Oligomeric reaction products of 4,4'-propane-2,2-diyldiphenol and 2-methyloxirane and 2-(chloromethyl)oxirane

Endpoint summary

Description of key information

Skin irritation: It was concluded that the test substance,  EP-4000S, with a mean tissue viability of 56.4 ± 7.5%, was predicted as non-irritant to 
the skin.
Eye irritation: The test substance, EP-4000S, elicited an In Vitro Irritancy Score of -0.4 ± 1.7 and was predicted to be a non-corrosive/ non-severe eye irritant.

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:

10 July 2012 - 28 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted in accordance with international guidelines.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Standard Operating Procedure, In Vitro Skin Irritation Test: Human Epidermis Model (L’Oreal 2009), supplied by L’Oreal (leading laboratory in the validation of the test for ECVAM).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EPISKIN three-dimensional human skin model.

Details on test animals or test system and environmental conditions:

The test involves the application of the test substance for 15 minutes to the EPISKIN three dimensional human skin model. The model consists of normal,
human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen.

After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum
has formed. The epidermis surface area supplied is 0.38cm2.
The EPISKIN kits include
assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.

Controls:

other: Please refer to above entry

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL applied over each tissue using a positive displacement pipette

Duration of treatment / exposure:

15 ± 0.5 minutes

Observation period:

After 15 ± 0.5 minutes of exposure of the test substance, each tissue was rinsed and the inserts were blotted on absorbent paper to remove remaining sterile Dulbeccos Phosphate Buffered Saline (DPBS), rinsing agent. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37°C ± 2 °C in a humidified atmosphere of 5% CO2 in air.
After 42 ± 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.

Number of animals:

The negative control was sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium.
The positive control was 5% Sodium Dodecyl Sulphate (SDS) in distilled water.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done):each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS)
- Time after start of exposure: 15 ± 0.5 minutes

Irritation / corrosion parameter:

other: other: Mean tissue viability

Value:

56.4

Remarks on result:

other:

Remarks:

Basis: mean. Remarks: Predicted as non-irritant. (migrated information)

Irritant / corrosive response data:

The test substance, EP-4000S/water solution and water control were colourless after the 15 minute shaking period. The test substance, EP-4000S, had not shown any potential for colouring water.

Other effects:

There was no change in the test substance, EP-4000S/MTT solution or the water control/MTT solution after three hours incubation in the dark at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The test substance had not interacted with the MTT.

The results of the assay are summarised below:

Sample	Tissue viability as percentage of mean OD negative control				Prediction MTT endpoint
	Replicate Tissues			Mean +/- SD
	a	b	c
Negative control	102.1	98.0	99.9	100.0 ± 2.0	Not applicable
Positive control	13.8	16.5	34.5	21.6 ± 11.3	Irritant
EP-4000S	60.8	60.7	47.7	56.4 ± 7.5	Non-irritant

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It was concluded that the test substance, EP-4000S, with a mean tissue viability of 56.4 ± 7.5%, was predicted as non-irritant to the 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:

14 August 2012- 15 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted in accordance with international guidelines.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine eyes

Details on test animals or tissues and environmental conditions:

The bovine eyes were excised and collected as soon after slaughter as possible (excised at 12.30 hours, 14 August 2012).
Excised eyes were maintained and transported to the laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v) penicillin/streptomycin solution, to cover all the eyes in the receptacle.
The eyes were used within 4 hours of slaughter.

Vehicle:

unchanged (no vehicle)

Controls:

other: Concurrent positive and negative control corneas were examined

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): neat

Duration of treatment / exposure:

10 minutes (± 30 seconds)

Observation period (in vivo):

Following incubation, the positive and negative controls were removed and the epithelial surface of the cornea washed, at least three times or until the wash medium (EMEM with phenol red) was clear and there was no discolouration. The wash medium was added via the holes on the top of the holder. After each wash, the wash medium was removed using a pipette tip attached to a vacuum pump.
As the test substance, EP-4000S, was very viscous, it was not possible to remove using the vacuum pump. The anterior glass was removed and the corneas were gently washed with EMEM without phenol red until there was no discolouration of the wash medium. The anterior glass was then replaced.

Following completion of the two hour incubation period, the medium was removed from both compartments and replaced with fresh cMEM. The posterior compartment was re-plugged and the opacity of each cornea measured and recorded.
The opacity values obtained at this stage were used in calculating the final In Vitro Irritancy Score.

Throughout the assay the corneas were examined for opaque spots or other irregularities.

Number of animals or in vitro replicates:

Corneas were treated in triplicate with either the test substance, positive control (1% w/v sodium hydroxide) or negative control (0.9% sodium chloride solution).

Details on study design:

The assay uses isolated bovine corneas as a means of assessing the ocular corrosivity or severe irritancy potential of test substances in vitro.
The isolated corneas were obtained as a by-product of the meat production industry. Two endpoints, corneal opacity and permeability,
were measured and combined to give an In Vitro Irritancy Score.

Irritation parameter:

other: Opacity

Basis:

mean

Time point:

other: 10 min

Score:

-0.333

Remarks on result:

other: SD=1.528

Irritation parameter:

other: Permeability OD 490

Basis:

mean

Time point:

other: 10min

Score:

-0.004

Remarks on result:

other: SD=0.013

Irritation parameter:

other: In Vitro Irritancy

Basis:

mean

Time point:

other: 10min

Score:

-0.4

Remarks on result:

other: In Vitro Irritancy Score (IVS) was calculated as: (Opacity +(15x Corrected OD 490)

Irritant / corrosive response data:

Throughout the assay the corneas were examined for opaque spots or other irregularities.
Following treatment with test substance, EP-4000S, the corneas were noted as clear. The corneas treated with the positive control, sodium hydroxide 1% (w/v), were very opaque and the corneas treated with the negative control, 0.9% saline, were clear.

The positive control, sodium hydroxide (1%), elicited an In Vitro Irritancy Score of 131.4.

This value was within the historical range (mean ± 2 x SD = 82.3 – 144.7) for the assays performed to date.

The negative control, 0.9% saline, opacity mean change value was 1.0 which was below the maximum acceptance value of 2.0.

The permeability mean of the negative control was 0.028 which was below the maximum acceptance value of 0.1.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance, EP-4000S, elicited an In Vitro Irritancy Score of -0.4 ± 1.7 and was predicted to be a non-corrosive/ non-severe eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

The key skin irritation/corrosion study was performed with EP-4000S according to OECD guideline 439 and in accordance with GLP.

The test substance was applied to EPISKIN human epidermis skin constructs. The constructs consisted of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify

irritant and non-irritant substances.

The test substance, EP-4000S, elicited a mean tissue viability of 56.4 ± 7.5% and was predicted as non-irritant to the skin.

Eye irritation

The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the ocular irritancy potential in vitro of the test substance, EP-4000S.The key eye irritation study was performed according to OECD guideline 437 and in accordance with GLP.

The assay uses isolated bovine corneas as a means of assessing the ocular corrosivity or severe irritancy potential of test substances in vitro. The isolated corneas were obtained as a by-product of the meat production industry. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score which can be used to classify and rank test substances as potential eye irritants according to OECD guideline 437 (OECD 437).

The test substance, EP-4000S, elicited an In Vitro Irritancy Score of 56.4 ± 7.5 and was predicted to be a non-corrosive / non-severe eye irritant.

Justification for selection of skin irritation / corrosion endpoint:
GLP compliant study conducted in accordance with international guidelines.

Justification for selection of eye irritation endpoint:
GLP compliant study conducted in accordance with international guidelines.

Justification for classification or non-classification

In-vitro studies assessing skin irritation and eye irritation are available. Each study concluded that EP-4000S would not be irritant to skin or eyes; on this basis it is concluded that EP-4000S does not meet the criteria for classification as irritant under the CLP Regulation.