N-methyldidecylamine

Endpoint summary

Description of key information

The dermal irritant/ corrosive potential of the substance was assessed using:
-  an in vitro skin corrosion test performed in the Episkin reconstituted human epidermis model according to OECD guideline 431 and Good Laboratory Practices (Warren, 2010a)
- an in vitro skin irritation test  performed using the Episkin reconstituted human epidermis model according to OECD guideline 439 and EU method B.46. the study was performed in compliance with the Good Laboratory Practices (Warren, 2010b).
- two in vivo acute dermal irritation studies  performed on rabbits according to OECD guidelines 404 (Guest, 1987 and 1991). 
The substance was found to be irritating to the skin in the two in vivo studies and thus require a classification to CLP (Reg. n° 1272/2008/EC) and directive 67/548/EEC.
The ocular irritant potential of the substance was assessed using:
-An in vitro ocular irritation test performed on isolated calf cornea according to the method described by Gautheron P. et al (1992) Fundam.Appl.Toxicol.18 p442-449 and Good Laboratory Practices (Gerbeix, 2010b)
The substance was found to be slightly irritating to the eyes and did not require a classification according to CLP (Reg. n° 1272/2008/EC) and directive 67/548/EEC.

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation/corrosion:

In vitro studies :

The dermal irritant/ corrosive potential of the substance was assessed sequentially:

The purpose of the first test (Warren, 2010a) was to evaluate the corrosivity potential of the test material using the EPISKIN^TMin vitroReconstituted Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to meet the requirements of the following guideline OECD Guideline for the Testing of Chemicals No. 431 “In VitroSkin Corrosion: Human Skin Model Test” (adopted 13 April 2004)

The EPISKIN^TMmodel is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied. As stated in regulation 1272/2008/EEC a validatedin vitrotest such asin vitroskin corrosion test in the EPISKIN^TMmodel can be used for classification and labelling. The EPISKIN^TMmodel is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) chemicals.

Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96‑well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

The relative mean viability of the test material treated tissues was:

240 minutes exposure	:	82.9%
60 minutes exposure	:	98.4%
3 minutes exposure	:	129.5%

The quality criteria required for acceptance of results in the test were satisfied.The test item was considered to be Non-Corrosive to the skin.

As the substance was found to be non-corrosive, the purpose of this second test (Warren, 2010b) was to evaluate the skin irritation potential of the test material using the EPISKIN^TMreconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42‑hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end‑point will be used to either confirm a non-irritant result or will be used to override the non‑irritant result.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre‑labelled 96‑well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

The quality criteria required for acceptance of results in the test were satisfied.

With the test item, the relative mean viability of the treated tissues was 112.1% after a 15‑minute exposure period. Due to the absence of cytotoxicity in the MTT assay it was necessary to determine the concentration of inflammatory mediator IL-1α in the retained culture medium for confirmatory purposes. The concentration of inflammatory mediator IL-1α was 31.085 pg/ml. This result confirmed the absence of cytotoxicity as determined in the MTT assay. Under these experimental conditions, the test material was considered to be non-irritating to the skin.

In vivo studies :

Two in vivo studies were provided after the performance of the in vitro skin corrosion/irritation tests.

The first one (Guest, 1987) was a study performed to assess the effects of didecylmethylamine on the irritancy potential to the skin of the 3 New Zealand White rabbits. The method used was based on that described in the OECD Guidelines for Testing of Chemicals (1981) No. 404 "Acute Dermal Irritation/Corrosion". A three minute, 1 hour and 4 hours, semi-occluded dermal application of the undiluted test material to the intact skin of three rabbits followed by a decontamination procedure with 3% (v/v) aqueous acetic acid followed by cotton wool in distilled water, produced irritation scores directly after patch removal and 1, 2 and 3 days afterwards. The mean scores over 24, 48 and 72 hours for individual animals were 2.7, 2.7, and 2.0 for erythema and 3.0, 2.3, and 2.0 for oedema. The erythema/eschar effects were not fully reversible within 21 days, but the oedema effects were within 14 days. There was no evidence of visible necrosis.

 

The second study (Guest, 1991) was an acute dermal irritation/corrosion study performed in rabbits (4 females and 2 males) according to OECD 404 and under GLP. Severe dermal responses were produced. The adverse skin reaction sometimes precluded accurate evaluation of erythema and oedema. Very slight to well-defined erythema with or without very slight oedema was noted one hour after patch removal. The responses increased in severity and at subsequent observations were identified as well-defined erythema, very slight to moderate oedema, blanching of the skin, haemorrhage of the dermal capillaries, a white exudate, white raised areas of skin, thickening of the skin, scabbing, fissuring, a dry straw-coloured crust (possible hyperkeratinisation), reduced re-growth of fur, glossy skin and desquamation. No corrosive effects were noted. In conclusion 2 animals had a erythema or oedema score ≥ 2.3 over 72 hours (mean score of 24, 48 and 72 hours) and after 14 days the erythema score could not be read due to the presence of other adverse dermal responses. The oedema score was fully reverible.

Consequently, although the in vitro skin corrosion/irritation tests (Warren, 2010a) performed on reconstituted humans epidermis indicated no irritant potential, the in vivo studies (Guest, 1987 and 1991) showed that didecylmethylamine produced positive criteria for skin irritation following a four-hour exposure period in all rabbits.

Therefore Didecylmethylamine requires classification to CLP (Reg. n° 1272/2008/EC) and directive 67/548/EEC.

Eye irritation:

The aim of the study of Gerbeix (2010b) was to evaluate the potential irritant properties of the test item for the isolated calf cornea. This study was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practices.

The corneas were obtained from the eyes of freshly slaughtered calves at the abattoir. They were mounted in the corneal holders with the endothelial side against the O-ring of the posterior half of the holder. Both compartments of the corneal holder were filled in excesswith Minimal Essential Medium Eagle completed with 1% fetal calf serum plus penicillin/streptomycin (cMEM), then the holders were preincubated for 1 hour at 32°C. Three corneas were used for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed using an opacitometer (determining the light transmission through the center of each mounted cornea). For the treatment, the test item was used in its original form.

 The test item was tested sequentially in two consecutive experiments.

As the meanin vitroscore at the 30-minute treatment was ≤ 10, the second experiment was undertaken using a 4-hour treatment.

 At the completion of the treatment period, the test item was removed from the front opening of the anterior part of the holder and the epithelium was washed.

 Following the 30-minute treatment, the corneas were incubated for 2 hours at. At the completion of the 2-hour incubation period, the second opacity measurement was performed.

Following the 4-hour treatment, the second opacity measurement was performed immediately without any further incubation after the rinsing of the dosage form.

After the second opacity measurement, the medium was removed from both compartments of each holder. The posterior compartment was refilled with cMEM at, while the anterior compartment received 1 mL of a 5 mg/mL fluoresceine solution in Dulbecco's Phosphate-Buffered Saline (DPBS). Then, the holders were incubated vertically for 90 minutes at 32°C.

 At the end of the 90-minute incubation, the optical density of the solution from the posterior compartment of the holder was measured at 490 nm in order to determine the permeability of the cornea. Then the cornea was removed from the holder and observed for opaque spots and other irregularities.

For each experiment, the acceptance criteria were fulfilled and the study was therefore considered to be valid. No notable opaque spots or irregularities were observed on negative control corneas, either following the 30-minute treatment or following the 4 -hour treatment.

Fluoresceine fixation was observed on test item-treated corneas, following both the 30-minute and 4-hour treatments. Residual test item was also noted on corneas following the 4-hour treatment.

Following the 30-minute treatment, the meanin vitroscore was -0.3. Then following the 4-hour treatment, the meanin vitroscore was 1.3.

 

Under these experimental conditions and according to both mean in vitro scores of the 30‑minute and 4-hour treatments, the test item

was slightly irritant for the isolated calf cornea and did not require classification according to CLP (Reg. n° 1272/2008/EC) and directive 67/548/EEC.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Skin irritation/corrosion:

Based on the results of the Guest (1987,1991) studies and according to the criteria laid down in EU directive 67/548/EEC and CLP (Reg. n° 1272/2008/EC), the substance is classified Xi, R38 (irritating to skin) or skin irritant cat. 2, H315, respectively.

Eye irritation:

Based on the result of the Gerbeix study (2010b) and according to the criteria laid down in EC regulation 1272/2008/EC and EU directive 67/548/EEC, the substance is not classified for eye irritation.