2,2,6,6-Tetramethylpiperidin-4-yl dodecanoate

Endpoint summary

Description of key information

A GLP-compliant in-vitro study for skin irritation was performed. Based on the relative tissue viability, the thresholds for classification as skin corrosive / irritating are not met. However, effects below the classification threshold were observed (BASF SE, 61V0731/11A525, 2012). The in-vitro assay for eye irritation showed that the substance is not highly irritating, but may well be irritating. This is supported by the measured pH of 9.7

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study that is suitable for classification and labelling

Qualifier:

according to guideline

Guideline:

other: OECD 439 and OECD 431

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

EPIDERM(TM) 200 kit from MaTek Corp, Ashland MA, USA
reconstructed epidermis, surface 0.6cm2 and cultured in vessels of 1 cm diameter.

Type of coverage:

open

Preparation of test site:

other: not applicale

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated skin tissue

Amount / concentration applied:

30 μl applied to each tissue, spread to match tissue size.

Duration of treatment / exposure:

60 min

Observation period:

48h (recovery period, including 3h MTT incubation)

Number of animals:

not applicable

Details on study design:

To determine whether the test substance is able to reduce MTT directly, the test substance was incubated with the substrate.
Three tissues were treated with each test substance, positive and negative controls, espectively.

Irritation / corrosion parameter:

other: other: viability of reconstituted epidermis (corrostion test)

Value:

97

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Score given in %. (migrated information)

Irritation / corrosion parameter:

other: other: viability of reconstituted epidermis (irritation test)

Value:

53

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Score given in %. (migrated information)

Irritation / corrosion parameter:

other: other: Absorbance 570 nm (OD) (corrosion test)

Value:

1.847

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 min. Reversibility: other: not applicable. Remarks: Negative Control: 1.909; Positive Control: 0.14. (migrated information)

Irritation / corrosion parameter:

other: other: Absorbance 570 nm (OD) (irritation test)

Value:

0.974

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 min. Reversibility: other: not applicable. Remarks: Negative Control: 1.83; Positive Control 0.07. (migrated information)

Irritation experiment		Tissue 1	Tissue 2	Tissue 3	mean	SD
Negative control	OD570 nm	1.839	1.850	1.787	1.826
	viability [% of negative control]				100	7.83
test substance	mean OD570 nm	0.952	0.979	0.99	0.974
	viability [% of negative control]				53	1.08
Positive control5% (w/v) sodium dodecyl sulfate	OD570 nm	0.061	0.07	0.08	0.07
	viability [% of negative control]				4	0.51

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance causes effects in the vitro-skin irritation assay. These effects are below the threshold for classification as a skin irritant (GHS Category 1 or 2).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Species:

other: in vitro test on isolated bovine cornea

Strain:

other: not applicable

Vehicle:

water

Controls:

other: not applicable

Amount / concentration applied:

TEST MATERIAL


Form of application: 10% emulsion in deionized water

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

not applicable (in vitro test)

Number of animals or in vitro replicates:

not applicable (in vitro test); each treatment group consisted of 3 corneas

Details on study design:

TEST SYSTEM:
- Isolated bovine cornea: Target system is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).

OBJECTIVE:
- Corneal opacity was measured quantitatively as the amount of light transmission through the cornea.
- Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea.
- Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas.

EXPERIMENTAL PROCEDURE:
- Preparation of the bovine corneas and measurement of initial corneal opacity:
• The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle's MEM (containing phenol red) and once with Eagle's MEM (without phenol red) . Both chambers were then refilled with fresh Eagle's MEM (without phenol red).
• After the equilibration period the medium in both chambers was replaced with fresh medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 512 opacity units were discarded.

- Application of the test substance and washing
• Before application the medium in the anterior chamber was removed using a syringe.
750 μL of the 10% (w/v) test-substance preparation (surfactant) was applied into the anterior chamber.

Control tissues:
• Negative control, NC: 750 μL of de-ionized water
• Positive control, PC: 750 μL of 100% ethanol I 100% dimethylformamide

• After the incubation period, NC and PC were removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
The epithelium of the test substance treated corneas was rinsed with the open chamber method.

- Measurement of final corneal opacity
• Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.

- Determination of permeability
• For determination of permeability the medium in the anterior chamber was replaced by 1 ml of sodium fluorescein solution (4 mg/mL for liquid test substances) and incubated for 90 ± 5 min at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

DATA EVALUATION
The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS).

- Calculation of the corneal opacity value:
First, the opacity was calculated using the opacitometer specific algorithm:
• opacity value = a * Io/I + b
a and b: device specific;
Io: illuminance (lux) through the empty corneal holder with windows and liquid
I: illuminance (lux) through the holder with the cornea

Then the opacity change per cornea was calculated by subtracting the initial from the final
• opacity (opacity change per cornea = final opacity - initial opacity).

Subsequently, the corrected opacity change was calculated by subtracting the mean opacity change of the negative control
• corrected opacity change = opacity change - mean opacity change of NC).

Finally, the mean opacity value for each test substance could be determined as the mean of all corrected opacity changes per treatment group
• mean opacity value = mean of all corrected opacity changes per group).

- Calculation of permeability value:
First, the OD490 value was calculated by subtracting the mean blank OD490 (blank = Eagle´s MEM w/o phenol red) from the OD490 of each cornea.
• OD490 value = OD490 - mean blank OD490

If the OD490 value of the treated cornea was above 1.5, the OD490 of a 1:5 dilution was used to calculate the OD490 value:
• OD490 value = 5 * (OD490 of a 1:5 dilution - mean blank OD490)

Subsequently, the corrected OD490 value was calculated by subtracting the mean OD490 value of the negative control.
• corrected OD490 value = OD490 value - mean OD490 value of NC

Finally, the mean OD490 value for each test substance could be determined as the mean of all corrected OD490 values per treatment group.
• mean OD490 value = mean of all corrected OD490 values per group

- Calculation of the In Vitro Irritancy Score (IVIS)
The IVIS could be calculated per treated cornea and finally the mean IVIS per treatment group ± standard deviation was determined:
• IVIS per cornea = corrected opacity change + 15 * corrected permeability OD change
• IVIS per treatment group = mean opacity value + 15 * mean permeability OD value

ACCEPTANCE CRITERIA
- In case one of the below given acceptance criteria is not covered, repetition of the test was considered.
• A study is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
• The negative control responses should result in opacity and permeability values that are less than the established upper limits.
• Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. If no clear prediction is possible, e.g. different predictions are obtained for single corneas, the test will be repeated.

EVALUATION OF RESULTS
- Rules for assessment:
IVIS > 55: risk of serious damage to the eyes
IVIS ≤ 55: no risk of serious damage to the eyes

Irritation parameter:

other: In Vitro Irritancy Score (IVIS)

Basis:

mean

Time point:

other: 10 min

Score:

25.7

Reversibility:

other: not applicable

Remarks on result:

other: in vitro test on isolated bovine cornea

Irritant / corrosive response data:

See figure

Interpretation of results:

other: no serious eye damage

Remarks:

Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™; BASF SE 61V0731/11A525, 2012; reliability score 1). The test was conducted according to GLP and OECD 431 and 439, respectively. For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues was compared to that of negative control tissues. The quotient of the values indicated the relative tissue viability. The test substance was not able to reduce MTT directly. In the corrosion test, the mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 103%, and it was 97% after an exposure period of 1 hour. In the irritation test, the mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 53%. Based on the observed results it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

Eye irritation:

The potential of 2,2,6,6-tetramethylpiperidin-4-yl dodecanoate to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 µL of a 10% test-substance preparation to theepithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation for 10 minutes followed by a 2-hours post-incubation period. This set-up was chosen because of the surface-active properties of the test substance.

 In addition to the test substance a negative control (NC; de-ionized water) and a positive control (PC; 100% ethanol) were applied to three corneas, each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.

The IVIS score of 25 was below the threshold of 55 for classification as a severe irritant and on this study alone, no decision on classification can be taken. However, a pH value of 9.7 was measured. In according to a model described in the draft ECHA guidance document r7.a of September 2014, a substance is very likely to be irritating if the pH is above 8.6. The combination of the high pH-value and the moderately high IVIS score, the substance is considered to be irritating to eyes.

Justification for selection of skin irritation / corrosion endpoint:
valid study

Effects on skin irritation/corrosion: slightly irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available in-vitro study is considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin irritation under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG. Based on pH and the OECD 437 study, the substance is classified as irritating to eyes (R36).

 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin irritation under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012). The substance is classified as irritating to eyes (GHS Cat 2) based on the IVIS score of 25 in the BCOP assay and pH.