2-[4-[(2-chloro-4-nitrophenyl)azo]-N-(2-cyanoethyl)anilino]ethyl acetate

Endpoint summary

Description of key information

Non skin sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From May 13th to 21st, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The read across approach is detailed into the document attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

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
- Housing: 4 per cage
- Diet: RM1, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days.

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

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 2.0 Ci/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.

Animals were checked at least once daily for signs of systemic toxicity.
The results were expressed as a counts per minute (cpm) value per lymph node for each group. The stimulation index for each test group was then calculated by dividing cpm value per lymph node by the equivalent value for the control (vehicle only) group.

The criterion for a positive response (sensitiser) was 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 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.

Parameter:

SI

Value:

0.83

Test group / Remarks:

3 % of test substance

Parameter:

SI

Value:

1.19

Test group / Remarks:

10 % of test substance

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.
Stimulation indices recorded were 0.83, 1.19 and 1.06 at the tested concentrations of 1, 10 and 30 %, respectively.

Concentration of test substance (% w/v)	Number of lymph nodes assayed	Counts per minute (cpm)	cpm per lymph node (×10^-^2)	Test control ratio
0 (vehicle control)	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:

other: not classified, according to the CLP Regulation (EC 1272/2008)

Conclusions:

Non skin sensitizer

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.

Conclusion

Non skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There are no information about the skin sensitisation potential of Disperse Red 054, thus the available information on the structural analogue Similar Substance 02 have been taken into consideration; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

A study was conducted to determine the skin sensitisation potential of the test substance, according to OECD guideline 429 (local lymph node assay) and 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.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.4.1.2.section, skin sensitiser means a substance that will lead to an allergic response following skin contact.

From the classification point of view, skin sensitisers can be sub-categorized into sub-category 1A, strong sensitisers, or sub-category 1B for other skin sensitiser; where data are not sufficient for sub-categorisation, skin sensitisers shall be classified in Category 1. In the case of local lymph node assay, a stimulation index of three or more is considered a positive response.

Stimulation indices recorded were 0.83, 1.19 and 1.06 at the tested concentrations of 1, 10 and 30 %, respectively. Therefore, the test item resulted to be not skin sensitizer.

In conclusion, Disperse Red 054 can be considered to not meet the criteria to be classified as skin sensitizer, according to the CLP Regulation (EC 1272/2008).