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 Aliphaticdiol diglycidyl ether is predicted to be non-irritant to the skin.

Eye irritation: The test substance, Aliphaticdiol diglycidyl ether, elicited an In Vitro Irritancy Score of  1.1 ± 2.3  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:

read-across from supporting substance (structural analogue or surrogate)

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.

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Common functional groups and similar composition.

EP-4000s and the registered substance are very similar. They are UVBC substances and both are oligomeric reaction products of 4,4'-propane-2,2-diyldiphenol and 2-methyloxirane and 2-(chloromethyl)oxirane where constituents are structurally related. EP-4000s contains less than 0.1% monochlorinated constituents while the registered substance contains approximately 10% monochlorinated constituents. As these are otherwise structurally similar to other constituents it can be reasonably expected that EP-4000s and the registered substance will have essentially the same properties.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

EP4000s:
Purity 79.7-90%, 3 main components F, E, C
Impurities:
5 known by-products 18%
6 unknown impurities 2.4%
Water 0.1%
Chlorinated impurities <0.1%

Aliphaticdiol diglycidyl ether:
Purity 92.57%, 3 main components F, E, C
Impurities:
5 known by-products (same identity as EP-400s) 14.37%
Water none
Two new impurities (I and J) 10.48% in total

Further details are provided in Section 13 Assessment Reports

3. ANALOGUE APPROACH JUSTIFICATION

EP-4000s has been tested and found to be not irritant to eyes or skin. Aliphaticdiol diglycidyl ether contains 10% of a primary alkyl halide which is not present in EP-4000s. Alkyl halides can hydrolyse and one of the hydrolysis products is hydrochloric acid. Therefore there is potential for irritancy in an aqueous environment. However, as an in vitro test for eye irritation was negative for Aliphaticdiol diglycidyl ether it can be reasonably be assumed it will not be a skin irritant due to hydrolsis and realease of HCL on the skin, thus allowing read across from available skin irritation data for EP-4000s.

Overall conclusion – no new information would be gained by testing Aliphaticdiol diglycidyl ether for skin irritation.

4. DATA MATRIX

Not applicable – read across based on close similarity of composition.

Reason / purpose for cross-reference:

read-across source

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:

The substance is predicted as non-irritant to the skin with a mean tissue viability of 56.4 ± 7.5%.

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:

2 June to 19 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study caried out in accordance with internationally recognised guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

other: isolated bovine corneas

Strain:

other:

Details on test animals or tissues and environmental conditions:

The bovine eyes, supplied by an abattoir, were excised and collected as soon after slaughter as possible. Eyes were obtained from cattle aged less than 30 months. Instructions were given to avoid damaging the corneas during excision. 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:

other: 0.9% saline.

Controls:

yes, concurrent vehicle

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit): 750 µL- Concentration (if solution): 10% v/v in vehicle

Duration of treatment / exposure:

10 minutes (± 30 seconds)

Observation period (in vivo):

2 hours ±10 minutes (opacity) then 90 ± 5 minutes for permeability

Number of animals or in vitro replicates:

Corneas treated in triplicate

Details on study design:

REMOVAL OF TEST SUBSTANCE - Washing:
Following incubation, the test substance, positive and negative controls were removed from the epithelial surface of the cornea by washing, at least three times or until the wash medium (Eagles Minimal Essential Medium with phenol red) was clear and there was no discolouration. The corneas were gently rinsed with the wash medium until the medium was clear and the colour unchanged. The test substance proved difficult to remove by this method, so the front glass was removed from the holder and the cornea carefully washed using a gentle stream of Minimal Essential Medium with phenol red from a syringe.

SCORING SYSTEM:
Opacity: The change in the opacity of each cornea was calculated by subtracting the initial basal opacity from the post-treatment opacity measurement. The mean change in opacity for the negative control corneas was calculated and was subtracted from the change in opacity of each treated cornea to obtain the corrected opacity value. The mean corrected opacity change value of each treatment group (of three corneas) was calculated from the individual corrected opacity values of the treated corneas. Permeability (OD490): The corrected permeability value (OD490) of each treated cornea was calculated by subtracting the mean negative control cornea value from the permeability value of each cornea. The mean corrected permeability value of each treatment group was calculated from the individual corrected permeability values of the treated corneas. The In Vitro Irritancy Score (IVIS) was calculated using the following formula: In Vitro Irritancy Score = Corrected Opacity Value + (15 x Corrected OD490 Value) The IVIS was calculated for each individual treatment and positive control cornea. The mean IVIS value for each treatment group was calculated from the IVIS of each individual cornea in the treatment group.

Permeability: Following the final opacity measurement, the medium was removed from the anterior compartment of the holder. One mL of sodium fluorescein solution was added to the anterior compartment using a micropipette.and the compartment plugged and the corneas incubated in a horizontal position at 32°C ± 1°C for 90 ± 5 minutes in a waterbath. Following incubation, the medium in the posterior compartment was mixed by drawing
approximately 2.5 mL gently up and down a 5 mL syringe, with a needle attached, three times. An aliquot of the mixed medium from the posterior compartment was removed and transferred to a 1 cm path length cuvette. A spectrophotometer was adjusted to read at 490 nm (OD490) and a sample of cMEM read (OD = 0.079). The spectrophotometer was blanked using this solution prior to reading the permeability samples. Any solution giving an
OD490 value above 1.8 was diluted 1 in 5 with complete Minimal Essential Medium.

The In Vitro Irritancy Score (IVIS) was calculated by combining the coeneal opacity and permeability using the following formula:

IVIS = Corrected Opacity Value + (15 x Corrected Permeability Value)

The IVIS was calculated for each individual treatment and positive control cornea. The mean IVIS value for each treatment group was calculated from the IVIS of each individual cornea in the treatment group.


TOOL USED TO ASSESS SCORE: spectrophotometer adjusted to read at 490 nm

Irritation parameter:

in vitro irritation score

Run / experiment:

EP-4000S

Value:

-0.4

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Sodium Hydroxide 1% (w/v)

Value:

131.4

Remarks on result:

positive indication of irritation

The pH of the test substance, Aliphaticdiol diglycidyl ether, as 10% (v/v) suspension in 0.9% saline, was approximately 7.5.

Throughout the assay the corneas were examined for opaque spots or other irregularities. Following treatment with test substance, Aliphaticdiol diglycidyl ether, the corneas were noted as clear. The corneas treated with the positive control, ethanol, were opaque and the corneas treated with the negative control, 0.9% saline, were clear. The results of the BCOP assay are summarised in the table below.

Sample	Opacity  ± SD	Permeability ± SD	In vitro irritancy Score  ± SD
Aliphaticdiol diglycidyl ether	1.000 ± 2.082	0.007 ± 0.015	1.1 ± 2.3
Ethanol	28.333 ± 1.732	1.146 ± 0.183	45.5 ± 2.7
0.9% Saline	0.667 ± 0.577	0.005 ± 0.002	Not applicable

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Eye irritation

The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the ocular irritancy potential in vitro of the test substance, Aliphaticdiol diglycidyl ether. 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, Aliphaticdiol diglycidyl ether, elicited an In Vitro Irritancy Score of 1.1 ± 2.3 and was predicted to be a non-corrosive / non-severe eye irritant.

Skin irritation/corrosion (read across)

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.

The value of mean tissue viability of 56.4 ± 7.5% for EP-4000s will be taken as the value for Aliphaticdiol diglycidyl ether.

Justification for read across justification - EP-4000s and Aliphaticdiol diglycidyl ether are very similar substances. They are UVBC subtances and both are oligomeric reaction products of 4,4'-propane-2,2-diyldiphenol and 2-methyloxirane and 2-(chloromethyl)oxirane where consituents are structurally related. EP-4000s contains less than 0.1% monochlorinated consitiuents while Aliphaticdiol diglycidyl ether contains approximately 10% monochlorinated constituents, but as these are otherwise structurally similar to other constituents it can be reasonably expected that EP-4000s and Aliphaticdiol diglycidyl ether will have essentially the same physicochemcial properties.

Aliphaticdiol diglycidyl ether contains 10% of a primary alkyl halide which is not present in EP-4000s. Alkyl halides can hydrolyse and one of the hydrolysis products is hydrochloric acid.  Therefore there is potential for irritancy in an aqueous environment. Therefore, as Aliphaticdiol diglycidyl etherwas shown not to be an irritant in vitro test for eye irritation the the negative test result for skin irritation of EP-4000s can be read across to Aliphaticdiol diglycidyl ether.

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 Aliphaticdiol diglycidyl ether would not be irritant to skin or eyes; on this basis it is concluded that Aliphaticdiol diglycidyl ether does not meet the criteria for classification as irritant under the CLP Regulation