N-(2-aminoethyl)-N'-[3-(trimethoxysilyl)propyl]ethylenediamine

Endpoint summary

Description of key information

In vivo skin irritation (OECD 404, read across CAS 1760 -24 -30): not irritating

In vivo eye irritation test (OECD 405): corrosive

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the attached justification below and the overall justification for grouping of substances attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 11 days

Remarks on result:

other: Desquamation from Day 7

Remarks:

CAS 1760-24-3, Dow Corning Corporation, 2000

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 13 days

Remarks on result:

other: Desquamation from Day 4

Remarks:

CAS 1760-24-3, Dow Corning Corporation, 2000

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

other: Desquamation from Day 5

Remarks:

CAS 1760-24-3, Dow Corning Corporation, 2000

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

7

Reversibility:

fully reversible within: 9 days

Remarks on result:

other:

Remarks:

CAS 1760-24-3, Dow Corning Corporation, 2000

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other:

Remarks:

CAS 1760-24-3, Dow Corning Corporation, 2000

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

other:

Remarks:

CAS 1760-24-3, Dow Corning Corporation, 2000

Four supporting studies are available with the source substance CAS 1760-24-3. The following results were observed:

Hazleton France, 1992:

erythema score: animal #1: 24/48/72h: score 2 of max. score 4: fully reversible within 7 days: There was a slight dryness of the skin at the application area at 72 hours. Slight desquamation of the epidermis on Day 7.

erythema score: animal #2: 24/48/72h: score 1 of max. score 4: fully reversible within 7 days: Slight desquamation of the epidermis on Day 7.

erythema score: animal #3: 24/48/72h: score 1 of max. score 4: fully reversible within 7 days: Slight desquamation of the epidermis on Day 7.

erythema score: animal #4: 24/48/72h: score 0.67 of max. score 4: fully reversible within 72 hours

erythema score: animal #5: 24/48/72h: score 2 of max. score 4: fully reversible within 7 days: There was a slight dryness of the skin at the application area at 72 hours. Slight desquamation of the epidermis on Day 7.

erythema score: animal #6: 24/48/72h: score 1 of max. score 4: fully reversible within 7 days

edema score: animal #1: 24/48/72h: score 1.67 of max. score 4: fully reversible within 7 days

edema score: animal #2: 24/48/72h: score 0.3 of max. score 4: fully reversible within 48 hours

edema score: animal #3: 24/48/72h: score 0.3 of max. score 4: fully reversible within 48 hours

edema score: animal #4: 24/48/72h: score 0.3 of max. score 4: fully reversible within 48 hours

edema score: animal #5/6: 24/48/72h: score 0 of max. score 4

Hüls, 1993:

erythema score: animal #1: 24/48/72h: score 2 of max. score 4: fully reversible within 8 days: Skin was dull up to 72 hours and there was some scaling after 72 hours and on Day 6.

erythema score: animal #2/3: 24/48/72h: score 2 of max. score 4: fully reversible within 14 days: Skin was dull up to 48 hours and there was some scaling after 24 hours up to Day 14. There were no signs of irritation by Day 17.

edema score: animal #1: 24/48/72h: score 1.3 of max. score 4: fully reversible within 6 days

edema score: animal #2: 24/48/72h: score 2 of max. score 4: fully reversible within 8 days

edema score: animal #3: 24/48/72h: score 1.3 of max. score 4: fully reversible within 8 days

TNO, 1998:

erythema score: animal #1/2/3: 24/48/72h: score 0.3 of max. score 4: fully reversible within 48 hours

edema score: animal #1/2/3: 24/48/72h: score 0.3 of max. score 4: fully reversible within 48 hours

Huntington Research Centre Ltd., 1993

erythema score: animal #1/3: 24/48/72h: score 0 of max. score 4

erythema score: animal #2: 24/48/72h: score 1.67 of max. score 4: fully reversible within 7 days

edema score: animal #1/3: 24/48/72h: score 0 of max. score 4

edema score: animal #2: 24/48/72h: score 1.67 of max. score 4: fully reversible within 7 days

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification is required according to Regulations (EC) No 1272/2008.

Conclusions:

Several studies similar or according to OECD 404 are available for the source sbstance N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3). All of them showed mild irritation but EU criteria for classification were not met. As explained in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in skin irritation potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-11-23 to 2016-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

02 Oct 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Eurofins BioPharma Product Testing

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: 22 - 23 weeks old
- Weight at study initiation: > 2 kg
- Housing: ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Diet: Altromin 2123 maintenance diet, ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): 100%

Duration of treatment / exposure:

Single application without washing

Observation period (in vivo):

Animals were observed for 21 days after exposure.

Number of animals or in vitro replicates:

1

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: See table below

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not reversible

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Irritant / corrosive response data:

During the 24 to 72 hour observation period conjunctival redness, chemosis, discharge and corneal effects were observed in the animal; these effects persisted until Day 21 of the observation period. Corneal lesions also were observed at the end of the prolonged observation period.

Other effects:

No mortality or significant clinical signs of toxicity, with the exception of local effects, were observed.

Interpretation of results:

other: CLP/EU GHS criteria are met, Category 1 (irreversible effects on the eye) classification required according to Regulations (EC) No 1272/2008.

Conclusions:

In an OECD 405 in vivo eye irritation study with the submission substance that was GLP compliant, severe irritation and corrosion were observed in one male rabbit up until the end of the 21-day observation period. Based on the serverity of effects (i.e., corneal lesions) that were not fully reversible within 21 days, the submission substance was considered to be corrosive. These findings results in a Category 1 classification based on CLP criteria.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-12-16

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)

Deviations:

no

GLP compliance:

yes

Species:

other: cattle

Strain:

other: Bovine eyes from young cattle

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Vitelco, 's Hertogenbosch, The Netherlands
- Transport medium and temperature conditions: physiological saline in a suitable container under cooled conditions

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularization): yes
- Dissection of the eyes and treatment: Isolated corneas were mounted in cornea holders
- Type of cornea holder used: MC2, Clermont-Ferrand, France
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea
- Test medium and temperature conditions used in the cornea holder: cMEM (Eagle’s Minimum Essential Medium) supplemented with 1% [v/v] fetal calf serum and 1% [v/v] L-glutamine; prior to use: pre-warmed to 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer
- Specification of the device: opacitometer (OP-KIT, MC2, Clermont-Ferrand, France)

Vehicle:

not specified

Controls:

other: number of eyes for the negative control: 3; number of eyes for the positive control: 3

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 750 µL
- Concentration (if solution): liquid: 61.5%

VEHICLE
- Substance: physiol. saline
- Concentration (if solution): 0.9% NaCl solution in deionised water
- Amount(s) applied in the test: 750 µL

POSITIVE SUBSTANCE
- Substance: Benzalkonium Chloride
- Concentration (if solution): 10% (w/v) Benzalkonium Chloride in physiol. saline
- Amount(s) applied in the test: 750 µL

Duration of treatment / exposure:

10 min

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

number of eyes for the test item: 3

Details on study design:

TEST CONDITIONS
- The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (10% (w/v) Benzalkonium Chloride) or test substance was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

POSTEXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed from the anterior chamber and the epithelium was washed.
- Medium for washing the corneas: MEM containing phenol red
- Medium for final rinsing: cMEM

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: After refilling fresh cMEM without phenol red in the anterior chamber and incubation for 120 ± 10 min the final opacity was measured.
- Specification of the device: OP-KIT, MC2, Clermont-Ferrand, France

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 490 nm recorded as optical density (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (4 mg/ml; dissolved in cMEM)
- Incubation time: 90 ± 5 min at 32 ± 1 °C
- Treatment for measuring: OD490 of a 360 µL aliquot was determined in a flat-bottomed 96-well plate
- Specification of the spectrophotometer: TECAN Infinite® M200 Pro Plate Reader

Irritation parameter:

in vitro irritation score

Value:

4.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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 (10% (w/v) Benzalkonium Chloride) was 125 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. N-(2-Aminoethyl)-N’-[3-(trimethoxysilyl)propyl]ethylenediamine induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 4.2 after 10 minutes of treatment. Since N-(2-Aminoethyl)-N’-[3-(trimethoxysilyl)propyl]ethylenediamine induced an IVIS > 3 = 55, no prediction on the classification can be made.

Table 1: Opacity values

Parameter	Initial opacity	Final opacity	Opacity change	Mean opacity change of NC	Corrected opacity change		Mean opacity value
Negative control	2	2	0	0.3		-	-
	1	2	1
	1	1	0
Test substance	1	4	3	-	2.7		3.0
	2	4	2		1.7
	1	6	5		4.7
Positive control	1	80	79	-		78.7	78.7
	3	85	82			81.7
	3	79	76			75.7

Table 2: Permeability values (optical density (OD) at 490 nm)

Parameter	OD490	Dilution factor	OD490 change	Mean OD490 change of NC	Corrected OD490 change		Mean OD490 value
Negative control	-0.001	1	-0.001	0.000		-	-
	-0.001	1	-0.001
	0.002	1	0.002
Test substance	0.064	1	0.064	-	0.064		0.083
	0.072	1	0.072		0.072
	0.113	1	0.113		0.113
Positive control	0.396	6	0.396	-		2.735	3.109
	0.327	6	0.327			1.969
	0.832	6	0.832			4.991

Table 3: In-Vitro Irritancy Score (IVIS) values

	IVIS	Mean IVIS
Negative control	-0.3	0.0
	0.7
	-0.3
Test substance	3.6	4.2
	2.7
	6.4
Positive control	114.3	125.3
	111.1
	150.5

Table 4: Historical control data for the BCOP studies

	Negative control			Positive control
	Opacity	Permeability	In Vitro Irritancy Score	In Vitro Irritancy Score
Range	-3 – 2	-0.020 – 0.021	-3.1 – 2.3	81 – 264
Mean	-0.14	0.00	-0.16	135
SD	0.89	0.01	0.94	24
n	106	95	100	101

SD = Standard deviation

n = number of observations

Interpretation of results:

study cannot be used for classification

Conclusions:

N-(2-Aminoethyl)-N’-[3-(trimethoxysilyl)propyl]ethylenediamine induced ocular irritation through cornea opacity (3.0) and permeability (0.083), resulting in a mean in vitro irritancy score of 4.2 after 10 minutes of treatment. Since the submission substance induced an IVIS > 3 = 55, no prediction on the classification can be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

Only non-reliable data on the skin irritation of N-(2 -aminoethyl)-N'-[3 -(trimethoxysilyl)propyl]-ethylenediamine (CAS 35141 -30 -1) are available. Therefore, the risk assessment was performed based on the available data from the source substance N-(3-trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3). In accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5 “Grouping of substances and read across” and following the Read across assessment framework (RAAF, ECHA 2017) read across from a analogue substance has been applied to support the human health hazard assessment of N-(2-aminoethyl)-N'-[3-(trimethoxysilyl)propyl]-ethylenediamine (CAS 35141-30-1). Further details are provided in the analogue justification attached to the respective target entry.

Beside a non-guideline study without GLP compliance (Carnegie-Mellon Institute of Research, 1975, reliability score 4) no further reliable data on skin irritation are available with N-(2-aminoethyl)-N'-[3-(trimethoxysilyl)propyl]-ethylenediamine (CAS 35141-30-1). Therefore, read across from the similar substance N-(3-trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3) was applied.

There are several skin irritation studies available for the structural analogue N-(3-trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3). The supporting studies supports the findings of the key study.

In the key skin irritation study conducted to EPA OPPTS 870.2500 (Acute Dermal Irritation) and to GLP (Dow Corning Corporation, 2000) N-(3-(trimethoxysilyl)propyl)ethylenediamine was mildly irritating to the skin of rabbits, but this irritation did not meet the EU criteria for classification. Well-defined erythema was seen in all three animals, as well as desquamation of the stratum corneum. Slight edema was noted in one of the three animals. Dermal reactions resolved completely in all animals by Day 10, 11 or 13. There were no deaths or clinical signs of toxicity.

Four reliable supporting studies for skin irritation were also available, all conducted according to current guidelines and in compliance with GLP. At most mild irritation was evident which was in all cases reversible and did not meet the EU criteria for classification (TNO, 1998, Hüls, 1993, Huntingdon 1993, Hazleton, 1992)

The skin irritation properties of N-(2-aminoethyl)-N'-[3-(trimethoxysilyl)propyl]-ethylenediamine (CAS 35141-30-1) were tested in a non-guideline study and without GLP compliance (Carnegie-Mellon Institute of Research, 1975). The test substance was applied in 0.01 mL amounts to clipped, uncovered intact skin of 5 rabbit abdomen either undiluted or in progressive dilutions of 10, 1, 0.1, and 0.01% in solvent. Animals were observed and evaluated according to a scoring system (grade 1 to 10). One of 10 grades was assigned based on appearance of moderate or marked capillary injection, erythema, edema, or necrosis within 24 h. The undiluted test item was openly applied and caused moderate and marked capillary injection on 2/3 animals, respectively. No skin reactions were observed in 3/5 rabbits. However, moderate and marked capillary injection (grade 2) was observed in one each of the other two animals, respectively. In conclusion, the test material was found to be a minor primary skin irritant.

The results found with registered substance support the results with the source substance and therefore N-(2-aminoethyl)-N'-[3-(trimethoxysilyl)propyl]-ethylenediamine (CAS 35141-30-1) is also considered to be not irritating to skin.

 

Eye irritation

The eye irritation properties of N-(2-aminoethyl)-N'-[3-(trimethoxysilyl)propyl]-ethylenediamine (CAS 35141-30-1) have been investigated in accordance with OECD TG 437 under GLP conditions using the bovine corneal opacity and permeability test (BCOP, WIL, 2015). For the assessment of the eye irritation properties 750 µL of the test substance was used to cover the corneal surface of the bovine eyes for 10 min. Positive and negative controls were included in the study and gave the expected results.N-(2-Aminoethyl)-N’-[3-(trimethoxysilyl)propyl]ethylenediamine induced ocular irritation through cornea opacity (3.0) and permeability (0.083), resulting in a mean in vitro irritancy score of 4.2 after 10 minutes of treatment. Since the test substance induced an IVIS > 3 = 55, no prediction on the classification can be made.

Because eye irritation classification could not be determined from the in vitro study, an in vivo study was conducted (Bioservice, 2016) with N-(2-aminoethyl)-N'-[3-(trimethoxysilyl)propyl]-ethylenediamine (CAS 35141-30-1). In an OECD 405 study, eye corrosion was evaluated in one rabbit. A single instillation of undiluted test material (0.1 mL) was applied into the conjunctival sac. The animal was observed and evaluated according to the Draize scoring system. Severe irritation and corrosion was observed in the animal up until the end of the 21-day observation period. Cornea opacity, conjunctivae, and chemosis scores for the 24 to 72 observation period were all 2 and not reversible. Based on the severity of effects (i.e., corneal lesions) that were not fully reversible within 21 days, the test material was considered to be dagaming to the eyes.

In a supporting study, irritation was also observed. The eye irritation properties of N-(2-aminoethyl)-N'-[3-(trimethoxysilyl)propyl]-ethylenediamine (CAS 35141-30-1) were tested in a non-guideline study and without GLP compliance (Carnegie-Mellon Institute of Research, 1975). Single instillation of 0.005, 0.02, 0.1, or 0.5 mL undiluted or 0.5 mL of 40, 15, 5, or 1% test material dilutions were applied into the conjunctival sac of 5 rabbits for 24 h. Animals were observed and evaluated according to a scoring system (grade 1 to 10) 24 h post-application. Moderate corneal injury with iritis from 0.5 mL undiluted test material per eye and mild or moderate corneal injury from 0.1 mL, with iritis, or 0.02 mL per eye were observed (mean out of all animals: grade 3). Hence, the test substance was concluded to be moderately irritating to the eyes. 

Justification for classification or non-classification

Based on reliable data from an in vivo study conducted with N-(2-aminoethyl)-N'-[3-(trimethoxysilyl)propyl]-ethylenediamine (CAS 35141-30-1) the substance is classified as a Category 1 eye irritant with the hazard statement 'H318: Causes serious eye damage' according to Regulation (EC) No 1272/2008.

Reliable data from in vivo studies conducted with the structural analogue N-(3-(trimethoxysilyl)propyl)ethylenediamine on skin irritation indicates that the registerd substance does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and the available data are therefore conclusive but not sufficient for classification.