Benzenemethanaminium, N,N,N-trimethyl-, 2-hydroxy-2-methylpropanoate (1:1)

Endpoint summary

Description of key information

The substance has no irritating potential in the in vitro Epiderm skin corrosion/ irritation test (OECD 431, GLP). No highly irritating potential was identified in the BCOP test (OECD 437, GLP). Based on the reduction of viability of cultivated human cornea, the substance is considered as irritating to eyes (Epiocular assay, GLP).

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:

03 April 2012 to 02 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and gudeline study

Qualifier:

according to guideline

Guideline:

other: OECD TG No. 431, April 13, 2004 “In vitro Skin Corrosion: Human Skin Model Test”

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD TG No. 439, July 22, 2010 “In vitro Skin Irritation: Reconstructed Human Epidermis Test Method"

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008, Part B: Methods for the determination of toxicity and other health effects: In Vitro Skin Corrosion: Human Skin Model Test; Official Journal of the European Union, No. L 142

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 761/2009 of 23 July 2009: Part B: Methods for the determination of toxicity and other health effects: In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test; Official Journal of the European Union, No. L 220

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed human epidermal model EpiDermTM

Strain:

not specified

Details on test animals or test system and environmental conditions:

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Type of coverage:

other: human epidermis skin model

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

66 mg per skin model

Duration of treatment / exposure:

Corrosion test: 3 min and 1 h
Irritation test: 1h and post-incubation period of 42h

Observation period:

Not applicable.

Number of animals:

Number of skin models:
Corrosion test: 2
Irritation test: 3

Details on study design:

Corrosion test:
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application. Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. 25 μL de-ionized water was applied first. As the test substance could not be applied with a sharp spoon, a metal pin was covered with ca. 66 mg of the solid ground test material and was applied with direct contact to the wetted tissue. Control tissues were concurrently applied with 50 μL of de-ionized water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC). The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours. After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation test:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. 25 μL sterile PBS was applied first. As the test substance could not be applied with a sharp spoon, a metal pin was covered with ca. 66 mg of the solid ground test material and was applied with direct contact to the wetted tissue. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation / corrosion parameter:

other: other: viability

Value:

102

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 min. Max. score: 0.0. Remarks: Score in % viability (corrosion test) - 100% viability indicates absence of irritation/corrosion.. (migrated information)

Irritation / corrosion parameter:

other: other: viability

Value:

93

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 h. Max. score: 0.0. Remarks: Score in % viability (corrsion test). (migrated information)

Irritation / corrosion parameter:

other: other: viability

Value:

113

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 43h. Max. score: 0.0. Remarks: Score in % viability (irritation test). (migrated information)

Irritant / corrosive response data:

Based on the observed results and applying the evaluation criteria it was concluded, that the substance does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

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:

03 April 2012 to 02 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

other: OECD TG No. 437, September 07, 2009 “Bovine Corneal Opacity and Permeability Test, Method for Identifying Ocular Corrosives and Severe Irritants"

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No. 1152/2010 of 8 December 2010 Part B: Bovine Corneal Opacity and Permeability Test, Method for identifying ocular corrosives and severe irritants; Official Journal of the European Union, No. L324

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: isolated bovine cornea

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The test system (target tissue) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months). Supplier: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim, Germany

Vehicle:

other: de-ionized water

Amount / concentration applied:

20% (w/v) solution in de-ionized water

Duration of treatment / exposure:

4h

Observation period (in vivo):

Not applicable.

Number of animals or in vitro replicates:

3 corneas per treatment group

Details on study design:

- Preparation of the bovine corneas and measurement of initial corneal opacity: Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consists of anterior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.
After the equilibration period the medium in both chambers was replaced with fresh prewarmed
medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 507 opacity units1 were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number on the chambers.

- Application of the test substance and washing: Each treatment group (test substance, NC and PC) consisted of 3 corneas. Before application the medium in the anterior chamber was removed using a syringe. 750 μL of the 20% (w/v) test-substance preparation (non-surfactant) was applied into the
anterior chamber using a pipette. Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (negative control, NC) or with 750 μL of 20% (w/v) solution of Imidazole in de-ionized water (positive control, PC) using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids). The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).

- Measurement of final corneal opacity: Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.

- Determination of permeability: For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (5 mg/mL for solid test substances) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

Irritation parameter:

other: Opacity score

Basis:

mean

Time point:

other: 4h

Score:

28

Reversibility:

other: not applicable

Irritation parameter:

other: Permeability score

Basis:

mean

Time point:

other: 4h

Score:

0.006

Reversibility:

other: not applicable

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

mean

Time point:

other: 4h

Score:

28

Reversibility:

other: not applicable

Remarks on result:

other: A score of 55 or higher indicates highly irritatig properties.

Irritant / corrosive response data:

Based on the observed results and applying the evaluation criteria it was concluded, that the substance does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

Interpretation of results:

other: not highly irritating

Remarks:

Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation and corrosion:

 

The potential to cause dermal corrosion/irritation was assessed by a single topical application of ca. 66 mg of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™, OECD 439). The study was performed under GLP.

For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiDerm™ skin corrosion/irritation test showed the following results:

The test substance is not able to reduce MTT directly.

Corrosion test:

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 102%, and it was 93% after an exposure period of 1 hour.

Irritation test:

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 113%.

Based on the observed results it was concluded, that BTMA-HIB does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

 

Eye irritation and corrosion:

Two studies were performed, and both of them are required for hazard assessment. The BCOP assay (OECD 431, GLP) allows to distinguish highly irritating substances from merely irritating or non-irritating substances. The EPIOCULAR assay is a non-guideline study which has been validated internally and is used to distinguish non-irritating from irritating substances.

EpiOcular:

The potential to cause ocular irritation was assessed by a single topical application of ca. 66 mg of the test substance to a reconstructed three dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissue samples were incubated with the test substance for 90 minutes followed by a 18-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiOcular™ eye irritation test showed the following results:

The test substance is not able to reduce MTT directly.

The mean viability of the test-substance treated tissues was 8%.

Based on the observed results it was concluded, that the substance shows an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen. The test method does not yet allow for the evaluation of serious eye damage. The result does not exclude a serious eye irritation potential of the test substance. For final assignment of a risk phrase at present, results from the BCOP assay are taken.

 

BCOP:

The potential to cause serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% test-substance preparation to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation for an exposure period of 4 hours.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas.

The BCOP test showed the following results:

Mean opacity value: 28.0

Mean permeability value: 0.006

In vitro Irritancy score: 28.1

Based on the observed results it was concluded, that BTMA-HIB does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

Justification for selection of skin irritation / corrosion endpoint:
GLP and guideline study

Justification for selection of eye irritation endpoint:
Both studies need to be chosen, but this is not possible in the current IUCLID 5.4 set up. The indicated study is a GLP and guideline study.

Effects on eye irritation: irritating

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available in-vitro studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin irritation and classified as an eye irritant (R36) under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.

 

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 with the exception that subclassification Cat 2A / 2B for eye irritation is not possible. The BCOP assay is accepted to exclude classification as highly irritating (R41, H218) to eyes. As a result the substance is not considered to be classified for skin rritation, but classified for eye irritation (Category 2, H319) under Regulation (EC) No. 1272/2008, as amended for the second time in Directive EC 286/2011.