Amides, tall-oil fatty, N,N-di-Me

Endpoint summary

Description of key information

An in vitro assay for skin irritation potential (and a separate in vitro assay for assessing potential dermal corrosivity) and an older in vivo eye irritation study are available for the submission substance.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-18 February 2013

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)

Version / remarks:

adopted 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Buckman Laboratories, Lot No. 12J46399
- Expiration date of the lot/batch: 01/10/2015
- Purity test date: 100%; certificate of analysis dated 05/11/2012

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: assumed stable for study duration

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult-human derived epidermal keratinocytes

Details on animal used as source of test system:

Not applicable - in vitro study

Justification for test system used:

RhE model specified in the OECD Guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN RHE Model (skinEthic Laboratories, Lyon, France)
- Delivery date: 12/02/2013
- Date of initiation of testing: 12/02/2013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of exposure each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's phosphate buffered saline (DPBS). Rinsing was achieved by filling and emptying each tissue inert for approx. 40 seconds using a constant soft stream of DPBS to gently remove residual test material.
- Observable damage in the tissue due to washing: None.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: none

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: water-killed tissues
- Procedure used to prepare the killed tissues (if applicable): untreated EPISKIN tissues were placed in a 12-well plate containing 2.2 mL of sterile distilled water, then incubated at 37°C, 5% CO2 in air for 48±1 hours.
- Result : Negligible intereference due to reduction of MTT by the test material occurred therefore the results were not used for quantitative correction of results

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 irritating to skin if the viability after 15 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritating to skin if the viability after 15 minutes exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The test material was used as supplied, 10 µL was applied to the surface of the epidermis.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Tissues treated in triplicate

Irritation / corrosion parameter:

% tissue viability

Value:

114.4

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean viability of the treated tissues was 114.4% after a 15 minute exposure period. The relative mean tissue viability for the positive control was 10.2% relative to the negative control and the standard deviation was 2.3%. The mean OD540 for the negative control was 0.834 and the standard deviation of the percent viability was 10.8%. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 8.5%. The acceptance criteria were therefore met for this assay.

An assessment found the test material was able to directly reduce MTT. An additional procedure using water-killed tissues was performed during the determination of skin corrosivity potential. The results obtained showed negligible interference due to direct MTT reduction. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Table 1. Mean OD540 values and percentage viabilities

Item	OD540 of tissues	Mean OD540 of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control	0.752	0.834	0.090	90.2	100*	10.8
	0.819			98.2
	0.930			111.5
Positive Control	0.106	0.085	0.019	12.7	10.2	2.3
	0.080			9.6
	0.068			8.2
Test material	0.912	0.954	0.071	109.4	114.4	8.5
	0.913			109.5
	1.036			124.2

SD = standard deviation; * = mean viability of the negative control is set at 100%

Interpretation of results:

other: non irritating

Conclusions:

The test material was considered to be non-irritating as the relative mean tissue viability was > 50% (114.4%).

Executive summary:

The skin irritating potential of DMATO was evaluated in the EPISKIN reconstructed human epidermis model, according to OECD Guideline 439 and GLP. Cell viability following exposure is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in treated tissues compared to relative controls.

10 µL of the test material was applied undiluted to the tissues for a 15 minute exposure period, followed by a 42 hour post-exposure incubation period. The test material, positive and negative controls were tested in triplicate. At the end of the post-exposure incubation period each tissue was taken for MTT loading. After MTT loading a total biopsy of each epidermis was made and placed into tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. At the end of the formazan extraction period each tubed was mixed and duplicate 200 µL samples were transferred to a 96 -well plate. The optical density was measured at 540 nm. The percentage viability is calculated from the MTT reduction in treated tissues relative to the negative control.

The relative mean viability of the treated tissues was 114.4%. The relative mean tissue viability was greater than 50%, therefore the test material is considered to be non-irritating under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1971

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Older proprietary study conducted prior to development of guidelines and GLP, limited information available on methods but adequate for the purposes of hazard classification.

Qualifier:

no guideline followed

Principles of method if other than guideline:

In vivo eye irritation study in rabbits, conducted prior to development of the guidelines

GLP compliance:

no

Remarks:

study conducted prior to development of GLP

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

The animals were adult albino New Zealand White rabbits.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL undiluted test material was applied

Duration of treatment / exposure:

Not applicable - single treatment

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

9

Details on study design:

Rabbits were placed in a collar to prevent rubbing of the treated eye. The test substance (0.1 mL) was instilled into one eye, the other eye served as an untreated control. Reactions were assessed at 24, 48 and 72 hours after instillation. The test material was not washed from the eyes following instillation, however any test material residue/discharge was flushed from the eye prior to scoring. Reactions were evaluated according to the degree of damage caused to the cornea, iris and the bulbar and palpebral conjunctivae.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

9 rabbits

Time point:

other: 24 hours

Score:

0

Irritation parameter:

iris score

Basis:

mean

Remarks:

9 rabbits

Time point:

other: 24 hours

Score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

9 rabbits

Time point:

other: 24 hours

Score:

0.3

Reversibility:

fully reversible within: 48h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

9 rabbits

Time point:

other: 24 hours

Score:

0

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

9 rabbits

Time point:

other: 48 hours

Score:

0

Irritation parameter:

iris score

Basis:

mean

Remarks:

9 rabbits

Time point:

other: 48 hours

Score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

9 rabbits

Time point:

other: 48 hours

Score:

0

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

9 rabbits

Time point:

other: 48 hours

Score:

0

Irritant / corrosive response data:

A score of 1 was assigned to 3 rabbits for conjunctival redness at the 24 hour time point. No other reactions were observed at the 24 hour reading. At 48 hours all readings were negative. Scores were not presented for the 72 hour time point.

Other effects:

No other effects reported.

No additional information available

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the results of this study the test material is considered to be non-irritating to the eye.

Executive summary:

The eye irritation potential of DMAD was evaluated in 9 New Zealand White rabbits. A quantity of 0.1 mL test material was instilled into one eye of the rabbits, the other eye remained untreated to serve as a control. Ocular reactions were evaluated at 24, 48 and 72 hours post-instillation; any residual test material or discharge was flushed from the eye prior to evaluations. The test material caused minimal conjunctival irritation in 3 of the 9 rabbits at 24 hours post-instillation. No other reactions were observed, and no signs of irritation were observed at the 48 hour time point. Based on the results of this study, the test material is considered to be non-irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

The skin irritating potential of DMATO was evaluated in the EPISKIN reconstructed human epidermis model, according to OECD Guideline 439 and GLP. The relative mean tissue viability (assessed by MTT reduction) was greater than 50%, therefore the test material is considered to be non-irritating under the conditions of the study.

The skin corrosion potential of DMATO was evaluated in the EPISKIN in vitro Reconstructed Human Epidermis (RHE) Model according to OECD guideline 431 and GLP. Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes. Cell viability following exposure is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in treated tissues compared to relative controls.Data were presented in the form of percentage tissue viability (MTT reduction in the test material treated tissues relative to negative control tissues). The relative mean viability of the test material was 97.9% after 240 minutes exposure; 110.6% after 60 minutes exposure, and 114.1% after 3 minutes exposure. The relative mean tissue viability for the positive control treated tissues was 6.3% relative to the negative control following the 240 minute exposure period. The mean OD540 for the negative control tissues was 0.811. Therefore, the test material was considered to be non-corrosive to the skin, and classification is not required.

The eye irritation potential of DMAD was evaluated in 9 New Zealand White rabbits. A quantity of 0.1 mL test material was instilled into one eye of the rabbits, the other eye remained untreated to serve as a control. Ocular reactions were evaluated at 24, 48 and 72 hours post-instillation; any residual test material or discharge was flushed from the eye prior to evaluations. The test material caused minimal conjunctival irritation in 3 of the 9 rabbits at 24 hours post-instillation. No other reactions were observed, and no signs of irritation were observed at the 48 hour time point. Based on the results of this study, the test material is considered to be non-irritating to the eye.

Justification for selection of skin irritation / corrosion endpoint:
In vitro assays for skin irritation and skin corrosivity both give negative results.

Justification for selection of eye irritation endpoint:
Only study available for this endpoint

Justification for classification or non-classification

Classification for skin or eye irritation according to the Dangerous Substances Directive (67/548/EEC) or the CLP Regulation (1272/2008) is not triggered by the results of the available studies.