Hydrogen chloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 05 March 2013 and 18 April 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

Study is an acceptable method to evaluate skincorosion potential and does not require the use of animals.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD guideline for the testing of chemicals 431

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU B40bis to Com Reg 440/2008

Deviations:

no

GLP compliance:

yes

Remarks:

Date of inspection: 10 July 2012. Date of Signature: 07 November 2013

Test material

Test material information

Constituent 1

Reference substance name:

Hydrochloric acid

IUPAC Name:

Hydrochloric acid

Test material form:

other: Liquid

Details on test material:

Sponsor's identification : Hydrochloric acid
Description : clear colourless liquid
Batch number : SZBC1030V
Purity : 37.5%
Date received : 08 October 2012
Expiry date : 24 September 2015
Storage conditions : room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Reconstructed human epidermis

Details on animal used as source of test system:

EPISKIN™ model

Justification for test system used:

Validated and internationally accepted testsystem

Vehicle:

water

Remarks:

deionised water

Details on test system:

EPISKIN™ Model Kit 0.38 cm2

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3, 60 and 240 minutes

Duration of post-treatment incubation (if applicable):

No

Number of replicates:

Duplicate

Test system

Amount / concentration applied:

TEST ITEM

Concentrations prepared:
Phase 1: 10%, 25% and 30% w/w in deionised water.
Phase 2: 15% w/w in deionised water*.

*The 15% w/w concentration of the test item was applied for an exposure period of 3 minutes only. It was considered that the results of the 10% w/w concentration tested at 60 and 240 minutes were adequate for classification purposes and therefore application of the 15% w/w for 60 and 240 minutes was unnecessary.

Amount(s) applied (volume or weight with unit):
50 µl of each test item concentration was applied topically to the epidermis surface.

VEHICLE
Deionised Water.

Duration of treatment / exposure:

3, 60 & 240 minutes post exposure incubation.

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE:
Area of exposure: 50 µl of each test item concentration was applied to the 0.38cm2 epidermis surface.

PERCENTAGE COVERAGE:
The test item was applied topically to the corresponding tissues ensuring uniform covering.

EXPOSURE TIME:
3, 60 AND 240 minutes.

REMOVAL OF TEST ITEM:
Washing: At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item.

Time after start of exposure:
3, 60 or 240 minutes post exposure.

SCORING SYSTEM:
The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = mean OD540 of test item concentration / mean OD540 of negative control x 100

Classification of corrosivity potential was based on relative viabilities for each test item concentration and exposure time according to the following prediction model (concise):
3 minutes exposure : <35% viability : Prediction = Corrosive
3/60 minutes exposure : ≥35 / <35 viability : Prediction = Corrosive
60/240 minutes exposure : ≥35 / <35 viability : Prediction = Corrosive
240 minutes exposure : ≥35 : Prediction = Non-Corrosive

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

10% / 3 minutes

Value:

93.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

10% / 60 minutes

Value:

27.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

10% / 240 minutes

Value:

5.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

25% / 3 minutes

Value:

30.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive

Results 5

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

25% / 60 minutes

Value:

6.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive

Results 6

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

25% / 240 minutes

Value:

6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive

Results 7

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

30% / 3 minutes

Value:

9.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive

Results 8

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

30% / 60 minutes

Value:

4.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive

Results 9

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

30% / 240 minutes

Value:

6.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Corrosive

Other effects / acceptance of results:

viability ≤ 35% are indication for corrosive effects.
Phase 1 Exposure period
Concentration 3 minutes 60 minutes 240 minutes
10% Test Item 93.3 27.6 5.4
25% Test Item 30.4 6.5 6.0
30% Test Item 9.5 4.1 6.6

Phase 2 Exposure period
Concentration 3 minutes
15% Test Item 92.3

In vivo

Other effects:

None

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

The MTT solution containing the undiluted Hydrochloric acid did not turn blue which indicated that the test item did not directly reduce MTT.

Test Item, Positive Control Item and Negative Control Item

For phase 1 of testing, the mean OD₅₄₀ values of individual tissues, mean OD₅₄₀ values of duplicate tissues and relative mean tissue viabilities for the negative, positive and vehicle controls are given in Table 1 and the values for the test item concentrations are given in Table 2. For phase 2 of testing, the mean OD₅₄₀ values of individual tissues, mean OD₅₄₀ values of duplicate tissues and relative mean tissue viabilities for the negative, positive, vehicle controls and the test item are given in Table 3.

Data are presented in the form of relative mean viability (percentage MTT reduction in the test item treated tissues relative to negative control tissues):

Phase 1:

	Exposure period
Concentration	3 minutes	60 minutes	240 minutes
10% Test Item	93.3	27.6	5.4
25% Test Item	30.4	6.5	6.0
30% Test Item	9.5	4.1	6.6

Phase 2:

	Exposure period
Concentration	3 minutes
15% Test Item	92.3

Quality Criteria

The relative mean tissue viability for the positive control treated tissuesin phases 1 and 2 were 0 to 20% relative to the negative control treated tissuesfollowing the 240-Minute exposure period. The positive control acceptance criterion was therefore satisfied in each phase of testing.

The mean OD₅₄₀ for the negative control treated tissues in phases 1 and 2 were ≥0.600 and ≤1.500. The negative control acceptance criterion was therefore satisfied in each phase of testing.

Table 1 : Mean OD₅₄₀ Values and Percentage Viabilities for the Negative, Positive and Vehicle Control Items for Phase 1 of Testing

Item	Exposure Period	Mean OD540 of individual tissues	Mean OD540 of duplicate tissues	Relative mean viability (%)
Negative Control Item	240 Minutes	0.969	0.928	100*
		0.887
Positive Control Item	240 Minutes	0.064	0.065	7.0
		0.065
Vehicle Control Item	240 Minutes	0.941	0.918	98.9
		0.895

*=     The mean viability of the negative control tissues is set at 100%

Table 2 : Mean OD₅₄₀ Values and Viabilities for the Test Item Concentrations for Phase 1 of Testing

Item	Exposure Period	Mean OD540 of individual tissues	Mean OD540 of duplicate tissues	Relative mean viability (%)
10% Test Item	240 Minutes	0.048	0.050	5.4
		0.051
	60 Minutes	0.307	0.256	27.6
		0.204
	3 Minutes	0.850	0.866	93.3
		0.881
25% Test Item	240 Minutes	0.064	0.056	6.0
		0.048
	60 Minutes	0.047	0.060	6.5
		0.072
	3 Minutes	0.279	0.282	30.4
		0.284
30% Test Item	240 Minutes	0.059	0.061	6.6
		0.063
	60 Minutes	0.031	0.038	4.1
		0.044
	3 Minutes	0.090	0.088	9.5
		0.085

Table 3 : Mean OD₅₄₀ Values and Percentage Viabilities for the Negative, Positive and Vehicle Control Items and the Test Item for Phase 2 of Testing

Item	Exposure Period	Mean OD540 of individual tissues	Mean OD540 of duplicate tissues	Relative mean viability (%)
Negative Control Item	240 Minutes	0.979	1.020	100*
		1.061
Positive Control Item	240 Minutes	0.039	0.039	3.8
		0.038
Vehicle Control Item	240 Minutes	1.136	1.153	113.0
		1.169
15% Test Item	3 Minutes	1.053	0.941	92.3
		0.829

*=     The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

other: Various concentrations, See below:

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

Based on the resulsults obtained, concentrations of Hydrochloric Acid in deionised water, were classified as follows:
10%: Category 1B, H314 “Causes severe skin burns and eye damage”.
15%: After a 3 minute exposure, a 15% w/w concentration did not induce a reduction of cell viability indicative of skin corrosion.
25% and 30%: Category 1A, H314 “Causes severe skin burns and eye damage”.

Executive summary:

Introduction: The purpose of this test was to evaluate the skin corrosivity potential of various concentrations of the test item, Hydrochloric acid, using the EPISKIN^TM in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to be compatible with the following:

• OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004)
• Method B.40bis of CommissionRegulation (EC) No. 440/2008

The EPISKIN^TM model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied.

Preparation of Test Item: The study was conducted in two phases. The result from the preceding phase was used to determine the concentration to be used in the subsequent phase. The sponsor was consulted prior to determination of the concentrations to be tested.

Concentrations prepared:

Phase 1: 10%, 25% and 30% w/w in deionised water.
Phase 2: 15% w/w in deionised water*.

* The 15% w/w concentration of the test item was applied for an exposure period of 3 minutes only. It was considered that the results of the 10% w/w concentration tested at 60 and 240 minutes were adequate for classification purposes and therefore application of the 15% w/w for 60 and 240 minutes was unnecessary.

Methods: Duplicate tissues were treated with the concentrations of the test item diluted w/w in deionised water for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 540 nm (OD₅₄₀).

Results: Data are presented in the form of relative mean viability (percentage MTT reduction in the test item treated tissues relative to negative control tissues):

Phase 1:

	Exposure period
Concentration	3 minutes	60 minutes	240 minutes
10% Test Item	93.3	27.6	5.4
25% Test Item	30.4	6.5	6.0
30% Test Item	9.5	4.1	6.6

Phase 2:

	Exposure period
Concentration	3 minutes
15% Test Item	92.3

Quality criteria: The quality criteria required for acceptance of results in each test phase were satisfied.

Conclusion: According to the Study Plan followed, concentrations of the test item, Hydrochloric Acid, were classified as follows:

10% w/w concentration in deionised water: EU DSD (67/548/EEC) Corrosive requires symbol “C” risk phrase R34 “CAUSES BURNS”. EU CLP (1272/2008/EC) H314 “Causes severe skin burns and eye damage” Category 1B.

15% w/w concentration in deionised water: After a 3 minute exposure, a 15% w/w concentration did not induce a reduction of cell viability indicative of skin corrosion. Given that the preceding 10% w/w concentration had induced a corrosive response after 60 and 240 minute exposures the following classification was considered implicit: EU DSD (67/548/EEC) Corrosive requires symbol “C” risk phrase R34 “CAUSES BURNS”. EU CLP (1272/2008/EC) H314 “Causes severe skin burns and eye damage” Category 1B.

25% and 30% w/w concentrations in deionised water: EU DSD (67/548/EEC) Corrosive requires symbol “C” risk phrase R35 “CAUSES SEVERE BURNS”. EU CLP (1272/2008/EC) and UN GHS Hazard statement H314 “Causes severe skin burns and eye damage” Category 1A.