L-menthan-3-one

Endpoint summary

Description of key information

L-menthan-3-one is deemed to be irritating to skin according to the read-across study in vitro testing Reaction mass with human skin model EpiSkin. 
A negative BOCP study of L-menthan-3-one and an additional negative test in rabbit eye using Isomenthone as read across to L-menthan-3-one allow to consider L-menthan-3-one as not irritant to eye compound. 
No study is required for respiratory 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:

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

Adequacy of study:

key study

Study period:

from 2012-05-14 to 2012-08-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD TG 439. Read-across from Reaction mass (76 % Menthone and 23.5 % Isomenthone)

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)

Details on test animals or test system and environmental conditions:

not relevant

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

15 min treatment + 42 h incubation

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with PBS
- Time after start of exposure: 15 min

SCORING SYSTEM:
In vitro result: In vivo prediction:
mean tissue viability ≤ 50 % → irritant (I), H315 (category 2)
mean tissue viability > 50 % → non-irritant (NI)

Irritation / corrosion parameter:

other: other: Relative Absorbance

Value:

10.4

Remarks on result:

other:

Remarks:

Basis: other: Test item on EpiSkin. Max. score: 100.0. Reversibility: no data. Remarks: % of negative control. (migrated information)

Results after treatment with 620103 Menthone / Iso Menthone Racemic and controls

Dose group	Treat-ment interval	Absor-bance 540 nm Tissue 1*	Absor-bance 540 nm Tissue 2*	Absor-bance 540 nm Tissue 3*	Mean Absorbance of 3 Tissue	Relative absor-bance [%] Tissue 1, 2+3**	Stan-dard De-viation [%]	Rel. Absor-bance [% of negative control]***
Negative Control	15 min	1.154	1.047	1.032	1.077	107.1 97.2 95.8	6.2	100.0
Positive Control	15 min	0.175	0.142	0.154	0.157	16.3 13.2 14.3	1.6	14.6
Test Item	15 min	0.106	0.145	0.086	0112	9.8 13.5 7.9	2.8	10.4

*Mean of two replicate after blank correction

**Relative absorbance per tissue [rounded values]: 100xabsorbance (tissue)/mean absorbance (negative control)

***Relative absorbance per treatment group [rounded values]: 100xmean absorbance (test item)/mean anbsorbance (negative control)

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 10.4% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

Executive summary:

This in vitro study was performed to assess the irritation potential of 620103 Menthone / Iso Menthone Racemic by means of the Human Skin Model Test according to OECD TG 439. Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. 10 μL of the liquid test item were applied to each tissue, spread to match the tissue size. 10 μL of either the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD  0.6 till ≤ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval thus ensuring the validity of the test system. Compared to the value of the negative control, after exposure of the test item 620103 Menthone / Iso Menthone Racemic the mean relative absorbance value decreased to 10.4%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item 620103 Menthone / Iso Menthone Racemic is irritant to skin according to UN GHS and EU CLP regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2012-05-14 to 2012-08-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD TG 437

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

not relevant

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

10 min incubation treatment; after rincing: 2 h incubation + 90 min incubation

Number of animals or in vitro replicates:

not relevant

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with saline
- Time after start of exposure: 10 min

SCORING SYSTEM:
In vitro irritation score: ≥ 55.1
In vitro Irritation scale (according to OECD 437): corrosive / severe irritant CLP/EPA/GHS (Cat 1)

TOOL USED TO ASSESS SCORE: opacitometer / fluorescein

Irritation parameter:

cornea opacity score

Remarks:

mean in vitro irritation score

Basis:

mean

Remarks:

3 corneae

Score:

1.76

Max. score:

81.52

Remarks on result:

other: Max score: positive control

Irritant / corrosive response data:

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 0.54). The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean in vitro irritation score 81.52) corresponding to a classification as corrosive / severe irritant to the eye (CLP/EPA/GHS (Cat 1)). Relative to the negative control, the test item Menthone-L / Isomenthone-D did not
cause any increase of the corneal permeability, and only very slight opacity effects could be observed. The calculated mean in vitro irritation score was 1.76. According to OECD 437 the test item is classified as not corrosive / not severe irritant to the eye.

Results after 10 minutes incubation time

Test Group	Opacity value = Difference (t130-t0) of opacity		Permeability 490 nm (OD490)		In vitro irritation score	Mean in vitro irritation score	Proposed in vitro irritation scale
		Mean		Mean		0.54	Non corrosive / non severe irritant
Negative Control	0	-0.33	0.054	0.058	0.81
	0		0.059		0.89
	-1		0.061		-0.09
Positive Control	77.33*		0.743*		88.48	81.52	Corrosive / severe irritant
	62.33*		1.134*		79.34
	70.33*		0.426*		76.72
Test Item	2.33*		-0.004*		2.27	1.76	Non corrosive / non severe irritant
	2.33*		-0.011*		2.50
	0.33*		0.012*		0.519

*corrected values

Interpretation of results:

other: not corrosive / not severe irritant to the eye

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

According to the current study and under the experimental conditions reported, the test item Menthone-L / Isomenthone-D is not corrosive / not severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Menthone-L / Isomenthone-D by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item Menthone-L / Isomenthone-D, the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. The posterior chamber contained MEM

medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium = cMEM). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were

incubated for another 120 minutes at 32 ± 1 °C in complete medium, and opacity was measured a second time (t130). After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as corrosive / severe irritant to the eye (CLP/EPA/GHS (Cat 1)). Relative to the negative control, the test item Menthone-L / Isomenthone-D caused a

very slight increase of the corneal opacity. Permeability effects did not occur. The calculated mean in vitro irritation score was 1.76. According to OECD 437 the test item is classified as not corrosive / not severe irritant to the eye. In conclusion, according to the current study and under the experimental conditions reported, the test item Menthone-L / Isomenthone-D is not corrosive / not severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion

Key study

L-menthan-3-one is considered to be irritating to skin. The read-across from Reaction mass result of fully reliable study in vitro (three tissues of human skin model EpiSkin) conducted according to OECD TG 439 and GLP showed decrease of mean relative absorbance value to 10.4 % (Heppenheimer 2012). This value is well below the threshold for irritancy of ≤ 50 %. Therefore the test item is considered to possess skin irritating potential.

Supporting information

In one supporting study (Kitahara 1993), 1.3 % of L-menthan-3-one in gel ointment showed a low irritating potential in rats. Since the test item has only a low percentage in the ointment, it could be supported the key study that the pure compound has an irritating potential to the skin.

Eye irritation / corrosion

WoE studies

L-menthan-3-one was reported as not corrosive / not severe irritant to eye based on the results from a fully reliable study (Heppenheimer 2012, according to OECD TG 437 and GLP). The in vitro study alone does not allow to classify L-menthan-3-one as not irritant to eye, the in vivo study with rabbits (Schreiter 1999) is considered as WoE read across study for L-menthan-3-one from Isomenthone. In this study Isomenthone is not irritant to eye. Therefore, we can assume that L-menthan-3-one is not irritating to eye as well.

Justification for selection of skin irritation / corrosion endpoint:
Read-across from Reaction mass (76 % L-Menthone and 23 % Isomenthone) GLP study performed according to OECD TG 439. Due to an important percentage of L-Menthone in Reaction mass, the read-across could be relevant.
This in vitro study was selected as the key study since the registration item is a cosmetic raw material and starting from March 11th, 2009, it was enacted to ban the tests of cosmetic raw materials on animals in the EU. Therefore, no additional in-vivo study shall be performed. In addition, according to ECHA Chapter R.7a (Version 2.0, November 2012, page 174), it is expected that the EPISKIN test will be validated and endorsed as a full replacement of the in vivo test. The available in vitro study is therefore considered acceptable to serve as the key study.

Justification for selection of eye irritation endpoint:
A BCOP study performed according to OECD TG 437 and GLP is available. The result of this study is negative. For classification, an additional in vivo test (rabbits) is required. A reliable study on Isomenthone performed according to OECD TG 405 is available, where Isomenthone is not irritating to the eye. As read across from Isomenthone is applicable for L-menthan-3-one, the substance is considered to be not irritant to eye in a WoE approach.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

- skin irritation / corrosion:

The results of the skin irritation / corrosion tests of L-menthan-3-one show that the test item is a skin irritant. Accordingly L-menthan-3-one need to be classified as "R38 Irritating to skin" according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and as Category 2, "Warning - H315: Causes skin irritation" according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

- eye irritation/corrosion:

Based on the above stated assessment of the irritating properties of L-menthan-3-one, the results from a reliable studies show that the substance is not corrosive or irritating to the eye. Accordingly the substance does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN GHS in the EU.