Allyl hexanoate

Endpoint summary

Description of key information

Not irritating to the skin and eyes

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

other: validated "in vitro" test method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-14 to 2009-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform GLP study

Qualifier:

according to guideline

Guideline:

other: Commission regulation (EC) No. 440/2008 B.46.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the testing of chemicals; Draft proposal for a new guideline, in vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method, 9 September 2009, 3rd WNT circulation, Vers. 7.6.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
15 µL of the undiluted test item were applied to each of triplicate tissues.
No further information on the amount/concentration applied was stated.

Duration of treatment / exposure:

15 ± 1 min

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CELL CULTURE:
EpiSkin TM kits (Lot No.: 09-EKIN-035) are purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin TM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin TM tissues (surface 0.38 cm^2) are cultured on specially prepared cell culture inserts.

TREATMENT:
The negative control (deionised water (lot no. 091009; 15 µL was applied to each of triplicate tissues)) and positive control (5% sodium lauryl sulphate (Fluka, batch no. 1353471 51508322) solution in deionised water; 15 µL was applied to each of triplicate tissues), and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues. The plates were placed into the incubator for 15 ± 1 min at 37 ± 1.5 °C, 5 ± 0.5% CO2.
After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 ± 1 hour at 37 ± 1.5 °C, 5 ± 0.5% CO2.

CELL VIABILITY TEST:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential.
After the treatment procedure (42 hours) was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to the plates containing 2 mL assay medium containing 0.3 mg/mL MTT per well. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for approx. 70 hours in the refrigerator at 2 – 8 °C.
Per each tissue sample 2 X 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, D-85737 Ismaning) at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS:
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item/ OD negative control] * 100
For the test item and the positive control the mean relative viability +/- standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 is predicted if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥ 0.6 till ≤ 1.5.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.
In case the standard deviations in between tissues of the same treatment group is ≤ 18%.

TEST FOR DIRECT MMT REDUCTION:
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 15 µL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
A colour change could not be observed in this study.
No further information on the study design was stated.

Irritation parameter:

other: relative viability (%)

Basis:

mean

Time point:

other: after 15 min incubation

Score:

79.8

Max. score:

95

Reversibility:

no data

Irritant / corrosive response data:

After treatment with the test item Allyl capronate (Sym09/611045) the relative absorbance values were decreased to 79.8%. This value is above the treshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Results after treatment with the test substance:

Dose group	Treat-ment Interval	Absor-bance 570 nm Tissue 1	Absor-bance 570 nm Tissue 2	Absor-bance 570 nm Tissue 3	Mean Absor-bance of 3 Tissues	Absorbance [%] Tissue 1, 2 + 3	Standard Deviation [%]	Rel. Absorbance [%] of Negative Control]
Nega-tive Con-trol	15 min	1.2513	1.0690	1.2215	1.1806	106 91 104	8.3	100.0
Posi-tive Con-trol	15 min	0.3342	0.4393	0.5269	0.4335	28 37 45	8.2	36.7*
Test Item	15 min	0.7239	1.1257	0.9766	0.9420	61 95 83	17.2	79.8

*The validity of the test system was not influenced by the fact, that the positive control absorbance value is above the range of the historical data, since the "OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, 9 September 2009, 3rd WNT circulation, Vers. 7.6" only demands a clear positive effect (≤40%) on the tissues after exposure to the positive control

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Historical data:

Positive Control		Negative Control
Number of Studies	48	Number of Studies	48
Period	March 2009 – March 2010	Period	March 2009 – March 2010
Mean Viability	17.0%	Mean Viability	1.063
Standard Deviation	11.0%	Standard Deviation	0.176
Range of Viabilities	6% - 28%	Range of ODs	0.8 – 1.3

- After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.6 (between 0.6 and 1.5) for the15 minutestreatment interval thus showing the quality of the tissues.

-Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 36.7% thus ensuring the validity of the test system.

-The standard deviations between the % variabilities of the test item, the positive and negative controls were below 17.3% thus ensuring the validity of the study.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

After treatment with the test item the relative absorbance values were decreased to 79.8%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.
The test item should not be classified and labeled as skin irritant according to regulation (EC) No. 1272/2008.

Executive summary:

This in vitro study was performed to assess the irritation potential of Allyl capronate (Sym09/611045) by means of the Human Skin Model Test (OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline in Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method 2009). Each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. 15 μL of the liquid test item were applied to each tissue, spread to match the tissue size. 15 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 above the required acceptability criterion of mean OD ≥ 0.6 (between 0.6 and 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. After treatment with the test item Allyl capronate (Sym09/611045) the relative absorbance values were decreased to 79.8%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential. It can be stated that in this study and under the experimental conditions reported, the test item Allyl capronate (Sym09/611045) is non irritant to skin and therefore, the test item should not be classified and labeled as skin irritant according to regulation (EC) No.: 1272/2008.

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 the official OECD Guideline No 405.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

other: Mol:Russian

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Möllegaard Breeding And Research Centre A/S, Ejby, Lille Skensved, Denmark
- Age at study initiation: not reported
- Weight at study initiation: 2.3 to 2.8 kg
- Housing: caged individually in PPO cages (area 2580 cm2) with perforated floor
- Diet (e.g. ad libitum): pelleted complete rabbit diet Altromin 2123 from Altromin, Lage/Lippe, Germany ad libitum
- Water (e.g. ad libitum): domestic quality drinking water 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 hours light/12 hours dark

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.1 mL of the neat test substance

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):

72 hours

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 materials and methods"

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein
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 ratings at 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

other: mean of ratings at 24, 48 and 72 hours

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: mean of ratings at 24, 48 and 72 hours

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: mean of ratings at 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritant / corrosive response data:

One hour after application of the test substance all animals showed weak conjunctival effects with conjunctival vessels being definitely injected. After 24, 48 and 72 hours all four animals were free of any signs of eye irritation.

Other effects:

No other effects reported

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance is not irritating to the rabbit eye.

Executive summary:

The potential for eye irritation was tested for allyl caproate (allyl hexanoate) in a GLP study according to OECD guideline No. 405. Four healthy SPF albino rabbits (Mol:Russian) weighing 2.3 to 2.8 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. All animals showed slight conjunctival effects at the examinations occurring 1 after treatment. All four animals were free of any signs of ocular irritation at the gradings following 24, 48 and 72 hours after treatment. 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

After treatment of the in vitro dermal test system with the test item the relative absorbance values were decreased to 79.8%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to not possess an irritant potential. The test item should not be classified and labelled as skin irritant according to regulation (EC) No. 1272/2008.

In the eye irritation study with rabbits, all four treated animals showed slight conjunctival effects at the examinations occurring 1 after treatment. All four animals were free of any signs of ocular irritation at the gradings following 24, 48 and 72 hours after treatment. On the basis of these observations it was concluded that the substance is non-irritating to the rabbit eye.

There are no data concerning the respiratory irritation potential of the substance. However, based upon the lack of irritation observed in the eye irritation study giving an indication of potential irritancy to mucosal tissue, it is considered unlikely that the registered substance will present a respiratory irritant hazard.

Justification for selection of skin irritation / corrosion endpoint:
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:
Reliable in-vivo eye irritation study was chosen as key study

Justification for classification or non-classification

The test method based on the EPISKIN assay allows the prediction of both irritant and non-irritant substances and can thus be considered as a standalone method to be used as replacement for the animal test. Therefore, allyl hexanoate is considered not to have skin irritating properties and should not be classified and labelled according to regulation (EC) No 1272/2008. The results of the eye irritation study show that the substance is not irritating to the rabbit eye. A classification according to Regulation (EC) No 1272/2008 is not warranted.