Bis(2,3-epoxypropyl) phthalate

Endpoint summary

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

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)

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Details on animal used as source of test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 24 May 2016
EpiDermTM Tissues (0.63cm2) lot number : 23338
Assay Medium lot number : 051916TMA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 uL

Duration of treatment / exposure:

3 minute
60 minute

Duration of post-treatment incubation (if applicable):

3 hours

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute

Value:

105.7

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute

Value:

95.5

Negative controls validity:

valid

Positive controls validity:

valid

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	5.1	105.7
60 minute	100*	4.7	95.5

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 21 June 2016
EpiSkinTM Tissues (0.38cm2) lot number : 16-EKIN-025
Maintenance Medium lot number : 16-MAIN3-040
Assay Medium lot number : 16-ESSC-025

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 uL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes

Value:

67.8

Negative controls validity:

valid

Positive controls validity:

valid

Mean OD₅₆₂ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.648	0.643	0.006	100.8	100+	0.9
	0.644			100.2
	0.637			99.1
Positive Control Item	0.158	0.133	0.022	24.6	20.7	3.4
	0.118			18.4
	0.122			19.0
Test Item	0.469	0.436	0.053	72.9	67.8	9.3
	0.464			72.2
	0.374			58.2

OD = Optical Density

SD = Standard deviation

∗ = The mean viability of the negative control tissues is set at 100%

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply: EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was

based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified

isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 67.8% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were

satisfied.

Conclusion

The test item was classified as non-irritant. The following classification criteria apply: EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 can not be determined).

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Animal Information
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.93 or 3.16 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

3 minute
1 hour
4 hours

Observation period:

1,24,48 and 72 hours

Number of animals:

2

Details on study design:

On the day before the test two rabbits were clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.

One rabbit was initially treated. Three suitable sites were selected on the back of the rabbit. A quantity of 0.5 mL of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. Each patch was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset for the duration of the exposure period.

One patch was removed at each of three time points: 3 minutes, 1 hour and 4 hours after application. Any residual test item was removed by gentle swabbing with cotton wool soaked in distilled water.

After consideration of the skin reactions produced in the first animal, an additional animal was treated with 0.5 mL oftest item. One patch was applied to the back of the rabbit and was allowed to remain in contact with the skin for a period of 4 hours.

Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the Draize scale.

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

24/48/72 h

Score:

0.8

Reversibility:

fully reversible

Skin Reaction	Observation Time (following patch removal)	Individual Scores		Total
		Rabbit Number and Sex
		75497 Female	75499 Male
Erythema/Eschar Formation	Immediately	0	0	(0)
	1 Hour	0	0	(0)
	24 Hours	1	1	2
	48 Hours	0	1	(1)
	72 Hours	0	0	0
Edema Formation	Immediately	0	0	(0)
	1 Hour	0	0	(0)
	24 Hours	1	0	1
	48 Hours	0	0	(0)
	72 Hours	0	0	0

Sum of 24 and 72-Hour Readings (S) 3

Primary Irritation Index (S/4) 3/4 = 0.8

Classification MILD IRRITANT

Interpretation of results:

GHS criteria not met

Conclusions:

The test item produced a primary irritation index of 0.8 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme.
The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item following single, 3-Minute, 1 and 4-Hour, semi-occluded applications to the intact rabbit skin.

Results

3-Minute and I-Hour semi-occluded applications of the test item to the intact skin of one rabbit produced no evidence of skin irritation.

A single 4-Hour, semi occluded application of the test item to the intact skin of two rabbits produced very slight erythema, with or without very slight edema. One treated skin site appeared normal at the 48-Hour observation and the other treated skin site appeared normal at

the 72-Hour observation. No corrosive effects were noted.

Conclusion

The test item produced a primary irritation index of 0.8 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)