations of 1, 3, and 10% w/v in acetone) controls were valid. The isotope incorporation was increased by less than a threefold at all concentrations of the test substance. Under the study conditions, the test substance was considered to be not sensitizing to mouse skin (Johnson, 2002).

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Publication

Principles of method if other than guideline:

The study was conducted following two protocols: sensitisation and sensitisation-challenge protocol.

GLP compliance:

not specified

Remarks:

The experimental study was published in a journal, however, the GLP status of the study was not specified by the author.

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

Source: Winkelmann, Borchen, Germany
Age: 7 weeks old
Temperature and relative humidity: 23+/-1°C and 45-55%, respectively
Light: artificial from 9:00 am to 9:00 pm
Feed and water: Altromin pellet feed and tap water, ad libitum

Vehicle:

dimethyl sulphoxide

Concentration:

10 and 30%

No. of animals per dose:

6

Details on study design:

1. Sensitisation protocol:

Animals received 25 µL of the test solution on the dorsum of the left ear for 3 consecutive days. The right ears were treated with equal volume of DMSO. 48 h after the last exposure, mice were euthanised. Ear thickness was measured, draining auricular lymph nodes were excised and weighed, and cell suspensions were prepared to count the number of cells.

2. Sensitisation-challenge protocol:
Animals were shaved over a surface of approximately 2 cm2 on their back. This surface was treated once daily on Days 1-3 with 50 µL of the test solution. Mice remained untreated on Days 4-14 and were then challenged with 25 µL of the test solution on Days 15-17. 48 h after the last exposure, mice were euthanised. Ear thickness was measured, draining auricular lymph nodes were excised and weighed, and cell suspensions were prepared to count the number of cells.

- Lymphocyte subpopulations (T-cells and B-cells, CD69 and 1A epitopes) were also analysed by flow cytometry.

-Results obtained in the treated mice were compared to those obtained with the vehicle alone.

Statistics:

In assays according to the sensitisation protocol, the treated left ears and lymph nodes were compared to the respective vehicle treated right ears and lymph nodes with regard to ear thickness, lymph node weight, lymph node cellularity and flow cytometric data using a paired non-parametric test, the Wilcoxon test.
In assays performed according to the sensitisation-challenge protocol, where both ears were treated, mean values were calculated for the ear thickness, lymph node weight and lymph node cellularity of each animal. Results were compared to those obtained from DMSO-treated control animals using the non-parametric Mann–Whitney test.
Statistical analysis was done with the software SPSS 10.0 (SPSS Inc., Chicago, USA). Statistical significance was assumed when two-sided P < 0.05.

Positive control results:

-

Key result

Parameter:

other: lymph node weight and cellularity, ear thickness, lymphocytes subpopulations

Test group / Remarks:

10 and 30% in DMSO

Remarks on result:

other: Not sensitizing in sensitisation protocol

Key result

Parameter:

other: lymph node weight and cellularity, ear thickness, lymphocytes subpopulations

Test group / Remarks:

10 and 30% in DMSO

Remarks on result:

other: Not sensitising in sensitisation-challenge protocol

Cellular proliferation data / Observations:

1. In the sensitisation protocol, a decrease in lymph node cell count (-20%) was observed but it was not signifiant.
2. In the sensitisation protocol, a non-significant increase in lymph node cellularity (+28%) was found in conjunction with only minor and inconsistent changes in cell surface markers.

Immunophenotypic results of the different leukocyte epitope markers obtained from mice treated according the sensitisation protocol

Cell marker	n	Dye	Concentration	Quartile	Median	Quartile	% Change of median
			(%)	1 (%)	(%)	3 (%)	(dye vs. DMSO)
CD4	10	DO30	10	64.08	67.03	69.26	0.65
	10	DMSO		64.98	66.60	68.63
CD8	10	DO30	10	15.22	17.58	19.48	0.09
	10	DMSO		15.91	17.57	21.22
CD45R	10	DO30	10	4.66	6.81	8.18	4.77
	10	DMSO		6.00	6.50	8.02
CD69	10	DO30	10	6.66	6.89	7.38	-10.87
	11	DMSO		7.10	7.73	8.74
1A	10	DO30	10	8.17	11.16	14.11	2.20
	11	DMSO		9.63	10.92	15.57

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was considered to be not sensitizing to mouse skin in both sensitisation and sensitisation-challenge protocols (LLNA).

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance. Modified protocols of the Local Lymph Node Assay i.e. sensitisation and sensitisation-challenge protocols, were used in this study. Female NMRI mice were exposed to the test substance at concentrations of 10 and 30% in DMSO. The negative control was the vehicle alone. In the sensitisation protocol, animals received 25 µL of the test solution on the dorsum of the left ear for 3 consecutive days. The right ears were treated with equal volume of DMSO. 48 h after the last exposure, mice were euthanised. Ear thickness was measured, draining auricular lymph nodes were excised and weighed, and cell suspensions were prepared to count the number of cells. In the sensitisation-challenge protocol, animals were shaved over a surface of approximately 2 cm2 on their back and this surface was treated once daily on Days 1 -3 with 50 µL of the test solution. Mice remained untreated on Days 4 -14 and were then challenged with 25 µL of the test solution on Days 15 -17. 48 h after the last exposure, mice were euthanised. Ear thickness was measured, draining auricular lymph nodes were excised and weighed, and cell suspensions were prepared to count the number of cells. Lymphocyte subpopulations (T-cells and B-cells, CD69 and 1A epitopes) were also analysed by flow cytometry. Results obtained in the treated mice were compared to those obtained with the vehicle alone. The test substance did not induce any changes regarding lymph node weight and cellularity, ear thickness, or lymphocytes subpopulations. Under the study conditions, the test substance was considered not sensitizing to mouse skin in both sensitisation and sensitisation-challenge protocols (Stahlmann, 2006).

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August 04, 1997 to September 15, 1997

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

- the substance used for the GPMT has a purity of 42.3% only with unknown composition - 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

Justification for type of information:

The study was conducted in 1997 before the requirement of in vitro studies was published.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A GPMT study was conducted and submitted before the requirement for LLNA testing was published.

Species:

guinea pig

Strain:

other: Albino

Sex:

female

Details on test animals and environmental conditions:

Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF (Harlan Nederland B.V.)
Acclimatization: 1 week
Age: 6-8 weeks
Weight: 410-509 g
Temperature and relative humidity: 22 ±3 °C and between 40-70 %, respectively
Light period: 12-h light, 12-h dark cycle
Diet and water: pelleted standard Nafag Ecosan 845 25W4, batch nos. 37/97 and 58/97 guinea pig breeding / maintenance diet ("Nafag", Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum and tap water, respectively

Route:

intradermal

Vehicle:

water

Remarks:

bi-distilled

Concentration / amount:

- Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test substance, diluted to 5 % with bi-distilled water.
3) The test substance diluted to 5 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
- Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Bi-distilled water.
3) 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Adequacy of induction:

other: maximally tolerated concentration of the test substance

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

bi-distilled

Concentration / amount:

50% in vehicle
(volume of test substance: 0.3g)

Day(s)/duration:

48h

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

bi-distilled

Concentration / amount:

50% in vehicle
(Volume of the test substance: 0.3g)

Day(s)/duration:

24h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

15 (10: test and 5: control)

Details on study design:

- Pre-test (intradermal injections - epidermal application):
This study was conducted to identify a maximally tolerated concentration of the test substance suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test substance, by the topical route of administration, was identified for the challenge application. The concentrations tested were for the epidermal application the most qualified to assure an optimum technical application procedure and for the intradermal injection the selected concentrations were tested up to 5%.
- Main study:
The intradermal induction of sensitization was carried out with a 5% dilution of the test substance in bi-distilled water and in an emulsion with Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted under occlusion (clipped, shaved skin) with the test substance at 50% in bi-distilled water. 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 50% in bi-distilled water 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. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 h after removal of the dressing.

Challenge controls:

Negative controls: vehicle on the right side of the animals

Positive control substance(s):

yes

Remarks:

alpha-hexylcinnamaldehyde

Positive control results:

The positive control alpha-hexylcinnamaldehyde (at a concentration of 25% in PEG400) induced skin reactions in 70% of the animals

Key result

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Erythema (scores 1 and 2)

Remarks on result:

positive indication of skin sensitisation

Key result

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Erythema (scores 1 and 2)

Remarks on result:

positive indication of skin sensitisation

Key result

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

25%

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

25%

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Interpretation:

The results obtained from test animals following the challenge applications were compared with the results seen in control animals. An allergic reaction was defined by visible reddening of the challenge site.

- If the dermal reactions of test animals following the challenge were more marked and/or persistent than those of the control animals, the animals were considered to show evidence of contact hypersensitivity.

- If the dermal reactions of test animals following the challenge were not clearly different from the reactions seen in the control group animals, the results for the test animals were considered "inconclusive".

- The test animals were considered to show no evidence of contact hypersensitivity if the dermal reactions to the challenge application were identical to or less marked and/or persistent than the reactions observed in the control animals.

A response of at least 30% positive animals is considered positive "R43": may cause sensitization by skin contact.

Results:

- Induction (intradermal): a normal development of the expected local symptoms was observed in the animals of the control and test group.

- Induction (epidermal): no erythematous or oedematous reaction was observed in the animals either when treated with bi-distilled water only or when treated with the test substance at 50% in bi-distilled water. As the test substance stained the skin red, it was not possible to determine whether erythema was present or not. However no oedema was observed.

- Challenge:

Control group: no positive reactions were observed in the animals either when treated with bi-distilled water only or when treated with the test substance at 50% in bi-distilled water. Red discoloration was noted directly after removal of the patch. To remove discoloration all animals were depilated approximately 3 h prior to challenge reading.

Test group: positive reactions were observed in all animals at the 24 - and 48 -h reading when treated with the test substance at 50% in bi-distilled water. No positive reactions were observed in the animals when treated with bi-distilled water only. Red discoloration was noted directly after removal of the patch. To remove discoloration all animals were depilated approximately 3 h prior to challenge reading.

Other observations:

No deaths, no symptoms of systemic toxicity, and no changes in body weights were detected during the study.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the study conditions, the test substance applied at a concentration of 50% in bi-distilled water was considered to be a sensitizer to guinea pig skin.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 406 and EU Method B.6 (Guinea pig maximization test), in compliance with GLP. Female guinea-pigs were exposed to the test substance at a concentration of 50% (in bi-distilled water). 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. In addition, a suitable non-irritant concentration of the test substance, by the topical route of administration, was identified for the challenge application. The concentrations tested were for the epidermal application the most qualified to assure an optimum technical application procedure and for the intradermal injection the selected concentrations were tested up to 5%. In the main study, the intradermal induction of sensitization was carried out with a 5 % dilution of the test substance in bi-distilled water and in an emulsion with Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted under occlusion (clipped, shaved skin) with the test substance at 50% in bi-distilled water. 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 50% in bi-distilled water 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. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 h after removal of the dressing. Mortality, symptoms of systemic toxicity, and body weights were also investigated. A normal development of the expected local symptoms was observed in the animals of the control and test group during the intradermal induction phase. No erythematous or oedematous reaction was observed in the animals either when treated with bi-distilled water only or when treated with the test substance at 50% in bi-distilled water in the epidermal induction phase. After the challenge phase, no positive reactions were observed in the animals either when treated with bi-distilled water only or when treated with the test substance at 50% in bi-distilled water in the control group. Positive reactions were observed in all animals at the 24 and 48 h readings when treated with the test substance at 50% in bi-distilled water. Under the study conditions, the test substance applied at a concentration of 50% in bi-distilled water was considered to be a sensitizer to guinea-pig skin (Arcelin, 1997).

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. Male CBA/Ca mice were exposed to the test substance at concentrations of 0, 3, 10 and 30% in acetone. Isotope (3H-methyl thymidine) incorporation was measured in lymph nodes. The negative (vehicle alone) and positive (hexylcinnamaldehyde at concentrations of 1, 3, and 10% w/v in acetone) controls were valid. The isotope incorporation was increased by less than a threefold at all concentrations of the test substance. Under the study conditions, the test substance was considered to be not sensitizing to mouse skin (Johnson, 2002).

A study was conducted to determine the skin sensitisation potential of the test substance. Modified protocols of the Local Lymph Node Assay i.e. sensitisation and sensitisation-challenge protocols, were used in this study. Female NMRI mice were exposed to the test substance at concentrations of 10 and 30% in DMSO. The negative control was the vehicle alone. In the sensitisation protocol, animals received 25 µL of the test solution on the dorsum of the left ear for 3 consecutive days. The right ears were treated with equal volume of DMSO. 48 h after the last exposure, mice were euthanised. Ear thickness was measured, draining auricular lymph nodes were excised and weighed, and cell suspensions were prepared to count the number of cells. In the sensitisation-challenge protocol, animals were shaved over a surface of approximately 2 cm2 on their back and this surface was treated once daily on Days 1 -3 with 50 µL of the test solution. Mice remained untreated on Days 4 -14 and were then challenged with 25 µL of the test solution on Days 15 -17. 48 h after the last exposure, mice were euthanised. Ear thickness was measured, draining auricular lymph nodes were excised and weighed, and cell suspensions were prepared to count the number of cells. Lymphocyte subpopulations (T-cells and B-cells, CD69 and 1A epitopes) were also analysed by flow cytometry. Results obtained in the treated mice were compared to those obtained with the vehicle alone. The test substance did not induce any changes regarding lymph node weight and cellularity, ear thickness, or lymphocytes subpopulations. Under the study conditions, the test substance was considered not sensitizing to mouse skin in both sensitisation and sensitisation-challenge protocols (Stahlmann, 2006).

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 406 and EU Method B.6 (Guinea pig maximization test), in compliance with GLP. Female guinea-pigs were exposed to the test substance at a concentration of 50% (in bi-distilled water). 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. In addition, a suitable non-irritant concentration of the test substance, by the topical route of administration, was identified for the challenge application. The concentrations tested were for the epidermal application the most qualified to assure an optimum technical application procedure and for the intradermal injection the selected concentrations were tested up to 5%. In the main study, the intradermal induction of sensitization was carried out with a 5 % dilution of the test substance in bi-distilled water and in an emulsion with Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted under occlusion (clipped, shaved skin) with the test substance at 50% in bi-distilled water. 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 50% in bi-distilled water 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. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 h after removal of the dressing. Mortality, symptoms of systemic toxicity, and body weights were also investigated. A normal development of the expected local symptoms was observed in the animals of the control and test group during the intradermal induction phase. No erythematous or oedematous reaction was observed in the animals either when treated with bi-distilled water only or when treated with the test substance at 50% in bi-distilled water in the epidermal induction phase. After the challenge phase, no positive reactions were observed in the animals either when treated with bi-distilled water only or when treated with the test substance at 50% in bi-distilled water in the control group. Positive reactions were observed in all animals at the 24 and 48 h readings when treated with the test substance at 50% in bi-distilled water. Under the study conditions, the test substance applied at a concentration of 50% in bi-distilled water was considered to be a sensitizer to guinea-pig skin (Arcelin, 1997).

The WoE approach based on the three skin sensitisation studies conducted leads to the conclusion that Disperse Orange 30 has no skin sensitising effect. This is based on the fact that

- neither the LLNA with a highly pure substance nor the one with a commercial product leads to any sensitising effects

- the substance used for the GPMT has a purity of 42.3% only with unknown composition

- 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

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.