[1S-(1α,2β,3α,5α)]-[2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]methylamine

Endpoint summary

Description of key information

Skin:

In two studies on skin irritation/corrosion (OECD 431, 439), a skin corrosive potential of the test substance was determined (BASF SE, 2016).

In a study on skin irritation/corrosion according to OECD 435 using the Corrositex® test system, no irritating/corrosive potential of the test substance was observed (BASF SE, 2016).

Eye:

In an in vivo eye irritation study in rabbits similar to OECD 405, the test substance caused irriversible eye damage (BASF SE, 1979).

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

Study period:

2016-02-25 to 2016-09-22

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)

Version / remarks:

2015-06-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: WE 20PA15

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS:
- Liquid / colorless, clear
- pH ~7, undiluted test substance

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: 00267

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm EPI-200, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue lot number: 23317

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min exposure at room temperature, 1 h exposure at 37 °C.
- Temperature of post-treatment incubation: 3 h at 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing steps: at least one washing step with PBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
- Viability: 1.567 ± 0.068 (MTT QC assay, 4 h, n = 3; acceptance criteria: OD(540 - 570 nm) [1.0 - 3.0])
- Barrier function: 6.7 h (ET-50 assay, 100 µL 1 % tritinX-100, 4 time points, n = 3, MTT Assay; acceptance criteria: ET-50 [4.77 -8.72 h])
- Contamination: No


NUMBER OF REPLICATE TISSUES:
2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed EPI-200 tissues
- Procedure used to prepare the killed tissues: killed by freezing at -20 °C
- No. of replicates : 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50 %. In addition, those materials with a viability of > 50 % after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 min, 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

13.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Exposure period 3 min: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation.

Test substance identification			Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Viable tissues	Mean OD570	1.550	1.836	1.693
		Viability [% of NC]	91.6	108.4	100	11.9	11.9
	KC tissues	Mean OD570	0.115			0.115
		Viability [% of NC]	6.8			6.8
Test substance	Viable tissues	Mean OD570	0.607	0.771	0.689
		Viability [% of NC]	35.9	45.5	40.7	6.8	16.8
	KC tissues	Mean OD570	0.695	0.054	0.695
		Viability [% of NC]	41.0	3.2	41.0
	Final mean viability of tissues after KC correction [% of NC]***:				0.0
PC	Viable tissues	Mean OD570	0.330	0.332	0.331
		Viability [% of NC]	19.5	19.6	19.5	0.1	0.5

* due to organizational reasons, only 1 KC-tissue was used for the NC

** the value of KC-tissue 2 is attributed to be an outlier, therefore it was not used for calculation

*** negative value is set to zero

Table 2: Exposure period 1 h: Individual and mean OD570 values, individual and mean viability values,

standard deviations and coefficient of variation

Test substance identification			Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Viable tissues	Mean OD570	1.701	1.716	1.708
		Viability [% of NC]	99.6	100.4	100.0	0.6	0.6
	KC tissues	Mean OD570	0.090	0.107	0.098
		Viability [% of NC]	5.3	6.2	5.8	0.7	11.9
Test substance	Viable tissues	Mean OD570	0.777	1.017	0.897
		Viability [% of NC]	45.5	59.5	52.5	9.9	18.9
	KC tissues	Mean OD570	0.628	0.718	0.673
		Viability [% of NC]	36.8	42.0	39.4	3.7	9.4
	Final mean viability of tissues after KC correction [% of NC]***:
PC	Viable tissues	Mean OD570	0.096	0.085	0.090
		Viability [% of NC]	5.6	4.9	5.3	0.5	8.6

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-04-15 to 2016-09-22

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)

Version / remarks:

2015-06-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: WE 20PA15

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS:
- Liquid / colorless, clear
- pH ~7, undiluted test substance

Test system:

artificial membrane barrier model

Details on test system:

SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
- Was the Corrositex® test kit used: yes
- Components: reconstituted collagen matrix
- Apparatus and preparation procedures:
The vial containing the biobarrier matrix powder was placed in a water bath at 64 – 68 ºC. The entire contents of the biobarrier diluent vial was added slowly to the matrix powder. The stir bar rotated slowly enough to avoid foaming of the solution. Two hundred μL of the solubilized matrix was pipetted into each of the membrane discs. The membrane discs were then refrigerated for at least 2 hours at 2 – 8 ºC. The biobarriers were wrapped and stored at 2 – 8 ºC for a maximum of 7 days.

WAS THE COMPATIBILITY TEST PERFORMED: yes

WAS THE TIMESCALE CATEGORY TEST PERFORMED: yes

TEMPERATURE USED DURING TREATMENT: Room teperature

METHOD OF DETECTION
- Chemical detection system: Chemical Detection System, Corrositex® kit, InVitro International, Irvine CA, USA

METHOD OF APPLICATION:
500 μL of the undiluted test substance was added onto the membrane disc.

NUMBER OF REPLICATES: 4

NUMBER OF INDEPENDENT EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive subcategory 1A to skin if the time elapsed between application of the test substance to the membrane barrier and barrier penetration is < 3 min.
- The test substance is considered to be corrosive subcategory 1B to skin if the time elapsed between application of the test substance to the membrane barrier and barrier penetration is > 3 - 30 min.
- The test substance is considered to be corrosive subcategory 1C to skin if the time elapsed between application of the test substance to the membrane barrier and barrier penetration is > 30 - 60 min.
- The test substance is not considered to be corrosive if the time elapsed between application of the test substance to the membrane barrier and barrier penetration is > 60 min.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 500 µL

NEGATIVE CONTROL
- Amount applied: 500 µL
- Concentration: 10 %

POSITIVE CONTROL
- Amount applied: one pellet

Duration of treatment / exposure:

> 60 min

Number of replicates:

4

Irritation / corrosion parameter:

penetration time (in minutes)

Value:

> 60

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Principles of method if other than guideline:

according to Fed. Reg. 38, 187, § 1500.42, 1973; Draize test

GLP compliance:

not specified

Specific details on test material used for the study:

OTHER SPECIFICS:

pH 9

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.3 - 2.8 kg
- Diet: ad libitum, standardized animal laboratory diet

Vehicle:

other: olive oil

Controls:

other: untreated eye of the same animal

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL
- Concentration: 95.3 % in olive oil (w/v)

Duration of treatment / exposure:

24 h

Observation period (in vivo):

8 d

Number of animals or in vitro replicates:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The test substance was not washed out.

SCORING SYSTEM:

TOOL USED TO ASSESS SCORE: Readings of the reaction of the palpebral conjunctiva and of the eye are made 1, 24, 48, and 72 h after instillation.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not reversible

Remarks on result:

other: value could not be exactly determined

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not reversible

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Irritant / corrosive response data:

Eight days after test substance instillation, purulent necrotic changes of the Bulbus oculi were observed.

Interpretation of results:

Category 1 (irreversible effects on the eye)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Additional information

Skin:

The objective of the studies was to assess the skin irritation and corrosion potential of (+)-3-Aminomethylpinan. Using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, three in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT, according to OECD Guideline 431), the Skin Irritation Test (SIT, according to OECD Guideline 439) and the in vitro membrane barrier method for skin corrosion (according to OECD Guideline 435).

The potential of (+)-3-Aminomethylpinan to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™).

For the corrosion test two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. The irritation test was performed with three EpiDerm™ tissues, 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 following results were obtained in the EpiDerm™ skin corrosion/irritation test:

The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control KC (freeze-killed control tissues) was introduced. In both tests high KC-values for direct MTT-reduction of the test substance were determined. Thus the final mean viabilities of the test-substance treated tissues are given after KC correction.

Results of the corrosion test:

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 0 %, and it was 13 % after an exposure period of 1 hour.

Results of the 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 0 %.

The KC-values for direct MTT-reduction of the test substance are out of the acceptance range. However, the evaluation is considered to be valid, due to the unambiguous result of the 3 minutes exposure without correction of the KC-value for MTT reduction.

Based on the observed results and applying the evaluation criteria it was concluded, that (+)-3-Aminomethylpinan shows a skin corrosive potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

In contrast, in the the in vitro membrane barrier method for skin corrosion using the Corrositex® test system (BASF SE, 2016), the test substance showed no corrosive potential after application of 500 µL test substance onto the test matrices and an incubation of > 60 min. According to the OECD Guideline 435 liquids with an pH in the range of 4.5 to 8.5 often do not qualify for this test. This could be the reason for the different outcome since the test substance has a pH of ~7.

In conclusion, taking into account the positive results of the in vitro skin irritation (OECD Guideline 439) and in vitro skin corrosion (OECD Guideline 431) assays, a classification into Category 1B is justified.

Eye:

In a primary eye irritation study, 0.1 mL of (+)-3-aminomethylpinan in olive oil was instilled into one conjunctival sac of 6 adult New Zealand white rabbits (2.3 - 2.8 kg) for 24 hours (BASF SE, 1979). The eyes were not washed thereafter. Animals were then observed for 8 days. Irritation was scored by the method of Draize.

Very severe, irreversible damages to the eyes were observed (mean scores of all observed functional units were at maximum). In this study, (+)-3-aminomethylpinan was corrosive to the eye based on the scores (mean of 24, 48, and 72 h).

 

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Eye and skin corrosive properties were documented. As a result the substance should be considered to be classified as eye irritant (category 1, H318) and skin corrosive (subcategory 1B, H314) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.