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]

Endpoint summary

Description of key information

The HB TM (Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethyl propane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol) was not found to be irritating to skin in an in vitro skin irritation study conducted according to OECD Test Guideline 439.

Based on read-across to eye irritation studies on TMP (propylidynetrimethanol) and Di-TMP (2,2’-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]) and lack of structural alerts for eye irritation for TMF (2,2'-[methylenebis(oxymethylene)] bis[2-ethylpropane-1,3-diol] in QSAR analysis, the HB TMP (Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol) is not predicted to have any potential to cause eye irritation. Taking the overall information together, also considering the two available inconclusive in vitro eye irritation studies on HB TMP, applying the method presented in OECD Guideline on Defined Approaches for Serious Eye Damage / Eye Irritation and also the precautionary principle, the Weight of Evidence stipulates HB TMP substance to be classified as Eye irritation Category 2.

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

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

Remarks:

The tested substance (TMP) is a main component of the registered multi-constituent substance HB TMP.

Adequacy of study:

weight of evidence

Justification for type of information:

This endpoint study record is part of a Weight of Evidence approach comprising both inconclusive in vitro studies on the registered substance HB TMP as well as a read-across to in vivo rabbit studies on two of the three main components of the registered mulit-constituent substance.

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The main components of HB TMP is Di-TMP (2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol], CAS: 23235-61-2) >20 - <70%, TMF (2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol], CAS: 93983-16-5) >15 - <40% and TMP (2-ethyl-2-(hydroxymethyl)propane-1,3-diol CAS: 77-99-6) >2 - <30%, it is therefore considered relevant to include the available information on two of the main components of HB TMP.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See information provided in the Test material section of the source and target records.

3. ANALOGUE APPROACH JUSTIFICATION
Taking the overall information together, the conclusive studies on HB TMP, TMP and Di-TMP as well as the lack of structural alerts for eye irritation for the read-across substance TMF, it is unlikely that HB TMP exhibits any potential to cause eye irritation. Considering also the two inconclusive in vitro eye irritation studies on HB TMP, applying the method presented in OECD Guideline on Defined Approaches for Serious Eye Damage / Eye Irritation and also the precautionary principle, the Weight of Evidence stipulates HB TMP substance to be classified as Eye irritation Category 2.

4. DATA MATRIX
See related endpoint study records.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 0 h

Score:

>= 0.1

Reversibility:

fully reversible within: 24 h

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 1, 2 and 7 days

Score:

0

Reversibility:

other: score = 0 at any time point

Interpretation of results:

GHS criteria not met

Remarks:

Not irritating

Conclusions:

Taking the overall information together, the conclusive studies on HB TMP, TMP and Di-TMP as well as the lack of structural alerts for eye irritation for the read-across substance TMF, it is unlikely that HB TMP exhibits any potential to cause eye irritation. Considering also the two inconclusive in vitro eye irritation studies on HB TMP, applying the method presented in OECD Guideline on Defined Approaches for Serious Eye Damage / Eye Irritation and also the precautionary principle, the Weight of Evidence stipulates HB TMP substance to be classified as Eye irritation Category 2.

Executive summary:

In a primary eye irritation study, 0.05 g of TMP was instilled into the conjunctival sac of the one eye of two New Zealand White rabbits for 7 days. Animals then were observed during these 7 days. In this study, TMP is not irritating to the eye.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

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

Remarks:

The tested substance (Di-TMP) is a main component of the registered multi-constituent substance HB TMP.

Adequacy of study:

weight of evidence

Justification for type of information:

This endpoint study record is part of a Weight of Evidence approach comprising both inconclusive in vitro studies on the registered substance HB TMP as well as a read-across to in vivo rabbit studies on two of the three main components of the registered mulit-constituent substance.

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The main components of HB TMP is Di-TMP (2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol], CAS: 23235-61-2) >20 - <70%, TMF (2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol], CAS: 93983-16-5) >15 - <40% and TMP (2-ethyl-2-(hydroxymethyl)propane-1,3-diol CAS: 77-99-6) >2 - <30%, it is therefore considered relevant to include the available information on two of the main components of HB TMP.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See information provided in the Test material section of the source and target records.

3. ANALOGUE APPROACH JUSTIFICATION
Taking the overall information together, the conclusive studies on HB TMP, TMP and Di-TMP as well as the lack of structural alerts for eye irritation for the read-across substance TMF, it is unlikely that HB TMP exhibits any potential to cause eye irritation. Considering also the two inconclusive in vitro eye irritation studies on HB TMP, applying the method presented in OECD Guideline on Defined Approaches for Serious Eye Damage / Eye Irritation and also the precautionary principle, the Weight of Evidence stipulates HB TMP substance to be classified as Eye irritation Category 2.

4. DATA MATRIX
See related endpoint study records.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Remarks:

(degree)

Basis:

mean

Time point:

other: 24, 48, 72 hours

Score:

ca. 0.6

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Mean value based on 24, 48, and 72 hour time points only.

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48, 72 hours

Score:

ca. 0.3

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Mean value based on 24, 48, and 72 hour time points only.

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48, 72 hours

Score:

ca. 1.1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Mean value based on 24, 48, and 72 hour time points only.

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Time point:

other: 24, 48, 72 hours

Score:

ca. 1.2

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Mean value based on 24, 48, and 72 hour time points only.

Irritant / corrosive response data:

Moderate conjuntival irritation (chemosis, redness, discharge) was observed in all rabbits 1 to 48 hours after exposure. Slight corneal opacity was seen in all rabbits 1 to 24 hours after exposure and in two rabbits 48 hours after exposure. At 72 hours no abnormalities were observed.

Interpretation of results:

GHS criteria not met

Remarks:

Not irritating

Conclusions:

Taking the overall information together, the conclusive studies on HB TMP, TMP and Di-TMP as well as the lack of structural alerts for eye irritation for the read-across substance TMF, it is unlikely that HB TMP exhibits any potential to cause eye irritation. Considering also the two inconclusive in vitro eye irritation studies on HB TMP, applying the method presented in OECD Guideline on Defined Approaches for Serious Eye Damage / Eye Irritation and also the precautionary principle, the Weight of Evidence stipulates HB TMP substance to be classified as Eye irritation Category 2.

Executive summary:

In a primary eye irritation study [OECD 405], 0.1 g of Di-TMP was instilled into the conjunctival sac of the left eye of three SPF albino rabbits for 24 hours. Flouresceing was then instilled and the eyes rinsed with saline solution. Animals then were observed for 72 hours.  Irritation was scored by the method presented in Technical Guidance OECD 405. In this study, Di-TMP is not irritating to the eye.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 March 2020 - 17 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This endpoint study record is part of a Weight of Evidence approach comprising both inconclusive in vitro studies on the registered substance HB TMP as well as a read-across to in vivo rabbit studies on two of the three main components of the registered mulit-constituent substance.

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)

Version / remarks:

9 October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

Guideline:

other: Guidance Document on ‘The Collection of Tissues for Histological Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160.

Version / remarks:

Adopted July 6, 2018 Paris

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was stored in the test facility in a tightly closed vessel at room temperature (20 ± 5 °C) under dry conditions in the dark.

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Industriestraße 42, 75217 Birkenfeld, Germany, on the date of treatment. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container within 1 hour and 15 minutes. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The corneas together with the holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C. After the initial incubation, the medium in cornea holders was completely changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item solution or positive control solution), three replicates were used.

Vehicle:

Hank's balanced salt solution

Remarks:

The test item with HBSS was warmed up in a water bath (80°C) until soluble and was tested with a concentration of 20 % in HBSS. Small pieces were rubbed off using a sharp item. Before applying onto the corneas, the test item solution was cooled down

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The respective substances (negative control solution, test item solution or positive control solution) were applied by pipetting 0.75 mL of the appropriate liquid through the refill hole in the anterior holder on the cornea. The controls and the test item were given on the epithelium that the cornea was evenly covered.

Duration of treatment / exposure:

Each holder was incubated at 32 ± 1 ºC for 4 hours

Duration of post- treatment incubation (in vitro):

The holders were incubated, in a horizontal position, at 32 ± 1 ºC for 90 minutes

Number of animals or in vitro replicates:

Triplicates (3 test item, 3 negative control, 3 positive control)

Details on study design:

After incubation or 4hr, the anterior chambers were rinsed three times with cMEM with phenol red and final rinsing with cMEM without phenol red (one time), the anterior chambers were filled with cMEM without phenol red and the final opacity value of each cornea was recorded. After the recording of the final opacity values, the cMEM without phenol red was removed from both chambers of each cornea holder. The posterior chamber, which interfaces with the endothelial side of the cornea was filled with fresh cMEM without phenol red. Then 1 mL sodium fluorescein solution was added to the front chamber of each cornea holder for the detection of permeability of the corneas. For the controls and the test item, a sodium fluorescein solution with a concentration of 5 mg/mL was used. After 90 more minutes of incubation, the content of each posterior chamber was thoroughly mixed and pipetted in a 96-well plate. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Item

Value:

35.32

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive Control

Value:

98.39

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative Control

Value:

1.36

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean. The mean IVIS of the negative control has to show an IVIS ≤ 3.

The value for the negative and positive control was within the range of historical data of the test facility. Therefore, the test system was acceptable.In the negative control, no signs of eye irritation were observed. (IVIS = 1.36) The positive control induced serious eye damage (IVIS = 98.39), which would be classified as GHS category I. The positive and negative control acceptance criterion was therefore satisfied.

The test item HB TMP showed effects on the cornea of the bovine eye. The calculated mean IVIS was 35.32. According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only. In this case no prediction can be made.

Data Interpretation

IVIS  UN GHS  < 3  No Category  >3; < 55  No prediction can be made  > 55  Category 1

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this test, the test item HB TMP showed effects on the cornea of the bovine eye (IVIS = 35.32). According to UN GHS Classification, the test item HB TMP cannot be given a classification under the conditions of the test.

Executive summary:

In an in vitro GLP study performed according to OECD 437 (9 October 2018), HB TMP (20% in HBSS) was tested onto the cornea of a bovine eye for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. Irritation was scored by the method of In Vitro Irritancy Score (IVIS).

Based on the IVIS score of 35.32 in this study, no prediction can be made for HB TMP regarding the classification for eye irritation or serious eye damage.

Reference 4

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 Sept 2019 - 14 Jan 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This endpoint study record is part of a Weight of Evidence approach comprising both inconclusive in vitro studies on the registered substance HB TMP as well as a read-across to in vivo rabbit studies on two of the three main components of the registered mulit-constituent substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 18 June 2019

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

* The test item was melted first on 09. Sep. 2019. But during the melting process it was observed that the test vessel was leaky and the amount of test item was too big for melting. This is why a smaller amount of test item in a new test vessel was received on 01. Oct. 2019. The new amount of test item was melted in the water bath (about 80°C) and its homogeneity was assessed. Then it was filled into aliquots. The study was conducted on the 01. Oct. 2019 received sample.

The test item was stored in the test facility in a tightly closed vessel at room temperature (20 ± 5 °C) under dry conditions in the dark.

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 15. Oct. 2019
Batch no.: 30630

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

For the pre- and main test the solid test item was rubbed off with a scalpel and was cut into small pieces. The amount of 50 ± 5 mg was enough to cover the complete tissue surface (48.5mg in tissue 1 and 49.4mg in tissue 2).

Duration of treatment / exposure:

After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 30 minutes. After dosing the last tissue, each plate was incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 6 hours.

Duration of post- treatment incubation (in vitro):

At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately.
It was observed that the test item became liquid during the incubation time.
The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately trans-ferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.
After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours and 10 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.

Number of animals or in vitro replicates:

Duplicate

Details on study design:

On the day of the start of the experiment, the MTT concentrate (5 mg/mL) was thawed. The MTT concentrate (5 mg/mL) was diluted with 8 mL assay medium directly before use (= MTT solution 1 mg/mL). The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 16 hours and 27 minutes.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Test Item

Value:

2.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Positive Control

Value:

34.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: Optical Density

Run / experiment:

Negative Control

Value:

1.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

After treatment with the test item, the mean value of relative tissue viability was reduced to 2.6% (2.5% tissue 1, 2.7% tissue 2, 0.2% variation). This value is below the threshold for eye irritation potential (≤ 60%). The variation within the replicates of the controls and the test item was acceptable (< 20%).

Assessment

Eye hazard potential is assessed using the criteria given in the following table:

% Viability	Assessment	UN GHS Classification
>60%	Non eye irritant	No Category
≤ 60 %	At least eye irritant	No prediction can be made (category 1 or 2)

Validity

Validity criteria and results are stated in the following table:

Criterion	Demanded	Found
Mean OD of negative control	>0.8 and < 2.5	1.8
% mean relative viability of positive control	< 50 % of negative control	34.9%
Variation within replicates	< 20 %	0.9% (negative) 9.3% (positive) 0.2% (test item)

All validity criteria were met. The values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the test, no prediction can be made for HB TMP in the EpiOcu-larTM Eye Irritation Test.

Executive summary:

In an in vitro GLP study performed according to OECD 492 (June 2019), HB TMP was tested on a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. After treatment the mean value of relative tissue viability was 2.6%. Under the conditions of the test, no prediction can be made for HB TMP regarding the classification for eye irritation or serious eye damage in the EpiOcularTM Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion
The potential of HB TMP (Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol) to cause skin irritation has been investigated in an in vitro skin irritation assay conducted according to OECD Test Guideline 439, ECVAM guidance and GLP using the EpiSkin irritation test and reconstructed human skin (Blackstock, 2012).

In the study, 10 mg ± 2 mg of the solid test material, HB TMP was applied to the exposed surface of three EpiSkin® reconstructed human epidermis (RhE) units of surface area 0.38 cm²for 15 min (i.e. application rate was ca 26.3 mg/cm²). The EpiSkin® units were then washed to remove the test material and incubated in a humidified incubator at 37^oC (5% CO₂) for a recovery period of 41.5 hours. Following incubation, the EpiSkin® units were transferred to assay medium containing methylthiazoldiphenyl-tetrazolium bromide (MTT) (0.3 mg/mL) and returned to the incubator for 3 hours. Biopsies of the EpiSkin® membranes were removed and tested for formazan production to determine cell viability). 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 whereas 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 the EpiSkin irritation test, a test substance is considered to be an irritant to the skin in accordance with GHS category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%. The test item may be considered to be a non-irritant if the tissue viability after exposure and post-treatment incubation was more than (>) 50%.

Based on this study, the HB TMP (Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol) is not irritating to skin according to Directive 67/548/EEC or Regulation 1272/2008/EC.

Eye irritation
HB TMP (Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol) has been tested in two in vitro studies for eye irritation potential, but without conclusive results. The testing approach used was the so-called bottom-up approach, in accordance with the OECD guidance document no 263 on “Integrated approaches to testing and assessment (IATA) for serious eye damage and eye irritation”. The “Bottom-Up” approach starts with an in vitro test method that can identify test chemicals not requiring classification for eye hazard (UN GHS No Cat.). Depending on the outcome from the first test other in vitro tests may be required if the result is inconclusive.

According to the “Bottom-Up” approach, HB TMP was tested in the in vitro EpiOcular RhCE (OECD Test Guideline 492) test, followed by the ex-vivo BCOP study (OECD Test Guideline 437).

OECD 492
In an in vitro GLP study performed according to OECD 492 (June 2019), HB TMP was tested in order to identify if it can induce serious eye damage and to identify if it requires classification for eye irritation or serious eye damage.

The test item HB TMP was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. After treatment of the tissues with controls and test item, the respective substance was rinsed from the tissue. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.8. The positive control should induce eye irritation; the relative tissue viabil-ity should be < 50%. The positive control showed clear eye irritating effects, the mean val-ue of the relative tissue viability was 34.9%. The variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 2.6%. This value is below the threshold for eye irritation potential (≤ 60%), therefore no prediction can be made.

According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required.

Under the conditions of the test, no prediction can be made for HB TMP in the Epi-OcularTM Eye Irritation Test.

OECD 437
In an in vitro GLP study performed according to OECD 437 (9 October 2018), HB TMP was tested in order to identify if it can induce serious eye damage and to identify if it requires classification for eye irritation or serious eye damage. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS). Test items inducing serious eye damage are classified as UN GHS Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category.

The test item was tested as 20% solution in HBSS. Negative and positive control items were tested concurrently. The test item HB TMP was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

All acceptance criteria were met and there were no deviations from the guideline. Under the conditions of this test, the test item HB TMP showed effects on the cornea of the bovine eye. The calculated mean IVIS was 35.32. In conclusion, since the substance induced an IVIS > 3 and ≤ 55, no classification for eye irritation or serious eye damage can be made.

According to UN GHS Classification, the test item HB TMP cannot be given a classification under the conditions of the test.

Justification for selection of skin irritation / corrosion endpoint:
Sole study providing data from a guideline compliant study. Under the conditions of this study, the test substance was not determined to be irritating to skin according to Directive 67/548/EEC or Regulation 1272/2008/EC.

Justification for selection of eye irritation endpoint:
Taking the overall information together, the conclusive studies on HB TMP, TMP and Di-TMP as well as the lack of structural alerts for eye irritation for the read-across substance TMF, it is unlikely that HB TMP exhibits any potential to cause eye irritation. Considering also the two inconclusive in vitro eye irritation studies on HB TMP, applying the method presented in OECD Guideline on Defined Approaches for Serious Eye Damage / Eye Irritation and also the precautionary principle, the Weight of Evidence stipulates HB TMP substance to be classified as Eye irritation Category 2 according to Directive 67/548/EEC or Regulation 1272/2008/EC.

 

Justification for classification or non-classification

Skin irritation/corrosion
The Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol did not cause skin irritation in an in vitro skin irritation study. The substance does not meet the criteria for classification for skin irritation according to Directive 67/548/EEC or Regulation 1272/2008/EC. 

 

Eye irritation

Taking the overall information together, the conclusive studies on HB TMP, TMP and Di-TMP as well as the lack of structural alerts for eye irritation for the read-across substance TMF, it is unlikely that HB TMP exhibits any potential to cause eye irritation. Considering also the two inconclusive in vitro eye irritation studies on HB TMP, applying the method presented in OECD Guideline on Defined Approaches for Serious Eye Damage / Eye Irritation and also the precautionary principle, the Weight of Evidence stipulates HB TMP substance to be classified as Eye irritation Category 2 according to Directive 67/548/EEC or Regulation 1272/2008/EC.