Reaction mass of 1-hydroxypentan-2-yl formate and 2-hydroxypentyl formate and pentane-1,2-diol and pentane-1,2-diyl diformate

Endpoint summary

Description of key information

Skin irritation (OECD 439): not irritating

Eye irritation (OECD 492 and OECD 437): irritating

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:

25 Feb - 21 Mar 2016

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)

Version / remarks:

adopted 28 Jul 2015

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:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (EPI-200)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE)
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23324
- Delivery date: 15 March 2016
- Date of initiation of testing: 15 March 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile bench

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.59 h.
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce the MTT solution, an additional test with freeze-killed tissues was not performed.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% aqueous solultion

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

41.5 h

Number of replicates:

triplicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 minutes exposure

Value:

56.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not interfere with the MTT assay (no reducing capacity).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.635, 1.976 and 1.715) was in the range of ≥ 0.8 and ≤ 2.8 for every exposure time.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 6.2% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The standard deviations of the 3 identical replicates of the test substance, positive and negative control in the main test were < 14%, thus meeting the acceptance criteria of the study.

Table 2. Results after treatment with the test substance and controls

Test group	Mean absorbance at 570 nm*			Rel. absorbance (%)**			SD (%)	Rel. absorbance (% of negative control)***
	Tissue 1	Tissue 2	Tissue 3	Tissue 1	Tissue 2	Tissue 3
Negative control	1.635	1.976	1.715	92.1	111.3	96.6	10.0	100.0
Positive control	0.124	0.095	0.111	7.0	5.3	6.3	13.2	6.2
Test substance	1.084	0.926	1.021	61.0	52.2	57.5	7.8	56.9

* Mean of three replicate wells after blank correction (blank = 0.037)

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

Interpretation of results:

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

Conclusions:

Under the conditions of the reconstructed human epidermis test the test substance does not possess any skin irritating potential.

Executive summary:

After treatment with the test item the mean relative absorbance value decreased to 56.9% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

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:

key study

Study period:

30 Mar - 28 Apr 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Jul 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species:

human

Strain:

other: EpiOcular™

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

120 min

Number of animals or in vitro replicates:

in duplicates for each treatment and control group

Details on study design:

- RhCE tissue construct used, including batch number: EpiOcular™ tissue (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia), batch number: 23707
- Viability: The quality of the final product was assessed by an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 15.12 min.
- Contamination: The cells used to produce the EpiOcular tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation at 37 °C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. Since the MTT solution colour did not turn blue/purple, the test substance is not presumed to have reduced the MTT solution. An additional test with freeze-killed tissues did not have to be performed.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength: 570 nm
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The test substance was considered to be not irritating to the eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is >60%.
- Acceptance criteria: The results are acceptable if the negative control OD is >0.8 and <2.5 and if the mean relative viability of the positive control is below 50% of the negative control viability.
- Reference to historical data of the RhCE tissue construct: Historical control data was used to assess the validity of the test.
- Acceptable variability between tissue replicates for the test chemical, positive and negative controls: The difference of viability between the two relating tissues of a single test substance is < 20% in the same run.

Irritation parameter:

other: % tissue viability

Remarks:

mean value out of 2 tissues

Run / experiment:

30 min exposure

Value:

20.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER OBSERVATIONS:
The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test substance in water or isopropanol did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.289 and 1.388) was in the range of > 1.0 and < 2.6.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the mean relative absorbance compared to the negative control to 32.5%, thus the validity of the test system is ensured.

The difference of viability between the two relating tissues of a single substance is < 20% (values differ about 0.3% to 7.3%) in the same run (for positive and negative control tissues and tissues of single test substance).

Table 2. Results after 30 min incubation time

Test group	Absorbance*		Mean absorbance of 2 tissues*	Rel. absorbance (%)**		Absolute value of the difference of the rel. absorbance (%) Tissue 1 and 2	Rel. absorbance (% of negative control)
	Tissue 1	Tissue 2		Tissue 1	Tissue 2
Negative control	1.289	1.388	1.338	96.3	103.7	7.3	100.0
Positive control	0.443	0.428	0.435	33.1	32.0	1.1	32.5
Test substance	0.278	0.282	0.280	20.7	21.1	0.3	20.9

* Mean of two replicate wells after blank correction (blank = 0.036)

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

Interpretation of results:

other: Eye Irrit. Cat. 2

Remarks:

according to Regulation (EC) 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance possessed irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.

Executive summary:

Irritating effects were observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (20.9%).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Feb 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in Hank's Buffered Salt Solution (HBSS) at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Applied volume: 0.75 mL

POSITIVE SUBSTANCE
- Applied volume: 0.75 mL
- Purity: 99%

NEGATIVE CONTROL
- Applied volume: 0.75 mL

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAS:
At the end of the equilibration period, the basal opacity was determined (t0). Each cornea with a value of a basal opacity >7 was discarded.

TREATMENT METHOD:
The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on the top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. The anterior compartment received the test substance or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 10 minutes.

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with saline.

- POST-EXPOSURE INCUBATION: 2 h in a vertical position

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP_KiT opacitometer, Electro Design, France).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (Versamax Molecular Devices) at 490 nm.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value of 3 corneae

Value:

27.23

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae was observed.
The positive control (2-ethoxyethanol) showed clearly increased opacity and distinctive permeability of the corneae fullfilling the criteria as severe irritating/corrosive.
Relative to the negative control, the test substance caused an increase of the corneal opacity and permeability.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in an IVIS which was within two standard deviations of the current historical mean.
- Acceptance criteria met for positive control: The positive control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Table 2. Results after 10 min incubation time.

Test group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS
		Mean		Mean
Negative control	1	0.33	0.084	0.079	2.26	1.52
	0		0.075		1.13
	0		0.079		1.19
Positive control	56.67*		1.146*		73.85	81.42
	62.67*		1.821*		89.98
	58.67*		1.452*		80.44
Test substance	28.67*		0.409*		34.80	27.23
	19.67*		0.252*		23.44
	21.67*		0.120*		23.46

*: corrected values

Interpretation of results:

other: non-corrosive

Remarks:

according to Regulation (EC) No 1272/2008

Conclusions:

The irritation/corrosive potential of the test substance was assessed in the BCOP assay. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 27.23. According to OECD Guideline 437 no prediction on the irritation potential can be made but the test substance does not have to be classified as serious eye irritant (Eye Dam. Cat. 1).

Executive summary:

Relative to the negative control, the test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 27.23. According to OECD 437 the test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1) but the test item’s hazard for eye damaging cannot be predicted.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a human skin model according to OECD Guideline 439 and in compliance with GLP (2016). In this study the test substance did not exhibit skin irritating properties.

 

Eye

The eye irritation potential of the test substance was determined by an in vitro eye irritation test using a human cornea model according to OECD Guideline 492 and in compliance with GLP (2016). In this study the test substance possessed irritating properties towards human-derived epidermal keratinocytes.

To exclude corrosive properties towards the eyes, a bovine corneal opacity and permeability (BCOP) test according to OECD Guideline 437 and in compliance with GLP was conducted (2016). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 27.23. According to OECD Guideline 437,  the test substance does not have to be classified as corrosive.

In conclusion, based on available results, the test substance is considered to possess irritating properties towards the eyes.

Justification for classification or non-classification

The available data on skin irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

The available data on eye irritation meet the criteria for classification as Eye Irrit. Cat. 2 (H319) according to Regulation (EC) 1272/2008.