2-[[4-[(2-cyano-3-nitrophenyl)azo]-m-tolyl](2-acetoxyethyl)amino]ethyl acetate

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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-[(2-cyano-3-nitrophenyl)azo]-m-tolyl](2-acetoxyethyl)amino]ethyl acetate

EC Number:

250-061-4

EC Name:

2-[[4-[(2-cyano-3-nitrophenyl)azo]-m-tolyl](2-acetoxyethyl)amino]ethyl acetate

Cas Number:

30124-94-8

Molecular formula:

C21H21N5O6

IUPAC Name:

2-[[4-[(2-cyano-3-nitrophenyl)azo]-m-tolyl](2-acetoxyethyl)amino]ethyl acetate

Test material form:

solid: particulate/powder

In vivo test system

Test animals

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.

Study design: in vivo (LLNA)

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.

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:

0.78

Test group / Remarks:

5%

Results 2

Key result

Parameter:

SI

Value:

0.85

Test group / Remarks:

10%

Results 3

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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..