4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

Endpoint summary

Description of key information

Results for eye irritation determined using OECD test guideline 437 and EU guidance B.47.
Result for skin irrtation determined using OECD test guideline 439 and EU guidance B.46.
Substance is not irritating to the skin or eye. 

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:

02 July 2012 to 27 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidance in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

other: EPISKIN Small Model

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Test system: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 12-EKIN-30).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Source: SkinEthic Laboratories, Lyon, France.

Type of coverage:

other: Detailed under study design

Preparation of test site:

other: Detailed under study design

Vehicle:

unchanged (no vehicle)

Controls:

other: negative and positive control performed

Amount / concentration applied:

11.0 to 14.3 mg

Duration of treatment / exposure:

15 minutes

Observation period:

42-hour post incubation period.

Number of animals:

Not applicable. Triplicate exposure were performed.

Details on study design:

Test substance preparation: No correction was made for the purity/composition of the test compound.
The solid test substance (11.0 to 14.3 mg) was applied directly on top of the skin tissue. 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was spread to match the size of the tissue.

Reference substances
Negative control:Phosphate buffered saline (PBS, Invitrogen Corporation, Breda, The Netherlands).
Positive control:5% (aq) Sodium dodecyl sulphate (SDS, Sigma Aldrich, Zwijndrecht, The Netherlands) [CAS Number 151-21-3] in PBS.

Preparation and preincubation
Tissues: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT medium: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).

Environmental conditions: All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 83 - 96%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.0 - 37.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Study design
Test for reduction of MTT by the test substance: 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 12.6 mg of the test substance was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

Application/Treatment of the test substance: The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (11.0 to 14.3 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µl PBS (negative control) and 3 tissues with 25 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Cell viability measurement: After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Irritation / corrosion parameter:

other: other: Mean adsorption

Value:

0.886

Remarks on result:

other:

Remarks:

Basis: mean. Time point: overall. Max. score: 0.893. Remarks: SD +/- 0.042. (migrated information)

Irritant / corrosive response data:

4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] did not interact with MTT.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] compared to the negative control tissues was 119%. Since the mean relative tissue viability for 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was above 50% 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 6%. The standard deviation value of the percentage viability of three tissues treated identically with the positive control and test substance was less than 5%. Although the SD of the percentage viability of three tissues treated identically with the negative control was 19% , the absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Mean absorption in the in vitro skin irritation test with 4,4’,4’’-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.750	0.853	0.576	0.727	±	0.140
Test substance	0.893	0.817	0.888	0.866	±	0.042
Positive control	0.045	0.057	0.024	0.042	±	0.016

Test substance = 4,4’,4’’-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

OD = optical density

SD = standard deviation

Triplicate exposure are indicated by A, B and C.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In vitro skin irritation test with 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] using a human skin model.

 

This report describes the ability of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was tested through topical application for 15 minutes.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines and performed in compliance with the Principles of Good Laboratory Practice.

 

Batch WCA1A0001 of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was a white powder with a purity of 97.6 Area%. Skin tissue was moistened with 5 µl of Milli-Q water and 11.0 to 14.3 mg of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was applied directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] compared to the negative control tissues was 119%. Since the mean relative tissue viability for 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was above 50% after 15 minutes treatment 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] is considered to be non-irritant.

 

The positive control had a mean cell viability of 6% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically with the positive control and test substance was less than 6%. Although the SD of the percentage viability of three tissues treated identically with the negative control was 19%, the absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

Finally, it is concluded that this test is valid and that 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

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:

21 May 2012 to 22 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines and in compliance with GLP and reported with a valid GLP certificate.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine eyes

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter on the same day.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco,'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (319 to 323 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.

Observation period (in vivo):

Not observation period specified

Number of animals or in vitro replicates:

A total of 9 cornea's used in the experiment.

Details on study design:

After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: overall

Score:

0.5

Max. score:

0.8

Reversibility:

not specified

Remarks on result:

other: In Vitro Irritancy Score (IVIS)

Other effects:

No further effects specified

INDIVIDUAL OPACITY, PERMEABILITY AND IN VITRO SCORES

Table 2: Opacity Score

Eye	Opacity before treatment	Opacity after treatment	Final Opacity^1	Negative control corrected Final Opacity	Mean Opacity
Negative control
1	1	2	1	0	0
2	1	2	1	0
3	1	3	2	1
Positive control
4	0	127	127	126	92
5	1	73	72	71
6	1	81	80	79
4,4’,4’’-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]
19	1	2	1	0	0
20	1	1	0	-1
21	1	1	1	0

¹Final Opacity = Opacity after treatment – Opacity before treatment.

 

Table 3: Permeability score individual values (uncorrected)

Eye	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control
1	1	0.012	0.013	0.011	0.012	0.012	0.015
2	1	0.020	0.021	0.018	0.020	0.020
3	1	0.011	0.018	0.013	0.014	0.014
Positive control
4	6	0.420	0.411	0.413	0.415	2.490
5	6	0.413	0.407	0.414	0.411	2.466
6	6	0.453	0.446	0.444	0.448	2.688
4,4’,4’’-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]
19	1	0.007	0.006	0.007	0.007	0.007
20	1	0.060	0.061	0.071	0.064	0.064
21	1	0.066	0.069	0.064	0.066	0.066

 

Table 4: Permeability score individual values (corrected)

Eye	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OD490Average	Negative control corrected final OD490	Average OD
Negative control
1	1	-0.003	-0.002	-0.004	-0.003	-0.003	0.000
2	1	0.005	0.006	0.003	0.005	0.005
3	1	-0.004	0.003	-0.002	-0.001	-0.001
Positive control
4	6	0.405	0.396	0.398	0.400	2.400	2.458
5	6	0.398	0.392	0.399	0.396	2.376
6	6	0.438	0.431	0.429	0.433	2.598
4,4’,4’’-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]
19	1	-0.008	-0.009	-0.008	-0.008	-0.008	0.031
20	1	0.045	0.046	0.056	0.049	0.049
21	1	0.051	0.054	0.049	0.051	0.051

¹OD490 values corrected for the mean final negative control permeability (0.015)

 

Table 5: In Vitro irritancy score

Eye	Negative control corrected Final Opacity	Negative control corrected Final OD490	In vitro Irritancy Score^1
Negative control
1	0	-0.003	0.0
2	0	0.005	0.1
3	1	-0.001	1.0
Positive control
4	126	2.400	162.0
5	71	2.376	106.6
6	79	2.598	118.0
4,4’,4’’-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]
19	0	-0.008	-0.1
20	-1	0.049	-0.3
21	0	0.051	0.8

¹In vitro irritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It is concluded that this test is valid and that 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tertbutyl-m-cresol] is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Executive summary:

Screening for the eye irritancy potential of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-mcresol] using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the ocular irritation properties of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tertbutyl-m-cresol] on an isolated bovine cornea. The possible ocular irritancy of 4,4’,4”-(1 -methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was tested through topical application for approximately 240 minutes.

The study procedures described in this report were based on the most recent OECD and EC Guideline. The study was performed in compliance with the Principles of Good Laboratory Practice (GLP) and reported with a valid GLP certificate.

Batch WCA1A0001 of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was a white powder with a purity of 97.6 Area%. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (319 to 323 mg).

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.

The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 129 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.5 after 240 minutes of treatment.

Finally, it is concluded that this test is valid and that 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tertbutyl-m-cresol] is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Read across by structural analogue to an in vivo study also supports the findings of the study used for the endpoint selection for eye irritation.

Justification for selection of eye irritation endpoint:
Study performed in accordance with test guidance OECD 437 and EU B.47

Justification for classification or non-classification

The data available demonstrate a lack of corrosivity and consequently a lack of irritation.