2,2'-[[4-[(3,5-dinitro-2-thienyl)azo]phenyl]imino]bisethyl diacetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

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

Test guideline 2

Qualifier:

according to guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

2-[4-[N-(4-acetoxybutyl)-N-ethyl]amino-2-methylphenylazo]-3-acetyl-5-nitrothiophene

EC Number:

404-830-8

EC Name:

2-[4-[N-(4-acetoxybutyl)-N-ethyl]amino-2-methylphenylazo]-3-acetyl-5-nitrothiophene

Cas Number:

122063-39-2

Molecular formula:

C21H26N4O5S

IUPAC Name:

4-({4-[2-(3-acetyl-5-nitrothiophen-2-yl)diazen-1-yl]-3-methylphenyl}(ethyl)amino)butyl acetate

Test material form:

solid: particulate/powder

Details on test material:

Disperse Blue 369

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Barriered Animal Breeding Unit at ICI Pharmaceuticals, Alderley Park, Cheshire, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:8-12 week
- Housing: group housing
- Diet (ad libitum): SDS PCD 5/8" pelleted diet
- Water (ad libitum): tap water
- Acclimation period: at least 2 days housed in groups of 10 as on delivery from the Breeders and at least 2 days in their study groups of 4 animals.

- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-2°C
- Humidity (%): 55+/-10
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 14 January 1990 to 23 January 1990

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

3%, 10%, 30% and 50% w/v

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 of choice because previous examination of strain differences in lymphocyte proliferation responses to contact sensitisers showed the CBA/Ca mice to exhibit the most vigorous response (Kimber and Weisenberger (1989)).

Test method
Groups of four female mice were used for this study. Approximately 25 μl of the vehicle or the different test substance preparations was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. The procedure was repeated daily for 3 consecutive days.
Five days following the initial dosing all mice were injected I.V. with approximately 250 µL of phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (specific activity 2.0 Ci/mmol). Approximately 5 hours later mice were sacrificed (inhalation of anaesthetic ['Fluothane'] followed by cervical dislocation) and the draining auricular lymph nodes were removed and pooled for each experimental group.
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.
The results are expressed as mean counts per minute (cpm) per lymph node for each experimental group. The activity of each test group was divided by the activity of the vehicle only (control) group to give a test/control ratio for each concentration tested.

Criteria for Positivity
For a chemical to be designated a potential sensitiser in the Local Lymph Node Assay it has to fulful two criteria:
1) The increase in isotope incorporation for at least one concentration tested must be 3-fold or more compared to the control (vehicle treated) mice.
2) The data generated must not be incompatible with a biological dose response.
If a chemical does not fulfil the above criteria it is designated as showing no evidence of sensitising potential

Positive control substance(s):

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

Statistics:

The results are expressed as a counts 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.

Results and discussion

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.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.08

Test group / Remarks:

3%

Results 2

Key result

Parameter:

SI

Value:

1.63

Test group / Remarks:

10%

Results 3

Key result

Parameter:

SI

Value:

1.63

Test group / Remarks:

30%

Results 4

Key result

Parameter:

SI

Value:

1.32

Test group / Remarks:

50%

Any other information on results incl. tables

Test results

Concentration of test substance (% w/v)	Number of lymph nodes assayed	Counts per minute (cpm)	cpm per lymph node	Test control ratio
0 (vehicle only)	8	1580	197.5	N/A
3% w/v	8	1711	213.88	1.08
10% w/v	8	2570	321.25	1.63
30% w/v	8	2576	322	1.63
50% w/v	8	2080	260	1.32

N/A – not applicable

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance showed no evidence of sensitising potential in the Local Lymph Node assay in mice

Executive summary:

The test substance was assessed for its skin sensitisation potential using the Local Lymph Node assay. The assay determines the level of T lymphocyte proliferation in the lymph node draining the site of chemical application,

by measuring the amount of radiolabelled thymidine incorporated into the dividing cells. The increase in isotope incorporation at all concentrations tested did not reach 3 times that of the control (vehicle treated) mice. Therefore under

the conditions of this assay the test substance showed no evidence of sensitising potential.