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Description of key information

The in vitro skin irritation study conducted according to OECD TG 439, and in compliance with GLP, reports the test item to be not irritating to human skin model EPISKIN-SMTM (WIL Research, 2016).

In the in vitro skin corrosion study, conducted according to OECD TG 431, and in compliance with GLP, the mean tissue viability was ≥ 50% after 3 minutes exposure and ≥ 15% after 1 hour exposure to the test item. It was concluded that the test item is not corrosive to skin under the conditions of the EpiDerm Skin Model test (Charles River, 2016).

In the in vivo skin irritation/corrosion study, conducted according to an appropriate OECD TG 404 and in compliance with GLP, Reaction Mass of N-[3-(trimethoxysilyl)propyl]-1,3-benzenedimethanamine and {3-[(2,2-dimethoxy-1,2-azasilolidin-1-yl)methyl]phenyl}methanamine was concluded to be not irritating to skin (Eurofins, 2019).

In the key in vitro Bovine Corneal Opacity and Permeability test, conducted according to an appropriate OECD guideline and in compliance with GLP, the test material was reported to induce an IVIS score greater than 55 and to be irritating to bovine corneas (Charles River, 2017).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-10-12 to 2015-10-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes
Species:
other: Human skin model
Strain:
other: EPISKIN Small ModelTM
Details on test animals or test system and environmental conditions:
- Test system: EPISKIN Small ModelTM is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes. The adult human-derived epidermal keratinocytes have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were grown for 13 days in culture, resulting in highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Preincubation: Tissues were preincubated with Maintenance Medium for 27 hours at 37°C.
- Killed tissues: Living epidermis was transferred into 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium.
- MTT medium: MTT concentrate was diluted in assay medium to result in final concentration of 0.3 mg/ml.
- Environmental conditions: All incubations, excluding incubation with test item were carried out in a controlled environment with humid atmosphere of 80 - 100% (actual range 70 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.3°C).
Vehicle:
unchanged (no vehicle)
Controls:
other: negative control: 25 μl PBS; positive control: 25 μl 5% SDS
Amount / concentration applied:
25 μl of undiluted test item
Duration of treatment / exposure:
15 +/- 5 min at room temperature
Observation period:
incubated for 42 hours at 37°C
Number of animals:
3 tissues per test item together with negative and positive controls
Details on study design:
TREATMENT:
- The test item was applied undiluted on top of the skin model.
- Three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT.
- The positive control was re-spread after 7 minutes contact time.
- After exposure period the tissues were washed with phosphate buffered saline to remove residual test item.
- After rinsing, the cell culture inserts were dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed.

CELL VIABILITY MEASUREMENT:
- After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml) and incubated for 3 h at 37°C and dried afterwards.
- Total biopsy was performed
- Epidermis was separated from the collagen matrix and both parts were placed in prelabelled microtubes and extracted with 500 μL isopropanol
- Tubes were stored refrigerated and protected from light for 72 hours
- The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 ± 0.5 minutes treatment
Value:
124
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Mean % tissue viability was >= 50 %

Irritant / corrosive response data

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the submission substance compared to the negative control tissues was 124%.

Other effects

-               Test for colour interference: a colour change was observed by adding MTT-medium it was concluded that the submission substance did interact with the MTT endpoint

-               Test for reduction of MTT by the test item: The non-specific reduction of MTT by the submission substance was -5% of the negative control tissues

Table 1: Mean absorption in the in vitro skin irritation test

 

Mean

(OD570)

SD

Negative control

0.878

+/- 0.122

Test substance

1.088

+/- 0.110

Positive control

0.107

+/- 0.028

OD = optical density

SD = Standard deviation

Table 2: Mean tissue viability in the in vitro skin irritation test

 

Mean tissue viability (percentage of control)

Negative control

100

Test substance

124

Positive control

12

Interpretation of results:
GHS criteria not met
Conclusions:
In the in vitro skin irritation study, the submission substance was concluded to be not irritating to human skin model EPISKIN-SMTM.
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-07-18 to 2016-07-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, flushed with nitrogen
- Stability under test conditions: Stable under test conditions
- Solubility and stability of the test substance in the solvent/vehicle: no solvent/vehicle was used.

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 24306 kit J
- Date of initiation of testing: 18-07-2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature during the 3-minute treatment; all other incubations were carried at 37.0 ± 1.0°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 ml MTT solution in 1 mg/ml phosphate buffered saline
- Incubation time: 1 hour at 37.0 ± 1.0ºC
- Spectrophotometer: TECAN Infinite M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter:
- Filter bandwidth:
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.761 +/- 0.111 [within the acceptable ranges of historical data]
- Barrier function: 6.41 h [within the acceptable ranges of historical data]
- Morphology: appropriate formation of the epidermal barrier, functional stratum corneum, a viable basal layer, and intermediate spinous and granular layers.
- Contamination: no contamination detected


NUMBER OF REPLICATE TISSUES: Duplicate tissues were used for each exposure

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Living epidermis was transferred to a freezer ( =< -15°C), thawed and then again transferred to a freezer ( =< -15°C). The freeze-killed epidermis was stored at =< -15°C prior to use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6-well plate on 0.9 ml DMEM medium. Further use of killed tissues was similar to living tissues.
- No. of replicates : duplicate
- Method of calculation used: The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl of undiluted test material


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl Milli-Q water


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl 8N Potassium hydroxide
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours at 37°C in air containing 5% CO2
Number of replicates:
Duplicate tissues were used for each exposure.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
69
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The absolute mean OD570 (optical density at 570 nm) was within the historical control data range
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
>= 50 %
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
52
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The absolute mean OD570 (optical density at 570 nm) was within the historical control data range
Positive controls validity:
valid
Remarks:
Mean tissue viability was 9%
Remarks on result:
no indication of irritation
Remarks:
>= 15 %
Other effects / acceptance of results:
Negative and positive control values were with the range of historical control data.
The non-specific reduction of MTT by the submission substance was 4.5% and 19% of the negative control tissues.
Interpretation of results:
GHS criteria not met
Conclusions:
In the in vitro skin corrosion study, conducted according to an appropriate OECD test guideline, and in compliance with GLP, the mean tissue viability was ≥ 50% after 3 minute exposure and ≥ 15% after 1 hour exposure to the test item. It was concluded that the test item is not corrosive to EpiDerm Skin Model under the conditions of the test.
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 September 2018 to 07 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: animal no. 1: approximately 12–13 weeks old; animal no. 2: approximately 25–27 weeks old; animal no. 3: approximately 28–29 weeks old
- Weight at study initiation: 2.5–3.7 kg
- Housing: Semi barrier in an air-conditioned room
- Diet (e.g. ad libitum): autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 +/- 3 °C
- Humidity (%): 55+/- 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): not applicable

Duration of treatment / exposure:
4 hours
Observation period:
Animal no. 1 was observed for 14 days
Animal no. 2 was observed for 9 days
Animal no. 3 was observed for 8 days
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: dorsal area
- % coverage: small area
- Type of wrap if used: a gauze patch, which was held in place with a non-irritating tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, removed with cottonseed oil
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) 24, 48 and 72 hours after the patch removal, then daily until up to 14 days post-application

SCORING SYSTEM:
- Method of calculation: Draize score
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 5 days
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 9 days
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 4 days
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Mild to moderate erythema (grade 1 or 2) was observed in all animals; this persisted until day 4 in Animal no.1, day 8 in Animal no. 2 and day 3 in Animal no. 3. No edema was observed in any of the test animals at any time point.
Other effects:
- Other adverse local effects: Signs of scaling were observed in all test animals post-application; these were fully reversed by days 7 and 8 in Animals 2 and 3. Scaling was not fully reversed in Animal 1 at day 14, when the study was terminated. No other effects were reported.
- Other adverse systemic effects: none reported. There were no changed to body weights.
Interpretation of results:
GHS criteria not met
Conclusions:
In an in vivo skin irritation/corrosion study, conducted according to an appropriate OECD TG 404 and in compliance with GLP, Reaction Mass of N-[3-(trimethoxysilyl)propyl]-1,3-benzenedimethanamine and {3-[(2,2-dimethoxy-1,2-azasilolidin-1-yl)methyl]phenyl}methanamine was concluded to be not irritating to skin.
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
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test material was applied undiluted.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was applied undiluted.
- Preliminary purification step (if any): No correction was made for the purity/composition of the test item.
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Vitelco, 's Hertogenbosch, The Netherlands
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): young cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: none specified
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750µl
- Concentration (if solution): not applicable

Duration of treatment / exposure:
10 +/- 1 minutes at 32 +/- 1°C
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes at 32 +/- 1°C
Number of animals or in vitro replicates:
Three corneas per treatment group.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The corneas were incubated with cMEM for the minimum of 1 hour at 32 +/- 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS: After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES: Three corneas per group

NEGATIVE CONTROL USED: Yes, physiological saline

SOLVENT CONTROL USED (if applicable): not applicable

POSITIVE CONTROL USED: Yes, ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750µl for 10 +/- 1 minutes at 32 +/- 1°C

TREATMENT METHO: not specified

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium) and thereafter with cMEM
- POST-EXPOSURE INCUBATION: 120 +/- 10 minutes at 32 +/- 1°C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: diminution of light passing through the cornea
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology):

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.
Irritation parameter:
cornea opacity score
Run / experiment:
treatment with 100% test material for 10 min
Value:
> 186 - <= 304
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
ranged from -2.2 to -1.0
Positive controls validity:
valid
Remarks:
ranged from 51 to 71
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
treatment with 100% test material for 10 min
Value:
228
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 61 and within two standard deviations of the current historical positive control mean (Appendix 3). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, within historical control ranges
- Acceptance criteria met for positive control: Yes, within historical control ranges
- Range of historical values if different from the ones specified in the test guideline: Not different
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In the Bovine Corneal Opacity and Permeability test, conducted according to an appropriate OECD guideline and in compliance with GLP, the test material was reported to induce an IVIS score greater than 55 and to be irritating to bovine corneas.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The in vitro skin irritation study, conducted according to OECD TG 439 and in compliance with GLP, reported the test item to be not irritating to human skin model EPISKIN-SMTM (WIL Research, 2016).

Following 15 +/- 5 min application of 25 μL of undiluted test item to human skin model EPISKIN-SMTM, the mean relative tissue viability was >50% (124%.). The test system was incubated for 42 hours at 37°C after removal of the test item. Positive and negative controls were included and gave the expected results. The mean tissue viability for positive and negative controls were 12 and 100% respectively. The test item was concluded to be not irritating to human skin model EPISKIN-SMTM under the conditions of the study.

The in vitro skin corrosion study, conducted according to OECD TG 431 and in compliance with GLP, reported the test item to be not corrosive to EpiDerm Skin Model under the conditions of the test (Charles River, 2016).

Following 3-minute and 1-hour exposures of 50 μL undiluted test item to EpiDerm Skin Model, the mean tissue viability was ≥50% and ≥15% respectively. Duplicate tissue cultures were used for each exposure. For cytotoxicity evaluation with MTT two freeze-killed tissues treated with the test item and two freeze-killed negative control treated tissues were used. After the 3-minute exposure period the treated tissues were washed with phosphate buffered saline to remove residual test item. No washing was done after the 1-hour exposure as the test item became solid. After the exposure period 300 μL of MTT-medium was added and the tissues were incubated for 3 hours at 37°C in air containing 5% CO2. Following incubation, the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature. The amount of the extracted formazan was determined using spectrometer at 570 nm. The relative mean tissue viability obtained after 3-minute and 1-hour exposures was 69% and 52% respectively. Positive and negative controls were included and gave the expected results. The test item was concluded to be not corrosive.

During a skin sensitisation pre-test some irritation skin reactions were observed in the test animals. Therefore, the registrant performed an in vivo skin irritation study (OECD TG 404) to confirm the presence or absence of irritation potential of the registered substance.

In an in vivo skin irritation/corrosion study, conducted according to an appropriate OECD TG 404 and in compliance with GLP, Reaction Mass of N-[3-(trimethoxysilyl)propyl]-1,3-benzenedimethanamine and {3-[(2,2-dimethoxy-1,2-azasilolidin-1-yl)methyl]phenyl}methanamine was concluded to be not irritating to skin (Eurofins, 2019).

Following, application of 0.5 mL of the test material onto intact rabbit skin for 4 hours under a semi-occlusive patch, the erythema and oedema grades were assessed for each of the three test animals. Erythema was observed in all of the animals, which was fully reversible within 9 days following patch removal. No oedema was observed in any of the test animals. Scaling was noted at the test site in all the animals, which was fully reversible for animals no. 2 and no. 3, but was fully reversed in animal no. 1 on day 14 post-application, when the study was terminated. In accordance with Annex I: 3.2.2.8.2 of Regulation (EC) No 1272/2008, the test item was concluded to be not irritating to skin since scaling persisted to day 14 in only one of the three test animals; scaling was fully reversed in the remaining two animals.

In the key in vitro Bovine Corneal Opacity and Permeability test, conducted according to an appropriate OECD guideline and in compliance with GLP, the test material was reported to induce an IVIS score greater than 55 and to be irritating to bovine corneas (Charles River, 2017).

Following a 10-minute exposure of triplicate cultures of corneas to 750 µL of undiluted test material, serious eye damage through one endpoint (opacity) was induced, resulting in a mean in vitro irritancy score of 228 after 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 61 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 


Justification for classification or non-classification

Based on the in vitro and in vivo data for the submission substance, no classification is required for skin irritation and classification Category 1 "H318: Causes serious eye damage" is required for eye irritation according to Regulation (EC) No. 1272/2008.