Registration Dossier

Administrative data

Description of key information

Substance is not irritating to the skin or to the eye.

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:
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
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Details on test animals 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
Number of animals:
not relevant
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:
Value:
105.3
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 menthyl acetate 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

1.175

1.050

1.178

1.134

109.0

97.5

109.4

6.8

105.3

*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:
not 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, did not decrease (105.3%; threshold for irritancy: ≤ 50%), consequently the test item was non irritant to skin.
Executive summary:

This in vitro study was performed to assess the irritation potential of Menthyl Acetate 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 Menthyl Acetate racemic the mean relative absorbance value did not decrease (105.3%; threshold for irritancy of ≤ 50%). Therefore, the test item is not 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 Menthyl Acetate racemic is non irritant to skin according to UN GHS and EU CLP regulation.

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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted under GLP-conditions in accordance with Guideline No 405.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
from February 1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
from January 1997
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland GmbH, Stolzenseeweg 32-36, 88353 Kisslegg
- Age at study initiation: not reported
- Weight at study initiation: 2.8 - 3.2 kg bw
- Housing: caged individually in PPO cages (floor area: 2576 cm2) with perforated floor
- Diet (e.g. ad libitum): pelleted complete rabbit diet Altromin 2123 from Altromin, D-32791 Lage/Lippe, Germany, ad libitum
- Water (e.g. ad libitum): free access to domestic quality drinking watet acidified with hydrochloric acid to pH 2.5
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: The right eye remained untreated and served as control.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
single administration; after 24 hour reading Fluorescein was instilled and treated eye was washed with 20 mL 0.9% sodium chloride solution
Observation period (in vivo):
The eyes were examined on the day before testing and 1, 24, 48 and 72 hours after the treatment.
Number of animals or in vitro replicates:
4
Details on study design:
REMOVAL OF TEST SUBSTANCE
- substance was not removed from the treated eye

SCORING SYSTEM:
see section "Any other information on materilas and methods incl. tables"

TOOL USED TO ASSESS SCORE:
Fluorescein was instilled after 24 hours, eyes were rinsed with 20 mL 0.9% sodium chloride solution and were examined using UV light
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: mean of scores at 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Time point:
other: mean of scores at 24, 48 and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: mean of scores at 24, 48 and 72 hours
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: mean of scores at 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24 h
Score:
1
Max. score:
3
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
48 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24 h
Score:
1
Max. score:
3
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
48 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24 h
Score:
1
Max. score:
3
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
48 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #4
Time point:
24 h
Score:
1
Max. score:
3
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Basis:
animal #4
Time point:
48 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #4
Time point:
72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
One hour after application of the test article all animals showed a diffuse crimson red conjunctiva with individual vessels not easily discernible and some swelling above normal. After 24 hours all animals showed some conjunctival vessels definitely injected. After 48 and 72 hours all animals were free of any signs of eye irritation.
Other effects:
No other effects were reported.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The substance is non irritating to the rabbit eye.
Executive summary:

The potential for eye irritation was tested for methyl acetate in a GLP study according to OECD guideline No. 405. Four female albino rabbits weighing 2.8 to 3.2 kg were used in the study. The day before testing both eyes of the animals were examined before and after the instillation of Fluorescein to ensure there were no defects or irritation. The next day an aliquot of 0.1 mL of the neat substance was placed in the left eyes of the animals by gently pulling the lower lid away from the eyeball. The lids were then gently hold together for one second. The right eye remained untreated and served as control. The eyes were examined and the grade of ocular reaction was recorded 1 and 24 hours later. After a first reading at 24 hours, Fluorescein was instilled and the treated eye was rinsed with 20 mL 0.9 % sodium chloride solution. Then the eye was examined again using UV-light to detect possible corneal damage. Further gradings were done after 48 and 72 hours after treatment. One hour after application of the test article all animals showed a diffuse crimson red conjunctiva with individual vessels not easily discernible and some swelling above normal. After 24 hours all animals showed some conjunctival vessels definitely injected. After 48 and 72 hours all animals were free of any signs of eye irritation. On the basis of these observations it was concluded that the substance is non-irritating to the rabbit eye.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

- skin irritation / corrosion

Menthyl acetate is considered to be not irritating to skin. The result from fully reliable study in vitro (three tissues of the human skin model EpiSkin) conducted according to OECD TG 439 and GLP does not showed a decrease of the mean relative absorbance value: 105.3 % (Heppenheimer 2012). This value is well above the threshold for irritancy of ≤ 50 %. Therefore, the test item is considered to be not irritating.

- eye irritation

The primary eye irritant effect of the registration item was investigated in a GLP study according to OECD TG 405. Four female albino rabbits were exposed to 0.1 mL of the test article in the left eye. The eyes were examined and the changes were graded according to a numerical scale 1, 24, 48 and 72 hours after dosing. After the treatment, only slight changes were observed in the eyes. The following mean values, based on the results from the 24, 48 and 72 hour readings, were calculated: cornea score: 0, iris score: 0, conjunctivae score: 0.3, chemosis score: 0. It is concluded that the test substance caused no eye irritation under the conditions of the test.


Justification for selection of skin irritation / corrosion endpoint:
GLP study according to OECD TG 439 (In vitro Skin Irritation: Reconstructed Human Epidermis Test Method).
GLP study performed according to OECD TG 439 (In vitro Skin Irritation: Reconstructed Human Epidermis Test Method).
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:
GLP study in vivo according to OECD TG 405.

Justification for classification or non-classification

- skin irritation/corrosion:

Based on the above stated assessment of the irritating properties of menthyl acetate, the results from reliable in vitro studies show that the substance is not irritating to the skin. Accordingly the test item 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.

 

- eye irritation/corrosion:

Based on the above stated assessment of the irritating properties of menthyl acetate, the results from reliable in vivo 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.