Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol]and propylidynetrimethanol and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3rd July - 19th November 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant and conducted in accordance with testing guidelines.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ECVAM Scientific Advisory Committee (ESAC) Statement on the Validity of In Vitro Tests for Skin Irritation (2007)

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ECVAM Skin Irritation Validation Study. Validation of the EpiSkin® Irritation Test – 42 Hours Assay for the Prediction of Acute Skin Irritation of Chemicals,

Deviations:

not specified

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ECVAM Skin Irritation Validation Study (SIVS). Performance Standards for Applying Human Skin Models to In Vitro Skin Irritation Testing (2007).

Deviations:

not specified

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,6-diethyl-2,6-bis(hydroxymethyl)heptane-1,3,5,7-tetrol; 2-ethyl-2-(hydroxymethyl)propane-1,3-diol; 2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol

EC Number:

904-155-3

Molecular formula:

C12H28O6 C12H26O5 C6H14O3

IUPAC Name:

2,6-diethyl-2,6-bis(hydroxymethyl)heptane-1,3,5,7-tetrol; 2-ethyl-2-(hydroxymethyl)propane-1,3-diol; 2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): HB TMP
- Analytical purity: 69%
- Composition of test material, percentage of components: 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] (CAS: 23235-61-2, EC No.: 245-509-0), Propylidynetrimethanol (CAS: 77-99-6, EC No.: 201- 074-9) and 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] (CAS: 93983-16-5, EC No.: 301-304-9).
- Lot/batch No.: 7LTQA002
- Expiration date of the lot/batch: 8th May 2014
- Storage condition of test material: Stored at ambient temperature in the dark when not in use.

Test animals

Species:

human

Strain:

not specified

Details on test animals or test system and environmental conditions:

Not applicable as no test animals were used.

Test system

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

other: ultra pure water.

Controls:

other: Control substances were used.

Amount / concentration applied:

Approximately 12mg HB TMP was applied.

Duration of treatment / exposure:

15 minutes.

Observation period:

Not applicable.

Number of animals:

Not applicable as study was conducted using EpiSkin Irritation test.

Details on study design:

Prior to conducting the irritation assay, a preliminary test was conducted to assess the intrinsic ability of the test item to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan. HB TMP did not reduce MTT to formazan.

12mg HB TMP was loaded onto the underside of 3 cylindrical metal weights using a microspatula. The HB TMP was applied to the cylindrical weights so that the entire underside was covered with the test item. After loading, the total weight of the HB TMP and cylindrical weight was recorded. Triplicate EpiSkin® units were dosed by applying the weights (HB TMP side down) onto the EpiSkin® surface. Prior to dosing, the EpiSkin surface was moistened by applying 5 μL of water. The weights were gently massaged onto the EpiSkin® surface over the course of the exposure (every 2 min) to ensure good contact between the HB TMP and the exposed EpiSkin® tissues. During the exposure period, most of the test item was transferred from the underside of the cylindrical weight onto the EpiSkin® surface, forming a homogenous liquid that was evenly distributed over the entire exposed area. The cylindrical weights were reweighed after dosing to confirm the dose transferred to the EpiSkin®. For all units the applied dose within the defined limits (10 mg ± 2 mg).
The surface area of the EpiSkin® was ca 0.38 cm2. Therefore, the mean application rate was 26.3 μL/cm2 for the controls and ca 26.3 mg/cm2 for HB TMP.

After the 15 min exposure period, the test item was washed from the surface of the EpiSkin® using Dulbecco’s phosphate-buffered saline (PBS).
The EpiSkin® was then incubated for a recovery period of 41.5 h in a humidified incubator, set to maintain temperature and CO2 levels of 37°C and 5%, respectively. Following incubation, the EpiSkin® units were transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for 3 h. Biopsies of the EpiSkin® membranes were removed, added to acidified isopropanol, and refrigerated for approximately 75 h in order to extract the formazan. The formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 550 nm. Three replicates of the positive control, aqueous sodium dodecyl sulphate (SDS) solution (5%, w/v), and the negative control, PBS were tested in parallel to demonstrate the efficacy of the assay. The viability of each
individual EpiSkin® tissue was calculated as a percentage of the mean negative control viability (defined as 100%).

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

For the HB TMP test, the results were similar for the three viable EpiSkin® units dosed with HB TMP. Exposure to HB TMP resulted in a mean EpiSkin® viability of 100.57% ± 3.17% of the negative control value.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Evaluation of Direct Reduction of MTT (Prelimiary Assay).

Treatment	Replicate ID	Weight/volume Applied	Colour change observed	Treatment reduces MTT?
HB TMP	Rep 1 Rep 2 Rep 3	10mg	No colour change	No
Water (negative control)	Rep 1 Rep 2 Rep 3	10µL	No colour change	No
Eugenol (positive control)	Rep 1 Rep 2 Rep 3	10µL	Purple colour change	Yes

Percentage Viability of EpiSkin® Tissues After 41.5 h Recovery Time

Treatment	Replicate ID	Relative Viability (%)	Mean Relative Viability per Tissue (%)	Mean Relative Viability per Test item (%)	SD (%)
PBS Solution (Negative Control)	Rep 1	96.13	95.78	100.0	3.83
		95.44
	Rep 2	105.21	103.26
		101.30
	Rep 3	103.03	100.96
		98.89
Aqueous SDS Solution (5%, w/v) (Positive Control)	Rep 1	13.57	13.80	14.22	3.01
		14.03
	Rep 2	11.15	11.44
		11.73
	Rep 3	17.82	17.42
		17.02
HB TMP	Rep 1	102.91	104.06	100.57	3.17
		105.21
	Rep 2	99.92	99.81
		99.69
	Rep 3	102.11	97.85
		93.60

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

Under the conditions of this study, HB TMP was determined to be a non-irritating to skin in accordance with GHS classification when tested in the EpiSkin in vitro irritation assay.

Executive summary:

The skin irritation potential of HB TMP was evaluated using the EpiSkin® in vitro irritation test in accordance with OECD Guideline 439 and ECVAM guidance and GLP compliance.

A preliminary test was conducted to assess the intrinsic ability of the test item to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan. HB TMP did not reduce MTT to formazan.

The dermal irritation potential was assessed by applying 10 mg ± 2 mg of the solid HB TMP to the exposed surface of three EpiSkin® reconstructed human epidermis (RhE) units for 15 min. Prior to applying the test item, the EpiSkin® surface was pre-moistened with sterile, ultra-pure water (5 μL). The surface area of the EpiSkin® was 0.38 cm2, therefore the application rate was ca 26.3 mg/cm2. After the 15 min exposure period, the test item was washed from the surface of the EpiSkin® using Dulbecco’s phosphate-buffered saline (PBS).

The EpiSkin® was then incubated for a recovery period of 41.5 h in a humidified incubator, set to maintain temperature and CO2 levels of 37°C and 5%, respectively. Following incubation, the EpiSkin® units were transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for 3 h. Biopsies of the EpiSkin® membranes were removed, added to acidified isopropanol, and refrigerated for ca 75 h in order to extract the formazan. The formazan production (cell viability) was assessed by measuring the optical

density of the extracts at a wavelength of 550 nm. Three replicates of the positive control, aqueous sodium dodecyl sulphate (SDS) solution (5%, w/v), and the negative control, PBS were tested in parallel to demonstrate the efficacy of the assay. The viability of each

individual EpiSkin® tissue was calculated as a percentage of the mean negative control viability (defined as 100%).

Exposure to HB TMP resulted in a mean EpiSkin® viability of 100.57% ± 3.17% of the negative control value. Exposure to the positive control, aqueous SDS solution (5%, w/v), resulted in a mean EpiSkin® viability of 14.22% ± 3.01% of the negative control value.

In conclusion, HB TMP was demonstrated to be non-irritant in accordance with GHS classification when tested in the EpiSkin® in vitro irritation assay.