Hexanoic acid

Endpoint summary

Description of key information

Skin irritation (OECD 404): corrosive (Kästner, 1984)

Eye irritation (OECD 437), 50%: corrosive (Lütkenhaus, 2012)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Oct - 13 Nov 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1981

Deviations:

yes

Remarks:

limited data; occlusive dressing

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: mean weight 3294 g

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 h

Observation period:

21 days
Reading time points: immediately, 1, 24, 48 and 72 h and 6, 8, 10, 13, 15, 17 and 21 days after patch removal

Number of animals:

5

Details on study design:

TEST SITE
- Area of exposure: dorsal area (3 cm x 3 cm)
The substance was applied on shaved skin and covered with a linen lobule.
- Type of wrap if used: polyethylene foil wrapped with an elastic bandage

REMOVAL OF TEST SUBSTANCE
- Washing: The skin was cleaned of residual test substance.
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize scoring system

Body weights of the rabbits were determined 7, 14 and 21 days after initiation of the study.

Irritation parameter:

other: necrosis and scar tissue

Basis:

other: all 5 animals

Time point:

other: 21 days

Reversibility:

not reversible

Remarks on result:

other: scar tissue was observed at the treated sites in all animals at the end of the observation period, indicating irreversible skin damage

Irritation parameter:

erythema score

Time point:

24/48/72 h

Remarks on result:

other: scores were not given

Irritation parameter:

edema score

Time point:

24/48/72 h

Remarks on result:

other: scores were not given

Irritant / corrosive response data:

Immediately after removal of the dressing, intensive erythema and edema was observed. The edema had disappeared after seven days, the erythema however persisted and passed into a full thickness necrosis.
10 days after initiation of the study eschar started to separate from treated skin. Another 7 days later eschar was completely separated from skin. 21 days after treatment a red scar tissue with single eschar rest had developed. Around the scar tissue, an intensified growth of hair appeared and on the treated skin area, there were just single flocci.
Based on the findings the test material is considered corrosive under the experimental conditions chosen. Corrosive effects are expected even if the study would had been conducted under semiocclusive conditions: As the test material has a very low vapour pressure, evaporating of the test material from the skin through the semiocclusive dressing is rather unlikely.

Other effects:

The body weights of the animals showed normal growth and were upto 3644 g at the end of the study.

Interpretation of results:

other: classification as Skin Corr. 1C, H314 required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: Skin Corr. 1C, H314

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Dec 2011 - 24 Jan 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline Study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (adopted 7 Sept 2009)

GLP compliance:

yes (incl. QA statement)

Remarks:

BAYERISCHES LANDESAMT FÜR GESUNDHEIT UND LEBENSMITTELSICHERHEIT, LANDESINSTITUT FÜR ARBEITSSCHUTZ UND PRODUKTSICHERHEIT, München, Germany

Species:

other: cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Attenberger Fleisch GmbH & Co. KG, Munich, Germany
- Date of eye collection: 14 Dec 2011
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) containing penicillin/streptomycin on ice

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Type of cornea holder used: MC2, Clermont, France
- Description of the cornea holder: the cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea.
- Test medium and temperature conditions used in the cornea holder: RPMI 1640 without phenol red supplemented with 1% [v/v] fetal bovine serum and 2 mM L-glutamine
- Equilibration time: 1 h at 32 ± 1 °C in a water bath
- Quality check of the equilibrated corneas: initial opacity measurement; corneas with an initial opacity above 7 in the opacitometer were discarded.

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Specification of the device: MC2, Clermont, France

Vehicle:

other: sesame oil

Controls:

other: 3 eyes each for the two negative controls (sesame oil and physiological saline 0.9% NaCl); 3 eyes for the positive control (70% ethanol)

Amount / concentration applied:

- Amount(s) applied in the test: 750 µL
- Concentration (if solution): 50%

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

3 eyes for the test item

Details on study design:

TEST CONDITIONS
- Short description of the method used: closed-chamber method.
The test substance or control substances were introduced into the anterior.

POST-EXPOSURE TREATMENT
- Removal of the test substance: Example: The test substance was removed after 10 min incubation and the epithelium washed at least three times. for the corneas treated with sesame oil as vehicle, the chambers were opened and the corneas were rinsed with 100 mL MEM.
- Medium for washing the corneas: MEM containing phenol red
- Medium for final rinsing: Example: RPMI 1640 without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: After refilling fresh RPMI 1640 without phenol red into the anterior chamber, the final opacity was measured after 2 hours incubation at 32 ± 1 °C
- Specification of the device: MC2, Clermont, France

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 490 nm recorded as optical density (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (4 mg/mL)
- Incubation time: 90 min at 32 ± 1 °C

Irritation parameter:

in vitro irritation score

Remarks:

mean of all 3 eyes

Run / experiment:

10 min exposure with the test substance

Value:

143.61

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

62.51

Irritation parameter:

other: opacity

Remarks:

mean of all 3 eyes

Run / experiment:

10 min exposure with the test substance

Value:

114

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

52.67

Irritation parameter:

other: permeability

Remarks:

mean of all 3 eyes

Run / experiment:

10 min exposure with the test substance

Value:

1.974

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

0.656

Other effects / acceptance of results:

For hexanoic acid at a concenration of 50% (v/v) an in vitro irritation score of 143.61 was calculated and therefore the test substance is considered as severe eye irritant.

Table 1: Opacity values

Parameter	Initial opacity	Final opacity	Opacity change	Mean opacity change of NC	Corrected opacity change	Mean opacity value
Negative control (0.9% NaCl)	4	5	1	2.33	-	-
	4	9	5
	4	5	1
Negative control (sesame oil)	4	7	3	2.33	-	-
	4	5	1
	5	8	3
Test substance	3	124	121	-	118.67	114.00
	3	112	109		106.67
	3	122	119		116.67
Positive control	5	56	51	-	48.67	52.67
	5	58	53		50.67
	5	66	61		58.67

 

 

Table 2: Permeability values (optical density (OD) at 490 nm)

Parameter	OD490 change	Mean OD490 change of NC	Corrected OD490 change	Mean OD490 value
Negative control (0.9% NaCl)	0.003	0.004	-	-
	0.009
	0.001
Negative control (sesame oil)	0.000	0.004	-	-
	0.003
	0.008
Test substance	1.842	1.977	1.838	1.974
	2.086		2.082
	2.004		2.00
Positive control	0.400	0.661	0.396	0.656
	0.770		0.766
	0.812		0.808

 

 

Table 3: In Vitro Irritancy Score (IVIS) values

	IVIS	Mean IVIS
Negative control (0.9% NaCl)	1.05	2.40
	5.14
	1.02
Negative control (sesame oil)	3.00	2.39
	1.05
	3.12
Test substance	146.24	143.61
	137.90
	146.67
Positive control	54.61	62.51
	62.16
	70.79

 

 

Interpretation of results:

corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

CLP: Cat. 1, H318

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

in vivo

Skin irritation by hexanoic acid was evaluated in a study performed according to OECD Guideline 404, where 0.5 mL of hexanoic acid was applied to the shaved skin of five New Zealand White animals under occlusion for 4 hours (Kästner, 1984). Immediately after removal of the plaster, intensive erythema and edema was observed. The edema had disappeared after seven days; the erythema, however, persisted and passed into a full thickness necrosis. 10 days after initiation of the study eschar started to separate from treated skin. Another 7 days later eschar was completely separated from skin. 21 days after treatment a red scar tissue with single eschar rest had developed. Around the scar tissue, an intensified growth of hair appeared and on the treated skin area, there were just singleflocci[AWE1] indicating the full thickness destruction of all epidermal layers. Based on the findings the test material is considered corrosive under the experimental conditions chosen. Corrosive effects are expected even if the study would had been conducted under semiocclusive conditions as the test material has a very low vapour pressure so that evaporating of the test material from the skin through the semiocclusive dressing is rather unlikely.

In addition Smyth et al. (1954) also reported strong skin injury (score 6) after application of hexanoic acid to the rabbit skin for 24 hours. In the used 10-grade ordinal scale the score of 6 indicates necrosis from the undiluted test substance.

in vitro

In the ECVAM International Validation study on in vitro tests for skin corrosivity during 1996 and 1997, hexanoic acid was one of 60 test chemicals (Fentem et al., 1998). The tests evaluated were inter alia, EPISKINTM, the in vitro membrane barrier test method for skin corrosion (CORROSITEX), and the rat skin transcutaneous electrical resistance (TER) assay.

In all three assays hexanoic acid revealed corrosive properties. A corrosive result was also obtained in a TER assay performed by Whittle et al. (1996), in which the application of hexanoic acid for 24 h to rat skin discs resulted in a TER value of 1.2 kΩ, which was well below the cut-off value for rat tissues of 5 kΩ. Whittle et al. (1996) also exposed human skin tissue samples to hexanoic acid for 24 h and evaluated the vitro skin corrosivity potential via TER measurements. The TER was decreased below 11 kΩ (cut-off-value for human tissues) in 5 out of 9 human tissue preparations.

Hexanoic acid in concentrations of 50, 60 and 70% (v/v) was tested in a human skin model test according to OECD Guideline 431 (EpiDerm) under GLP conditions (Lehmeier, 2011). Hexanoic acid was diluted to the corresponding concentrations with sesame oil. 50 µL of the test items were topically applied 3 minutes and 1 hour to a three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum. Afterwards the cell viability was assessed via MTT reduction assay.

For hexanoic acid at a concentration of 70%, the mean relative tissue viability was decreased below 50% (37%) after 3 min treatment compared to the negative control (distilled water) and thus showed corrosive effects. 50% and 60% hexanoic acid showed no clear corrosive effects. Since, the relative mean tissue viability after 3 min treatment was ≥ 50% (72% for 50% hexanoic acid and 57% for 60% hexanoic acid, respectively) and after 1-hour treatment ≥ 15% (15% for 50% and 60% hexanoic acid), hexanoic acid at concentrations of 50% and 60% are considered as non-corrosive. Hexanoic acid led to a dose-dependent colouring of the tissues after the 3 min and 1-hour treatment. To assess the influence of this extractable colour and to avoid false-negative results, the “Check-method to detect coloring test substance ability to stain tissues” according to the L´Oréal Standard Operating Procedure EpiSkinTM skin irritation method was performed in a follow-up study for 50% and 60% hexanoic acid. The non-specific colour for 50% and 60% hexanoic acid was below 5% (2.9% for both test items after 3 min and 2.7% and 3.0% in the 1-hour experiments, respectively) and therefore no correction of the results are necessary according to the L´Oréal Standard Operating Procedure (Lehmeier, 2011).

Human data

Hexanoic acid at concentrations of 0.5 M (5.8% (w/v)) and 1 M (11.6% (w/v)) were applied daily under occlusive patch tests to human skin until detectable erythema appeared or until the experiment terminated after 10 consecutive days of applications (Stillman et al., 1975). Daily dermal application of 1 M hexanoic acid resulted in erythematous responses in 7 out of 10 subjects until the termination of the study and one out of 10 volunteers showed erythema after daily dermal application of 0.5 M hexanoic acid. Based on these results, hexanoic acid is considered to produce skin irritation in humans after chronic exposure in a concentration-dependent manner.

In conclusion, the results of in vivo and in vitro studies demonstrated, that hexanoic acid is corrosive. Concentrations of ≤ 60% hexanoic acid are non-corrosive on a regulatory point of view.

 

Eye 

in vivo

Smyth et al. (1954) reported strong eye injury (score 8) after application of hexanoic acid to rabbit eyes. In the 10-grade ordinal scale used, the grade 5 describes severe burn from 0.005 mL of the undiluted test item and grade 10 indicates severe burn from 0.5 mL of a 1% solution.

in vitro

The eye irritancy potential of hexanoic acid in a concentration of 50% was investigated in the bovine corneal opacity and permeability assay (BCOP) according to OECD Guideline 437 under GLP conditions (Lütkenhaus, 2012). 50% hexanoic acid in sesame oil was applied to the epithelial surface of three cattle corneas for 10 min at 32 ± 1 °C by addition to the anterior chamber of the corneal holder. The corneas were washed and the damage by the test substance was assessed two hours later by quantitative measurement of changes in corneal opacity with an opacitometer. In addition the permeability of the corneas was measured with a visible light spectrophotometer after incubation of the corneas with sodium fluorescein solution for 90 min. The results of the opacity and permeability measurement resulted in an in vitro irritation score (IVIS) of 143.61. Test substances with an IVIS ≥ 55.1 are regarded as severe eye irritants.

No further testing is required in accordance with Column 2 of Annex VIII, Section 8.2.1, of Regulation (EC) No 1907/2006, since hexanoic acid is classified as corrosive to skin and the registrant classifies hexanoic acid as severe eye irritant.

Justification for classification or non-classification

The available data on irriration / corrosion of hexanoic acid at a concentration > 60% meet the criteria for classification as Skin Cat. 1C (H314) according to Regulation (EC) 1272/2008.

The following concentration limits are assigned:

CLP (Regulation (EC) 1272/2008)

>60% Skin Cat. 1C;H314, Eye Cat.1; H318

≥ 3% - ≤60% Eye Cat.1;H318 and Skin Cat. 2;H315

≥1% - < 3% Eye Cat. 2;H319 and Skin Cat. 2;H315