6,6'-(Ethane-1,2-diylbis(azanediyl))bis(dibenzo[c,e][1,2]oxaphosphinine-6-oxide)“

Endpoint summary

Description of key information

Skin irritation:

Three tissues of the human skin model EpiDerm^TMwere treated with EDA-DOPO for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size.

DPBS-buffer was used as negative control, 5% SDS solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 2.0. The positive control showed clear irritating effects. Relative absorbance was reduced to 2.9% (required: < 20%).

Variation within the tissue replicates was acceptable (required: ≤ 18%).

After the treatment with the test item, the relative absorbance values were reduced to 93.6 %. This value is above the threshold for skin irritation potential (50%).

Therefore, EDA-DOPO is considered as non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Eye irritation:

Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old.

The test itemEDA-DOPOwas brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for4 hours at32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 1.92.

20% Imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 132.32.

Under the conditions of the study, the test itemEDA-DOPOshowed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 0.18.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

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:

2016-09-23 - 2016-10-14

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 (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:EpiDermTM-Kit, procured by MatTek
- Tissue batch number(s):23362
- Delivery date:11. Oct. 2016
- Date of initiation of testing:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:37 ± 1°C
- Temperature of post-treatment incubation (if applicable):37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:1 mg/mL
- Incubation time:3 hours
- Spectrophotometer:plate spectrophotometer
- Wavelength:570 nm

Control samples:

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

negative control: 30 µL DPBS buffer
positive control: 30 µL 5% SDS-solution
test item: 25.6 mg, 24.9 mg,25.7 mg

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

23 hours and 27 minutes at 37 ± 1°C

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item

Value:

93.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

positive control

Value:

2.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

% Formazan Production

Designation	EDA-DOPO	Positive Control
% Formazan production (tissue 1)	99.2%	2.9%
% Formazan production (tissue 2)	87.8%	2.9%
% Formazan production (tissue 3)	94.0%	2.8%
% Formazan production (mean)	93.6%	2.9%
± SD of mean Formazan production (%)	5.7%	0.1%

Interpretation of results:

GHS criteria not met

Conclusions:

EDA-DOPO is considered as non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Executive summary:

Three tissues of the human skin model EpiDerm^TMwere treated with EDA-DOPO for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size.

DPBS-buffer was used as negative control, 5% SDS solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 2.0. The positive control showed clear irritating effects. Relative absorbance was reduced to 2.9% (required: < 20%).

Variation within the tissue replicates was acceptable (required: ≤ 18%).

 

After the treatment with the test item, the relative absorbance values were reduced to 93.6 %. This value is above the threshold for skin irritation potential (50%).

 

Therefore, EDA-DOPO is considered as non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

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:

2016-09-23 - 2016-10-20

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany
- Number of animals:3
- Characteristics of donor animals (e.g. age, sex, weight):between 12 and 60 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):The eyes were transported to the test facility in Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 h.
- indication of any existing defects or lesions in ocular tissue samples:None of the corneas showed tissue damage; therefore, all corneas were used.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

Amount / concentration applied:

750 µL negative control solution and positive control solution
432.4 mg, 431.3 mg, 431.7 mg test item

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Irritation parameter:

in vitro irritation score

Run / experiment:

test item

Value:

0.18

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

negative control

Value:

1.92

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control

Value:

132.32

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

IVIS Values

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS-solution	1.29	1.92	29.23%
	2.11
	2.35
Test Item EDA-DOPO	1.22	0.18	596.48%
	-0.91
	0.22
Positive Control 20% imidazole solution	146.85	132.32	10.77%
	131.76
	118.36

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study, the test item EDA-DOPO showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 0.18.

Executive summary:

Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old.

The test item EDA-DOPO was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for4 hours at32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 1.92.

20% Imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 132.32.

Under the conditions of the study, the test item EDA-DOPO showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 0.18.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or 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

According to the CLP Regulation, the substance has not to be classified as skin or eye irritant.

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