Fatty acids, C16-18 and C18-unsatd., branched and linear, reaction products with diethylenetriamine and tall-oil fatty acids, Me maleates

Endpoint summary

Description of key information

The test substance is a skin irritant,but not corrosive based on the outcome of the in vitro EPISKIN test (Harlan 2012h and Harlan 2013e). No eye irritation was found in both an in vitro assay (Harlan 2012i) and an in vivo skin irritation test in rabbits (Harlan 2013f)

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:

11-04-2012 to 17-04-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed according to the guideline and under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

exposure period 15 minutes with post-exposure incubation period of 42 hours

GLP compliance:

yes (incl. QA statement)

Details on study design:

The EpiSKIN assay uses reconstructed human epidermis to assess skin irritation. For the viability test enzymatic conversion of MTT to formazan is used.

In a pre-test direct interaction of the test substance with the detection chemical MTT is assessed.
In the main study triplicate tissues were treated with 30 uL of the test substance during 15 minutes. In addition a negative control (DPBS) and a positive control (5% SDS) were included. After treatment tissues were rinsed, incubated in maintenance fluid for 42 hours at 37 °C and treated with MTT during 3 hours (at 37 °C). After MTT loading a total biopsy of each epidermis (epidermis and collagen) was made, that was suspended in acidified iso-propanol for 3 days and kept in the refrigerator at 1-10 °C. Thereafter the optical density of the extracted MTT solutions was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control.
Tissue viability > 50% means non-irritant
Tissue viability ≤ 50% means irritant

Other effects:

The test substance did not interfere with the reduction of MTT to blue formazan salt.
In the main test after rinsing, the test substance was found to remain on the tissue surface due to the extreme viscosity.

	Mean OD540 ± SD	Relative mean viability ± SD
Negative control:	0.883 ± 0.087	100 ± 9.8%
Test substance:	0.090 ± 0.051	10.2 ± 5.8%
SDS:	0.053 ± 0.018	6.0 ± 2.1%

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: tissue viability > 50%: non-irritant

Conclusions:

The test substance is irritant in the EPISKIN reconstructed human epidermal model.

Executive summary:

In this assay triplicate reconstructed human skin tissues were treated with 30 uL of the test substance during 15 minutes. After treatment tissues were rinsed, kept for 42 hours and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control. The viability was 10.2%, which is indicative for irritant substances.

The test substance is irritant in the EPISKIN reconstructed human epidermal model.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

15-04-2013 to 25-04-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study according to the guidelines under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: 12 to 20 weeks
- Weight at study initiation: 2.50 to 2.71 kg
- Housing: individually
- Diet: ad libitum (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon)
- Water; ad libitum
- Acclimation period: minimal 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): ≥15/hr
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.1 mL applied to the conjunctival sac of the right eye

Observation period (in vivo):

1, 24, 48 and 72 hour observations

Number of animals or in vitro replicates:

2

Details on study design:

ANAESTHETICS AND ANALGESICS
– doses and times when topical anaesthetics and systemic analgesics were administered:
systemic: buprenorphine 0.01 mg/kg sc 60 minutes prior to dosing
local: 0.5% tetracaine hydrochloride (2 drops ineach eye) 5 minutes prior to dosing
systemic: buprenorphine 0.01 mg/kg sc and meloxicam 0.5 mg/kg sc 8 hours after dosing
- interaction with test substance: none reported
- inititial pain score: not reported

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: standard ophthalmoscope (no fluorescein used)

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: average 24-72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

mean

Time point:

other: average 24-72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: avarage 24-72h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: average 24-72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

not irritant

Other effects:

No effects on body weight

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

The test substance is not irritating to the eyes

Executive summary:

In an in vivo test two rabbits were treated with the test substance (0.1 mL applied in the conjunctival sac of one eye) after systemic and local analgesia/anaesthesia . No signs of irritatiion except for very slight effects on the conjunctivae after 1, 24 and 48 hours became apparent. After 72 hours no effects were noted. Therefore it is concluded that the test substance is not irritating to the eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

In an in vitro assay (EpiSKIN reconstructed human epidermal model) for irritation, triplicate skin tissues were treated with 30 uL of the test substance during 15 minutes. After treatment tissues were rinsed, kept for 42 hours and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. The viability was 10.2%, which is indicative for irritant substances (Harlan 2012h).

In the EpiSKIN test for corrosion, duplicate reconstructed human skin tissues were treated with 50 uL of the test substance during 3, 60 and 240 minutes. After treatment tissues were rinsed and incubated with MTT for 3 hours (at room temeprature). Thereafter tissues were allowed to dry, epidermis and collagen were separated, suspended (after vortexing) in isopropanol and kept overnight (all procedures in dark). Optical density of homogene solutions of MTT treated tissues was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control. The viability was 68.7 -97.0% for the different exposure times, which is indicative for non-corrosive substances (Harlan 2013e).

Eye irritation

In the SkinEthic test tissues were treated with 30 uL of the test substance during 10 minutes. After treatment tissues (2 per treatment) were rinsed and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. The viability was 60.2%, which is indicative for non-irritant substances. As the assay is not formally validated, the results need to be regarded as preliminary (Harlan 2012i).

In an in vivo test two rabbits were treated with the test substance (0.1 mL applied in the conjunctival sac of one eye) after systemic and local analgesia/anaesthesia . No signs of irritatiion except for very slight effects on the conjunctivae after 1, 24 and 48 hours became apparent. After 72 hours no effects were noted. Therefore it is concluded that this test confirms the results of the in vitro assay and the test substance is not irritating to the eyes (Harlan 2013f).

Justification for selection of skin irritation / corrosion endpoint:
study according to the guidelines under GLP with positive result

Justification for selection of eye irritation endpoint:
In view of the negative results in the in vitro eye irritation study and the positive in vitro test for skin irritation, the in vivo test was performed to confirm the negative outcome.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

The substance needs to be classified for skin irritation based on the outcome of the in vitro skin irritation test (H315). The substance is not corrosive (Harlan 2013e). No classification is necessary for eye irritation as both tests available did not show any effects of the test substance on the eye.