Allyl heptanoate

Endpoint summary

Description of key information

Allyl heptanoate is not irritating to the skin or the eyes.

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:

2009-10-14 to 2009-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

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

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiSkin™ 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™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

15 µL of the undiluted test item were applied to each of triplicate tissues.
For the positive and negative controls 15 µL were dosed per tissue.

Duration of treatment / exposure:

15 ± 1 min at 37 ± 1.5 °C, 5 ± 0.5% CO2

Duration of post-treatment incubation (if applicable):

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.

Number of replicates:

three replicate tissues with two replicate wells each per exposure and control group

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 TM 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 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 viablity 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 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. Tissues 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 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, category 2 is predicted if the mean relative tissue viability of three individual tissues is reduced below or equal to 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 MTT 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.
No further information on the study design was stated.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three tests with two replicate wells each

Value:

>= 54.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

5% SLS (Sodium lauryl sulphate)

Remarks on result:

no indication of irritation

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]
Negative Control	15 min	1.2513	1.0690	1.2215	1.1806	106 91 104	8.3	100.0
Positive Control	15 min	0.3342	0.4393	0.5269	0.4335	28 37 45	8.2	36.7*
Test Item	15 min	0.6414	0.5758	0.7288	0.6486	54 49 62	6.5	54.9

* 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

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

After treatment with the test item Allyl heptanoate (Sym09/611041) the relative absorbance values were decreased to 54.9 %. 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.

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

Study period:

March 05, 1999 - July 05, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD TG 405

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Details on test animals or tissues and environmental conditions:

Animals
Four female SPF albino rabbits weighing 2.3 - 2.5 kg of the stock Mol:Russian from Møllegaard Breeding and Research Centre AIS, Ejby, DK-4623 Lille Skensved were used.
The animals were eannarked on arrival.

Housing
The study took place in animal room No. 5 and 6 provided with filtered air at a temperature of 20°C ± 3°C, relative humidity of 55% ± 15% and air changes 10 times/hour. The rooms were illuminated to give a cycle of 12 hours light and 12 hours darkness. Light was on from 06 to
18 h.
During a pre-period of at least one week and throughout the experiment the rabbits were caged individually in PPO cages (floor area: 2576 cm2) with perforated floor.

Diet
A pelleted complete rabbit diet “Altromin 2123” from Altromin, D-32791 Lage, Lippe, was available ad libitum. Analyses for major nutritive components and relevant possible contaminants are performed regularly on the diet. Certificates of analysis are retained.

Drinking water
The animals had free access to bottles with domestic quality drinking water acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth. Analyses for relevant possible contaminants are performed regularly. Certificates of analysis are retained.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Four female albino rabbits were exposed to 0.1 ml of the test article in the left eye

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

The eyes were examined and the changes were graded according to a numerical scale 1, 24, 48 and 72 hours after dosing

Number of animals or in vitro replicates:

Four female albino rabbits

Details on study design:

The day before testing both eyes of the animals were examined with a hand held inspection lamp fitted with white and IJV-ligbt and magnifying glass with 2 x magnification to ensure there were no defects or irritation. The examination was performed before and after instillation of Fluorescein.
Only the left eye was treated. The right eye remained untreated and sewed as control.
0.1 ml of the test article was placed in the left eye of the rabbits by gently pulling the lower lid away from the eyeball to form a cup into which the article was dropped. The lids were then gently held together for 1 second.

The eyes were examined and the grade of ocular reaction was recorded 1 and 24 hours later. After the first 24 hour reading Fluorescein was instilled. After rinsing with 20 ml 0.9% sodium chloride solution the eyes were examined again using UV-light to detect possible corneal damage.
The eyes were also examined 48 and 72 hours after the treatment.

READING REACTIONS
The reactions were scored according to the following grades for ocular lesions:

SCORING SYSTEM
Cornea :
When Fluorescein was used the cornea reactions were scored both before and after the instillation of Fluorescein.

Opacity degree of density (Area_most dense taken for_reading)
0 = No ulceration or opacity
1 = Scattered or diffuse area of opacity (other than slight dulling of normal luster), details of iris clearly visible
2 = Easily discernible translucent areas, details of iris slightly obscured
3 = Nacreous areas, no details of iris visible, size of pupil barely discernible
4 = Complete comeal opacity, iris not discernible

Area of cornea involved
1 = One quarter (or less) but not zero
2 = More than 1 quarter, but less than half
3 = More than half, but less than 3 quarters
4 = More than 3 quarters, up to whole area

Iris:
0 = Normal.
1 = Markedly deepened folds, congestion, swelling, moderate circumcomeal injection (any of these or combination of any thereof), iris still reacts to light (sluggish reaction is positive)
2 = No reaction to light, haemorrhage, gross destruction (any or all of these)

Conjunctiva:

Redness (refers to palpebral and bulbar conj unctivae excluding cornea and iris)
0 = Vessels normal
1 = Some vessels definitely injected
2 = Diffuse, crimson red, individual vessels not easily discernible
3 = Difflise beefy red

Chemosis :
0 = No swelling
1 = Any swelling above normal (includes nictitating membrane)
2 = Obvious swelling with partial eversion of lids
3 = Swelling with lids about half closed
4 = Swelling with lids more than half closed

Discharge:
0 = No discharge
1 = Any amount different from normal (does not include small amounts observed in inner canthus of nonnal animals)
2 = Discharge with moistening of the lids and hairs just adjacent to lids
3 = Discharge with moistening of the lids and hairs, and considerable area around the eye

Irritation parameter:

cornea opacity score

Remarks:

degree

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

area

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

degree

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

area

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

degree

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

area

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

degree

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

area

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

One hour after application of the test article animal #1 and #3 showed some conjunctival vessels definitely injected and some amount of discharge different from normal. Some conjunctival vessels definitely injected and discharge with moistening of the lids and hairs just adjacent to lids were observed in animal #2 and #4.
After 24 hours some conjunctival vessels definitely injected were observed in all animals. Some amount of discharge different from normal was seen in animal #4.
After 48 and 72 hours all animals were free of any signs of eye irritation.
The following mean values were obtained from the data presented in the results:
cornea opacity 0.0
iris lesion 0.0
redness of conjunctiva 0.3
oedema of conjunctiva (chemosis) 0.0

Other effects:

Conjunctiva Discharge :
Animal#1 : from 0 to 1 fully reversible within 24 hours
Animal#2 : from 0 to 2 fully reversible within 24 hours
Animal#3 : from 0 to 1 fully reversible within 24 hours
Animal#4 : from 0 to 2 fully reversible within 48 hours

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the directive of the EEC commission 93/21/EEC of April 27, 1993 HR 99/611041 , Batch No. 9034305, shall not be classified as eye irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

- Skin irritation

The test performed in 2009 according to 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." shows after a treatment with the test item allyl heptanoate a relative absorbance values decreasing to 54.9 %. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess a skin irritating potential.

Another study performed in 1988 emphasizes the result of the cutaneous irritation potential of allyl heptanoate (0.5 ml of the test material as supplied). The concentration of the test material is ignored. Even if the method doesn’t follow current guidelines, it may be qualified as relevant seeing the conditions of the test. The average scores calculated from the numerical values given to the irritation observed (e.g. 24, 48 and 72 hour) were 1.7 for erythema and 0.4 for oedema. Then, the test item of allyl heptanoate in this study doesn’t deserve any classification (while at the score 2.3, positive classification is required according to CLP).

- Ocular irritation

The ocular irritation potential of allyl heptanoate up to 100% according to the current OECD guideline 405 was assessed. The means of Draize scores (in four female albino rabbits) were recorded after 24, 48, and 72 h. Only slight signs of irritation were observed among the rabbits. There was no reaction in the iris and the corneal observed in all cases.

Then, allyl heptanoate shall not be classified as eye irritating.

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:
Valid and reliable in vitro study supported by in vivo data

Justification for selection of eye irritation endpoint:
Valid and reliable in vivo study

Justification for classification or non-classification

- Skin irritation

The substance allyl heptanoate does not meet the criteria for classification and labelling for this endpoint, as set out in Regulation (EC) No. 1272/2008 and Directive 67/548/EEC accordingly. Based on valid in vitro and in vivo studies performed, the substance showed no irritating properties. The in vivo test showed maximum Draize scores of 2 which reduced over time and were fully reversible within the observation period.

- Ocular irritation

According to CLP and DSD, the substance does not meet the criteria for classification and labelling for this endpoint as all scores were below the limits specified for eye irritation Category 2 as set out in Regulation (EC) No.1272/2008 and Directive 67/548/EEC accordingly.