Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

Endpoint summary

Description of key information

Based on the results of the available in vitro and in vivo skin and eye irritation studies, Alkaterge E is not considered to be a skin or eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2017 - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Material Name: Alkaterge E
CAS Number: 68140-98-7
Batch nr.: D598F47BC1
Appearance (physical state, color): Liquid, brown, viscous
Molecular Weight: 365.60 g/mol

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Details on test system:

The EpiDerm™ Skin Model
Upon receipt of the EpiDerm™ Skin Bioassay Kit, the solutions were stored as indicated by the manufacturer. The EpiDerm™ tissues were stored at 2-8ºC until used. On the day of dosing, an appropriate volume of EpiDerm™ assay medium was removed and warmed to approximately 37ºC. Nine-tenths (0.9) mL of assay medium were aliquotted into the wells of each 6-well plate. The six-well plates were labeled to indicate test article and exposure time. The EpiDerm™ tissues were inspected for air bubbles between the agarose gel and cell culture insert prior to opening the sealed package. Tissues with air bubbles covering greater than 50% of the cell culture insert area were not used. The 24-well shipping containers were removed from the plastic bag and their surfaces were disinfected with 70% ethanol. The EpiDerm™ tissues were transferred aseptically into the 6-well plates. The EpiDerm™ tissues were then incubated in the dark at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) for at least one hour. The medium was then aspirated and 0.9 mL of fresh medium were added to each assay well below the EpiDerm™ tissues. The plates were returned to the incubator until treatment was initiated. Upon opening the bag, any remaining unused tissues were briefly gassed with an atmosphere of 5% CO2/95% air and placed back at 2-8ºC for later use.

Assessment of Direct Test Article Reduction of MTT
Each test article was added to a 1.0 mg/mL MTT (Sigma) solution in warm Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2 mM L-glutamine (MTT Addition Medium) to assess its ability to directly reduce MTT. Approximately 50 μL of the test articles, DMMOPA (aka RS-12803.00) and Alkaterge E, were each added to 1 mL of the MTT solution, and the mixture was incubated at standard culture conditions for at least one hour. Approximately 25 mg of the test article, AMPD, were added to 1 mL of the MTT solution, and the mixture was incubated at standard culture conditions for at least one hour. A negative control, 50 μL of sterile, deionized water (Quality Biological), was tested concurrently. If the MTT solution color turned blue/purple, the test articles were presumed to have reduced the MTT.

In cases where the test articles were shown to reduce MTT, only those test articles that remained bound to the tissue after rinsing, resulting in a false MTT reduction signal, could present a problem. To evaluate whether residual test article was binding to the tissue and leading to a false MTT reduction signal, a functional check (using freeze-killed control tissue) was performed as described in the section labeled “Killed Controls (KC)”. The test article was observed to directly reduce MTT in the absence of viable cells. The positive control, 8N potassium hydroxide (8N KOH), is known to directly reduce MTT in the absence of viable cells. Therefore, a killed control experiment was performed concurrently in the definitive assay to determine the extent of the direct MTT reduction (if any) by the test articles and the positive control in non-viable freeze-killed tissues.

Assessment of Colored or Staining Materials
Approximately 50 μL o Alkaterge E was added to 2.0 mL isopropanol in 6-well plates and placed on an orbital place shaker for 2-3 hours at room temperature. After shaking, 200 μL aliquots of the isopropanol solutions and two blank samples of isopropanol were transferred to a 96-well plate and the absorbance was measured with a plate reader at the MTT measurement wavelength (550 nm).The absorbance of the test article sample was determined by subtracting the mean isopropanol blank value from the absorbance of the test article samples. If the OD550 of the test article samples were > 0.08, the materials had to be considered as possibly interacting with the MTT measurement. The test article was not considered to have probable photometric MTT interference.

pH Determination
The pH of Alkaterge E was measured using pH paper (EMD Millipore Corporation). Initially, the test article was added to pH paper with a 0-14 pH range in 1.0 pH unit increments to approximate a narrow pH range. Next, the test article was added to pH paper with a narrower range of 0-6.0 pH units with 0.5 pH unit increments, to obtain a more accurate pH value.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test article was tested neat. Fifty (50) μL of Alkaterge E was applied directly on the tissue so as to cover the upper surface (epithelial side). The tissues designated to the negative control were treated with 50 μL of sterile, deionized water. The tissues designated to the positive control, 8N KOH (Sigma), were tested using the same method.

Duration of treatment / exposure:

The test and control articles were tested by treating four EpiDerm™ tissues per material. Two tissues were used to assess viability after the 3-minute exposure, and two were used to assess viability after the 60-minute exposure. Fifty (50) microliters of the test article was applied topically on the EpiDerm™ tissue. The negative and positive controls were treated as described above. The three-minute exposure time began as soon as the material was spread onto the tissue. This short exposure time precluded treating more than a small number of tissues at once. The cultures exposed for 3 minutes were held at room temperature during dosing, while the cultures exposed for the 60 minutes were incubated at standard culture conditions until the completion of the exposure time.

1.0 mg/mL solution of MTT in warm MTT Addition Medium was prepared no more than 2 hours before use. Three hundred (300) μL of MTT reagent solution were added to designated wells in a pre-labeled 24-well plate. The plate was held in the incubator until tissues were added. After the appropriate exposure time, the EpiDerm™ tissues were extensively rinsed with warm (approximately 37ºC) Calcium and Magnesium-Free Dulbecco's Phosphate Buffered Saline (Ca++Mg++-Free DPBS) and the wash medium was decanted. The EpiDerm™ tissues were transferred to the appropriate wells after rinsing. The plates were incubated at standard culture conditions for 3 ± 0.1 hours.

Duration of post-treatment incubation (if applicable):

After the incubation period with MTT solution, the EpiDerm™ tissues were blotted on absorbent paper, cleared of excess liquid, and transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were covered with paraffin film and stored in the refrigerator (2-8ºC) until the last exposure time was harvested. Then the plates were shaken for 2 - 3 hours at room temperature. At the end of the extraction period, the liquid within the cell culture inserts was decanted into the well from which the cell culture insert was taken. The extract solution was mixed and 200 μL were transferred to the appropriate wells of a 96-well plate. Two hundred (200) μL of isopropanol were placed in the two wells designated as the blanks. The absorbance at 550 nm (OD550) of each well was measured with a Molecular Devices Vmax plate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

97.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

97.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Alkaterge E was predicted to be non-corrosive to the skin in the in vitro Skin Corrosion Assay Using Epiderm™ Skin Model (Epi-200), and thus would not be classified or labeled as corrosive according to the UN GHS classification system.

Executive summary:

The in vitro Skin Corrosion Assay Using Epiderm™ Skin Model (Epi-200): 3- And 60-Minute Exposure Protocol was used to assess the potential skin corrosivity of Alkaterge E. The skin corrosion potential was evaluated using the protocol that is consistent with the OECD guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test. Test materials which reduce tissue viability to <50% within 3 minutes are considered corrosive by this method. In addition, test materials which result in tissue viability of ≥50% after a 3-minute exposure, but result in tissue viability of <15% after a 60-minute exposure are also classified corrosive. Test materials which result in tissue viabilities of ≥50% after a 3-minute exposure and ≥15% after a 60-minute exposure are classified non-corrosive. Furthermore, sub-classification of corrosive materials is possible using the 3 minute exposure time as follows: a sub-category classification of 1A is assigned if the viability is <25%, and 1B/1C if the viability is ≥ 25%. According to the current prediction model, Alkaterge E was predicted to be non-corrosive to the skin, and thus would not be classified or labeled as corrosive according to the UN GHS classification system.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2017 - March 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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Material Name: Alkaterge E
CAS Number: 68140-98-7
Batch nr.: D598F47BC1
Appearance (physical state, color): Liquid, brown, viscous
Molecular Weight: 365.60 g/mol

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Details on test system:

The EpiDerm™ Skin Model
Upon receipt of the EpiDerm™ Skin Kit (MatTek Corporation), the solutions were stored as indicated by the manufacturer. The EpiDerm™ tissues were stored at 2-8ºC until use. On the day prior to testing, EpiDerm™ Maintenance Medium was set to room temperature prior to use. Nine-tenths mL of Maintenance Medium were aliquotted into the appropriate wells of 6-well plates. Each 6-well plate was labeled with the test article, positive control, or negative control. Each EpiDerm™ tissue was inspected for air bubbles between the agarose gel and cell culture insert prior to opening the sealed package. Tissue inserts with air bubbles covering greater than 50% of the cell culture insert area were not used. The 24-well shipping containers were removed from the plastic bag and their surfaces were disinfected with 70% ethanol. The EpiDerm™ tissues were transferred aseptically into the 6-well plates. The EpiDerm™ tissues were then incubated at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) for 60±5 minutes. After 60±5 minutes, the EpiDerm™ tissues were transferred to appropriate wells containing 0.9 mL of fresh warmed (to 37ºC) Maintenance Medium. The plates were returned to the incubator for 18±3 hours to acclimate the tissues.

Assessment of Test Article/Nylon Mesh Compatibility
Prior to performing the assay, the compatibility of the test article with the nylon mesh was evaluated. Nylon meshes (Elko) were placed on a slide and 30 μL of the test article were applied. A negative control, 30 μL of sterile, Calcium and Magnesium Free Dulbecco’s Phosphate Buffered Saline (CMF-DPBS) (Gibco), was tested concurrently. The slides holding the treated meshes were placed into a covered petri dish and incubated at standard culture conditions for 60±1 minutes. Using a microscope, each mesh was checked after 60±1 minutes of exposure to assess any interaction between the test article and the mesh.
The test article Alkaterge E was not observed to interact with the nylon mesh, and therefore a nylon mesh was used to aid in the spreading of the test article after dosing the EpiDerm™ tissues.

Assessment of Direct Test Article Reduction of MTT
The test article was added to a 1.0 mg/mL MTT (Sigma) solution in warm Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2 mM L-glutamine (MTT Addition Medium) to assess its ability to directly reduce MTT. Approximately 30 μL of the test article was added to 1 mL of the MTT solution and the mixtures were incubated in the dark at standard culture conditions for at least one hour. A negative control, 30 μL of sterile, Calcium and Magnesium Free Dulbecco’s Phosphate Buffered Saline (CMF-DPBS), was tested concurrently. If the MTT solution color turned blue/purple, the test article was presumed to have reduced the MTT. Water insoluble test materials may show direct reduction (darkening) only at the interface between the test article and the medium.

In cases where the test article was shown to reduce MTT, the test article that remained bound to the tissue after rinsing, resulting in a false MTT reduction signal, could present a problem. To evaluate whether residual test article was binding to the tissue and leading to a false MTT reduction signal, a functional check (using freeze-killed control tissue) was performed.The test article, Alkaterge E, was not observed to directly reduce MTT in the absence of viable cells.

Assessment of Colored or Staining Materials
Prior to conducting any assays with viable tissues, the test article’s ability to interfere with the photometric MTT measurement was assessed. The test article was checked for its colorant properties (i.e., their ability to absorb light significantly at the wavelength used for the MTT determination. Approximately 30 μL was added to 2.0 mL isopropanol in 6-well plates and placed on an orbital place shaker for 2-3 hours at room temperature. After shaking, 200 μL aliquots of the isopropanol solutions and two blank samples of isopropanol were transferred to a 96-well plate and the absorbance was measured with a plate reader at the MTT measurement wavelength (570 nm). The absorbance of the test article samples was determined by subtracting the mean isopropanol blank value from the absorbance of the test article sample. If the OD570 of the test article sample was > 0.08, the material has to be considered as possibly interacting with the MTT measurement. Alkaterge Ewas not considered to have probable photometric MTT interference.

pH Determination
The pH of the liquid test article was measured using pH paper (EMD Millipore Corporation). Initially, test article was added to pH paper with a 0-14 pH range in 1.0 pH unit increments to approximate a narrow pH range. Next, the test article was added to pH paper with a narrower range of 0-6 pH units with 0.5 pH unit increments, to obtain a more accurate pH value.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Alkaterge E was tested in one valid definitive trial. After the overnight incubation for 18±3 hours, the 6-well plates containing the EpiDerm™ tissues were removed from the incubator and placed at room temperature for at least 5 minutes prior to dosing.

The EpiDerm™ tissues were treated in triplicate with each test article for 60±1 minutes. Thirty microliters of thetest article was applied to each of three tissues at 1 minute intervals per tissue. A nylon mesh was placed gently over the dose to spread the test article. If necessary, the mesh was gently pressed down to assure even spreading. The EpiDerm™ tissues were tested in triplicate with the positive or negative control for 60±1 minutes. Thirty microliters of each control were applied to each of three tissues at 1 minute intervals per tissue. Immediately after control administration onto the tissue, a nylon mesh was placed gently over the dose to spread the negative and positive controls. The plates with dosed tissues were kept in the laminar flow hood until the last tissue was dosed. After the last tissue was dosed, all of plates were transferred to the incubator for 35±1 minutes at standard culture conditions. After 35±1 minutes, all of the plates were removed from the incubator, placed into the laminar flow hood and kept at room temperature until the exposure period was completed for the first dosed tissue.

Duration of treatment / exposure:

After 60±1 minutes of test or control article exposure, the tissues were rinsed with sterile, CMF-DPBS by filling and emptying the tissue insert 15 times. A stream of CMF-DPBS was directed onto the tissue surface. For the test and control articles where a mesh was used, the mesh was carefully removed with forceps (if necessary) after the 5th rinse. After the removal of the mesh, the rinsing procedure of the tissue continued for 10 times. After the 15th rinse, each of the 3 inserts per treatment group (test article, positive and negative control) was completely submerged, gently swirled, and rinse media dumped in a beaker containing approximately 150 mL of CMF-DPBS and specifically assigned for each treatment group; this procedure was repeated three times for each insert of each treatment group. Finally, the tissues were rinsed once more on the inside and outside of the tissue insert with sterile CMF-DPBS from the wash bottle, and the excess CMF-DPBS was decanted. The bottoms of the tissue inserts were blotted on sterile paper towels and the inserts were transferred to new 6-well plates containing 0.9 mL of fresh warmed (to 37ºC) Maintenance Medium. The tissue surface was carefully blotted with sterile cotton-tipped applicators to remove any excess moisture, and the tissue surface was visually observed for residual test article using a dissecting scope. The tissues were then placed into the incubator at standard culture conditions for a post-treatment expression incubation of 42±2 hours. After an initial 24±1 hours of incubation, the 6-well plates were removed from the incubator and the tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh Maintenance Medium warmed to approximately 37ºC. The tissues were placed back into the incubator at standard culture conditions for an additional 18±1 hours for the remainder of the 42±2 hour post-treatment expression incubation.

Duration of post-treatment incubation (if applicable):

A 10X stock of MTT prepared in PBS (filtered at time of batch preparation) was thawed and diluted in warm MTT Addition Medium to produce a 1.0 mg/mL solution no more than two hours before use. Three hundred microliters of the MTT solution were added to each designated well of a pre-labeled 24-well plate.
After the total 42 ± 2 hours post-exposure expression incubation, the 6-well plates were removed from the incubator. Each tissue was blotted on a sterile paper towel and transferred to an appropriate well containing 0.3 mL of MTT solution. The 24-well MTT plates were incubated at standard culture conditions for 3±0.1 hours.
After the 3±0.1 hours incubation, the EpiDerm™ tissues were submerged, gently swirled, and rinse media decanted in a beaker containing approximately 150 mL of CMF-DPBS three times. The tissue was then blotted on absorbent paper, cleared of excess liquid, and transferred to a prelabelled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plate was covered with parafilm and shaken for 2 - 3 hours at room temperature to extract the MTT. At the end of the extraction period, the insert was gently agitated up and down in its extractant well. The tissues were pierced with forceps to allow the extract to flow back into the well from which the insert was removed, and the cell culture inserts were discarded. The extract solution was mixed (homogenized by pipetting up and down three times) and two x 200 μL aliquots were transferred to the appropriate wells of a 96-well plate. Two hundred μL of isopropanol were added to the wells designated as blanks. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader with the AUTOMIX function selected.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Only one experiment was performed

Value:

94.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Alkaterge E was predicted to be non-irritating to the skin in the Skin Irritation Test (SIT) using the EpiDerm™ Skin Model , and thus would not require classification and labelling for skin irritation according to the UN GHS classification system (No Category).

Executive summary:

The Skin Irritation Test (SIT) Using the EpiDerm™ Skin Model was used to assess the skin irritation potential of Alkaterge E. The skin irritation potential was evaluated using the protocol that is consistent with the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439). A test article was predicted to be an irritant (GHS Category 1 or 2) when the mean relative viability of the three treated tissues was less than or equal to 50% of the mean viability of the negative control. According to the current prediction model, the test articles, Alkaterge E was predicted to be non-irritating to the skin, and thus would not require classification and labelling for skin irritation according to the UN GHS classification system (No Category).

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981-1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 81-5 (Acute Dermal Irritation)

GLP compliance:

no

Specific details on test material used for the study:

Test Material Name: Alkaterge E
Chemical name: 2-heptadecenyl-4-ethyl-2-oxazolin-4-methanol
CAS Number: 68140-98-7
Appearance (physical state, color): Liquid, brown, viscous
Molecular Weight: 365.60 g/mol

Composition of ALKATERGE-E:
- Free Fatty Acids :0.69 % (wt)
- Free Amino Alcohols: 0.52% (wt)
- Amide Index: 18.31 % (wt)
- Oxazoline 80.48 % (wt)

Species:

rabbit

Strain:

other: albino

Type of coverage:

occlusive

Preparation of test site:

other: The skin on the left side of the middorsal line was left intact while the skin on the right side was abraded.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 ml

Duration of treatment / exposure:

After 24 h exposure, the bindings and patches were removed and the treated sites were gently cleaned.

Observation period:

14 days

Number of animals:

6

Details on study design:

The primary skin irritation test was conducted on a group of six albino rabbits weighing ca. 2.1 kg. The skin from the back area of each rabbit was clipped free of hair. The skin on the left side of the middorsal line was left intact while the skin on the right side was abraded by making minor epidermal incisions in a tic-tac-toe pattern with a blunt hypodermic needle. The abrasions were minor incisions through the stratum corneum, but not deep enough to disturb the derma or produce bleeding. A 0.5 ml of P-780, as supplied, was applied to each site and covered with a gauze pad. The entire trunk was then wrapped with a rubberized impervious cloth and a flexible wire screen held in place by tape. After 24 h exposure, the bindings and patches were removed and the treated sites were gently cleaned. The skin reactions were scored immediately (24 h) and at the end of 72 h (48 h after the first scoring).

Irritation parameter:

erythema score

Remarks:

intact skin

Basis:

mean

Remarks:

6 rabbits

Time point:

24 h

Score:

0.75

Max. score:

1

Remarks on result:

probability of mild irritation

Irritation parameter:

erythema score

Remarks:

intact skin

Basis:

mean

Remarks:

6 rabbits

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

erythema score

Remarks:

intact skin

Basis:

mean

Remarks:

6 rabbits

Time point:

72 h

Score:

0.83

Max. score:

1

Remarks on result:

probability of mild irritation

Irritation parameter:

erythema score

Remarks:

intact skin

Basis:

mean

Remarks:

6 rabbits

Time point:

14 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Remarks:

intact skin

Basis:

mean

Remarks:

6 rabbits

Time point:

24 h

Score:

0.75

Max. score:

1.5

Remarks on result:

probability of mild irritation

Irritation parameter:

edema score

Remarks:

intact skin

Basis:

mean

Remarks:

6 rabbits

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Remarks:

intact skin

Basis:

mean

Remarks:

6 rabbits

Time point:

72 h

Score:

1.67

Max. score:

3

Remarks on result:

probability of moderate irritation

Irritation parameter:

edema score

Remarks:

intact skin

Basis:

mean

Remarks:

6 rabbits

Time point:

14 d

Score:

0

Max. score:

0

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Remarks:

abraded skin

Basis:

mean

Remarks:

6 rabbits

Time point:

24 h

Score:

1

Max. score:

1

Remarks on result:

probability of mild irritation

Irritation parameter:

erythema score

Remarks:

abraded skin

Basis:

mean

Remarks:

6 rabbits

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

erythema score

Remarks:

abraded skin

Basis:

mean

Remarks:

6 rabbits

Time point:

72 h

Score:

0.92

Max. score:

1

Remarks on result:

probability of mild irritation

Irritation parameter:

erythema score

Remarks:

abraded skin

Basis:

mean

Remarks:

6 rabbits

Time point:

14 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Remarks:

abraded skin

Basis:

mean

Remarks:

6 rabbits

Time point:

24 h

Score:

1

Max. score:

1

Remarks on result:

probability of mild irritation

Irritation parameter:

edema score

Remarks:

abraded skin

Basis:

mean

Remarks:

6 rabbits

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Remarks:

abraded skin

Basis:

mean

Remarks:

6 rabbits

Time point:

72 h

Score:

1.75

Max. score:

3

Remarks on result:

probability of moderate irritation

Irritation parameter:

edema score

Remarks:

abraded skin

Basis:

mean

Remarks:

6 rabbits

Time point:

14 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Executive summary:

The primary skin irritation test was conducted on a group of six albino rabbits weighing ca. 2.1 kg. The skin from the back area of each rabbit was clipped free of hair. The skin on the left side of the middorsal line was left intact while the skin on the right side was abraded by making minor epidermal incisions in a tic-tac-toe pattern with a blunt hypodermic needle. The abrasions were minor incisions through the stratum corneum, but not deep enough to disturb the derma or produce bleeding. A 0.5 ml of P-780, as supplied, was applied to each site and covered with a gauze pad. The entire trunk was then wrapped with a rubberized impervious cloth and a flexible wire screen held in place by tape. After 24 h exposure, the bindings and patches were removed and the treated sites were gently cleaned. The skin reactions were scored immediately (24 h) and at the end of 72 h (48 h after the first scoring). The test material produced slight erythema and mild to severe edema on all the intact and abraded skin sites. The average skin irritation score for the six rabbits was 2.22. Since the material produced skin irritation, the animals were held for further observation. All the treated skin sites were normal by day 14. The average body weight gains of the treated animals were normal.

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:

experimental study

Adequacy of study:

key study

Study period:

6 June 2017-15 January 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)

GLP compliance:

yes

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals (J.W. TREUTH & SONS, Inc., Baltimore, MD). The eyes were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice packs. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Details on study design:

Preparation of Corneas
The eyes were grossly examined for damage and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised such that a 2 to 3 mm rim of sclera was present around the cornea. The isolated corneas were then stored in a petri dish containing HBSS until they were mounted in a corneal holder. The corneas were mounted in the holders with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and the screws were tightened. Starting with the posterior chamber, the two chambers were then filled with Minimum Essential Medium (EMEM) without phenol red, containing 1% fetal bovine serum and 2 mM L-glutamine (Complete MEM (without phenol red)). Each corneal holder was uniquely identified with a number written in permanent marker, on both the anterior and posterior chambers. The corneal holders were incubated at 32 ± 1ºC for a minimum of 1 hour.

Test Substance Preparation
Alkaterge E, was administered to the test system without dilution.

Test Substance pH Determination
The pH of the test substance was determined using pH paper (EMD Millipore Corporation; Billerica, MA). Initially, each test substance was added to 0-14 pH paper with 1.0 pH unit increments to approximate a narrow pH range. Next, the test substances were added to 0-6 and 5-10, or 7.5-14.0 pH paper with 0.5 pH unit increments, to obtain more accurate pH values.

Bovine Corneal Opacity and Permeability Assay
The liquid test substances Alkaterge E was tested on 17 July 2017. After a minimum of 1 hour of incubation, the corneas were removed from the incubator. The medium was removed from both chambers and replaced with fresh Complete MEM (without phenol red). The initial opacity was determined for each cornea using an Electro Designs OPKIT opacitometer. Any cornea with an initial opacity greater than 7 was not used in the assay. The treatment of each cornea was identified with the test substance number written in permanent marker on colored tape, affixed to each holder. The medium was then removed from the anterior chamber and replaced with the test substance, positive control, or negative control.

Alkaterge E was tested neat. An aliquot of 750 μL of the test substance, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea. Each treated cornea was completely covered with the test substance.
Three corneas were incubated in the presence of the negative or positive control at 32 ± 1ºC for 10 minutes. Four corneas were incubated in the presence of each test substance at 32 ± 1ºC for 10 minutes. After the 10-minute exposure times, the control or test substance treatments were removed. The epithelial side of the corneas was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test substances. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior chambers were refilled with fresh Complete MEM (without phenol red) and an opacity measurement was performed. The corneas were returned to the incubator for approximately 2 hours after which a final measure of opacity was obtained. After the final opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was filled with fresh Complete MEM (without phenol red) and 1 mL of a 4 mg/mL fluorescein solution was added to the anterior chamber. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1ºC. At the end of the 90-minute incubation period, the medium was removed from the posterior chamber and placed into tubes numbered corresponding to chamber number. Aliquots of 360 μL from the numbered tubes were placed into their designated wells on a 96-well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplate reader. If the OD490 value of a control or test substance sample was 1.500 or above, a 1:5 dilution of the sample was prepared in Complete MEM (without phenol red) (to bring the OD490 value within the linear range of the platereader). A 360 μL sample of each 1:5 dilution was transferred to its specified well on the 96-well plate. The plate was read again and the final reading was saved to a designated print file.

Fixation of Corneas
After the medium was removed for the permeability determination, each cornea was carefully separated from its corneal holder and transferred to an individual prelabeled tissue cassette containing a biopsy sponge. The endothelial surface of each cornea was placed on the sponge to protect it. The cassettes were placed in 10% neutral buffered formalin to fix the corneal tissue for at least 24 hours.

Irritation parameter:

in vitro irritation score

Run / experiment:

Alkaterge E

Value:

2.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Ethanol (positive control)

Value:

46.3

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is not irritating to isolated bovne eyes.

Executive summary:

The Bovine Corneal Opacity and Permeability Assay with Optional Histology was used assess the potential ocular corrosivity or severe irritancy of Alkaterge E to isolated bovine corneas. The ocular irritancy potential of the test substances were evaluated using the protocol that is consistent with the OECD Test Guideline 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage”. A test substance was predicted as a GHS Category I if the In Vitro Score was > 55. A test substance was predicted to not require classification or labelling for eye irritation or serious eye damage (GHS No Category), if the In Vitro score was ≤ 3.0. According to the current prediction model, Alkaterge E, was predicted to not require classification or labelling for eye irritation or serious eye damage (GHS No Category).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1981 - March 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 81-4 (Acute Eye Irritation)

GLP compliance:

no

Species:

rabbit

Strain:

other: allbino, no further specification

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.1 m l of the test material The eyelids were held closed for one to two seconds to prevent any loss of the material. The right eye served as an untreated control.

Duration of treatment / exposure:

The eyes of the first six rabbits were not treated further after the instillation of the test material (unwashed), but the eyes of the remaining three rabbits were irrigated with 50 ml of lukewarm tap water after 20 to 30 seconds exposure to the test material (washed).

Observation period (in vivo):

The eyes were examined at 24, 48, and 72 hours and at day 7 post treatment. At 24 hours and on the 7th day, a drop of sodium fluorescein (0.24%) was placed on the cornea of each treated eye. The excess fluorescein was flushed with sterile saline (0.85%) and the treated eye was examined under a UV light for corneal lesions.

Irritation parameter:

cornea opacity score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

24 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

24 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

24 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

24 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

24 h

Score:

0.33

Max. score:

1

Remarks on result:

probability of weak irritation

Irritation parameter:

conjunctivae score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

24 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

24 h

Score:

0.167

Max. score:

1

Remarks on result:

probability of weak irritation

Irritation parameter:

chemosis score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

24 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

48 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

48 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

48 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

48 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

48 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

48 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

48 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

48 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

7 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

7 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

7 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

7 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

7 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

7 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

unwashed eyes

Basis:

mean

Remarks:

mean of 6 rabbits

Time point:

7 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Remarks:

washed eyes

Basis:

mean

Remarks:

mean of 3 rabbits

Time point:

7 d

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is not irritating to the eyes of rabbits.

Executive summary:

The eye irritation test was carried out in nine albino rabbits weighing ca. 2.3 kg, free of eye defects and irritation. A 0.1 m l of the test material was instilled into the lower conjunctival sac of the left eye. The eyelids were held closed for one to two seconds to prevent any loss of the material. The right eye served as an untreated control. The eyes of the first six rabbits were not treated further after the instillation of the test material (unwashed), but the eyes of the remaining three rabbits were irrigated with 50 ml of lukewarm tap water after 20 to 30 seconds exposure to the test material (washed). The eyes were examined at 24, 48, and 72 hours and at day 7 post treatment. At 24 hours and on the 7th day, a drop of sodium fluorescein (0.24%) was placed on the cornea of each treated eye. The excess fluorescein was flushed with sterile saline (0.85%) and the treated eye was examined under a UV light for corneal lesions. At 24 h two rabbits in the unwashed group showed slight irrtation in the conjunctivae, but the eyes were normal by 48 h. When checked by fluorescein, none of' the treated eyes showed any corneal lesion.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of all available studies with Alkaterge E, the data on skin and eye irritation do not meet the criteria for classification according to the CLP/GHS Regulation (EC) 1272/2008, and therefore the substance does not need to be classified for skin or eye irritation.