Fatty acids, C16-18, reaction products with diethylenetriamine

Endpoint summary

Description of key information

in vitro skin corrosion, OECD431: not corrosive

in vitro skin irritation, OECD 439: not irritant

in vitro eye irritation, OECD 437: not classified

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Study period:

26 August 2015 - 28 August 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item without emulsifier was investigated.

Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2018
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

water

Details on test system:

After pre-incubation of the EpiDerm tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each
tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 µL of sterile water was added for wetting of the test item. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period. When dosing for the 60-Minute exposure period was complete, the same procedure was repeated
for the 3-Minute exposure period.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

3-Minute and 60-Minute exposure period

Duration of post-treatment incubation (if applicable):

Not applicable, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow
for the time taken to rinse each tissue with DPBS following exposure.

Number of replicates:

Triplicate tissues were treated with the test item, positve and negative control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative Mean Viability (%) - 3 minutes exposure period

Value:

97.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative Mean Viability (%) - 60 minutes exposure period

Value:

83.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean OD562 for the negative control treated tissues was 2.145 for the 3 Minute exposure period and 2.127 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 3.2% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20-100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Assessment of Color Interference with the MTT endpoint:
The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Direct MTT Reduction:
The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

In a Klimisch 1 GLP study from Warren (2015) the corrosivity potential using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes were investigated. The test item showed no skin corrosivity potential.

The relative mean  viability of the test item treated tissues was 97.5%  after a 3-Minute exposure period and 83.7% after a 60-Minute exposure period. The test item was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Study period:

30 September 2015 - 05 October 2015

Reliability:

2 (reliable with restrictions)

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

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:

Test item without emulsifier was investigated.

Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2018
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

The DBPS rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

Number of replicates:

Triplicate tissues were treated with the test item, positve and negative control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15-Minute exposure period - followed by 42-Hour post-exposure incubation period

Value:

105.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean tissue viability for the positive control treated tissues was 14.8% relative the negative control treated tissues and the standard deviation value of the viability was 5.4%. The positive control acceptance criteria were therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.739 and the standard deviation value of the viability was 10.2%. The negative control acceptance criteria were thereforesatisfied.

The standard deviation calculated from individual tissue viabilities of the three identically item treated tissues was 10.5%. The test item acceptance criterion was therefore satisfied.

Assessment of Color Interference with the MTT endpoint:
The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Direct MTT Reduction:
The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no skin irritation potential using the EPISKIN Reconstructed Human Epidermis Model. The test item is therefore classified as “non-irritant” in accordance with UN GHS and EU CLP 'No Category'.

Executive summary:

In a Klimisch 1 GLP study from Warren (2015) the skin irritation potential using the EPISKIN Reconstructed Human Epidermis Model was investigated. The test item showed no skin irritation potential.

The relative mean  viability of the test item treated tissues was 105.2%  after a 15-Minute exposure period and 42-Hour post-exposure incubation period. It was considered  unnecessary to perform IL-alpha analysis as the results of the MTT test were unequivocal.

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:

other: read across from analogue substance

Adequacy of study:

key study

Study period:

16 April 2015 - 06 December 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item without emulsifier was investigated.

Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2018
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared
immediately on arrival.

Vehicle:

other: 20% w/v solution in 0.9% w/v sodium chloride solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

the test item was ground to a powder and prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution. 0.75 mL of the test item preparation or control items were applied to the appropriate corneas.

Duration of treatment / exposure:

Each cornea holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red.
A post-treatment opacity reading was taken and each cornea was visually observed.
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Number of animals or in vitro replicates:

Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Details on study design:

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.

Irritation parameter:

in vitro irritation score

Run / experiment:

240 minutes incubation

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

In Vitro Irritancy Score: 1.8

Positive controls validity:

valid

Remarks:

In Vitro Irritancy Score: 68.8

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The positive control In Vitro Irritancy Score was within the range of 73.8 – 103.6. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤4.7 and permeability ≤0.085. The negative control acceptance criterion was therefore satisfied.

The corneas treated with the test item were clear post treatment.

The corneas treated with the negative control item were clear post treatment.

The corneas treated with the positive control item were cloudy post treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

No category. Not requiring classification to UN GHS or EU CLP.

Executive summary:

In a Klimisch 1 GLP study from Warren (2016) the potential of the test item to induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage using the Bovine Corneal Opacity and Permeability (BCOP) test method was investigated. The test item's In Vitro Irritancy Score was < 3. The test item was not considered to induce serious eye damage and is not requiring classification to UN GHS or EU CLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The analogue substance was tested for skin corrosion and skin and eye irritation following up-to-date in vitro testing (2015). The substance did not show any skin corrosion or irritation potential nor eye irritation pontetial.

Based on the read across considerations same results apply to Stearamide DETA.

Justification for classification or non-classification

Annex VII of the REACH Regulation includes a requirement for in vitro tests for skin corrosion (section 8.1.1) and for skin irritation (8.1.2) as well as a requirement for in vitro tests for serious eye damage/eye irritation (Annex VII, section 8.2.1). An in vivo eye/skin irritation/corrosion study shall only be considered at Annex VIII level (section 8.2) in case the in vitro serious eye damage/eye irritation test(s) are not applicable for the substance or the results obtained are not adequate for classification and risk assessment

Three validated in-vitro tests (OECD431, OECD439 and OECD437) were performed on the analogue substance for skin corrosion and skin and eye irritation. No criteria for classification under CLP were met for all the three different endpoints, therefore the substance is considered not skin corrosive or irritant, nor eye irritant.

Based on the read across considerations same classification is applied to Stearamide DETA.