(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-amine

Endpoint summary

Description of key information

OECD 435: The potential of 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- to cause dermal corrosion cannot be assessed in the Corrositex® assay and it cannot be excluded that the test substance has a corrosive potential (BASF, 2020).

OECD 431: 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- shows an intermediate or weak corrosion potential (BASF, 2020).

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:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name of test substance: 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)-
Test substance No.: 19/0201-1
Batch identification: B6943 v. 10.04.2019
CAS No.: 752984-24-0
Content: 99.9 area-% (96.6 area-% trans isomer, 3.3 area-% cis diastereomer)
Identity: Confirmed (for details see Final Report, Study code: 19L00134)
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: Expiry date: 09 Apr 2021
The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test system:

artificial membrane barrier model

Justification for test system used:

The Corrositex® assay is a standardized in vitro corrosion test. The Corrositex® assay kit is available commercially from InVitro International. The Corrositex® bio barrier membrane is a test system consisting of a reconstituted collagen matrix. The assay is based on the time that the test substance requires to penetrate through the Corrositex® bio barrier membrane and produce a change in the Chemical Detection System (CDS). The Corrositex® assay is used to determine the corrosive potential of test substances. The assay is limited to testing materials that cause detectable pH changes in the CDS.

Vehicle:

unchanged (no vehicle)

Details on test system:

Following the acceptance of the positive control, the Corrositex® assay was performed for the test substance. Four vials containing the CDS were used for the test substance. In addition, one vial was used for the PC, the NC and the color (blank) control each. A membrane disc coated with the bio barrier matrix was placed into one vial containing the CDS. 500 μL undiluted test substance were added onto the membrane disc. An electronic time clock was started with the application. The vial was observed for three minutes for any change in the CDS. If no color change was observed within three minutes, the remaining membranes were treated with the test substance. An electronic time clock was started with each application. The vials were observed continuously for the first ten minutes. Thereafter, the vials were observed for approximately ten minutes around the time points relevant for evaluation, or until breakthrough of the test substance occurred. The elapsed time between test substance application and the first change in the indicator solution (i.e. barrier penetration) was recorded.

The Corrositex® assay was accepted if the breakthrough time for the positive control substance was in the historic control range (mean ± 2-3x standard deviations, see chapter 4.3.). The expected breakthrough time of the concurrent positive control (Sodium Hydroxide solid) should be between 8 - 16 min. In order to demonstrate the functional integrity of the membrane barrier, the acceptance criterium for the negative control was not to induce membrance breakthrough within a 60-minute observation period.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

500 µL

Duration of treatment / exposure:

up to 60 min

Number of replicates:

5

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

first experiment vial #1

Value:

3.37

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

first experiment vial #2

Value:

> 60

Remarks on result:

other: no breakthrough

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

first experiment vial #3

Value:

> 60

Remarks on result:

other: no breakthrough

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

first experiment vial #4

Value:

3.06

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

first experiment vial #5

Value:

> 60

Remarks on result:

other: no breakthrough

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

first experiment: Positive Control (PC)

Value:

14.49

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

first experiment: Negative Control (NC)

Value:

> 60

Remarks on result:

other: no breakthrough

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

second experiment vial #1

Value:

20.09

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

second experiment vial #2

Value:

1.56

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

second experiment vial #3

Value:

6.12

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

second experiment vial #4

Value:

9.38

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

second experiment: Positive Control (PC)

Value:

10.39

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

second experiment: Negative Control (NC)

Remarks on result:

other: no breakthrough

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the inconclusive result observed and by applying the evaluation criteria the potential of 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- to cause dermal corrosion cannot be assessed in the Corrositex® assay and it cannot be excluded that
the test substance has a corrosive potential. For final assignment of a risk phase at present, results from another study would be needed.

Executive summary:

The Corrositex® assay was conducted in order to assess the potential of 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- to cause dermal corrosion by a single topical application of 500 μL test substance to the Corrositex® bio barrier membrane. The Corrositex® bio barrier membrane is a test system consisting of a reconstituted collagen matrix. The assay is based on the time that the test substance requires to penetrate through the Corrositex® bio barrier membrane and produce a change in the Chemical Detection System (CDS).

In addition to the test substance, a positive and a negative control were run in the assay. The Corrositex® assay showed the following results: The qualification screen demonstrated that the test substance is able to react with the CDS and produce a visible color change. Therefore, the membrane barrier test method was determined to be suitable for the evaluation of the corrosive potential of the test substance. A timescale category test was carried out to distinguish between weak and strong acids or bases. The test substance was assigned to timescale category 2 (having a low acid/alkaline reserve).

In the main test, five Corrositex® bio barrier membranes were treated with the undiluted test substance. Two CDS showed a minimal reddish discoloration on the surface shortly after test substance application, while the other two CDS did not show any discoloration within the 60-minute observation period. A fifth CDS first used for color comparison and then applied additionally to the four CDS with test substance did not show any discoloration within the 60-minute observation period either. Due to the nontypical observations without concrete breakthrough, no conclusive result could be determined. The negative control, 10% citric acid monohydrate, did not produce any reaction within 60 minutes after application. Sodium hydroxide (solid) applied as positive control showed a reaction time of 14 minutes and 49 seconds after application and was assigned accordingly Thus, the controls fulfill the acceptance criteria and demonstrate the validity of the assays.

A second test run was performed for clarification. In the second test run, four Corrositex® bio barrier membranes were treated with the undiluted test substance. All of the CDS showed a minimal reddish discoloration on the surface shortly after test substance application. Due to the recurrent nontypical observations without concrete breakthrough, no conclusive result could be determined in the second test run as well. The negative control, 10% citric acid monohydrate, did not produce any reaction within 60 minutes after application. Sodium hydroxide (solid) applied as positive control showed a reaction time of 10 minutes and 39 seconds after application and was assigned accordingly. Thus, the controls fulfill the acceptance criteria and demonstrate the validity of the assays. Based on the inconclusive results observed and by applying the evaluation criteria, the potential of 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- to cause dermal corrosion can not be assessed in the Corrositex® assay and it can not be excluded that the test substance has a corrosive potential.

For final assignment of a risk phase at present, results from another study would be needed.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name of test substance: 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)-
Test substance No.: 19/0201-1
Batch identification: B6943 v. 10.04.2019
CAS No.: 752984-24-0
Content: 99.9 area-% (96.6 area-% trans isomer, 3.3 area-% cis diastereomer)
Identity: Confirmed (for details see Final Report, Study code: 19L00134)
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: Expiry date: 09 Apr 2021
The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The objective of this in vitro test was to assess the skin corrosion potential of the test substance using the reconstructed human epidermal model EpiDermTM.
The present test is based on the experience that corrosive chemicals produce cytotoxicity in human reconstructed epidermis after a short-term topical exposure. The test is designed to predict skin corrosion potential of a chemical by using the three-dimensional human epidermis model EpiDermTM. After application of the test material to the stratum corneum surface of the EpiDermTM tissue for 3 minutes and 1 hour, the induced cytotoxicity (=loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored watersoluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol extraction of the formazan from the tissue, the optical density of the extract is spectrophotometrically determined. Optical density of the extracts of the tissues treated with the test substance is compared to negative control values from tissues treated with deionized water and expressed as relative tissue viability.

Details on test system:

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

EXPERIMENTAL PROCEDURE
Pretest for mesh compatibility
For liquid test substances, a nylon mesh can be used as spreading aid. In order to exclude a reaction of the test substance with the mesh, the compatibility of the test substance with the nylon mesh was checked in a pretest (experimental conduct in accordance with GLP, but without a GLP status). The test substance and the mesh are brought together on a slide, and the reaction was observed after a 60-minute exposure. An interaction between test substance and mesh was not noticed. However, it was judged that the use of a mesh was not necessary for the test substance.

Pretest for direct MTT reduction
The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. In order to assess the ability of the test material to reduce MTT directly, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed as described below. The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently. If the color of the MTT solution or (in case of water-insoluble test substances) the border to the water-phase turned blue / purple, the test substance was presumed to reduce MTT directly. In case of direct MTT reduction, two freeze-killed control tissues (KC) per exposure time were treated additionally with each the test article and the negative control in the same way. Based on the result of the pretest, it was judged that application of killed control tissues is
necessary.

Basic procedure
Several test substances were tested in parallel within the present test (test no. 119) by using the same control tissues (NC and PC). 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 by 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. In addition, two killed control tissues per exposure time were treated with the test substance and the NC, respectively, to detect direct MTT reduction. Fifty microliters (50 μL) of the undiluted liquid test substance were applied using a pipette. Control tissues were concurrently treated with 50 μL of deionized water (NC, NC KC) or with 50 μL 8 N potassium hydroxide solution (PC) or test substance (KC)1. A nylon mesh was placed carefully onto the tissue surface of the NC and NC KC afterwards. 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, the tissues were washed with PBS to stop the MTT incubation. The formazan that was metabolically produced by the tissues was extracted by overnight incubation of the tissues in isopropanol in the refrigerator. The optical density at a wavelength of 570 nm (OD570) of the extracts was spectrophotometrically determined. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Data evaluation
Table(s) and/or figure(s) of measured parameters were produced using a PC-based tabular calculation software. The mean and individual data were not always rounded but the significant digits were produced by changing the display format. Consequently, calculation of mean values by using the individual data presented in the report will in some instances yield minor variations in value. Principle The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether a test material is corrosive.

Calculation of individual and mean optical densities
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way is calculated. Application of measurements using killed control tissues In case of direct MTT reduction by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the tissues treated with the test substance (mean corrected OD570 KC). Since a killed tissue might still have a residual enzyme activity that is able to produce some formazan, the net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero, it is subtracted from the respective mean OD570 to result in the mean corrected OD570 KC. The mean corrected OD570 KC represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance. Tissue viability The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean corrected OD570 KC if applicable) divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA
In case one of the acceptance criteria below was not met, repetition of the test was considered.

Barrier function and Quality control (QC)
The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must
fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD guidelines.
Lower acceptance limit: ET50 = 4.0 hours
Upper acceptance limit: ET50 = 8.7 hours

Acceptance criteria for the negative control (NC)
The absolute OD570 of the negative control tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8 The mean OD570 of the NC should not exceed 2.8.

Acceptance criteria for the positive control (PC)
8 N potassium hydroxide solution is used as positive reference. A tissue viability of ≤ 30% is acceptable for the 3-minute exposure. Mean viability of the tissues exposed for 1 hour should be <15%.

Acceptance criteria for tissue variability
For every treatment, 2 tissues are treated in parallel. In the range of 20% and 100% viability, variability between the tissues is considered to be acceptable if the coefficient of variation (CV) of % viability is ≤ 30%.

Acceptance criteria for the killed controls (KC)
The OD570 of the tissues for the KC of the NC should be ≤ 0.35. The OD570 value for direct MTT reduction of a test substance should be ≤ 30% of the OD570 of the NC.

EVALUATION OF RESULTS
Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after a 3-minute treatment compared to the negative control tissues concurrently treated with deionized water. A chemical is considered as "corrosive" if the mean relative tissue viability after the 3-minute treatment with test material is decreased below 50%. In addition, materials with a viability of 50% after the 3-minute treatment are considered as "corrosive" if the mean relative tissue viability after a 1-hour treatment with test material is decreased below 15%. A single test composed of at least two tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in case of borderline results such as non-concordant replicate measurements and/or mean percent tissue viability equal to ± 5% of the cut-off values cited below, a second test should be considered as well as a third one in case of discordant results between the first two tests.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3 min and 1 hour

Number of replicates:

2 tissues were used per treatment period

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min treatment

Run / experiment:

test substance: mean of tissue 1 and 2

Value:

58.2

Remarks on result:

other:

Remarks:

Due to the ability of the test substance to directly reduce MTT, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (relative mean viability: 2.2% of NC). Thus, the final relative mean viability of the test substance is given after KC correction.

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min treatment

Run / experiment:

negative control: mean of tissue 1 and 2

Value:

100

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min treatment

Run / experiment:

posivie control: mean of tissue 1 and 2

Value:

9

Irritation / corrosion parameter:

% tissue viability

Remarks:

1 hour treatment

Run / experiment:

test substance: mean of tissue 1 and 2 after KC correction

Value:

4.8

Remarks on result:

other:

Remarks:

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (relative mean viability: 25.7% of NC). Thus, the final relative mean viability of the test substance is given after KC correction.

Irritation / corrosion parameter:

% tissue viability

Remarks:

1 hour treatment

Run / experiment:

negative control: mean of tissue 1 and 2

Value:

100

Irritation / corrosion parameter:

% tissue viability

Remarks:

1 hour treatment

Run / experiment:

positive control: mean of tissue 1 and 2

Value:

5.2

Other effects / acceptance of results:

At the 1-hour exposure, all tissues treated with the test substance were swollen and discolored severely whitish after incubation and washing.

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

Based on the results observed and by applying the evaluation criteria described in chapter 3.8., it was concluded that 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- shows an intermediate or weak corrosion potential in the EpiDerm™ in vitro skin corrosion test in under the test conditions chosen and should be assigned to UN GHS skin corrosivity subcategories Cat 1B or 1C. A differentiation between GHS subcategories 1B or 1C is not possible by the present type of study.

Executive summary:

The potential of 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- to cause dermal corrosion was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™), so that the surface f the epidermis model was completely covered with the test substance. Two EpiDerm™ tissues were incubated with the test substance for

3 minutes and 1 hour, each. In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and of a positive control (8 N KOH) were applied to two tissues each per exposure period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure 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 epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability.

The following results were obtained in the EpiDermTM skin corrosion test: The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

At the 1 hour-exposure, the tissues treated with the test substance were swollen and discolored severely whitish after incubation and washing. The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 58.2%, and it was 4.8% after an exposure period of 1 hour. The variability between the results of the tissues is within the acceptance range. The mean OD570 of the negative control (deionized water) fulfills the acceptance criteria and demonstrates the validity of the assay. The tissues treated with the positive control (8 N KOH) showed a relative mean viability of 9.0% (3-minute exposure) and 5.2% (1-hour exposure) reflecting the expected sensitivity of the tissues.

Based on the results observed and by applying the evaluation criteria, it was concluded that 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- shows an intermediate or weak corrosion potential in the EpiDerm™ in vitro skin corrosion test in under the test conditions chosen and should be assigned to UN GHS skin corrosivity subcategories Cat 1B or 1C. A differentiation between GHS subcategories 1B or 1C is not possible by the present type of study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

skin irritation

The Corrositex® assay (OECD 435) was conducted in order to assess the potential of 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- to cause dermal corrosion by a single topical application of 500 μL test substance to the Corrositex® bio barrier membrane. The Corrositex® bio barrier membrane is a test system consisting of a reconstituted collagen matrix. The assay is based on the time that the test substance requires to penetrate through the Corrositex® bio barrier membrane and produce a change in the Chemical Detection System (CDS).

In the main test, five Corrositex® bio barrier membranes were treated with the undiluted test substance. Two CDS showed a minimal reddish discoloration on the surface shortly after test substance application, while the other two CDS did not show any discoloration within the 60-minute observation period. A fifth CDS first used for color comparison and then applied additionally to the four CDS with test substance did not show any discoloration within the 60-minute observation period either. Due to the nontypical observations without concrete breakthrough, no conclusive result could be determined. The negative control, 10% citric acid monohydrate, did not produce any reaction within 60 minutes after application. Sodium hydroxide (solid) applied as positive control showed a reaction time of 14 minutes and 49 seconds after application and was assigned accordingly Thus, the controls fulfill the acceptance criteria and demonstrate the validity of the assays.

A second test run was performed for clarification. In the second test run, four Corrositex® bio barrier membranes were treated with the undiluted test substance. All of the CDS showed a minimal reddish discoloration on the surface shortly after test substance application. Due to the recurrent nontypical observations without concrete breakthrough, no conclusive result could be determined in the second test run as well. The negative control, 10% citric acid monohydrate, did not produce any reaction within 60 minutes after application. Sodium hydroxide (solid) applied as positive control showed a reaction time of 10 minutes and 39 seconds after application and was assigned accordingly. Thus, the controls fulfill the acceptance criteria and demonstrate the validity of the assays. Based on the inconclusive results observed and by applying the evaluation criteria, the potential of 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- to cause dermal corrosion can not be assessed in the Corrositex® assay and it can not be excluded that the test substance has a corrosive potential. For final assignment of a risk phase at present, results from another study would be needed. Therefore, an additional EpiDerm study (OECD 431) was conducted.

The potential of 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- to cause dermal corrosion was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™), so that the surface f the epidermis model was completely covered with the test substance. Two EpiDerm™ tissues were incubated with the test substance for

3 minutes and 1 hour, each. In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and of a positive control (8 N KOH) were applied to two tissues each per exposure period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure 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 epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability.

The following results were obtained in the EpiDermTM skin corrosion test: The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

At the 1 hour-exposure, the tissues treated with the test substance were swollen and discolored severely whitish after incubation and washing. The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 58.2%, and it was 4.8% after an exposure period of 1 hour. The variability between the results of the tissues is within the acceptance range. The mean OD570 of the negative control (deionized water) fulfills the acceptance criteria and demonstrates the validity of the assay. The tissues treated with the positive control (8 N KOH) showed a relative mean viability of 9.0% (3-minute exposure) and 5.2% (1-hour exposure) reflecting the expected sensitivity of the tissues.

Based on the results observed and by applying the evaluation criteria, it was concluded that 1H-Inden-1-amine, 2,3-dihydro-2,6-dimethyl-, (1R,2S)- shows an intermediate or weak corrosion potential in the EpiDerm™ in vitro skin corrosion test in under the test conditions chosen and should be assigned to UN GHS skin corrosivity subcategories Cat 1B or 1C. A differentiation between GHS subcategories 1B or 1C is not possible by the present type of study. For that reason and as a precautionary approach, the substance is classified as skin corrosion cat. 1B.

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. As a result the test substance is considered to be classified as corrosive with cat. 1B under Regulation (EC) No 1272/2008.