2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

Endpoint summary

Description of key information

The test substance does not give an indication of an irritant property to skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Sep 2014 - 21 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006, Part B: Methods for the determination of toxicity and other health effects: In Vitro Skin Corrosion: Human Skin Model Test

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, 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 EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, 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 EpiDermTM tissues (surface 0.6 cm²) are cultured on specially
prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Control samples:

other: Control tissues were concurrently treated with 50 μL of de-ionized water (negative control, NC) or with 50 μL of 8 N potassium hydroxide (positive control, PC).

Amount/concentration applied:

25 μL de-ionized water was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.

Duration of treatment / exposure:

3 minutes at room temperature or 1 hour in the incubator

Number of replicates:

Two tissues per exposure time.

Details on study design:

The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.
Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure; tissue 1

Value:

107.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure; tissue 2

Value:

107.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure; tissue 3

Value:

108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h exposure; tissue 1

Value:

96.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h exposure; tissue 2

Value:

106.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h exposure; tissue 3

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test substance is not able to reduce MTT directly, as determined in a pretest.

Individual and mean OD570 values, individual and mean viability values

		Exposure: 3 min			Exposure: 1 hour
Test substance		tissue 1	tissue 2	mean	tissue 1	tissue 2	mean
NC	mean OD570	1.689	1.716	1.703	1.987	1.880	1.934
	viability [% of NC]	99.2	100.8	100	102.8	97.2	100
test article	mean OD570	1.828	1.838	1.833	1.866	2.052	1.959
	viability [% of NC]	107.4	107.9	108	96.5	106.1	101
PC	mean OD570	0.341	0.383	0.362	0.190	0.224	0.207
	viability [% of NC]	20.0	22.5	21	9.8	11.6	11

Interpretation of results:

GHS criteria not met

Conclusions:

The test article does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Executive summary:

The potential of the test article to cause dermal corrosion/irritation was assessed by a single topical application of 25 μL bulk volume (about 16 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. 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 testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin corrosion/irritation test showed the following results: The test substance is not able to reduce MTT directly. Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 108%,and it was 101% after an exposure period of 1 hour. Based on the observed results it was concluded, that the test article does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Food and Drug Administration of the U.S.A. in The Federal Register (17 September, 1964 § 191.12),

Deviations:

no

GLP compliance:

no

Species:

rabbit

Details on test animals or tissues and environmental conditions:

no data

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

40 mg of the test item were instilled into each eye.

Duration of treatment / exposure:

single treatment

Observation period (in vivo):

daily

Number of animals or in vitro replicates:

6 animals

Details on study design:

SCORING SYSTEM: Ocular reactions were scored by the method described by J.H. Draize in "Appraisal of the safety of Chemicals in foods, Drugs and Cosmetics" p 51.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 4 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritant / corrosive response data:

Temporary mild conjunctival reactions were observed in four animals only. In the remaining two animals there was no observable response to treatment. A maximum group mean reaction score of 1 was established on day 1.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The potential for corrosive activity and skin irritation of the test article was assessed in vitro. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT). The potential of the test item to cause dermal corrosion/irritation was assessed by a single topical application of 25 µl bulk volume (about 16 mg) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). 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 is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin corrosion/irritation test showed the following results: The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 108%, and it was 101% after an exposure period of 1 hour. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 101%. Based on the observed results and applying the evaluation criteria it was concluded, that the test article does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

This finding was supported by the results of an old summary report investigating the dermal irritation potential. Here, twenty-four hours after application to the unabraded skin of rabbits, there was no discernible evidence of erythema or edema with any of the materials.

Eye irritation

Aim of the study was to determine the degree of eye irritation/corrosion of 6 rabbits caused by the test substance in 7 days. To that end, 40 mg of the test item were instilled into each eye. The eyes were examined at 24, 48, 72 hours, then 1 week after the application. One day after the treatment the test item caused conjunctivae irritant effects in 4 of 6 animals. No other symptoms were observed. 1 week after the treatment, all animals were free of symptoms, so the study was terminated. The animals individual mean scores (considering readings at 24, 48, and 72 hours after the treatment) were as follows: Cornea: 0 (mean value from all animals); Iris: 0 (mean value from all animals); Conjunctivae: 2, 0.66, 0, 0.66, 0.66, 0. In conclusion, the test item applied to the rabbits' eye mucosa caused slight conjunctivae irritant effects, fully reversible within 4 days.

Justification for classification or non-classification

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 (CLP). As a result the substance is not considered to be classified for skin irritation and for eye irritation under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.