Reaction mass of C12-14 tert-alkylamines and dimethyl hydrogen phosphate and methyl dihydrogen phosphate

Endpoint summary

Description of key information

Eye irritation

Key study

In a study performed to the standardized guidelines OECD 437 and EU Test Method B.47., under GLP conditions, the test item is classified as Category 1 eye irritant according to GHS (Envigo, 2018).

Skin irritation

Key study

In a study performed to the standardized guidelines OECD 439, under GLP conditions, the study does not allow the conclusion on whether the test item is Category 1 or Category 2 of GHS (Envigo, 2018).

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:

31 October 2017 to 16 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Purity: >98% (nominal); This substance has a Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Light yellow liquid

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

Application of Test Item and Rinsing (Day 1):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm^2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3):
Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker at room temperature for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.

2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the LabTech LT-4500 microplate reader.

Control samples:

other: Negative Control: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ and Positive Control: Sodium Dodecyl Sulphate (SDS)

Amount/concentration applied:

The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm^2) of the test item was applied to the epidermis surface.

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

Number of replicates:

Three.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 Hours post exposure incubation period.

Value:

5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

The relative mean viability for tissues treated with 5% (w/v) SDS aqueous solution (positive control) was 6.0% relative to the negative control treated tissues and the standard deviation value of the viability was 1.2% (≤18%). The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control (DPBS) treated tissues was 0.839, within the range of 0.6 - 1.5 and the standard deviation value of the viability was 5.9% (≤18%). The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 0.6% (≤18%). The test item acceptance criterion was therefore satisfied.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

based on suuportive evidence in 14 day and 28 day repeat dose oral testing.

Conclusions:

The test item has been determined oto be a skin irritant category 2 based on supporting evidence from other studies.

Executive summary:

A study to evaluate the skin irritation potential of the test item using the EPISKIN^TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The study has been performed to the standardized guideline OECD 439, under GLP conditions.

 

The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator IL-1α determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.

 

Data was presented in the form of percentage viability (MTT reduction in the test item or positive control treated tissues relative to negative control tissues).

 

The relative mean viability of the test item treated tissues was 5.0% after the 15-minute exposure period and 42-hours post-exposure incubation period.

 

The quality criteria required for acceptance of results in the test were satisfied.

 

Under the conditions of the study, the relative mean viability of tissues treated with the test item was 5%, less than 50%, and therefore, the test item demonstrated the ability to cause a positive response as skin irritant. In addition, this study does not allow the conclusion on whether the test item is Category 1 or Category 2 of Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP) and United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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:

02 February 2018 to 19 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Purity: >98% (nominal); this substance has a Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Light yellow liquid

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.

Vehicle:

unchanged (no vehicle)

Controls:

other: other: Negative control: Sodium chloride 0.9% w/v; Positive control: Ethanol

Amount / concentration applied:

0.75 mL of the test item or control items were applied to the appropriate corneas

Duration of treatment / exposure:

Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three

Details on study design:

Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the corneaThe isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Irritation parameter:

cornea opacity score

Value:

10.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Corneal Epithelium Condition
The corneas treated with the test item were cloudy post treatment and cloudy post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the historical range of 29.6 to 65.1. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤2.3 and permeability ≤0.041. The negative control acceptance criteria were therefore satisfied.

In Vitro Irritancy Score

Treatment	In Vitro Irritancy Score
Test Item	86.5
0.9% (w/v) Sodium Chloride (Negative Control)	0.7
Neat Ethanol (Positive Control)	37.1

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the results of the BCOP test, the test item is classified as Category 1 eye irritant according to GHS.

Executive summary:

In a study performed to the standardized guidelines OECD 437 and EU Test Method B.47., under GLP conditions, the undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative (0.9% (w/v) Sodium Chloride) and positive (Neat Ethanol) control items were tested concurrently. 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). Based on the results of the BCOP test, the test item is classified as Category 1 eye irritant according to GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available