4-nitrotoluene-2-sulphonic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report Date:

2010

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

other: OECD 431

Principles of method if other than guideline:

EpiDerm-Test

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): 4-Nitrotoluene-2-sulphonic acid
- Analytical purity: 84.2 %
- Lot/batch No.: 090330

Test animals

Species:

other: in vitro test

Strain:

other: in vitro test

Test system

Type of coverage:

other: in vitro test

Preparation of test site:

other: in vitro test

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):25 µl of the solid test substance

Duration of treatment / exposure:

3 minutes and 1 hour

Details on study design:

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm Ø) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
TEST PROCEDURE
- For the corrosion test two tissue samples were uses for 3 minutes and 1 hour, the irritaion test was perfomed with tree tissue sampels, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.
- After 3 minutes (room temperature) or after 1 hour (incubator) tissue contact with the test material, the tissues were washed with buffer to remove residual test material. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours. After incubation tissues were washed with buffer and formazan was extracted with Isopropanol over night at room temperature. The optical density at a wavelength of 570 nm (OD570) of extracted formazan was determined spectrophotometrically and cell viability was calculated for each tissue as % of the mean of the negative control tissues. Skin corrosivity potential of the test material was classified according to remaining cell viability obtained after test material treatment with either of the 2 exposure times.


CONTROLS
- Negative control (NC): Doubly distilled water
- Positive control (PC): 8 N potassium hydroxyde (Sigma-Aldrich, Munich, Germany)


ASSAY ACCEPTANCE CRITERIA:
- Negative control (NC ): mean OD570 of the NC per exposure time 2.5
- Positive control (PC ): mean relative tissue viability of the 3 min positive control is <= 30 %
- Tissue variability: The inter tissue variability is considered to be acceptable if the difference of the OD570 values of the two tissues is <= 0.3

Results and discussion

In vivo

Irritant / corrosive response data:

See result tables. Based on the observed results and applying the evaluation criteria cited, it was concluded, that 4-Nitrotoluene-2-sulphonic acid shows a skin irritation potential in the EpiDerm TM skin corrosion/irritation test under the test conditions chosen.

Any other information on results incl. tables

Corrosion test

		Exposure: 3 min			Exposure: 1 hour
Test Substance		Tissue 1	Tissue 2	Mean	Tissue 1	Tissue 2	mean
NC	Mean OD570	1.6233	1.8758	1.7496	1.5827	1.8112	1.6969
	Viability [% of NC]	92.8	107.2	100	93.3	106.7	100
09/0641-1	Mean OD570	1.9028	1.6308	1.7668	0.2857	0.2772	0.2814
	Viability [% of NC]	108.8	93.2	101	16.8	16.3	17
PC	Mean OD570	0.3493	0.3223	0.3358	0.1902	0.1907	0.1904
	Viability [% of NC]	20.0	18.4	19	11.2	11.2	11

 

Irritation Test

Test Substance		Tissue 1	Tissue 2	Tissue 3	Mean	SD
NC	Mean OD570	1.8535	2.3000	*	2.0768
	Viability [% of NC]	89.3	110.7	*	100	15.20
09/0641-1	Mean OD570	0.1445	0.1385	0.1635	0.1488
	Viability [% of NC]	7.0	6.7	7.9	7	0.63
PC	Mean OD570	0.1005	0.1140	0.1000	0.1048
	Viability [% of NC]	4.8	5.5	4.8	5	0.38

* For the NC two tissues were valid for evaluation, only (details are available with the raw data)

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information