L-(+)-lactic acid

Endpoint summary

Description of key information

There are reliable studies available for the assessment of the skin irritation and eye irritation endpoints with the target substance, L(+)-lactic acid. In accordance with the harmonized classification of the target substance, classification as Skin Corr. 1C, H314; Eye. Dam 1, H318 and supplementary labelling as EUH071 is warranted.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

yes

Species:

guinea pig

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: applied neat

Controls:

not required

Amount / concentration applied:

Concentration: 88 % aqueous solution

Duration of treatment / exposure:

3 min, 1h, 4 h

Observation period:

72h after patch removal

Number of animals:

6

Details on study design:

The animals were divided into 2 groups of 3. The hair was clipped from both flanks of all animals approximately 24 h prior to patch application. Care was taken to avoid abrading the skin. In Group l, 0.5 mL of the test material was applied under 2 gauze patches each measuring 2.5 cm x 2.5 cm (6 cm²). The patches were applied to the intact skin and covered with Micropore tape. The whole trunk was then bound with Elastoplast elastic bandage to give a semi-occlusive covering.
Group 2 animals were treated in a similar fashion except that the material was applied under one patch.
For Group l animals the first patch was removed 3 min after application and the skin gently washed with water to remove any residual test material. Reactions were scored. One hour after application the second patch was removed, the skin washed and reactions scored. The patch for Group 2 animals was left in place for 4 h before removal and scoring.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 1h

Score:

1

Max. score:

8

Reversibility:

fully reversible within: 24h

Remarks on result:

other: Only observed with 4h exposure

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 24h

Score:

0

Max. score:

8

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 48h

Score:

0

Max. score:

8

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 72h

Score:

0

Max. score:

8

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 1h

Score:

1

Reversibility:

fully reversible within: 24 h

Remarks on result:

other: Only observed with 4h exposure

Irritation parameter:

erythema score

Basis:

mean

Time point:

24 h

Score:

0

Irritation parameter:

erythema score

Basis:

mean

Time point:

48 h

Score:

0

Irritation parameter:

erythema score

Basis:

mean

Time point:

72 h

Score:

0

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 1h

Score:

0

Irritation parameter:

edema score

Basis:

mean

Time point:

24 h

Score:

0

Irritation parameter:

edema score

Basis:

mean

Time point:

48 h

Score:

0

Irritation parameter:

edema score

Basis:

mean

Time point:

72 h

Score:

0

Irritant / corrosive response data:

No irritation was noted in Group l animals at either 3 min or 1 h at the exposed sites. In Group 2, for which exposure was 4h, very slight erythema was noted in all animals. By 24 h after exposure, skin appeared normal. It is concluded from the test results that Lactic Acid Q88 is not corrosive to guinea pig skin. It is also noted that irritation after 4 h exposure was transient and limited to very mild erythema.

Interpretation of results:

other: not irritating

Conclusions:

Under the given conditions, the test item is not irritating to the skin.

Executive summary:

The corrosivity potential of a test material, Lactic Acid Q88, was investigated by means of a test in guinea pigs. The test was designed to assess irritancy and/or corrosivity by means of topical application of the test material to intact skin under semi occlusive conditions for various exposure times: 3 min, 1h and 4h. Two groups of 3 animals were used. In group 1 exposures of 3 min and 1h were investigated while 4 h exposures were investigated in group 2.

No irritation or corrosion were noted in animals exposed to Lactic Acid Q88 for 3 min and 1h. Responses in animals exposed to the test material for 4 h were limited to very slight erythema which was noted at patch removal and 1 h after patch removal. Skin appeared normal at 24 h after patch removal.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Purity: 88%
- Appearance: Clear colourless liquid

Species:

rabbit

Strain:

New Zealand White

Type of coverage:

semiocclusive

Preparation of test site:

other: intact skin and abraded skin

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

21 days in total

Number of animals:

6 rabbits treated on the intact skin and 6 rabbits treated on abreded skin

Details on study design:

The test was carried out according to the OECD Guideline no. 404, acute dermal irritation/corrosion, adopted by the OECD Council on May 12, 1981. After an acclimatization period of six days, i.e. one day prior to the start of the experiment, the hair was removed from the back and flanks of the animals, using electric clippers in a way as to avoid abrasions.

Six rabbits were treated on the intact skin and on the abraded skin. The abrasions were minor incisions of the stratum corneum, but not sufficiently deep to disturb the underlying derma or to produce bleeding. An around of 0.5 ml of the test material was brought onto the intact and the abraded skin under a surgical patch measuring 1 inch x 1 inch. The patches were fixed to the application site by means of adhesive tape and the entire trunk of the rabbits was wrapped with an impervious material to maintain the test patches in position and to retard evaporation ofvolatile substances. After an exposure period of 4 hours the patches and the material applied were removed and the resulting skin reactions were evaluated by the method of Draize et al. (J. Pharmacol. exptl. Therap. 82 (1944) 377-390). A transcript of the grading system by Draize et al. is given in an appendix to this report. Further readings were made after 28, 52 and 76 hours and after 7 and 21 days.

Irritation parameter:

overall irritation score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 4h

Score:

5.8

Max. score:

8

Irritation parameter:

overall irritation score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 28h

Score:

5.5

Max. score:

8

Irritation parameter:

overall irritation score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 52h

Score:

3.8

Max. score:

8

Irritation parameter:

overall irritation score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 76h

Score:

3.7

Max. score:

8

Irritation parameter:

erythema score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 4h

Score:

3.7

Max. score:

4

Irritation parameter:

erythema score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 28h

Score:

3.7

Max. score:

4

Irritation parameter:

erythema score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 52h

Score:

3.7

Max. score:

4

Irritation parameter:

erythema score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 76h

Score:

3.7

Max. score:

4

Irritation parameter:

edema score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 4h

Score:

2.2

Max. score:

4

Irritation parameter:

edema score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 28h

Score:

1.8

Max. score:

4

Irritation parameter:

edema score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 52h

Score:

0.2

Max. score:

4

Irritation parameter:

edema score

Remarks:

Intact skin

Basis:

mean

Time point:

other: 76h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Remarks:

Intact skin

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Remarks:

Other time points measured

Irritation parameter:

edema score

Remarks:

Intact skin

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Remarks:

Other time points measured

Irritant / corrosive response data:

See table 1 in the section "Any other information on results incl. tables".

After 4 hours the dermal effects generally observed in all rabbits consisted of very slight to slight ischemic necrosis, moderate to severe haemorrhages and slight or moderate oedema. After 28 hours the dermal effects observed generally consisted of very slight to slight ischemic necrosis, moderate haemorrhages, slight or moderate incrustation and slight: oedema. During the course of the following two days ischemic necrosis, haemorrhages and oedema were no longer observed. The application sites generally became crater-shaped with a central sunken area which was moderately or severely encrusted, and a surrounding, raised border of non-necrotic skin showing well-defined erythema. After 7 days this picture had hardly changed, apart from the clearance of erythema. The central sunken areas of the application sites generally showed moderate to severe incrustation. At the end of the observation period, after 3 weeks, some signs of healing were observed at the edges of the encrusted skin areas which had been in contact with the test material. In the new skin visible under the crust edges coming off from the treated skin, formation of scar tissue could be observed whereas hair growth was absent. There were no distinct differences between reactions of the intact skin and those of the abraded skin.

On the basis of observations in earlier experiments with comparable results, performed at the performing laboratory, the authors state that it is expected that the old necrotic skin will be ejected and that: the formation of scar tissue will be continued. This scar tissue forned already or yet to be formed is not considered a reversible skin alteration.

Table 1: INDIVIDUAL AND AVERAGE SKIN IRRITATION SCORES OF LACTIC ACID (88%), AFTER 4, 28, 52 AND 76 HOURS

Rabbit no.	Intact skin
	4 h		28 h		52 h		76 h
	A	B	A	B	A	B	A	B
3231	4	2	4	2	4	0	4	0
3232	4	2	4	2	4	0	4	0
3233	4	3	4	2	4	0	4	0
3234	4	3	4	2	4	0	4	0
3235	2	1	2	1	2	1	2	0
3236	4	2	4	2	4	0	4	0
average (A+B)	5.8		5.5		3.8		3.7

Rabbit no.	Abraded skin
	4 h		28 h		52 h		76 h
	A	B	A	B	A	B	A	B
3231	4	2	4	2	4	0	4	0
3232	4	2	4	2	4	0	4	0
3233	4	3	4	2	4	0	4	0
3234	4	2	4	2	4	0	4	0
3235	4	2	4	2	4	0	4	0
3236	4	2	4	2	4	0	4	0
average (A+B)	6.2		6.0		4.0		4.0

A = erythema

B = oedema

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

Under the given conditions, the test item is corrosive to the skin.

Executive summary:

A sample of lactic acid (88%) was examined for acute dermal irritating/corrosive properties in an experiment (OECD 404) with six albino rabbits.

After 4 hours the dermal effects generally observed in all rabbits consisted of very slight to slight ischemic necrosis, moderate to severe haemorrhages and slight or moderate oedema. After 28 hours the dermal effects observed generally consisted of very slight to slight ischemic necrosis, moderate haemorrhages, slight or moderate incrustation and slight: oedema. Three weeks after treatment, moderate to severe incrustation, formation of scar tissue and absence of hair growth were observed.

On the basis of the results obtained it was concluded that:

- lactic acid (88%) is severely irritating to the skin of albino rabbits

- lactic acid (88%) is corrosive to the skin of albino rabbit

Based on the results and in accordance with the harmonised classification for L-(+)-lactic acid, the test substance is considered to be corrosive to the skin.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

yes

Species:

pig

Strain:

other: F1 from Large White (GY) x Dutch Landrace (NL)

Details on test animals or test system and environmental conditions:

Healthy male, young pigs, F1 from Large White (GY) x Dutch Landrace (NL), born on 25th November 1986 and bred in the test laboratory, were used as the experimental animals. The body weight range at the start of the experiment was 40-50 kg.
The pigs were identified by earmarking, and housed, three together, in a stable with partially slatted floors. The stable was controlled for temperature (20 ± 3 °C), relative humidity (40-70%) and ventilation (air changes amounted to 0.5 - 1 m³/kg body weight/hour), and semi-controlled for light (external day light supplemented with 10 hours artificial light (TL) during the day time). A standard pig diet and tap water were provided ad libitum.

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

other: applied neat

Controls:

not required

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

21 days

Number of animals:

3

Details on study design:

Just prior to the start of the experiment, the hair was removed from the back of the animals using electric clippers in a way as to avoid abrasions. The test material was brought into contact with three small separate areas of intact skin (0.5 mL test material per application site). Each of the application sites was covered with an occlusive patch measuring 1 inch x 1 inch. The patches were fixed to the application sites by means of adhesive tape to maintain the patches in position and to prevent evaporation of the test liquid. The three patches were removed after a dermal contact period varying from 3 minutes to 4 hours, i.e. one patch was removed after 3 minutes, the second patch after 60 minutes, and the third patch after 4 hours. Immediately after removing the test patches the treated skin areas were cleaned with lukewarm water, and one hour later the resulting skin reactions were evaluated by the method of Draize et al. (J. Pharmacol. Exp. Ther. 82 (1944) 377-390). Further readings were made after 1 day, and after 2, 3, 7, 14 and 21 days.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 1h and 1, 2, 3, 7, 14, 21d

Score:

0

Max. score:

8

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Remarks:

After application duration of 3 minutes, 1 hour or 4 hours

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Remarks:

After application duration of 3 minutes, 1 hour or 4 hours

Irritation parameter:

erythema score

Basis:

mean

Time point:

7 d

Score:

0

Remarks on result:

no indication of irritation

Remarks:

After application duration of 3 minutes, 1 hour or 4 hours

Irritation parameter:

erythema score

Basis:

mean

Time point:

14 d

Score:

0

Remarks on result:

no indication of irritation

Remarks:

After application duration of 3 minutes, 1 hour or 4 hours

Irritation parameter:

erythema score

Basis:

mean

Time point:

21 d

Score:

0

Remarks on result:

no indication of irritation

Remarks:

After application duration of 3 minutes, 1 hour or 4 hours

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Remarks:

There was some minor superficial wounds and slight small crusts at application site and non-treated skin areas observed in 2 pigs at 24-28 h after 1-4 h of application duration. They were no longer detectable by 72 h after exposure.

Irritation parameter:

edema score

Basis:

mean

Time point:

7 d

Score:

0

Remarks on result:

no indication of irritation

Remarks:

After application duration of 3 minutes, 1 hour or 4 hours

Irritation parameter:

edema score

Basis:

mean

Time point:

14 d

Score:

0

Remarks on result:

no indication of irritation

Remarks:

After application duration of 3 minutes, 1 hour or 4 hours

Irritation parameter:

edema score

Basis:

mean

Time point:

21 d

Score:

0

Remarks on result:

no indication of irritation

Remarks:

After application duration of 3 minutes, 1 hour or 4 hours

Interpretation of results:

other: not irritating

Conclusions:

Under the given conditions the test item is not irritating to the skin of pigs.

Executive summary:

In a dermal irritation study, a sample of lactic acid (88%) was examined for acute dermal irritating/ corrosive properties with three pigs. In each animal, the test substance was brought into contact with three separate areas of shaved dorsal skin for 3 and 60 minutes and for 4 hours, respectively. The test sample did not cause any skin irritation when it was brought into contact with the dorsal skin of pigs for 3 or 60 minutes or for 4 hours. On the basis of the results; obtained in the present study with pigs, it was concluded that, according to the EEC-standards, lactic acid (88%) is not irritating or corrosive to skin.

Reference 4

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted according to a protocol entitled "Protocol for an in vitro skin irritation study in rabbit and human skin organ cultures after 30 minutes exposure to lactic acid and lactic acid esters", approved by the study director on May 28, 1996.
The toxicity of the test substances and the reference substance were determined in both human and rabbit skin organ cultures. The compounds were applied topically for a period of 30 minutes. The following two endpoints were determined 48 hr after removal of the compounds: epidermal cell proliferation and conversion of the tetrazolium salt MTT. Each experimental group consisted of six (unexposed control group) or four skin discs.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Test substance: HS88
- Chemical name: L(+)-lactic acid adduct aqueous solution
- Content of active ingredient: 88% in aqueous solution
- Supplier: PURAC Biochem B.V., Gorinchem, NL
- Batch No.: AP 5074L
- CAS: 79-33-4
- Date of arrival: 15-02-1996
- Appearance: clear colourless, yellowish liquid
- Storage conditions: room temperature

Test system:

isolated skin discs

Remarks:

in vitro rabbit skin and human skin organ culture

Source species:

rabbit

Details on animal used as source of test system:

Rabbit skin was obtained from one New Zealand White male albino rabbit. The animal was clipped free of hair one day before the start of the study. Approval for the use of one rabbit for this study has been given by the TNO Animal Experimentation Committee (DEC no. 015).
Human skin was obtained directly after surgery from one female caucasian donor of 23 years old (code TNA/9605) at the Department of Plastic and Recontructive Surgery (Academie: Hospital Utrecht, the Netherlands). The skin was transported on ice to the laboratory within 1 h after dissection. Directly after arrival, skin discs were isolated for culture. Approval for the use of human skin for in vitro studies has been given by the TNO Medical Ethical Committee (MEC-TNO code 95/17).
Skin was cultured in a two-compartment skin organ culture model as described by Rutten et al. (1990) and Van de Sandt et al (1993, 1995a,b). Briefly, circular discs (2 cm²) were punched out from the isolated skin after careful removal of subcutaneous fat. Skin discs were washed three times for ca. 15 min in medium supplemented with bactericides and fungicides to prevent microbiological contamination of the cultures. Skin explants were cultured in 6-well plates on a microporous membrane (Millicel-HA insert unit, Millipore, Bedford, MA), which allows transport of culture medium via the dermis into the epidermis, while the epidermal side of the skin remains free of contact with the culture medium. The skin discs were cultured in a humidified incubator gassed with 5% CO2 and 40% O2 at 32 °C. To obtain optimal culture conditions and a homogeneous distribution of the receptor fluid the 6-well plates were rocked on a platform ca. 9 times per min.

Vehicle:

unchanged (no vehicle)

Details on test system:

Before starting the culture of the skin discs, a sample of non-cultured skin was fixed in 70% (v/v) aqueous ethanol (4-8 °C) (encoded TO). At 48 h after removal of the test substances, each skin sample was cut in two with a scalpel knife. One half was fixed in 70% (v/v) aqueous ethanol (4-8 °C) for immunohistochemical analysis of epidermal cell proliferation, the other half was placed back into the original well for measuring the conversion of the tetrazolium salt MTT.

Control samples:

yes, concurrent no treatment

Amount/concentration applied:

100 µL

Duration of treatment / exposure:

30 min

Duration of post-treatment incubation (if applicable):

None - cells were harvested and tested directly upon end of exposure.

Number of replicates:

not specified

Details on study design:

Topical application of the test substance in vitro:
One day after the isolation of the skin, the culture medium was removed and fresh medium was added to the cultures. The epidermal side of the skin discs were carefully dried using sterile prefilters (Millipore filter, type AP10, 1.3 cm", sterilized). Subsequently, fresh sterile prefilters were put on the epidermal side together with 100 µl of the test solution. After a treatment period of 30 minutes, the filters and the culture medium were removed. By using fresh prefilters, traces of test substances remaining on the skin cultures were removed. Subsequently, each skin disc was washed three times with 4 mL sterile 0.1 M phosphate- buffered saline (pH= 7.2) (Flow Laboratories, Irvine, Scotland) while remaining in the insert units. Subsequently, fresh culture medium was added to the skin discs.

Sampling and measurements
Collection of skin tissue:
Before starting the culture of the skin discs, a sample of non-cultured skin was fixed in 70% (v/v) aqueous ethanol (4-8 °C) (encoded TO). At 48 h after removal of the test substances, each skin sample was cut in two with a scalpel knife. One half was fixed in 70% (v/v) aqueous ethanol (4-8°C) for immunohistochemical analysis of epidermal cell proliferation, the other half was placed back into the original well for rneasuring the conversion of the tetrazoHum salt

Determination of epidermal cell proliferation:
Determination of epidermal cell proliferation was carried out according to the method described by van de Sandt et al. (1995b). In brief, after a 24-h incubation period with 5-bromo-2'-deoxyuridine (BrdU, 10 µL from a stock solution of 2 mg BrdU/mL phosphate-buffered saline, 0.1 M), the skin discs were fixed in 70% (v/v) aqueous ethanol at 4-8 °C for at least 72 h. The tissues required for microscopic examination were embedded in paraffin wax and sectioned at 4 µm. For examination of proliferating epithelial cells, tissue sections were stained immunorustochemically and counterstained with haematoxylin. Cell proliferation was assessed by counting epidermal cells containing a BrdU-positive nucleus. Hair follicles and the epiboly, standardized as 1 mm from both edges of the skin disc, were excluded from the counting procedure. Cell proliferation was scored by one person in a "blind" fashion.

MTT assay:
The MTT assay was carried out 48 h after removal of the test substances. Cul- ture medium without serum containing 2.0 mg MTT (3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltei:razolium bromide) per ml was added to the half skin discs (4 ml/well). After a 4-h incubation period at 32 °C, 40% O2 and 5% CO2, the skin tissue was washed twice in prewarmed PBS (37 °C). The MTT-formazan precipitate was extracted from the tissue in polysterene tubes containing 5 mL acidified isopropanol (0.04 M HCl) for 4 days in the dark, after which the optical density was measured at 560 nm (OD56Ü) on a Cary l E UV-visible spectrophotometer (Varian Analytical Instruments, Houten, the Netherlands). The skin tissue was dried overnight at 37°C and weighed on an analytical balance. Data of the MTT assay were presented as OD5w/g tissue.

Deviations from the protocol
The following minor deviations were made in the method for immunohisto- chemical staining of cells labelled with 5-bromo-2'-deoxyuridine:
- endogenous peroxidase was inhibited using 0.3% H2O2 in methanol instead of demineralized water.
- the DAB solution contained 0.5 mg/mL instead of 5 mg/mL DAB. - no Tween-20 was used during incubation of the sections with the antibodies.

Irritation / corrosion parameter:

other: MTT conversion

Remarks:

in OD560 per gram dry weight

Run / experiment:

30 min exposure; Rabbit skin

Value:

ca. 18.48

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: no statistically significant deviation from control

Irritation / corrosion parameter:

other: MTT conversion

Remarks:

in OD560 in gram per dry weight

Run / experiment:

30 min exposure; Human skin

Value:

ca. 37.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: Statistically significantly lower than the non-exposed control group (p<0.05)

Irritation / corrosion parameter:

other: Epidermal cell proliferation in skin organ cultures

Remarks:

in BrdU+ cells per cm epidermis

Run / experiment:

30 min. exposure; Human skin

Value:

ca. 59

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: no statistically significant deviation from control

Irritation / corrosion parameter:

other: Epidermal cell proliferation in skin organ cultures

Remarks:

in BrdU+ cells per cm epidermis

Run / experiment:

30 min exposure; Rabbit skin

Value:

ca. 55

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: no statistically significant deviation from control

Interpretation of results:

other: not irritating

Conclusions:

The effect of lactic acid on rabbit and human skin was studied in vitro in skin organ cultures. Toxicity was measured by epidermal cell proliferation and by the conversion of a tetrazolium salt (MTT). Based on the MTT assay and inhibition of epidermal cell proliferation, rabbit skin was clearly more sensitive to the test item than human skin.

Executive summary:

Lactic acid was examined for in vitro skin toxicity in skin organ cultures. Toxicity was determined by measuring epidermal cell proliferation and the conversion of the tetrazolium salt MTT. In rabbit skin, MTT conversion was statistically significantly reduced after exposure to HS88. Possible species-specific irritant effects of lactic acid were tested in vitro by comparing rabbit skin to human skin. Based on the MTT assay and inhibition of epidermal cell proliferation, rabbit skin was clearly more sensitive to HS88 than human skin. A possible explanation for this difference is a lower skin absorption of the test substance in human skin, since rabbit skin is generally more permeable for topically applied chemicals than human skin (ECETOC, 1993). The anionic surfactant sodium dodecyl sulfate (SDS) was used as a reference substance to enable comparison of the in vitro results of this study to previous data obtained with the skin organ culture model. Exposure of rabbit skin for 30 minutes to 5% SDS induced a decrease of MTT conversion of approximately 15%. It has been reported that 5% SDS is a moderate irritant in rabbits (Gad et ai, 1986) and human volunteers (Willis et al, 1988). In conclusion, this in vitro skin toxicity study revealed that rabbit skin was more sensitive to HS88 than human skin.

Reference 5

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-5 (Acute Dermal Irritation)

GLP compliance:

yes

Specific details on test material used for the study:

- Name of the test material: SY-83
- Appearance: liquid

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Young adult, New Zealand White albino rabbits were obtained from Langshaw Farms, Route l, Box 256, Augusta, MI 49012. The albino rabbit is the species preferred in the EPA/OPP Guidelines (1982) for determining the dermal irritation potential of test articles. Animals were housed individually in stainless steel, wire-bottomed cages that conformed to the size standards specified in DHEW Publication (NIH) 78.23. The cages on each rack were numbered in a Standard manner and a list of random numbers was generated by computer program for each rack of cages. Upon receipt, each animal was removed from the shipping container and housed in the appropriate randomly selected cage.
Each animal was then assigned a sequential animal number unique within ToxiGenics and identified with an ear tag bearing this animal number. The sequential animal number was listed on a cage card that was affixed to the front of the animal's cage.
The animals were quarantined for 2 weeks after receipt. During the quarantine period, Veterinary Sciences' personnel observed the animals at least once each day for mortality, morbidity, and abnormal signs. Animals were examined during quarantine and only those considered to be in good health were used in this study. The quarantine and study room (241) was cleaned daily and cages were cleaned and sanitized as specified in ToxiGenics' Standard Operating Procedures. Urine and feces fail through the wire mesh floor onto animal caging board. The cage boards were changed at least 3 times a week.
The animal room was well ventilated and air-conditioned, and the temperature and humidity were monitored daily in this room during the quarantine and study periods. The temperature ranged from 66 to 70 °F and the relative humidity varied from 37 to 64 percent except for one day when a relative humidity value of 72 percent was recorded.
The animal room was lighted from approximately 6:00 a.m. to 6:00 p.m. (12-hour light/12-hour dark cycle) using automatic timers. The test article applications were completed at 12:05 p.m. on November 8, 1983. Purina Certified Rabbit Chow 5322 was fed to the animals ad libitum during the quarantine and study periods. Filtered tap water was provided ad libitum through an automatic watering system and was analyzed periodically as specified in ToxiGenics' Standard Operating Procedures.

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped and abraded

Vehicle:

other: applied neat

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

24 hours

Observation period:

60 minutes

Number of animals:

6

Details on study design:

The duration of the study was one day. One group consisting of 3 males and 3 females was used. Animals were assigned to the study by sequential animal number. However, any animal deemed unsuitable by the Study Director and any animal exhibiting dermal lesions or irritation (-24 hour evaluations) was not used, and the next acceptable sequentially numbered animal was used.
Two test sites were prepared on each side of the spinal column in the thoracic región of each animal by closely clipping the hair with Oster electric clippers equipped with a number 40 (surgical) blade. Approximately 24 (+ 2) hours after clipping, 0.5 milliliter of the test article was applied to each 2.5 centimeter square surgical gauze patch. While each animal was manually immobilized, 2 application sites on each animal After a 24-hour exposure period, the impervious binders and patches were removed. Each test site was gently wiped with gauze sponges moistened with water to remove remaining test article. All animals were observed for mortality and abnormal clinical signs twice daily during the study in the early morning and late afternoon. The skin condition of each test site was evaluated for erythema, edema, and other lesions at 30 to 60 minutes after test article removal. Dermal reaction scores were assigned using the grading system presented in Table l of this report. Representative photographs of lesions observed were taken after the 30 to 60 minute evaluations. One set of photographs were submitted to the Sponsor as an addendum report.
After the 30- to 60-minute evaluations, all animals were euthanized by administration of an intraveneus overdose of a barbiturate and discarded. The study was terminated due to the severity of the dermal reactions observed.

Irritation parameter:

erythema score

Basis:

other: at 10 of 12 abraded sites

Time point:

other: 30 to 60 minutes

Score:

ca. 4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Remarks:

24-hour exposure period

Irritation parameter:

edema score

Basis:

other: at 11 of 12 intact test sites

Time point:

other: 30 to 60 minutes

Score:

ca. 4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Remarks:

24-hour exposure period

Irritation parameter:

erythema score

Basis:

other: number of abraded sites

Time point:

24/48/72 h

Reversibility:

not specified

Remarks on result:

not measured/tested

Remarks:

Only evaluated 30 to 60 minutes after test item removal

Irritation parameter:

edema score

Basis:

other: number of intact test sites

Time point:

24/48/72 h

Reversibility:

not specified

Remarks on result:

not measured/tested

Remarks:

Only evaluated 30 to 60 minutes after test item removal

Irritant / corrosive response data:

The following were observed at the evaluations done at 30 to 60 minutes after test article removal (Grading system according to Draize 1979):
- Severe erythema (grade 4) at 10 of the 12 abraded test sites (on 5 animals) and at 7 of the 12 intact test sites on 4 of the same animals. Moderate to severe erythema (grade 3) at the 2 remaining abraded and 5 remaining intact test sites (all sites on one animal and one or both intact sites on 2 animals).
- Severe edema (grade 4) at 11 of 12 intact and 11 of 12 abraded test sites, and slight edema (grade 2) at one intact site and one abraded site on one animal.
- Blanching at both abraded sites on each animal, and at both intact sites on 5 animals.
- Yellow-brown color of skin at all sites on 3 animals, at both abraded sites on one animal, and at one intact site and both abraded sites on a fifth animal.
- Red exudate at one intact site on one animal.
- Skin missing at all sites on one animal, at one intact site and both abraded sites on one animal, and at one intact site or one abraded site on 2 other animals. No other dermal reactions were observed at the evaluations done at 30 to 60 minutes after test article removal.
One animal (BB5196) was observed by an attending veterinarian and the following comments were noted:
Both lesions in the left lateral thoracic and abdominal wall were circular and approximately 4 cm. in diameter. Tags of necrotic skin were observed around the circumference while the entire center (greater than a 3 cm. circle) was totally denuded of all layers of skin. The peritoneum was intact, and hemorrhage from the peritoneal vessels and skin vessels around the circumference was evident. Without medical treatment, these and similar lesions were expected to become secondarily infected
resulting in the animals death. Termination of the study was recommended by the veterinarian. No abnormal clinical signs were observed and no mortalities occurred prior to sacrifice after the 30- to 60-minute evaluations.

Interpretation of results:

other: highly irritating

Conclusions:

Under the given conditions, the test item is highly irritating to the skin of rabbits.

Executive summary:

L(+)-lactic acid, was evaluated for primary dermal irritation potential when applied to 2 intact and 2 abraded test sites on the skin of 6 albino rabbits and covered with impervious bandages for 24 hours. These 24 test sites were evaluated for erythema, edema, and other lesions at 30 to 60 minutes after test article removal. This study was designed to comply with the procedures described in the EPA/OPP Guidelines, 1982. The following were observed at 30 to 60 minutes after test article removal: Severe erythema was observed at all test sites on 3 animals, and at both abraded sites on 2 other animals and at one intact site on one of these 2 animals. Moderate to severe erythema was observed at all test sites on one animal, and at one or both intact sites on 2 animals. Severe edema was observed at all test sites on 5 animals and at one intact site and one abraded site on the sixth animal. Slight edema was observed at the other 2 sites on the sixth animal. Blanching was observed at both abraded sites on all animals and at both intact sites on 5 of these animals. Yellow-brown colour of the skin was observed at all sites on 3 animals and at either 2 or 3 test sites on 2 animals. A red exudate was observed at one intact site on one animal. Skin was missing at all test sites on one animal, at one intact and both abraded sites on one animal, and at one intact site or one abraded site on 2 other animals. No other dermal reactions were observed during the study. This study was terminated after the 30- to 60-minute evaluations upon the recommendation of an attending veterinarian due to the severity of the reactions observed. No abnormal clinical signs were observed, and no mortalities occurred prior to sacrifice after the 30- to 60-minute evaluations. Based on the results, the test item is considered to be highly irritating to the skin of rabbits.

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:

1996-01-18 to 1996-03-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

Study performed prior to validation and publication of the test guideline.

Deviations:

yes

Remarks:

No positive controls

GLP compliance:

yes

Specific details on test material used for the study:

- Name of the test material: Purac HS88
- Appearance: clear colourless liquid
- Batch No.: AP 5074L
- Purity: ca. 88%
- Stotrage: ambient temperature

Species:

other: Chicken Enucleated Eye Test

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

Concentration: 100%
Amount applied: 0.03 mL

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

3

Details on study design:

Approximately 7 weeks old, male or female chickens (ROSS, spring chickens), body weight range approximately 2.5-3.0 kg, were used as eye-donors. Heads of these animals were obtained from poultry slaughterhouse v.d. Bor, Amersfoortseweg 118, Nijkerkerveen, the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes (3 heads per box) on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature. Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium BP 2% w/v (Minims, Smith & Nephew Ltd., Romford, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline of ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 CN, Haag-Streit AG, Liebefeld-Bern, Switzerland), to ensure that the cornea was not damaged. If undamaged, the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (TNO, Zeist, the Netherlands). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a rate of ca 0.10-0.15 mL/min (peristaltic pump, Desaga STA 131900, Heidelberg, Germany). The chambers of the superfusion apparatus as well as the saline were temperature controlled at 32 ± 1.5 °C (waterpump, Thermomix 1441, B. Braun Melsungen AG, Melsungen, Germany). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment no. II for the Haag-Streit slit-lamp microscope. Thickness of the cornea was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, or eyes that were unacceptably stained with fluorescein (score higher than 0.5), indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced, if necessary. Per test sample, three eyes were selected for testing, whereas one additional eye was rinsed with isotonic saline only and served as a control of the experimental conditions. After an equilibration period of 45-60 minutes, the corneal thickness of the eyes were measured again to determine the zero reference value for corneal swelling calculations. At time t = 0, i.e. immediately after the zero reference measurement, each of the three test samples was applied to the designated test eyes. The following procedure applied for each test eye:
The clamp holding the eye was placed on paper tissues outside the chamber with the cornea facing upwards. Each of the two liquid samples was applied in an amount of 0.03 mL from a micropipette (Nichiryo Co., Ltd., model 8100, Tokyo, Japan), in such a way that the entire surface of the cornea was bathed with the test material. With the solid sample, the cornea was powdered with 0.03 g of the test material. After a total exposure period of 10 seconds, the corneal surface was rinsed thoroughly with 20 mL of isotonic saline of ambient temperature. After rinsing, each eye in the holder was returned to its chamber.
The control eye and test eyes were examined at 0, 30, 75, 120, 180 and 240 minutes after treatment, using the criteria and scoring system, given in the annex. Fluorescein retention was only scored at 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.

Irritation parameter:

cornea opacity score

Remarks:

after 240 min

Run / experiment:

1

Value:

ca. 4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

after 240 min

Run / experiment:

2

Value:

ca. 4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

after 240 min

Run / experiment:

3

Value:

ca. 4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Irritation parameter:

percent corneal swelling

Remarks:

after 240 min

Run / experiment:

1

Value:

ca. 31

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Irritation parameter:

percent corneal swelling

Remarks:

after 240 min

Run / experiment:

2

Value:

ca. 26

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Irritation parameter:

percent corneal swelling

Remarks:

after 240 min

Run / experiment:

3

Value:

ca. 28

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Remarks:

after 30 min

Run / experiment:

1

Value:

ca. 3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

not determinable

Irritation parameter:

fluorescein retention score

Remarks:

after 30 min

Run / experiment:

2

Value:

ca. 3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Remarks:

after 30 min

Run / experiment:

3

Value:

ca. 3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In conclusion, based on the given conditions the test item is considered severely irritating to eyes.

Executive summary:

L-lactic acid 88% (sample code HS88), was examined undiluted for eye irritating/corrosive potential in an ex vivo bioassay, namely the Enucleated Eye Test with chicken eyes (CEET). The eyes were collected as waste material from a slaughterhouse for chickens, which were killed for human consumption. HS88 induced severe corneal effects. On the basis of the results obtained with this in vitro (ex vivo) assay, HS88 can be considered severely irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are reliable studies available for the assessment of the skin irritation and eye irritation endpoints with the target substance, L(+)-lactic acid. In in vivo and in vitro tests with L(+)-lactic acid, the skin irritation potential of the test item was assessed using the weight of evidence approach.

Based on the results from in vivo studies conducted with rabbits, the test substance must be considered as corrosive to the rabbit skin; however, it is not irritating at all to guinea pig skin or porcine skin and in an in vitro test it was shown that lactic acid 88 % was toxic to a skin cell patch of rabbit skin cells, but not to human skin cells. Therefore, it can be stated that rabbit skin is more sensitive to lactic acid irritation than other skin types, including human skin.

In accordance with the harmonized classification for the target substance, classification as Skin Corr. 1C, H314 is warranted.

An in vitro enucleated eye test with chicken eyes was conducted equivalent to OECD TG 438 with L(+)-lactic acid (88%). The eyes were collected as waste material from a slaughterhouse for chickens, which were killed for human consumption. The test item induced severe corneal effects. Therefore classification for eye damage (Eye Dam.1, H318) is warranted according to CLP Regulation 1272/2008.

In summary and according to Annex VI of CLP Regulation 1272/2008, classification as Skin Corr. 1C, H314, Eye Dam. 1, H318 and supplementary labelling as EUH071 (corrosive to the respiratory tract) is warranted.

Justification for classification or non-classification

According to Annex VI of CLP Regulation 1272/2008, classification as Skin Corr. 1C, H314, Eye Dam. 1, H318 and supplementarily labelling as EUH071 (corrosive to the respiratory tract) is warranted.