2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol hydrochloride

Endpoint summary

Description of key information

In an experimental study according to OECD test guideline 439 under GLP conditions, the read-across substance did not reduce cell viability (96 % with an SD of 14.52) and is considered not irritating to skin (reference 7.3.1 -1).

In an in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model (OECD 438) with the read-across substance, no ocular corrosion potential was observed. The overall ICE score was 1xI, 2xII (reference 7.3.2 -1).

In an experimental study according to OECD test guideline 492 under GLP conditions, the read-across substance reduced the cell viability in two experiments (55.8 % and 29.0 %) (reference 7.3.2 -2).

Based on the results of both studies the read-across substance is considered to have potential to cause eye irritation but not serious damage to the eye.

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

Justification for type of information:

Please refer to section 13 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

18

Remarks on result:

no indication of irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to section 13 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

percent corneal swelling

Run / experiment:

mean (240 min)

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

28

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

4

Irritation parameter:

fluorescein leakage

Run / experiment:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

3

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to section 13 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

other: % viability

Run / experiment:

Experiment 1 (mean)

Value:

55.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

39.5

Irritation parameter:

other: % viability

Run / experiment:

Experiment 2 (mean)

Value:

29

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

32.4

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

No study data with the test item is available for skin irritation. Therefore, a read-across to the read-across substance with a very similar chemical structure and comparable physico-chemical parameters is used to evaluate the skin irritation potential of the test item.

 

An EpiSkinTM SM test with the read-across substance has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439.

Triplicates of the human skin model EPISKIN (Reconstructed Human Epidermis, SkinEthic Laboratories) were treated with read-across substance and incubated for 15 minutes at room temperature. Exposure of the reconstructed human epidermis skin units with the read-across substance was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the read-across substance did not show significantly reduced cell viability in comparison to the negative control (mean value: 96 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the read-across substance reveals no skin irritation potential under the utilised testing conditions. The read-across substance is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

 

Eye irritation

No study data with the test item is available for eye irritation. Therefore, a read-across to the read-across substance with a very similar chemical structure and comparable physico-chemical parameters is used to evaluate the eye irritation potential of the test item.

 

To determine the corrosion or irritation potential of the read-across substance for the eye a stepwise weight of evidence approach was done. An Isolated Chicken Eye Test (ICET) according to OECD 438 (version 2013) was conducted resulting in “No prediction can be made” and therefore, neither classification in Category 1 (UN GHS) nor no classification (UN GHS: No Category) was triggered. Therefore an eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 (version 2015) was conducted in accordance with the top down approach described in the Guidance on an integrated approach on testing and assessment (IATA) for serious eye damage and eye irritation (Series on testing and assessment No. 263).

 

An Isolated Chicken Eye Test (ICET) according to OECD 438 (version 2013) was conducted to evaluate the potential ocular corrosivity and irritancy of the read-across substance by its ability to induce toxicity in enucleated chicken eyes. The read-across substance was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes and rinsed after 10 seconds with saline. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points. Imidazole (positive control) was ground before use in the study. The read-across substance and positive control were applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the read-across substance or positive control. Three read-across substance treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, the read-across substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 2xII.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

According to the guideline OECD 438 (version 2013), the read-across substance overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438 (version 2013), the read-across substance has been categorized as “No prediction can be made”.

 

A study according to OECD 492 (version 2015) was conducted to evaluate the eye irritation potential of the read-across substance. Two experiments were performed. The first experiment was not sufficient to determine the properties of the read-across substance due to non-concordant replicates. The result of the second experiment was unequivocal and led to the same classification as the mean value of the first experiment.

The solid test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of at least 6 hours. After treatment, the 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 0.8≤mean OD≤2.5, OD was 1.5 (1st experiment) and 1.8 (2nd experiment). The positive control showed clear eye irritating effects (< 50 %), the relative tissue viability was reduced to 39.5 % (1st experiment) and 32.4 % (2nd experiment).

Variation within tissue replicates was acceptable in both experiments (range≤20 %).

After treatment with the read-across substance, the mean value of relative tissue viability was 55.8 % (1st experiment) and 29.0 % (2nd experiment). Both values are below the threshold for eye irritation potential (≤60 %).

Under the conditions of the test system, the read-across substance is considered as eye irritant / inducing serious eye damage in the EpiOcularTM Eye Irritation Test according to OECD 492 (version 2015).

 

Conclusion

Based on the result from the Isolated Chicken Eye Test according to OECD 438 (version 2013) a classification as No category according to UN GHS was not possible due to the result of “No predication can be made”. The second in vitro study, the eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 (version 2015), determined that the read-across substance induces eye damage, but that no prediction regarding classification as Category 1 or Category 2 of the read-across substance can be made.

In the meantime new guideline versions for both OECD test guidelines were published and the impact of the changes of the guidelines were checked for the registration of the target substance (Summer 2020). The current guideline versions are OECD TG 438 of 27th June 2018 and OECD TG 492 of 18th June 2019. In both guidelines the procedures for the conduction of the described experiments remained the same, as did the criteria for evaluation of the results (e.g. ICE class for OECD 438). However, in both guidelines the prediction models according to the UN GHS classification changed due to new scientific knowledge. In OECD 438 version 2018 the overall ICE class of 1xI, 2xII is considered as “No category” (previously “No predication can be made”). For OECD 492 the predicted classification besides “No category” changed to “No predication can be made” (from previously “potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1)”.

Considering the experimental results of the studies under the new guideline versions the following is determined:

According to the guideline OECD 438 (version 2018), the read-across substance has been categorized as “No category”.

Under the conditions of the test system, the read-across substance is considered as “No predication can be made” in the EpiOcularTM Eye Irritation Test according to OECD 492 (version 2019).

 

Using the top down approach described in the Guidance on IATA for serious eye damage and eye irritation an overall conclusion for the read-across substance can be reached based on the updated guidelines. The weight of evidence with the two conducted studies determined that the read-across substance is not to be classified as UN GHS Category 1. With the results of “No Category” and “No predication can be made”, it was decided to assume the worst-case classification in order to avoid an in vivo study. Therefore, the read-across substance is considered to be eye irritating and classified into Category 2 (UN GHS).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin irritation, the test item does not require classification for skin irritation or corrosion according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.

 

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on the potential to cause damage to the eye, the test item requires classification for causing eye irritation into Category 2 (H319: Causes serious eye irritation) according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.