2-methylpropyl-(R)-2-hydroxypropanoate

Endpoint summary

Description of key information

Isobutyl-R-lactate was tested negative for dermal irritating properties in accordance to OECD 439. As no data was available for eye irritating properties of the target substance isobutyl-R-lactate, data from lactic acid and several structurally similar lactate esters were used in a read-across and weight-of-evidence approach. All compounds tested induced eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-25 to 2015-07-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: human epidermis model:Episkin

Details on test animals or test system and environmental conditions:

Test system:
The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit: 10 µL (26.3 µL/cm²)

PREPARATION OF THE TEST ITEM:
The test item was applied undiluted

CONTROLS:
Controls were set up in parallel to the test item in order to confirm the validity of the test.
Negative control: DPBS (Gibco, Cat. No. 14040-091, Lot No.: 1672813).
Positive control: 5 % sodium dodecyl sulfate (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3, Lot No.: 1V010396) in aqua dest.

Duration of treatment / exposure:

15 ± 0.5 min

Details on study design:

TEST SITE
- Area of exposure: EPISKIN-SM tissue
- size per reconstructed epidermis unit: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing (if done): after 15 ± 0.5 min with DPBS

STUDY DESIGN:
Upon receipt of the EPISKIN-SM tissues, the tissues were transferred into 12-well plates containing 2 mL pre-warmed maintenance medium per well, and were incubated in a humidified incubator at 37 ± 1 °C, 5.0 % CO2 for at least 24 h.
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the tissues were incubated at room temperature for 15 ± 0.5 min. The positive control was re-spread after 7 min contact time. Afterwards, the tissues were washed with DPBS to remove any residual test item. Excess DPBS was removed by blotting bottom with blotting paper. The inserts were placed in a prepared 12-well plate containing 2 mL pre-warmed fresh maintenance medium and post-incubated at 37 ± 1 °C, 5.0% CO2 for 42 ± 1 h.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 12-well plate containing 2 mL pre-warmed MTT medium and further incubated for 3 h ± 5 min. at 37 ± 1 °C, 5.0 % CO2.
After the 3 h MTT incubation period the tissues were placed on blotting paper to dry. Afterwards a total biopsy of the epidermis using the special biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 μL of acidic isopropanol were added. The plugged tubes were mixed using a vortex mixer. Extraction was carried out protected from light over the weekend at 2-8 °C.
At the end of the formazan extraction period the tubes were mixed by vortexing until solution colour became homogeneous.
If any visible cell/tissue fragments were in suspension, the tubes were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
Per each tissue 2 × 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer.

SCORING SYSTEM:
The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 [18] and UN GHS [16], [17] “Category 2”, if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50 %. The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is higher than 50 %.

Irritation / corrosion parameter:

other: other: mean relative tissue viability

Value:

ca. 88.5

Remarks on result:

other:

Remarks:

Basis: mean (of triplicates). Time point: 15 min treatment and 42h post-incubation. Max. score: 100.0. Remarks: Max. score: 100 % viability obtained by the negative control. (migrated information)

Table 1: Results of the test item isobutyl-R-lactate

Name		Negative Control			Positive Control			Test Item
Tissue		1	2	3	1	2	3	1	2	3
Absolute OD 570		0.815	0.726	0.989	0.083	0.093	0.070	0.650	0.911	0.693
		0.835	0.725	1.008	0.085	0.106	0.073	0.673	0.898	0.715
OD570 (Corrected)		0.775	0.687	0.950	0.043	0.053	0.030	0.610	0.872	0.653
		0.796	0.686	0.968	0.045	0.067	0.033	0.633	0.859	0.675
Mean OD 570 (Corrected)		0.810*			0.045			0.717
SD OD 570		0.138			0.014			0.130
Relative Tissue Viabilities [%]		97.0	84.7	118.4	5.5	7.4	3.9	76.8	106.8	82.0
Mean Relative Tissue Viability [%]		100 %			5.6**			88.5
SD Tissue Viability [%]***		17			1.7			16.0

*Corrected mean OD 570 of the negative control corresponds to 100 % absolute tissue viability

**Mean relative tissue viability of the three positiv control tissues is ≤ 40 %

***Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18 %.

Table 2: Test acceptance criteria

	Value	Cut off	pass/fail
Mean OD570 nm Blank	0.039	< 0.1	pass
Mean Absolute OD570 nm NK	0.85	0.6 ≤ NK ≤1.5	pass
Mean Relative Viability [%] PC	5.6	≤ 40 %	pass
SD of % Viability [%]	1.7–17.0	≤ 18 %	pass

NK: negative control

PC: positive control

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, in the human skin model EPISKIN-SM (SkinEthic) no irritant effects after treatment with isobutyl-R-lactate were observed.

Executive summary:

In the present study 10 µL of undiluted isobutyl-R-lactate (99.7 % purity) was applied directly atop the EpiSkin-SMTM tissue for 15 min followed by 42 h post incubation period and immediate determination of cytotoxic effects via MTT reduction assay in accordance to OECD guideline 439.

Irritant potential from the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control. No irritant effects were observed. The mean relative tissue viability (% negative control) was > 50 % (88.5 %) after 15 min treatment and 42 h post-incubation. The controls confirmed the validity of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1994-06-14 to 1994-09-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. 2-ethylhexyl lactate used as read-across partner.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Broekman Instituut BV, Someren, the Netherlands
- Age at study initiation: young adult, sex not determined
- Date of arrival: 1994-05-18 (animal number 29 and 30) or 1994-06-01 (animal number 37)
- Date of dosing: 1994-06-14 (animal number 37) and 1994-06-15 (animal number 29 and 30)
- Weight at study initiation: 2250-2940 g
- Housing: individually in a stainless steel cage, fitted with a perforated floor
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 and 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 42-82.5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 1994-06-01 to 1994-07-08

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated, serving as a control.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): min. 97 %

Duration of treatment / exposure:

An amount of 0.1 mL of the test substance was instilled in the conjunctival cul-de-sac of the right eye. After administration, the upper and lower eye lid were carefully closed and subsequently held together for at least one second before releasing, to prevent loss of material.

Observation period (in vivo):

1, 24, 48 and 72 hours; 7, 14, 21 and 25 days

Number of animals or in vitro replicates:

3

Details on study design:

Pre-test:
Prior to the in vivo testing, the test substance was also examined in the Chicken Enucleated Eye Test (CEET). The test substance caused severe reactions in the CEET.

Main Test:
Based on the results from this CEET it was decided to start with one rabbit. As severe signs of irritation were not observed in this rabbit, it was decided to continue the test with additional two rabbits.

SCORING SYSTEM: See Table 1.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of three animals

Time point:

other: 24, 48 and 72 hours

Score:

1.66

Max. score:

2

Reversibility:

fully reversible within: 21 days

Remarks on result:

other: The cornea score for two animals was 0 after 7 days

Irritation parameter:

iris score

Basis:

mean

Remarks:

of three animals

Time point:

other: 24 , 48 and 72 hours

Score:

0.66

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: The iris score of two animals was 0 after 72 hours

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

of three animals

Time point:

other: 24, 48 and 72 hours

Score:

2.44

Max. score:

3

Reversibility:

fully reversible within: 21 days

Remarks on result:

other: The conjunctivae score of two animals was 0 after 14 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of three animals

Time point:

other: 24, 48 and 72 hours

Score:

1.22

Max. score:

2

Reversibility:

fully reversible within: 21 days

Remarks on result:

other: The chemosis score of two animals was 0 after 14 days

Irritation parameter:

other: ocular discharge

Basis:

mean

Remarks:

of three animals

Time point:

other: 24, 48 and 72 hours

Score:

1.55

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: The ocular discharge score of two animals was 0 after 72 hours

Irritant / corrosive response data:

At 1 hour after treatment, the eye effects observed in the three rabbits consisted of slight corneal opaicity, slight iritis, slight redness and moderate swelling of the conjunctivae, and severe ocular discharge. At 24 hours after treatment, the eye effects observed consisted of slight or moderate corneal opacity, slight iritis, moderate or severe redness and slight swelling of the conjunctivae, and slight or severe ocular discharge. At 48 hours after treatment, the eye effects observed consisted of slight or moderate corneal opacity, slight iritis (two rabbits), moderate or severe redness and slight or moderate swelling of the conjunctivae, and slight or severe ocular discharge. At 72 hours after treatment, the eye effects observed consisted of slight or moderate corneal opacity, slight iritis (one rabbit), slight, moderate or severe redness and slight or moderate swelling of the conjunctivae, and moderate ocular discharge (one rabbit). At 7 days after treatment, the eye effects observed consisted of slight corneal opacity and vascularization of the cornea in one rabbit, and slight redness and slight swelling of the conjunctivae in two rabbits. At 14 days after treatment, eye effects were still observed in one rabbit and consisted of slight corneal opacity, vascularisation of the cornea, and slight redness and slight swelling of the conjunctivae. At 21 days, these eye effects except minor vascularisation of the cornea had cleared completely. At 25 days, this eye effect had also cleared completely.

Other effects:

N.A.

Table 2: Individual scores awarded to the ocular lesions elicited by 2-etyhlhexyl lactate
rabbit number	corneal opacity	iris effects	conjunctivae		ocular discharge
			redness	chemosis
after one hour
37	1 (4)	1	1	2	3
29	1 (4)	1	1	2	3
30	1 (4)	1	1	2	3
after 24 hours
37	2 (4)	1	3	1	3
29	1 (4)	1	3	1	3
30	2 (2)	1	2	1	1
after 48 hours
37	2 (2)	1	3	1	1
29	2 (2)	1	3	2	3
30	1 (2)	0	2	1	1
after 72 hours
37	2 (2)	0	2	1	0
29	2 (4)	1	3	2	2
30	1 (1)	0	1	1	0
after 7 days
37	0	0	1	1	0
29	1 (4)*	0	1	1	0
30	0	0	0	0	0
after 14 days
37/30	0	0	0	0	0
29	1 (3)*	0	1	1	0
after 21 days
29	0*	0	0	0	0
after 25 days
29	0	0	0	0	0
(): area of opacity, 1= one quarter (or less) but not zero, 2= half area, 3= three quarter, 4= entire area
*= vascularization of the cornea; the degree diminshed gradually

Interpretation of results:

Category II

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

2-ethylhexyl-lactate is distinctly irritating for the eyes of rabbits.

Executive summary:

In a primary eye irritation study (OECD 405), 0.1 mL of  2-etyhlhexyl lactate (purity ≥ 97 %) was instilled into the conjunctival sac of the right eye of three young adult New Zealand White rabbits. Animals then were observed for a maximum of 25 days. Irritation was scored by the method according to OECD guideline 405. The test substance caused moderate corneal opacity, slight iritis, moderate or severe redness and moderate swelling of the conjunctivae, and severe ocular discharge in the three rabbits. These adverse irritation effects were fully reversible within 21 days. In accordance to the CLP regulation 2-etyhlhexyl lactate needs to be classified in Category 2 (irritating to eyes).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Isobutyl-R-lactate (target substance) was tested negative for dermal irritating properties in accordance to OECD test guideline 439.

As no data was available for eye irritating properties of the target substance, data from lactic acid and several structurally similar lactate esters were used in a read-across and weight-of-evidence approach. All compounds tested induced eye irritation. In the key study (OECD test guideline 405) the source substance ethylhexyl lactate induced irritating effects, which were completely reversible within 21 days.

Based on the available data, the target substance isobutyl-R-lactate can be considered as not irritating to the skin, but irritating to the eye.

Justification for selection of skin irritation / corrosion endpoint:
GLP guideline study according to OECD 439.

Justification for selection of eye irritation endpoint:
GLP guideline study according to OECD 404.

Effects on eye irritation: irritating

Justification for classification or non-classification

Isobutyl-R-lactate did not cause any skin effects in an acute dermal irritating study in accordance to OECD test guideline 439. Therefore, the substance does not warrant classification for skin irritation.

Based on the available data from the read-across substances lactic acid, n-propyl lactate, 2-ethylhexyl lactate and n-butyl lactate the target substance isobutyl-R-lactate can be considered as severely irritating, but the effects are reversible. Thus, the substance meets the criteria for being classified for eye irritation (Category 2, H319). The classification into category 2, based on the available data, is further supported as isobutyl-R-lactate was tested negative for irritating properties in accordance to OECD 439.