Maleic anhydride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Characterization in mice of the immunological properties of five allergenic acid anhydrides

Author:

Dearman R.J. et al.

Year:

2000

Bibliographic source:

Journal of Applied Toxicology 20, 221-230

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

(no positve controls, mice strain BALB/c used)

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Maleic anhydride

EC Number:

203-571-6

EC Name:

Maleic anhydride

Cas Number:

108-31-6

Molecular formula:

C4H2O3

IUPAC Name:

2,5-dihydrofuran-2,5-dione

Details on test material:

- Name of test material (as cited in study report): maleic anhydride
- Analytical purity: 99%

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Seralab, Bicester, Oxfordshire, UK
- Age at study initiation: 6-12 weeks old
- Housing: 10 per cage (acclimation period), 4-5 per cage (during treatment)
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 0.1,0.25, 0.5, 1, 2.5% (w/v)

No. of animals per dose:

4

Details on study design:

MAIN STUDY
Groups of mice (n= 4) were exposed topically on the dorsum of both ears to 25 µL of various concentrations of acid anhydrides or to vehicle (acetone:olive oil, AOO) alone, daily for three consecutive days. Five days after the initiation of exposure all mice were injected intravenously via the tail vein with 20 µCi of [3H] methyl thymidine in 250 µL of phosphate-buffered saline (PBS). Five hours later, mice were killed, and the draining auricular lymph nodes were excised and pooled for each experimental group. A single cell suspension of LNC was prepared by gentle mechanical disaggregation through 200-mesh stainless-steel gauze. Cells were washed twice with PBS and precipitated in 5% trichloroacetic acid (TCA) at 4 °C overnight. Pellets were then resuspended in 1 mL of 5% TCA and transferred to 10 mL of scintillation fluid. Incorporation of 3HTdR was measured by ß-scintillation counting as disintegrations per min (dpm) per node for each experimental group. In each case a stimulation index (SI) relative to the concurrent vehicle-treated control was derived. The estimated concentration of chemical required to induce SI= 3 relative to concurrent vehicle-treated controls i.e. the EC3 value was derived by linear interpolation.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

0.16

Results 2

Parameter:

SI

Value:

1.91

Test group / Remarks:

0.1% w/v

Results 3

Parameter:

SI

Value:

4.86

Test group / Remarks:

0.25 % w/v

Results 4

Parameter:

SI

Value:

6.32

Test group / Remarks:

0.5% w/v

Results 5

Parameter:

SI

Value:

14

Test group / Remarks:

1% w/v

Results 6

Parameter:

SI

Value:

15.98

Test group / Remarks:

2.5% w/v

Cellular proliferation data / Observations:

For detailed results please refer to Table 1 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Local lymph node assay responses to maleic anhydride

Concentrations (% w/v)	DPM per node	SI
0	245	1
0.1	467	1.91
0.25	1181	4.86
0.5	1548	6.32
1	3431	14
2.5	3915	15.98
5	ND
10	ND

ND = not determined

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

In this study under the given conditions the EC3 was calculated to be 0.16% w/v = 0.016 M. Therefore, the test item in this test can be considered to be a skin sensitizer category 1A according to the CLP Regulation (EC) 1272/2008.

Executive summary:

In a dermal sensitization study conducted equivalent to OECD Guideline 429 young adult Balb/c mice (4 females per dose) were treated with 0.1, 0.25, 0.5, 1 and 2.5% maleic anhydride in acetone/olive oil (4:1, v/v). Five days after the initiation of exposure all mice were injected intravenously via the tail vein with 20 µCi of [3H] methyl thymidine in 250 µL of phosphate-buffered saline (PBS). Five hours later, mice were killed, and the draining auricular lymph nodes were excised and pooled for each experimental group. Incorporation of 3HTdR was measured by ß-scintillation counting as disintegrations per min (dpm) per node. Furthermore, in each case a stimulation index (SI) was derived.

The stimulation indices (SI) were 1.91 (0.1%), 4.86 (0.25%), 6.32 (0.5%), 14.00 (1%) and 15.98 (2.5%). Based on the results, the extrapolated EC3 value was determined to be 0.16%. Thus, maleic anhydride must be considered as a dermal sensitizer and classification as Skin Sens 1A, H317 according to the criteria of the CLP Regulation (EC) 1272/2008 is warranted.