Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

Endpoint summary

Description of key information

Skin irritation: not irritating
Eye irritation: not irritating (based on in vitro data from the target substance and read-across from propane-1,2,3-triyl trisheptanoate [CAS 620-67-7])

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:

28 Aug - 03 Sep 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Species:

human

Strain:

other: EPISKIN™ reconstructed human epidermis model

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: SkinEthic Laboratories, Nice, France
- Description: The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

ADAPTATION TO CELL CULTURE CONDITIONS
2 mL of maintenance medium, warmed to appr. 37 °C was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

INCUBATION CONDITIONS (Incubator)
- Temperature (°C): 37
- CO2 gas concentration (%): 5

Type of coverage:

other: open in vitro system

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+ served as negative controls, positive controls were exposed to 5% SDS

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL

NEGATIVE CONTROL
- Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+
and Mg2+
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Positive control substance: Sodium Dodecyl Sulphate (SDS), 5% w/v

Duration of treatment / exposure:

15 min

Observation period:

Not applicable. Post-treatment incubation period: 42 h

Number of animals:

Not applicable. The test was performed in triplicates for each treatment and control group.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca2+ and Mg2+. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Time after start of exposure: 15 min

CELL VIABILITY MEASUREMENTS
- Method: colourimetric MTT reduction assay
- Principles of method: Cell viability is measured by enzymatic reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells. The formazan product is extracted from the tissue with acidified isopropanol and the optical density of the extracts is measured at 540 nm using acidified isopropanol as blank.
- Time after exposure: 42 h (MTT loading/Formazan extractin) and on Day 6 (Absorbance/Optical density measurements)

INTERPRETATION OF RESULTS
For the test item, the relative mean tissue viabilities obtained after the 15 min exposure period followed by the 42 h post-exposure incubation period were compared to the mean of the negative control tissues (n = 3). The relative mean viabilities were calculated as follows:

Relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x 100

Classification of irritation potential was based upon relative mean tissue viability following the 15 min exposure period followed by the 42 h post-exposure incubation period according to the following criteria:

Relative mean tissue viability ≤ 50%: Irritant (I) R38
Relative mean tissue viability ≥ 50%: Non-irritant (NI)

The results were evaluated according to EU Commission Directive 2001/59/EC.

QUALITY CRITERIA
The results of the assay were considered acceptable if the following assay acceptance criteria are achieved:
- Positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤ 40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤ 18%.
- Negative control: The assay establishes the acceptance criterion for an acceptable test if the mean OD540 for the negative control treated tissues was ≥ 0.6, and the standard deviation value of the percentage viability is ≤ 18%.
- Test item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tisues is ≤ 18%.

Irritation / corrosion parameter:

% tissue viability

Value:

101.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

QUALITY CRITERIA

The relative mean tissue viability for the positive control treated tissues was 12.2% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.6%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.784 and the standard deviation value of the percentage viability was 6.7%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 7.9%. The test item acceptance criterion was therefore satisfied.

The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viability values and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test item treated tissues was 101.6% after a 15-minute exposure period.

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Table 1. Mean OD540 values and percentage viabilities for the negative control item, positive control item and test item

Item	OD540 of tissues	Mean OD540 of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of relative mean viability
Negative control	0.754	0.784	0.053	96.2	100*	6.7
	0.845			107.8
	0.754			96.2
Positive control	0.115	0.096	0.020	14.7	12.2	2.6
	0.097			12.4
	0.075			9.6
Test item	0.801	0.796	0.062	102.2	101.6	7.9
	0.856			109.2
	0.732			93.4

 

SD: standard deviation

*: The mean viability of the negative control tissue is set at 100%

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required 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

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Principles of method if other than guideline:

In vitro eye irritation test using the SkinEthic reconstructed Human Corneal Epithelium (HCE) model

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

TEST TISSUE
- Test model: SkinEthic reconstructed Human Corneal Epithetial model (SkinEthic HCE)
- Source: HCE, SkinEthic Laboratories, Nice, France
- Description: Transformed human corneal epithelial cells of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The test item is applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.
- Age of tissue at study initiation: 6 days
- Tissue storage/maintenance: Upon arrival, tissues were stored at room temperature prior to transferring into 24-well plates designated “arrival plates” containing 300 µl of maintenance medium. It was ensured that there were no air bubbles present under the tissue inserts.

INCUBATION CONDITIONS
- Temperature (°C): 37
- CO2 (%): 5

PREPARATION OF TISSUE
Prior to treatment, 7-day old tissues were transferred from the “arrival plates” to 6-well plates designated “treatment plates” (for test item, negative and positive controls) containing 1 mL maintenance medium at room temperature.

Vehicle:

unchanged (no vehicle)

Controls:

other: yes, negative control solution (Solution A)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 30 µL

NEGATIVE CONTROL
- Identity: Solution A (0.142 g/L Na2HPO4; 1.802 g/L Glucose; 7.149 g/L HEPES; 0.224 g/L KCl; 7.597 g/L NaCl)
- Concentration: as such
- Amount(s) applied in the test: 30 µL

POSITIVE CONTROL SUBSTANCE
- Identity: Sodium Dodecyl Sulphate (SDS)
- Concentration: 2% (w/v)
- Amount(s) applied in the test: 30 µL

Duration of treatment / exposure:

10 min at 37°C

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

3 tissues

Details on study design:

PRE-TEST
ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT
The MTT Assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
The possible interference of the test item with the MTT assay by its potential ability to directly reduce MTT was tested prior to the main test. 30 µL of the test item was added to 1 mL of a 0.5 mg/mL MTT solution and incubated at 37 °C, 5% CO2 in air for 3 h. Untreated MTT solution was used as a control. If the MTT solution turned blue, the test item was presumed to have reduced MTT.

MAIN TEST
POST-EXPOSURE TREATMENT
- Removal of the test item: At the end of the exposure period, tissue inserts were removed from the wells and rinsed with Dulbecco’s Phosphate Buffered Saline without Ca++ and Mg++.
- Post-exposure incubation conditions: Tissues were placed into 24-well plates designated “holding plates” containing 300 µL of maintenance medium (at room temperature) until all tissues had been rinsed.

MTT ASSAY
- Loading: Following rinsing, tissues were transferred to 24-well plates (“MTT Loading plates”), each well containing 300 µL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium.
- Incubation conditions: 3 h at 37 °C, 5% CO2 in air
- Extraction: At the end of the incubation period, tissues were rinsed twice with phosphate buffered saline, blotted on absorbent paper to remove residual MTT and transferred to 24-well plates (“MTT extraction plates”) containing 750 µL isopropanol per well. Further 750 µL isopropanol were added to each well and plates were sealed to prevent evaporation.
- Extraction conditions: formazan crystals were extracted from the tissues overnight at room temperature protected from light.
- Optical density measurement: At the end of the extraction period, triplicate 200 µL aliquots of the extraction solutions were transferred to a 96-well plate. The optical density was measured at 540 nm (OD540) using the Anthos 2001 microplate reader for determination of tissue viability. Isopropanol served as blank.

TISSUE HISTOLOGY
One tissue per treatment group (test item, negative and positive controls) was retained for possible tissue histopathology.
The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a 1.5 mL Eppendorf tube containing 1 mL of 10% formalin and stored at room temperature.

INTERPRETATION OF RESULTS
- Tissue viability: The mean OD540 values of the duplicate tissues were calculated. Each of these values had already been corrected for blanks by the microplate reader. The relative mean tissue viability (percentage of the negative control) was calculated as follows:

Relative mean tissue viability (%) = [(mean OD540 of test item)/(mean OD540 of negative control)] x 100

SCORING SYSTEM
The mean tissue viability for the test item was compared to the negative control and classified according to the following criteria:

Irritant (I): If the relative mean tissue viability (percentage of negative control) was < 60.
Non-Irritant (NI): If the relative mean tissue viability (percentage of negative control) was ≥ 60.

ASSAY ACCEPTANCE CRITERION
The results of the assay were considered acceptable if the following assay acceptance criterion was achieved:
- Assay acceptance criterion: Positive control
The assay meets the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is < 60% relative to the negative control treated tissues.

Irritation parameter:

other: Relative Mean Viability (%)

Value:

92.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

The individual and mean OD540 values and mean viabilities for each treatment group are given in Table 1.

The relative mean viability of the test item treated tissues after a 10 min exposure period was 92.6%

It was considered unnecessary to proceed with tissue histopathology.

The MTT solution containing the test item remained yellow which indicated that the test item did not directly reduce MTT.

Table 1. Assessment of eye irritation potential – Viability of HCE tissues

Item	OD540 of individual tissue	Mean OD540	Relative Mean Viability (%)
Negative control	0.812	0.838	100*
	0.863
Positive control	0.152	0.111	13.2
	0.069
Test item	0.779	0.776	92.6
	0,773

* The mean viability of the negative control tissues was set at 100%

 

Assay Acceptance Criterion

The relative mean viability of the positive control treated tissues after a 10 min exposure period was 13.2%. Thus, the quality criterion required for the acceptance of results in the test was satisfied.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Source: CAS 620-67-7, Hüls, 1993

Irritation parameter:

iris score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Source: CAS 620-67-7, Hüls, 1993

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Source: CAS 620-67-7, Hüls, 1993

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: Source: CAS 620-67-7, Hüls, 1993

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Executive summary:

The eye irritation potential of the target substance is determined based on an in vitro study with the target substance and on an adequate and reliable in vivo study of the structural analogue source substance propane-1,2,3-triyl trisheptanoate. In both studies (in vitro and in vivo) no eye irritation potential was detected. As explained in the analogue justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the eye irritation potential. Therefore, no significant eye irritation potential is identified for the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In a GLP-compliant study according to OECD guideline 439, the skin irritation potential of propane-1,2,3-triyl 3,5,5-trimethylhexanoate was evaluated using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 min followed by a post-exposure incubation period of 42 h (Harlan, 2012a). The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item (10 µL/tissue) by means of the colourimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls (treated with Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+and Mg2+). Positive control tissues were treated with Sodium Dodecyl Sulphate (SDS), 5% w/v. After the 15 min exposure period, the relative mean tissue viability for the test item and positive control treated tissues compared to the control was 101.6% (± SD = 7.9%) and 12.2% (± SD = 2.6%), respectively. The quality criteria required for acceptance of results in the test were satisfied. Based on the study results, the test item was considered to be non-irritant in vitro.

Eye irritation / corrosion

Justification for grouping of substances and read-across

There are no data available on the in vivo eye irritation potential of propane-1,2,3-triyl 3,5,5-trimethylhexanoate (CAS 56554-53-1). In order to fulfil the standard information requirements set out in Annex VIII, Item 8.2, in accordance with Annex XI, Item 1.5, of Regulation (EC) No. 1907/2006, read-across from a structurally related substance is conducted. In accordance with Article 13(1) of Regulation (EC) No. 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No. 1907/2006, whereby physicochemical, toxicological and ecotoxicological properties may be predicted from data for reference substance(s) by interpolation to other substances on the basis of structural similarity, propane-1,2,3-triyl trisheptanoate (CAS 620-67-7) is selected as reference substance for assessment of the in vivo eye irritation potential of propane-1,2,3-triyl 3,5,5-trimethylhexanoate. The read-across is based on structural similarity between the source and target substances, which are triesters of glycerol and fatty acids with carbon chain lengths from C7-isoC9. A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Overview of eye irritation

CAS#	56554-53-1	620-67-7
Chemical name	Propane-1,2,3-triyl 3,5,5-trimethylhexanoate	Propane-1,2,3-triyl trisheptanoate
Molecular weight	512.78	428.61
Eye irritation in vitro	Experimental result	--
Eye irritation in vivo	RA: CAS 620-67-7	Experimental result: Not irritating

--: Data lacking

 Eye irritation in vitro

The eye irritation potential of propane-1,2,3-triyl 3,5,5-trimethylhexanoate was tested in vitro in a GLP-compliant study using the SkinEthic reconstructed Human Corneal Epithelium model (HCE) (Harlan, 2012b). The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently toxic to cause cell death. Tissue viability after exposure is determined by means of the colourimetric MTT reduction assay and serves as parameter for the eye irritation potential. Triplicate SkinEthic tissues were treated with 30 µL of the test item, negative control (Solution A: 0.142 g/L Na2HPO4; 1.802 g/L Glucose; 7.149 g/L HEPES; 0.224 g/L KCl; 7.597 g/L NaCl) or positive control (2% w/v Sodium Dodecyl Sulphate) for 10 min. Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test item treated tissues (quantitative measurement of tissue viability) relative to the negative control. For each treatment group, two tissues were used for viability measurement, while one tissue per group was retained for possible histopathology. The test item was considered to be an irritant if the relative mean tissue was < 60%; otherwise it was considered to be a non-irritant. The results of the assay were considered acceptable if the relative mean tissue viability of the positive control was < 60%. After a 10 min exposure, the relative mean viability of the test item and positive control treated tissues was 92.6%. It was considered unnecessary to proceed with tissue histopathology. The test item was considered to be a non-irritant. The relative mean tissue viability of the positive control treated tissue was 13.2%, thus satisfying the acceptance criterion.

Eye irritation in vivo

CAS No. 620-67-7

The eye irritation potential of propane-1,2,3-triyl trisheptanoate was investigated according to OECD guideline 405 and in compliance with GLP (Hüls, 1993). The undiluted test material (0.1 mL) was placed into the conjunctival sac of one eye of 3 male Small White Russian (Chbb:HM) rabbits each. The other eye remained untreated and served as control. The eyes were examined and scored 1, 24, 48 and 72 h after application. Within one hour, the administration of the test substance resulted in marked hyperaemia of some blood vessels in all 3 animals. After 24-72 h, no further symptoms of irritation to the eye mucosa and cornea were observed in any of the animals. Only the skin of the outer eyelids showed slight erythema after 24 h in all animals, which subsided after 48 h. Test substance instillation into the rabbit eye induced no iridial effects at any observation time point. No further local or systemic toxic effects were observed. The mean cornea, iris, conjunctivae and chemosis scores after 24, 48, and 72 h were 0 for all 3 animals, respectively. Thus, the test substance was not considered as eye irritant.

Justification for selection of eye irritation endpoint:
Hazard assessment is conducted based on the WoE from substance specific in vitro data and read-across from a structural analogue substances. The study is adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

Justification for classification or non-classification

Based on substance specific studies and read-across following an analogue approach, the available data on the skin and eye irritation potential of propane-1,2,3-triyl 3,5,5-trimethylhexanoate do not meet the classification criteria according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification. There are no data available on respiratory irritation.