Registration Dossier

Administrative data

Description of key information

Skin: Category 1 (Corrosive), in vitro skin corrosion test, OECD Guideline 431; GLP, RL1

Eye: Category 1 (irreversible effects on the eye), OECD Guideline 405, GLP, RL2; read-across: Amides, C8-18 even numbered, N-[3-(dimethylamino)propyl]

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physicochemical, toxicological and ecotoxicological properties because
- they are manufactured from similar resp. identical precursors under similar conditions
- they share structural similarities with common functional groups: tertiary amines, amides, and fatty acid chains with comparable length and degree of saturation.
- the metabolism pathway leads to comparable products: identical amine backbone and long chain fatty acids with slight differences in chain length distribution and degree of (un)saturation
As their origin is from natural sources, the used fatty acids may have a mixed and slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 amounts may be
included. Consequently, the target and source substance differ by their carbon chain length distribution and the degree of unsaturation in the fatty acid moiety.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance
Reference substance name: Amides, C8-18 even numbered, N-[3-(dimethylamino)propyl]
Carbon chain length distribution in starting material:
C8: <= 10%
C10: 3-10%
C12: 40-65%
C14: 10-26%
C16: 6-14%
C18: 2-24%
C18 unsatd.: <= 2%
Purity: 86.8-98.6 %

Target substance
Reference substance name: Amides, C8-18, C18 unsatd, N-[3-(dimethylamino)propyl]
Carbon chain length distribution in starting material:
C8: 0-10%
C10: 2-16%
C12: 40-55%
C14: 10-22%
C16: 5-15%
C18: 0-5%
C18 unsatd.: 5-20 %
Purity: > 80 %

3. ANALOGUE APPROACH JUSTIFICATION
The read across from the source substance Amides, C8-18 even numbered, N-[3-(dimethylamino)propyl] is justified:
a) Based on the information given in section 1, it can be concluded that source and target substance are similar in structure, since they are manufactured from similar resp. identical precursors under similar conditions and all contain the same functional groups. Thus a common mode of action can be assumed.
b) The only deviation within this group of substances is a minor variety in their fatty acid moiety, which is not expected to have a relevant impact on intrinsic toxic or ecotoxic activity and environmental fate.
c) The target and source substance are all expected to undergo hydrolysis of the amide bond by amidases which would result in free fatty acids of of differing only slightly in length and degree of (unsaturation and dimethylaminopropylamine.

The read across hypothesis is based on structural similarity of target and source substance. The respecive data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

4. DATA MATRIX
see attached document
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: Human Skin Model Test (adopted 13 April 2004)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
(8 June 2000)
Deviations:
no
Species:
other: in vitro test on a human three dimensional epidermal model (EpiDerm (EPI-200))
Details on test animals or test system and environmental conditions:
In vitro Test system EpiDerm Skin Model (EPI-200, Lot no.: 6454, kit C, source MatTek Corporation, Ashland MA, USA) used.

The 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 multi-Iayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Cell culture:
- Tissues:
-- On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM medium.

- Media:
-- DMEM (Dulbecco's Modified Eagle's Medium) = Supplemented DMEM medium, serum-free supplied by MatTek Corporation.
-- MTT medium = MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation..

- Environmental conditions:
-- All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 78 - 89%), containing 5.0 ±0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 - 37.0°C). Temporary deviations from the humidity (with a maximum of 20%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
the test item was heated to 37°C and applied undiluted (50 µL) directly on top of the skin tissue.
Duration of treatment / exposure:
The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test substance; exposure time: 3 minutes
Value:
45
Vehicle controls validity:
not applicable
Negative controls validity:
not specified
Positive controls validity:
not specified
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test substance; exposure time: 1 hour
Value:
45
Vehicle controls validity:
not applicable
Negative controls validity:
not specified
Positive controls validity:
not specified
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative control (water): exposure time: 1 hour
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
not specified
Positive controls validity:
not specified
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control (potassium hydroxide); exposure time: 3 minutes
Value:
7
Vehicle controls validity:
not applicable
Negative controls validity:
not specified
Positive controls validity:
not specified

Mean absorption in the in vitro skin corrosion test with test item

 

 3 min application

1 hour application

 

A

B

mean

SD

A

B

mean

SD

Negative control

1.476

1.487

1.481

±0.008

1.446

1.387

1.417

±0.042

Test substance

0.687

0.658

0.673

±0.020

0.655

0.624

0.639

±0.022

Positive control

0.097

0.099

0.098

±0.001

0.089

0.090

0.090

±0.001

 

SD = Standard deviation

 

In this table the values are corrected for background absorption. Isopropanol was used to measure the background absorption.

 

Mean tissue viability in the in vitro skin corrosion test with test item

 

3 min application viability (percentage of control)

1 hour application viability (percentage of control)

Negative control

100

100

Test substance

45

45

Positive control

7

6

 

This table shows the mean tissue viability obtained after 3 minutes and 1 hour treatment with test item compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with the test item compared to the negative control tissues was 45% for both treatment times. Since the mean relative tissue viability for the test item was below 50% after 3 minutes treatment it is considered to be corrosive.

The absolute mean OD540(Optical density) of the negative control was higher than 0.8. The mean relative tissue viability of the 3 minutes exposure of the positive control was less than 30%. The maximum inter tissue variability in viability between two tissues treated identically was less than 30% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 15%. It was therefore concluded that the test system functioned properly.

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Conclusions:
It is concluded that this test is valid and that the source test substance Amides, C8-18, N-[3-(dimethylamino)propyl] is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

In the In Vitro Skin Corrosion: Human Skin Model Test according to OECD Guideline 431 (adopted 13 April 2004) and EU Method B.40: "Skin corrosion" (8 June 2000), the topical application of the source substance Amides, C8-18, N-[3-(dimethylamino)propyl] on the skin tissue for a 3 minutes and 1 hour exposure was performed, followed by immediate determination of the cytotoxic (corrosive) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. It can be expected that the results are also aplicable for the target substance Amides, C8-18, C18 unsatd, N-[3-(dimethylamino)propyl].

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with the test item compared to the negative control tissues was both 45%. Since the mean relative tissue viability for the test item was below 50% after 3 minutes treatment with the test item is considered to be corrosive.

The positive control had mean relative tissue viability after 3 minutes exposure of less than 30%. The negative control had a mean OD540 (optical density at 540 nm) higher than 0.8. The maximum inter tissue variability in viability between two tissues treated identically was less than 30% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 15%, indicating that the test system functioned properly.

It is concluded that this test is valid and that the test substance is corrosive in the in vitro skin corrosion test under the experimental conditions described.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For the assessment of the skin and eye irritating potential of Amides, C8-18, C18 unsatd, N-[3-(dimethylamino)propyl] the following studies are available:

- in vivo skin irritation study in rabbits similar to OECD Guideline 404 with the target substance Amides, C8-18, C18 unsatd, N-[3-(dimethylamino)propyl] (no data on GLP, RL3 due to insufficient documentation)

- in vitro skin corrosion test according to OECD Guideline 431 (GLP, RL1)

- in vivo eye irritation test in rabbits similar to OECD Guideline 405 with the target substance Amides, C8-18, C18 unsatd, N-[3-(dimethylamino)propyl] (no data on GLP, RL3 due to insufficient documentation)

- in vivo eye irritation test in rabbits according to OECD Guideline 405 with the closely related source substance Amides, C8-18, C18 unsatd, N-[3-(dimethylamino)propyl] (GLP, RL2)

A justification for read-across is given in IUCLID section 13.

 

Skin irritation

In the In Vitro Skin Corrosion: Human Skin Model Test according to OECD Guideline 431 (adopted 13 April 2004) and EU Method B.40: "Skin corrosion" (8 June 2000), the topical application of Amides, C8-18, C18 unsatd, N-[3-(dimethylamino)propyl] on the skin tissue for a 3 minutes and 1 hour exposure was performed, followed by immediate determination of the cytotoxic (corrosive) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with the test item compared to the negative control tissues was both 45%. Since the mean relative tissue viability for the test item was below 50% after 3 minutes treatment with the test item is considered to be corrosive.

The positive control had mean relative tissue viability after 3 minutes exposure of less than 30%. The negative control had a mean OD540 (optical density at 540 nm) higher than 0.8. The maximum inter tissue variability in viability between two tissues treated identically was less than 30% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 15%, indicating that the test system functioned properly.

It is concluded that this test is valid and that the test substance is corrosive in the in vitro skin corrosion test under the experimental conditions described.

 

In a supporting study the substance Amides, C8-18, C18 unsatd, N-[3-(dimethylamino)propyl] was tested for primary skin irritation similar to OECD guideline 404 with 5 rabbits. The contact time of the test item was reduced to 30 min. The following symptoms were observed after application:

Intense erythema, crust development, necrotic skin alteration, moderate edema, healing with scars.

Based on this results the undiluted product has to be classified as corrosive.

 

Eye irritation

In a primary eye irritation study according to OECD Guideline 405 and EU Method B.5, 0.1 g of Amides, C8-18 even numbered, N-[3-(dimethylamino)propyl] was instilled into the conjunctival sac of six White New Zealand rabbits. The eyes of three animals were rinsed 4 seconds after substance application. Animals then were observed for 72 hours. Irritation was scored by the method of Draize as stipulated by OECD Guideline 405.

In unrinsed eyes severe eye reactions were seen at the 60 minutes and the 24 hour scoring:

-- Cornea scores of 0 or 1

-- Iris scores of 1 or 2

-- Conjunctivae redness scores of 2 or 3

-- Conjunctivae chemosis scores of 3 or 4.

Due to the severe reactions these animals were killed after the 24 hour scoring.

In eyes rinsed 4 seconds p.a. the test substance induced

-- Cornea scores of 0

-- Iris scores of 0 or 1

-- Conjunctivae redness scores of 2 or 3

-- Conjunctivae chemosis scores of 2 or 3

The observation time did not extend beyond 72 hours, even though severe eye responses were still present at these times, therefore, the reversibility of the eye effects cannot be evaluated.

The test substance was judged to be irritating to the eye based on the effects of the rinsed eye (washed out 4 secondes p.a.) within 72 hours.

Whereas an evaluation of the animals with unwashed eyes was not possible because they were killed 24 h p.a. due to the severe eye reactions.

 

In a supporting study the eye irritation potential of Amides, C8-18, C18 unsatd, N-[3-(dimethylamino)propyl] was tested with 4 male white rabbits. Therefore 0.1 mL 1% test substance diluted in dest. water was applied to the eye without later washout. Reaction were recorded for 48 h. The following symptomes were observed:

No alteration on cornea and iris, moderate conjunctivia reaction. 1 and 6 hours after application 45 and 40% of the maximal possible reaction, respectively. No reaction after 48 h.

In result a solution of 1% test item in dest. water has to rated as irritant to eye.


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

Justification for selection of eye irritation endpoint:
read-across from guideline study, GLP

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: corrosive

Justification for classification or non-classification

Based on the available data Amides, C8-18, C18 unsatd, N-[3-(dimethylamino)propyl] has to be classified as corrosive to the skin (Category 1) and causing irreversible effects on the eye (Category 1) and labelled with H314 (Causes severe skin burns and eye damage) according to regulation (EC) 1272/2008.