Fatty acids, C18 (even numbered) and C18 unsatd., reaction products with triethanolamine, di-Me sulfate quaternized

Endpoint summary

Description of key information

Skin irritation
Not irritating to skin, OECD TG 404, in vivo, rabbit, RL1; GLP (4 h semiocclusive, intact skin; grade 1-2 edema + erythema; fully reversible within 14 d); read-across: partially unsaturated TEA-Esterquat
Eye irritation
Not irritating to eye, weight of evidence:
- OECD TG 437, in vitro, BCOP, RL1; GLP (IVIS = 6; slight increase of the corneal opacity and permeability; not severely irritating)
- OECD TG 405, in vivo, rabbit, RL1; GLP (slight to moderate ocular reactions, fully reversible within 7 d; not irritating to eyes); read-across: partially unsaturated TEA-Esterquat
- HET-CAM, in vitro, RL2; GLP (score: 0.67 of maximum 21; vascular injection on the CAM of 4 eggs 5 minutes post application; no other effect detected; not irritating); read-across: partially unsaturated TEA-Esterquat

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because
• they share structural similarities with common functional groups: One quaternised ethanolamine moiety, one to three, mainly two ester groups with a typical UVCB distribution with long-chain fatty acids of natural origin. The molecular structure is almost identical.
• they are manufactured from similar resp. identical precursors (triethanolamine, long-chain fatty acids, dimethyl sulphate) under similar conditions. Therefore common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident
• A constant pattern in the changing of the potency of the properties across the TEA-Esterquats by chain-length and the grade of esterification is not observed, because the fatty acid chain-length distribution is too narrow and similar and the distribution of mono-, di-, and tri-esters is identical. Some variation caused by variation in C=C double bonds may occur and will be discussed at the relevant endpoint.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See general justification for read-across attached to chapter 13 of this IUCLID file.

3. ANALOGUE APPROACH JUSTIFICATION
See general justification for read-across attached to chapter 13 of this IUCLID file.

4. DATA MATRIX
See general justification for read-across attached to chapter 13 of this IUCLID file.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Species:

rabbit

Strain:

New Zealand White

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: The right flank remained untreated and served as a control.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g undiluted

Duration of treatment / exposure:

4 hours

Observation period:

1 hour and 24, 48, 72 hours 7, 14 and 21 days after application of the test substance.

Number of animals:

3 animals, 1 male and 2 females

Details on study design:

TEST SITE
- Area of exposure: Samples of 0.5 g of the test substance have been put on a gauze pad and spread over an area of 2 cm x 3 cm; the gauze pad then was applied to the skin.
- % coverage: One gauze pad of 4 cm x 5 cm size (Askina® Brauncel, B. Braun Petzold GmbH, Melsungen, Germany) with test substance was applied to the left flank of one animal. It was held in place by strips of Micropore (3M company, St. Paul, USA). The semi-occlusive dressing was bandaged with Acrylastic (Beiersdorf AG, Hamburg, Germany) and fixed with Leukoplast (Beiersdorf AG, Hamburg, Germany).


REMOVAL OF TEST SUBSTANCE
- Washing (if done): after exposure skin was cleaned carefully with lukewarm water
- Time after start of exposure: 4 hours

SCORING SYSTEM: as stipulated by OECD guideline 404, 2002

Irritation parameter:

erythema score

Basis:

animal: #1, #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal: #1, #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Symptoms of skin irritation (erythema and edema) were observed until 7 days after application. Well-defined erythema and slight edema formation (grading 1 or 2 in each case) were observed within 72 hours after patch removal in two animals. In one animal only very slight, barely perceptible erythema and edema occurred. 7 days after patch removal, very slight erythema and edema (grading 1) were still existent in two animals, which was reversible after 14 days. In addition, slight desquamation was observed in all three animals at observations at 72 hours, 7 days and 14 days after application. 21 days after application, all three animals were without any sign of skin irritation.

Other effects:

No symptoms of toxicity caused by the test substance or any unusual finding were observed.

Irritant/corrosive response data for each animal at each observation time up to removal of each animal from the test

Score at time point / Reversibility	Erythema	Edema
	Max. score: 4	Max. score: 4
60 min	1/2/1	0/0/0
24 h	2/2/1	1/1/0
48 h	2/2/1	2/2/1
72 h	2/2/1	2/2/1
7 days	1/1/0	1/1/0
14 days	0/0/0	0/0/0
Average 24h, 48h, 72h	2/2/1	1.67/1.67/0.67
Reversibility*)	c/c/c	c/c/c
Average time (unit) for reversion	14 days	14 days

*) Reversibility: c. = completely reversible; n.c. = not completely reversible; n. = not reversible

Desquamation persists at the 14 day observation, but was fully reversible at study day 21.

Interpretation of results:

GHS criteria not met

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:

weight of evidence

Study period:

2010-04-07 to 2010-04-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2002

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL test substance.
As the test substance is a firm solid paste it was melted in a water bath at 60 °C resulting in the formation of a smooth paste. Subsequently it was held at room temperature until it cooled down to 40 °C. Then 0.1 mL of the paste was applied by means of a syringe to the eye of one animal.

Observation period (in vivo):

1 hour and 24, 48, 72 hours and 7 days after application of the test substance.

Number of animals or in vitro replicates:

3 animals, 1 male and 2 females

Details on study design:

SCORING SYSTEM: as outlined in the OECD guideline 405, 2002

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein
Examinations of ocular reactions were facilitated without any device. After recording the observations at 24 hours, the eyes of all rabbits were further examined with the aid of fluorescein to look for damage of the cornea.
One drop of a 0.5 % (w/v) fluorescein solution in saline was poured into both eyes and washed off with saline solution 30 seconds later. Afterwards
scoring was executed by use of an UV-light lamp. This procedure was repeated at all observation time points until cornea damage was no longer
observed.

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal: #1, #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal: #2, #3

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

All 3 animals showed slight to moderate ocular reaction 1 hour after application of the test substance. After 24 hours slight to moderate ocular reactions were still observed in all three animals. Slight cornea opacity was observed in one animal at the 24 hour reading. The cornea damage was no longer observed 48 hours after treatment. Slight irritation of the conjunctivae persists until 72 hours but was fully reversible 7 days after application in all three animals.

Other effects:

No symptoms of systemic toxicity were observed during the whole study.

Interpretation of results:

GHS criteria not met

Conclusions:

Classification is based on effects on the cornea, iris and conjunctiva calculated as mean scores following grading at 24, 48 and 72 hours after instillation of the test material, in consideration of reversibility.
No classification for serious eye damage/eye irritation is justified for the partially unsaturated TEA-Esterquat according to CLP, EU GHS (Regulation (EC) No 1272/2008).

Executive summary:

In a primary eye irritation study according to OECD Guideline 405, 2002 0.1 mL of the partially unsaturated TEA-Esterquat was instilled into the conjunctival sac of three New Zealand White rabbits. Animals then were observed for 7 days. Irritation was scored by the method stipulated by the OECD Guideline 405.

All three animals showed slight to moderate ocular reaction 1 h after application of the test substance. 24 h after application slight to moderate ocular reactions were still observed in all three animals. Slight cornea opacity was observed in one animal 24 h after application. The cornea damage was not further observed 48 h after treatment. Slight irritation of the conjunctivae persists until 72 h but 7 days after application all three animals were without any sign of ocular irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For the assessment of the eye irritation potential of the target substance C18 and C18 unsatd. TEA-Esterquat an in vitro BCOP assay is available as well as a HET-CAM test and an in vivo study conducted with the closely related source substance partially unsaturated TEA-Esterquat.

No experimental data are available for the assessment of the skin irritation potential of the target substance C18 and C18 unsatd. TEA-Esterquat. However, reliable relevant data are available for the closely related source substance partially unsaturated TEA-Esterquat. A justification for read-across is attached to iuclid section 13.

 

Skin irritation

In a primary dermal irritation study performed according to OECD Guideline 404, three New Zealand White rabbits, were semi-occlusive dermally exposed to 0.5 g of the partially unsaturated TEA-Esterquat for 4 hours to approximately 6 cm² of body surface area. Irritation was scored by the method as stipulated by OECD Guideline 404. Animals were than observed for 21 days.

Symptoms of skin irritation (erythema and edema) were observed until 7 days after application. Well-defined erythema and slight edema formation (grading 1 or 2 in each case) were observed within 72 hours after patch removal in two animals. In one animal only very slight, barely perceptible erythema and edema occurred. 7 days after patch removal, very slight erythema and edema (grading 1) were still existent in two animals, which was reversible after 14 days. In addition, slight desquamation was observed in all three animals at observations at 72 hours, 7 days and 14 days after application. 21 days after application, all three animals were without any sign of skin irritation.

 

Due to technical difficulties in the in vitro skin irritation test, it was impossible to apply the test item at 35°C homogeneously and without damaging the skin tissue because of its pasty and sticky texture. Thus, no test on the substance itself could be performed.

However, as described in the justification for read-across in more detail, there are only slight differences in composition between the target substance C18 and C18 unsatd. TEA-Esterquat and the source substance partially unsaturated TEA-Esterquat.

The source substance partially unsaturated TEA-Esterquat contains considerable amounts of C16, whereas the target substance consists predominantly of C18 satd. C-chains. As fatty acids with chain lengths of C16 and C18 have in general a low irritation potential (HERA, 2002; Stillman, 1975; Aungst, 1989), this slight difference is not considered of relevance for the endpoint irritation.

There are also slight differences in the degree of unsaturation. However, the amount of unsaturated fatty acid moieties is slightly higher in the source substancepartially unsaturated TEA-Esterquat compared to the target substanceC18 and C18 unsatd. TEA-Esterquat. As a slightly higher irritation potential has been associated with C18 unsaturated fatty acid moieties compared to fully saturated moieties of the same chain length(HERA, 2002; Stillman, 1975; Aungst, 1989), the source substance may be considered as worst case.

For that reason, the result obtained with thesource substance partially unsaturated TEA-Esterquat is applicable also to the target substance C18 and C18 unsatd. TEA-Esterquat. 

 

Eye irritation

An in vitro study was performed to assess the corneal irritation and damage potential of C18 and C18 unsatd. TEA-Esterquat by means of the BCOP assay using fresh bovine corneas according to OECD guideline 437, adopted adopted on 26 July 2013.

As the test substance is a surfactant, it was applied at 10% in water in accordance with the testing guideline. The corneas were incubated with the test substance and controls for 10 min. After rinsing with culture medium, the corneas were incubated for another 2 h at 32±1°C. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS <3 does not need to be classified; a substance inducing an IVIS > 55 is defined as a corrosive or severe irritant.

The test item caused a slight increase of the corneal opacity and permeability. The calculated mean in vitro score was 6.

The positive control (ethanol) increased the opacity and permeability of the corneas (mean in vitro score 39).

Since the mean in vitro irritancy score of the test substance was <3 and >55, C18 and C18 unsatd. TEA-Esterquat was not severely irritating / corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

 

Further data from the closely related source substance partially unsaturated TEA-Esterquat are used in a weight-of-evidence approach:

 

In a primary eye irritation study according to OECD Guideline 405, 2002 0.1 mL of partially unsaturated TEA-Esterquat was instilled into the conjunctival sac of three New Zealand White rabbits. Animals then were observed for 7 days. Irritation was scored by the method stipulated by the OECD Guideline 405.

All three animals showed slight to moderate ocular reaction 1 h after application of the test substance. 24 h after application slight to moderate ocular reactions were still observed in all three animals. Slight cornea opacity was observed in one animal 24 h after application. The cornea damage was not further observed 48 h after treatment. Slight irritation of the conjunctivae persists until 72 h but 7 days after application all three animals were without any sign of ocular irritation.

 

In a hen's egg chorioallantoic membrane test (HET-CAM), an accepted alternative method to the Draize test, the potential to cause irritation upon first contact with eyes or mucous membranes was assessed by application of the test substance onto the chorioallantoic membrane (CAM). 200 mg of partially unsaturated TEA-Esterquat were applied to an area of approximately 1 cm x 1 cm of the chorioallantoic membrane (CAM) of six fertilized and incubated eggs, strain SPAFAS.

The occurrence of vascular injection, haemorrhage and coagulation was recorded 30 seconds, 2 minutes and 5 minutes after application.

To assure the evaluation of the treated CAM the test substance was detached with tweezers after 30 seconds, 2 minutes and 5 minutes, respectively, because the test substance hindered continuous observation of the CAM. After 30 seconds evaluation and 2 minutes evaluation the same area of the CAM was covered with the same test substance again.

Treatment with the test substance resulted in the appearance of vascular injection on the CAM of 4 eggs 5 minutes post application. No other effect was detected after treatment (0.67 scores of total 21 possible scores). Thus, partially unsaturated TEA-Esterquat was graded as not irritating according to the evaluation scheme.

Application of tap water onto the CAM showed no effects; the irritation index therefore was 0 of 21 total possible scores.

Treatment with a 5 % active substance preparation of the reference substance Texapon ASV 70 Spezial resulted in an irritation index of 10.3 of 21 total possible scores.

For an anticipation of the irritation potential in-vivo,the results obtained with the Texapon ASV 70 Spezial are taken into consideration. A 5% active substance preparation of Texapon ASV 70 Spezial is known to be only slightly irritating in-vivo (INVITOX Protocol Number 96, 1994, p4). A 5 % active substance preparation of Texapon ASV 70 Spezial reached 10.3 of 21 total possible scores. From this it can be concluded that partially unsaturated TEA-Esterquat will be not irritating in vivo if the result obtained with the reference substance were taken into consideration.

 

The result from the BCOP alone would not be sufficient conclude on the eye irritation potential ofC 18 and C18 unsatd. TEA-Esterquat as the score was slightly above the cut-off value for non-classification. In this case no further testing is required as data from a closely related source substance are available and are used in a weight of evidence approach.

The source substance partially unsaturated TEA-Esterquat contains considerable amounts of C16, whereas the target substance consists predominantly of C18 satd. C-chains. There are also slight differences in the degree of unsaturation. However, the amount of unsaturated fatty acid moieties is slightly higher in the source substancepartially unsaturated TEA-Esterquat compared to the target substanceC18 and C18 unsatd. TEA-Esterquat.

Fatty acids with chain lengths of C16 and C18 have in general a low irritation potential. Nevertheless,a slightly higher irritation potential has been associated with C18 unsaturated fatty acid moieties(HERA, 2002; Stillman, 1975; Aungst, 1989), thus, the source substance is considered as a worst case.

Considering all available data together, C18 and C18 unsatd. TEA-Esterquat is considered to be not irritating to eyes.

 

 

Respiratory irritation

No data on the respiratory irritation of C18 and C18 unsatd. TEA-Esterquat are available.

 

There are no data gaps for the endpoint irritation/corrosion. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

 

Similar results were obtained with the source substance MDEA-Esterquat C16-18 and C18 unsatd.: The substance is not classified for skin or eye irritation. These data are included into the dossier to demonstrate, that the substances have a similar toxicological profile.

 

Justification for classification or non-classification

Based on the available data, C18 and C18 unsatd. TEA-Esterquat does not need to be classified for skin and eye irritation according to regulation (EC) 1272/2008. Thus, no labelling is required.