trisodium 4-hydroxy-6-(sulfonatomethylamino)-5-(2-(2-sulfatoethylsulfonyl)phenylazo)naphthalene-2-sulfonate

Endpoint summary

Description of key information

Under the study conditions, the test substance was shown to be a potential skin sensitiser when administered at a concentration of > 3%. The EC3 value was calculated to be 3.8%.

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:

From November 19, 1997 to November 25, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

Ola/Hsd

Sex:

male

Details on test animals and environmental conditions:

Source: Harlan UK, Oxon
Specification: young adults
Husbandry: A maximum of 4 mice was housed per cage, in cages suitable for animals of this strain and weight range.
Temperature: 22±3 °C
Relative humidity: 30-70%
Air: A minimum of 15 changes per hour
Light cycle: 12 h/ 12 h
Diet: (R&M No. 1), supplied by Special Diet Services Limited, UK and mains water, supplied by automatic system were available as libitum.
Acclimatization period: 5 days

Vehicle:

propylene glycol

Concentration:

25 µL of a 1, 3 and 10 % w/v of the test substance in propylene glycol

No. of animals per dose:

4

Details on study design:

Approximately 25 µL of a 1, 3 and 10 % 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 buffer saline containing approximately 20 µCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were sacrificed 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 was added prior to β-scintillation counting using a Packard Tri-Card 2500TH liquid Scintillation Counter.

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

Positive control substance(s):

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

Statistics:

The results are expressed as a count per minute (cpm) 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 1%, 3%, and 10% w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at concentrations of 3 and 10% w/v. Therefore, hexylcinnamaldehyde was shown to be a skin sensitizer, confirming the validity of the protocol used for the study.

Parameter:

SI

Value:

1.84

Test group / Remarks:

1 % of the test substance in propylene glycol

Remarks on result:

other: no idication of skin sensitisation

Key result

Parameter:

SI

Value:

2.92

Test group / Remarks:

3 % of the test substance in propylene glycol

Remarks on result:

other: no indication of skin sensitisation

Key result

Parameter:

SI

Value:

3.59

Test group / Remarks:

10 % of the test substance in propylene glycol

Remarks on result:

other: greater than 3-fold

Remarks:

potential skin sensitizer

Key result

Parameter:

EC3

Value:

3.8

Cellular proliferation data / Observations:

The application of the test substance at concentrations of 1, 3 and 10% w/v in propylene glycol resulted in an isotope incorporation which was greater than 3-fold at the 10% w/v concentration. Consequently, the test substance was shown to be a potential skin sensitizer.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the study conditions, the test substance was shown to be a potential skin sensitiser when administered at a concentration of > 3%. The EC3 value was calculated to be 3.8%.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance in CBA/Ca mice according to OECD Guideline 406 (Local Lymph Node Assay), in compliance with GLP. The assay determined 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. Four male mice were used per test group. Approximately 25 µL of a 1, 3 and 10% w/v solution of test substance in propylene glycol was applied using a variable volume micro-pipette to the dorsal surface of each ear. Propylene glycol served as vehicle control and 25 µL of a 1, 3 and 10% w/v of hexyl cinnamic aldehyde served as positive control. The procedure was repeated daily for 3 consecutive days. Animals were checked at least once daily for signs of systemic toxicity. Three days after the last application, all animals were injected with 3H-methyl thymidine and, after 5 hours the draining (auricular) lymph nodes were excised and pooled for each animal. After the samples preparation the lymph node suspensions were transferred to scintillation vials and 10 mL of scintillate was added prior to β-scintillation counting using a Packard Tri-Card 2500TH liquid scintillation counter. The substance was shown to have the capacity to cause skin sensitization when applied as a 10% w/v preparation in propylene glycol. In the positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitization when applied as 3 and 10% w/v preparations in acetone, confirming the validity of the protocol. Under the study conditions, the test substance was sensitizing to skin (Johnson, 1998).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A study was conducted to determine the skin sensitisation potential of the test substance in CBA/Ca mice according to OECD Guideline 406 (Local Lymph Node Assay), in compliance with GLP. The assay determined 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. Four male mice were used per test group. Approximately 25 µL of a 1, 3 or 10% w/v solution of test substance in propylene glycol was applied using a variable volume micro-pipette to the dorsal surface of each ear. Propylene glycol served as vehicle control and 25 µL of a 1, 3 or 10% w/v of hexyl cinnamic aldehyde served as positive control. The procedure was repeated daily for 3 consecutive days. Animals were checked at least once daily for signs of systemic toxicity. Three days after the last application, all animals were injected with 3H-methyl thymidine and, after 5 hours the draining (auricular) lymph nodes were excised and pooled for each animal. After the samples preparation the lymph node suspensions were transferred to scintillation vials and 10 mL of scintillate was added prior to β-scintillation counting using a Packard Tri-Card 2500TH liquid scintillation counter. The substance was shown to have the capacity to cause skin sensitization when applied as a 10% w/v preparation in propylene glycol. In the positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitization when applied as 3 or 10% w/v preparations in acetone, confirming the validity of the protocol. Under the study conditions, the test substance was sensitizing to skin (Johnson, 1998).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of in vivo sensitization testing in rats (LLNA), the substance require classification as Skin Sens. 1 – H314 (may cause an allergic reaction) according to CLP (EC 1272/2008) criteria.