4,4'-(4-methylpentane-2,2-diyl)bis((heptyloxy)benzene)

Endpoint summary

Description of key information

In a reliable in vitro skin corrosion study human skin tissue (eipdermis keratinocytes) was exposed to undiluted substance for a period of 3 and 60 minutes. There was 95% and 102% tissue viability following the 3 and 60 minutes of exposure, respectively. Resultantly, the substance is considered to be non-corrosive to human skin.

In a reliable in vitro skin irritation study conducted on the substance the mean value of relative tissue viability was 100% following a 15-minutes exposure to undiluted substance. This value is above the threshold for skin irritation (50%). Therefore, the substance is considered to be a non-irritant to human skin.

In a reliable in vitro eye irritation study employing bovine cornea, exposure to the undiluted substance for 10 minutes resulted in a mean in vitro irritancy score (IVIS) of -0.7. Resultantly, as the substance induced an IVIS ≤ 3, the test material is considered to be non-irritant to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17th December 2018 - 21st December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch (Lot) Number: AS455433
Expiry date: 01 November 2020 (retest date)
Physical Description: Colourless to light yellow viscous liquid
Purity/Composition: 98.5%
Storage Conditions: At room temperature protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

Test system:

EpiDerm Skin Model (EPI-200, Lot no.: 29674 Kit K and L, Appendix 4).
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Excessive amount of the undiluted substance
50 µL Milli-Q water (negative control)
50 µL 8N KOH (positive control)

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours (incubation with MTT).

Number of replicates:

Substance and controls: 2 for each time point

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit equal to or lower than 2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 6.6%.

Mean Absorption in the in vitro Skin Corrosion Test with the Substance.

	3 -minute application viability (%)				1 -hour application viability (%)
	A (OD570)	B (OD570)	Mean (OD570)	SD (+/-)	A (OD570)	B (OD570)	Mean (OD570)	SD (+/-)
Negative control	1.825	1.732	1.779	0.066	1.736	1.620	1.678	0.083
Substance	1.643	1.733	1.688	0.064	1.670	1.739	1.704	0.049
Positive control	0.102	0.124	0.113	0.015	0.108	0.112	0.110	0.003

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0424). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in the in vitro Skin Corrosion Test with the Substance.

	3-minute application viability (% of control)	1-hour application viability (% of control)
Negative control	100	100
Substance	95	102
Positive control	6.3	6.6

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is considered to be non-corrosive to skin based on a reliable in vitro skin corrosion study.

Executive summary:

In a reliable in vitro skin corrosion study, conducted according to the OECD Guideline 431, 'In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method', the undiluted substance (50 µL) was applied onto reconstructed human skin tissue (epidermal model, EpiDerm (EPI-200)) in duplicate for a period of 3 or 60 minutes.

Skin corrosion is expressed as the remaining cell viability after exposure to the substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the substance compared to the negative control tissues was 95% and 102%, respectively. Because the mean relative tissue viability for the substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the substance is considered to be not corrosive.

In conclusion, the substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this study.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29th January 2019 - 4th February 2019

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 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch (Lot) Number: AS455433
Expiry date: 01 November 2020 (retest date) (taken from label)
Physical Description: Colourless to light yellow viscous liquid
Purity/Composition: 98.5%
Storage Conditions: At room temperature protected from light
Test item handling: No specific handling conditions required

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize 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).

Vehicle:

other: not applicable

Details on test system:

Test system

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 19 EKIN 005, See Appendix 4).
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.

Source

SkinEthic Laboratories, Lyon, France.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

undiluted (25 µL)

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean

Run / experiment:

1

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Mean Absorption in the In Vitro Skin Irritation Test with the substance.

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		± SD
Negative control	1.136	1.180	1.287	1.201	±	0.078
Substance	1.152	1.042	1.258	1.151	±	0.108
Positive control	0.048	0.072	0.074	0.064	±	0.014

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.045). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in the In Vitro Skin Irritation Test with the substance.

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	6.5
Test item	96	9.0
Positive control	5.4	1.2

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is considered to be a non-irritant to skin based on an in vitro study conducted according to OECD TG 439.

Executive summary:

In an in vitro skin irritation study conducted according to OECD TG 439 ‘In vitro Skin Irritation: Reconstructed Human Epidermis Test Method’ human skin tissue (eipdermis keratinocytes) was exposed to the undiluted substance for 15-minutes. Following the exposure, the substance was rinsed and incubated for further 42 hours in fresh medium. There was 100 % tissue viability following the 15-minute exposure point. This value is above the threshold for skin irritation (50%). Resultantly, the substance is considered to be a non-irritant to human skin.

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:

19 December 2018 - 19 December 2018

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted October 09, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch (Lot) Number: AS455433
Expiry date: 01 November 2020 (retest date) (taken from label)
Physical Description: Colourless to light yellow viscous liquid
Purity/Composition: 98.5%
Storage Conditions: At room temperature protected from light
Test Facility test item number: 209996/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System:

Bovine eyes were used as soon as possible after slaughter.

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 under cooled conditions.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 uL of undiluted substance, positive control (Ethanol) and negative control (physiological saline).

Duration of treatment / exposure:

10 +/- 1 minutes.

Duration of post- treatment incubation (in vitro):

210 minutes.

Number of animals or in vitro replicates:

3

Details on study design:

Experimental design:

The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1 °C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 °C.

After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading was determined and recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of Corneas and Opacity Measurements:

The medium from the anterior compartment was removed and 750 uL of either the negative control, positive control (Ethanol) or substance was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the substance over the entire cornea. Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1 °C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 +/- 10 minutes at 32 +/- 1 °C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Permeability Determinations.

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader).
Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the substance was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

-1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

-0.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean 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. Altough one of the negative control treated corneas was translucent, the mean results are within the acceptability range therefore this has no impact on the study result. The mean in vitro irritancy score of the positive control (Ethanol) was 47 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 

Summary of Opacity, Permeability and In Vitro Scores.

Treatment	Mean Opacity^1	Mean Permeability^1	Mean In vitro Irritation Score^1, 2
Negative control	2.6	0.002	2.7
Positive control (Ethanol)	18	1.906	47
Substance	-0.9	0.011	-0.7

¹    Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.

²    In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

Interpretation of results:

GHS criteria not met

Conclusions:

The results of an in vitro eye corrosion/irritation study employing isolated bovine cornea exposed to undiluted substance for 10-minutes indicate that it is a non-irritant to the eye.

Executive summary:

In a reliable in vitro eye irritation study, conducted according to OECD Guideline 437, ‘Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage’, the ability of the substance to induce opacity and permeability in an isolated bovine cornea were determined.

 

The substance was applied undiluted at 750 µL onto corneas (n=3) for a period of 10 minutes, followed by rinsing of the substance and further incubation for 2 hours. The opacity and permeability of the corneas were determined after 90 minutes incubation with fluorescein.

The substance did not induce ocular irritation according to opacity and permeability, resulting in a mean in vitro irritancy score (IVIS) of -0.7. In conclusion, since the substance induced an IVIS ≤ 3, the substance is considered to be a non- irritant to the eye and not expected to cause serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the findings of a reliable in vitro skin irritation, skin corrosion and eye irritation studies conducted on the substance, classification of the substance is not justified.