1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with C18-unsatd. fatty acids, Me sulfates (salts)

Endpoint summary

Description of key information

Skin irritation: irritating (OECD TG 404/EU method B.4; GLP), applied to the intact skin for 4 hours, mean erythema scores at 24, 48 and 72 h after patch removal were >/=2.3 for 2/3 animals; mean edema scores were >/=2.3 for all three animals
Eye irritation: serious eye damage (OECD TG 437/EU method B.47; GLP), IVIS = 99.30 (100% test substance);
20% test substance: serious eye damage (OECD TG 437/EU method B.47; GLP: IVIS = 52.59 / OECD TG 405/EU method B.5; GLP: 0.1 mL of 20% test substance in olive oil applied to the eye of 2 rabbits; iridial inflammation (grade 1), moderate conjunctival irritation (grade 2 redness and grade 2 chemosis),corneal vascularisation in one animal considered irreversible 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-11-24 to 2010-11-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(23 July 2009)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(22 July 2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material: 1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me-sulfates (salts)
- Physical state: liquid
- Analytical purity: 100%

Species:

other: in vitro test

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Controls:

other: positive, negative and quality control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL

Duration of treatment / exposure:

60 ± 1 min

Observation period:

42 ± 2 h

Number of animals:

triplicates

Details on study design:

TEST SYSTEM
EpiDerm EPI-200-SIT (MatTek Corporation, 200 Homer Avenue, Ashland, MA 01721, USA); Lot: 13885

APPLICATION
- test substance was applied topically for 60 min (± 1 min) to the EpiDerm RHE
- after washing: post incubation period of a total of 42 h (± 2 h) at 37°C (± 1°C) and 5% CO2 (± 1%)
- cell viability was determined by using the standard MTT assay

Negative control: 30 µL ultrapure water, 60 ± 1 min
Positive control: 30µL 5% SDS, 60 ± 1 min
Quality control: 30 µL 1% Triton X-100, 2 h ± 15 min

REMOVAL OF TEST SUBSTANCE
- after 60 min rinsing with PBS (15 times)

MTT ASSAY
- at the end of the 42 h (± 2 h) post incubation period, tissues were transferred into 24 well plate prepared for the Standard MTT Assay, each well containing 300 µL MTT medium
- the plates were incubated for 3 h (± 5 min) at 37°C (± 1°C) and 5 % CO2 (± 1 %)
- formazan dye was extracted from the tissues with Isopropanol for 2 h at room temperature while shaking at approx. 120 rpm
- from each tissue 2 x 200 µL aliquots of the blue formazan solution were transferred into a 96 well microtiter plate; OD was determined at 550 nm

DATA ANALYSIS
- viability in % of the treated tissues was calculated in relation to the corresponding negative control

SCORING SYSTEM:
- cell viability measurement (MTT assay, OD at 550 nm)
- mean tissue vialbility > 50% = non-irritant
- mean tissue viability

Irritation / corrosion parameter:

% tissue viability

Value:

82.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Although residual test substance adhered to the reconstructed epidermis after the rinsing procedure, the overall performance of the study was not affected, because it has been demonstrated that the test substance did not chemically reduce MTT and the cell viability was not decreased below the defined threshold limits.
The positive control (5% SDS) decreased the cell viability to 7.8%. The quality control (1% Triton X-100) experiment resulted in a tissue viability of 95.9% demonstrating intact barrier function of the test system.

No direct MTT reduction capacity of the test substance has been observed.

 

Viability of epidermal models:

 

	Quality control - Skin Barrier 1% Triton X-100	Negative control	Positive control 5% SDS	Test substance
60 min exposure; 42 h post exposue	-	100.0 (± 1.2)	7.8 (± 1.3)	82.7 (± 1.5)
2 h exposure	95.5 (± 1.4)	100.0 (± 4.0)	-	-
result	valid	valid	valid	Not irritant

 

Interpretation of results:

GHS criteria not met

Conclusions:

MDIPA Esterquat C18 unsatd. is not irritating in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In a dermal irritation study according to OECD Guideline 439 (In Vitro Skin Irritation) (22 July 2010) and EU method B.46 (In vitro skin irritation: reconstructed human epidermis model test) (23 July 2009), 30 µL MDIPA Esterquat C18 unsatd. (100% a.i.) was applied in triplicates for 60 min to a three-dimensional human epidermis model (EpiDerm EPI-200-SIT, MatTek Corporation).

After 60 minutes exposure at 37°C, the tissues were rinsed 15 times with phosphate buffered saline to remove residual test substance. Subsequently the skin tissues were incubated for 42 ± 2 h at 37°C.

Cytotoxic (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Positive (5% SDS), negative (ultrapure water) and quality (1% Triton X-100) control gave the appropriate response.

The relative mean tissue viability obtained after 60 minutes treatment with MDIPA Esterquat C18 unsatd. compared to the negative control tissues was 82.7 ± 1.5%. Since the mean relative tissue viability for the test substance was above 50%, MDIPA Esterquat C18 unsatd. is considered to be not irritating.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-02-21 to 2011-03-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

(30 May 2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

(24 April 2002)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material: 1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me-sulfates (salts)
- Physical state: liquid
- Analytical purity: 100%

Species:

rabbit

Strain:

other: New Zealand White, CRL:KBL (NZW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2499.9 g and 2469.2 g (female), 2382.6 g (male)
- Housing: individually in steel cages with a plastic bottom mould
- Diet (e.g. ad libitum): pelleted diet type "2023" from Altramin International, Lage, Germany, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3°C
- Humidity (%): 30 to 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated right flank served as control region

Amount / concentration applied:

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

Duration of treatment / exposure:

4 h

Observation period:

21 d

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 6 cm²
- Type of wrap if used: a gauze pad was held in place by strips of Micropore; semi-occlusive dressings were bandaged with Acrylastic and fixed with Leukoplast

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with lukewarm soapy water
- Time after start of exposure: 4 h

SCORING SYSTEM: OECD guideline 404

Irritation parameter:

erythema score

Basis:

animal: #1, #2

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

fully reversible within: 21 d

Remarks on result:

other: on day 14 grade 1 erythema still present

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 d

Remarks on result:

other: on day 14 grade 1 erythema still present

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

edema score

Basis:

animal: #2, #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritant / corrosive response data:

Well-defined to moderate erythema and slight to moderate edema (grading 2 or 3) was observed within 72 hours after patch removal in all three animals. 7 days after patch removal very slight to slight erythema and edema (grading 1 or 2) was observed and 14 days after patch removal slight erythema only was still existent in all animals. 21 days after application all three animals were without any sign of skin irritation.
In addition strong desquamation and atrophy of the skin was observed in all three animals beginning 7 days p.a. These skin reactions declined with time as well. Grade 1 atrophy was still present in 1 animal on day 21.

Other effects:

No systemic signs of toxicity were observed; body weight development was positive and within normal range.

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
60 min	2/2/2	1/2/1
24 h	2/2/2	2/2/2
48 h	3/3/2	3/3/3/
72 h	3/3/2	2/3/3
7 d	2/2/1	1/1/1
14 d	1/1/1	0/0/0
21 d	0/0/0	0/0/0
Average 24, 48, 72 h	2.67/2.67/2	2.33/2.67/2.67

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this study MDIPA Esterquat C18 unsatd. was irritating to the skin.
Mean erythema scores from observations at 24, 48 and 72 h after patch removal were >/=2.3 for 2/3 animals; mean edema scores were >/=2.3 for 3/3 animals. Very slight erythema was still present in all animals on day 14, but reversible within 21 d. Edema was fully reversible in all 3 animals within 14 days.

Executive summary:

In a primary dermal irritation study according to OECD guideline 404 (24 April 2002) and EU method B.4 (30 May 2008) young adult New Zealand White, CRL:KBL (NZW) rabbits (2 female, 1 male) were dermally exposed to 0.5 mL of undiluted MDIPA Esterquat C18 unsatd. (100% a.i.) for 4 hours to 6 cm² body surface area. Animals then were observed for 21 days.  Irritation was scored by the method of Draize.

Well-defined to moderate erythema and slight to moderate edema (grade 2 or 3) was observed within 72 hours after patch removal in all three animals. 7 days after patch removal very slight to slight erythema and edema (grading 1 or 2) was observed and 14 days after patch removal very slight erythema (grade 1) was still existent in all animals. 21 days after application all three animals were without any sign of skin irritation.

Additionally, strong desquamation and atrophy of the skin was observed in all three animals beginning on day 7. These skin reactions declined with time as well. Grade 1 atrophy was still present in one animal on day 21.

Mean erythema scores from observations at 24, 48 and 72 h after patch removal were >/=2.3 for 2/3 animals; mean edema scores were >/=2.3 for all three animals.

In this study MDIPA Esterquat C18 unsatd. was irritating to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-12-06 to 2012-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

24 April 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Chemical name: 1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me-sulfates (salts)
- Name of test material (as cited in study report): HH-2010-324
- Physical state: liquid
- Analytical purity: 100%

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12 – 20 weeks
- Weight at study initiation: 2.64 - 2.94 kg
- Housing: individually in suspended cages
- Diet (e.g. ad libitum): 2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

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

Vehicle:

other: olive oil

Controls:

other: the contralateral eye was treated with 0.1 mL olive oil as vehicle control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): 20% in olive oil

Duration of treatment / exposure:

the eyes were not rinsed after treatment

Observation period (in vivo):

21 d

Number of animals or in vitro replicates:

2

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the eyes were not rinsed

SCORING SYSTEM: Draize scale, OECD guideline 405

TOOL USED TO ASSESS SCORE: light source from a standard ophthalmoscope

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: vascularisation was still present in animal #2 at day 14 and 21

Irritation parameter:

iris score

Basis:

animal: #1, #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 to 14 d

Irritation parameter:

conjunctivae score

Basis:

animal: #1, #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: grade 1 redness still present in animal #2 on day 14, reversible within 21 d

Irritation parameter:

chemosis score

Basis:

animal: #1, #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritant / corrosive response data:

Test item:
Scattered or diffuse corneal opacity (grade 1) was noted in one treated eye one hour after treatment and in both treated eyes at the 24, 48, 72 h and 7 d observations. Vascularisation, with a localised ingrowth of vessels at the lower edge of the cornea, was noted in one treated eye at the 14 and 21 d observations.
Iridial inflammation (grade 1) was noted in one treated eye one hour after treatment, in both treated eyes at the 24, 48, 72 h observations and in one treated eye at the 7 d observation.
Moderate conjunctival irritation (grade redness and grade 2 chemosis) was noted in both treated eyes one hour after treatment and at the 24, 48 and 72 h observations. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 7 d observation. Minimal conjunctival irritation was noted in one treated eye at the 14 d observation. Petechial haemorrhage over the nictitating membrane was noted in one treated eye at the 72 h observation. Alopecia around the treated eye was noted in one animal at the 7 d observation.
One treated eye appeared normal at the 14 d observation. The corneal vascularisation noted in one treated eye was considered to be irreversible.

Vehicle control:
No corneal or iridial effects were noted during the study. Minimal conjunctival irritation was noted in both treated eyes one hour after treatment. Both treated eyes appeared normal at the 24 h observation.

Other effects:

Both animals showed expected gain in bodyweight during the study.

Irritant/corrosive response data for each animal at each observation time: test substance

Timepoint	Cornea		Iris	Conjunctivae		Discharge
	score	area		redness	swelling
1 h	0/1	0/2	0/1	2/2	2/2	2/2
24 h	1/1	2/2	1/1	2/2	2/2	3/2
48 h	1/1	2/2	1/1	2/2	2/2	2/2
72 h	1/1	1/3	1/1	2/2Pt	2/2	1/2
7 days	1/1	1/2	0/1	1Al/2	1/1	1/1
14 days	0/0V	0/0	0/0	0/1	0/0	0/1
21 days	-/0V	-/0	-/0	-/0	-/0	-/0
Average 24h, 48h, 72h	1/1	Not calculated	1/1	2/2	2/2	Not calculated

  - = not assessed

Al = Alopecia around the treated eye

Pt = Petechial haemorrhage over the nictitating membrane

V =Vascularisation, with a localised ingrowth of vessels at the lower edge of the cornea

Irritant/corrosive response data for each animal at each observation time: vehicle control

Timepoint	Cornea		Iris	Conjunctivae		Discharge
	score	area		redness	swelling
1 h	0/0	0/0	0/0	1/1	1/0	0/0
24 h	0/0	0/0	0/0	0/0	0/0	0/0
48 h	0/0	0/0	0/0	0/0	0/0	0/0
72 h	0/0	0/0	0/0	0/0	0/0	0/0
7 days	0/0	0/0	0/0	0/0	0/0	0/0
14 days	0/0	0/0	0/0	0/0	0/0	0/0
Average 24h, 48h, 72h	0/0	Not calculated	0/0	0/0	0/0	Not calculated

 

Interpretation of results:

Category 1 (irreversible effects on the eye)

Conclusions:

In this in vivo eye irritation test, MDIPA Esterquat C18 unsatd. (20% dilution in olive oil) produced irreversible effects to the eye and thus has to be classified as Category 1 (irreversible effects on the eye).

Executive summary:

In a primary eye irritation study according to OECD guideline 405 (24 April 2002) and EU method B.5 (31 May 2008) 0.1 mL of MDIPA Esterquat C18 unsatd.

(20% in olive oil) was instilled into the conjunctival sac of the right eyeof 2 male New Zealand White rabbits. The contralateral eye was treated with 0.1 mL olive oil as vehicle control. The eyes were not rinsed after application. Animals were then observed for 21 days.  Irritation was scored by the method of Draize.

No corneal or iridial effects were noted in the vehicle control, only minimal conjunctival irritation was noted in both treated eyes one hour after treatment. Both eyes appeared normal at the 24 h observation. The test substance produced scattered or diffuse corneal opacity (grade 1) in one eye one hour after treatment and in both treated eyes for up to 7 days. Vascularisation with a localised ingrowth of vessels at the lower edge of the cornea, was noted in one treated eye at the 14 and 21 d observations. Iridial inflammation (grade 1) was observed, which was reversible within 7 or 14 days.

Moderate conjunctival irritation (grade 2 redness and grade 2 chemosis) was noted in both treated eyes. This was reversible in one animal within 14 d. But minimal conjunctival irritation (grade 1 redness) was still present in one treated eye at the 14 d observation.

One treated eye appeared normal at the 14 d observation. The corneal vascularisation observed in one treated eye was considered to be irreversible.

Thus, a 20% dilution of MDIPA Esterquat C18 unsatd. in olive oil

produced irreversible effects to the eye and thus has to be classified as Category 1 (irreversible effects on the eye).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-08-12 to 2011-10-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

December 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material: 1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me-sulfates (salts)
- Physical state: liquid
- Analytical purity: 100%

Species:

cattle

Details on test animals or tissues and environmental conditions:

Test System: Freshly isolated bovine cornea
Rationale: Recommended by INVITTOX, UK, protocol no. 98
Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany

Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with Lglutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, annex III.

The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its
natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for one hour at 32 ± 1°C in a water-bath.

Vehicle:

other: olive oil

Controls:

other: Positive control: 2-Ethoxyethanol; Negative control: 0.9% (w/v) NaCl in deionised water, Solvent control: olive oil

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% in olive oil

Duration of treatment / exposure:

10 min ± 30 seconds

Observation period (in vivo):

2 h

Number of animals or in vitro replicates:

3 corneae per group (first experiment: test item, negative control, positive control; second experiment: solvent control, negative control, positive control)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): after 10 min exposure rinsing with saline

SCORING SYSTEM:
In vitro Irritation Score (IVIS) = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
according to OECD guideline 437: 98: IVIS ≥ 55.1 severely irritating to the eye/corrosive

TOOL USED TO ASSESS SCORE:
- Opacitometer
- permeability was assessed using fluorescein

Irritation parameter:

in vitro irritation score

Run / experiment:

20% dilution

Value:

52.59

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

A 20% dilution of the test substance in olive oil caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 52.59.
The positive control (2-Ethoxyethanol) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 65.46 (1st experiment) and 76.72 (2nd experiment)).
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score 0.65 (1st experiment) and 0.21 (2nd experiment)).
The solvent control olive control did not show relevant effects. The irritation score was 0.65.

Results of the 1st Experiment after 10 Minutes Incubation Time

Test group	Opacity	Permeability at 490 nm	In vitro Irritation Score	MeanIn vitro Irritation Score	in vitro Irritation Scale
Negative control	-1	0.086	0.29	0.65	Not severely irritating
	0	0.088	1.32
	-1	0.089	0.34
Positive control	47.67	0.710	58.32	65.46	Severely irritating
	50.67	1.228	69.09
	54.67	0.953	68.97
Test substance, 20% in olive oil	41.67	0.521	49.49	52.59	Not severely irritating
	52.67	0.492	60.05
	41.67	0.438	48.24

 

Results of the 2nd Experiment after 10 Minutes Incubation Time

Test group	Opacity	Permeability at 490 nm	In vitro Irritation Score	MeanIn vitro Irritation Score	in vitro Irritation Scale
Negative control	-1	0.064	-0.04	0.21	Not severely irritating
	-1	0.055	-0.18
	0	0.056	0.84
Positive control	63.67	0.319	68.45	76.72	Severely irritating
	62.67	1.071	78.73
	60.67	1.489	83.00
Solvent control	1.67	-0.004	1.60	0.65	Not severely irritating
	-0.33	-0.007	-0.44
	0.67	0.008	0.78

 

 

 

Interpretation of results:

other: not severely irritating/not corrosive (20% dilution)

Conclusions:

A 20% dilution in olive oil of MDIPA Esterquat C18 unsatd. is considered to be not severely irritating /not corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Executive summary:

This in vitro study was performed to assess them corneal irritation and damage potential of MDIPA Esterquat C18 unsatd. (20% in olive oil) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 7 September 2009 and EU method B.47, December 2010.

The corneae were incubated with the test substance and controls for 10 min. After rinsing with saline, the corneae 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 ≥ 55.1 is defined as a corrosive or severe irritant.

A 20% dilution of the test substance in olive oil caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 52.59.

The positive control (2-Ethoxyethanol) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 65.46 (1st experiment) and 76.72 (2nd experiment)). 

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score 0.65 (1st experiment) and 0.21 (2nd experiment)). The solvent control olive control did not show relevant effects. The irritation score was 0.65.

Since the mean in vitro irritancy score of the test substance was <55.1, a 20% dilution of MDIPA Esterquat C18 unsatd.

in olive oil is considered to not be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

December 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material: 1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me-sulfates (salts)
- Physical state: liquid
- Analytical purity: 100%

Species:

cattle

Details on test animals or tissues and environmental conditions:

Test System: Freshly isolated bovine cornea
Rationale: Recommended by INVITTOX, UK, protocol no. 98
Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany

Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with Lglutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, annex III.

The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its
natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for one hour at 32 ± 1°C in a water-bath.

Vehicle:

unchanged (no vehicle)

Controls:

other: Positive control: 2-Ethoxyethanol; Negative control: 0.9% (w/v) NaCl in deionised water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

10 min ± 30 seconds

Observation period (in vivo):

2 h

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): after 10 min exposure rinsing with saline

SCORING SYSTEM:
In vitro Irritation Score (IVIS) = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
according to OECD guideline 437: 98: IVIS ≥ 55.1 severely irritating to the eye/corrosive

TOOL USED TO ASSESS SCORE:
- Opacitometer
- permeability was assessed using fluorescein

Irritation parameter:

in vitro irritation score

Run / experiment:

100% substance

Value:

99.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro score was 99.30.
The positive control (2-Ethoxyethanol) increased the opacity and permeability of the corneae (mean in vitro score 56.02) corresponding to a classification as corrosive to the eye. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score 0.00).

Results after 10 min incubation:

 

Test group	Opacity	Permeability at 490 nm	In vitro Irritation Score	MeanIn vitro Irritation Score	in vitro Irritation Scale
Negative control	0	0.052	0.78	0.00	Not severely irritating
	-2	0.063	-1.06
	-1	0.075	0.13
Positive control	36	0.395	41.92	56.02	Severely irritating
	37	1.008	52.12
	54	1.336	74.04
Test substance	104	0.972	118.58	99.30	Severely irritating
	79	0.703	89.54
	73	1.120	89.80

 

 

 

Interpretation of results:

Category 1 (irreversible effects on the eye)

Conclusions:

In this BCOP assay, MDIPA Esterquat C18 unsatd. caused an increase of the corneal opacity and permeability. The calculated mean in vitro score was 99.30, which corresponds with the classification as corrosive to the eye.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of MDIPA Esterquat C18 unsatd. (100% a.i.) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 7 September 2009 and EU method B.47, December 2010.

The corneae were incubated with the test substance and controls for 10 min. After rinsing with saline, the corneae 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 ≥ 55.1 is defined as a corrosive or severe irritant.

 

The test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro score was 99.30.

The positive control (2-Ethoxyethanol) increased the opacity and permeability of the corneae (mean in vitro score 56.02) corresponding to a classification as corrosive to the eye. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score 0.00).

Since the mean in vitro irritancy score of the test substance was >55.1, MDIPA Esterquat C18 unsatd. is considered to be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Reference 4

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-12-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Journal Officiel de la Republique Francaise. 1996, pp. 19137-19138

Principles of method if other than guideline:

Prof. Dr. N.-P. Lüpke, Institute for Pharmacology and Toxicology, University of Münster (for example in H. Schöffl, Ersatz- und Ergänzungsmethoden zu Tierversuchen: Möglichkeiten und Grenzen der Reduktion von Tierversuchen, Springer Verlag, 1992).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material: 1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C18 unsatd., Me-sulfates (salts)
- Physical state: liquid
- Analytical purity: 100%

Species:

chicken

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Age at study initiation: <2 days at delivery, test was conducted 9 days after insertion into incubator
- Weight at study initiation: > 60 g

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control (tap water); positive control (5% Texapon ASV 70 Special)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 200 µL

Duration of treatment / exposure:

5 min

Observation period (in vivo):

5 min (scoring at 30 s, 2 and 5 min)

Number of animals or in vitro replicates:

6

Irritation parameter:

other: mean irritation index (HET-CAM)

Value:

8.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Treatment with the test substance resulted in the appearance of haemorrhage on the CAM of 5 eggs 5 minutes post application and in the appearance of coagulation on the CAM of 4 eggs 2 minutes and of 2 eggs 5 minutes post application. No other effect was detected after treatment (8.8 scores of total 21
possible scores).
Application of the negative control onto the CAM showed no effects; the irritation index therefore was 0.0 of 21 total possible scores. Treatment with a 5% preparation of the positive control substance Texapon ASV 70 Special resulted in an irritation index of 10.3 of 21 total possible scores.

Interpretation of results:

other: moderately irritating according to the evaluation scheme

Conclusions:

In this HET-CAM test MDIPA Esterquat C18 unsatd. was moderately irritating.

Executive summary:

MDIPA Esterquat C18 unsatd. (100% a.i.) was tested in the hen's egg chorioallantoic membrane test (HET-CAM), an accepted alternative method to the Draize test, to evaluate the acute irritation potential. 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) of 6 fertilised and incubated eggs (strain SPAFAS).

200 µL of the test substance, negative (tap water) and positive (5% Texapon ASV 70 Special) control were applied onto the membrane. Observations for vascular injection, haemorrhage and coagulation were made after 30 seconds, 2 and 5 minutes.

Application of the negative control onto the CAM showed no effects; the irritation index was 0.0 of 21 total possible scores. Treatment with the positive control resulted in an irritation index of 10.3 of 21 total possible scores.

 

Treatment with the test substance resulted in the appearance of haemorrhage on the CAM of 5 eggs 5 minutes post application and in the appearance of coagulation on the CAM of 4 eggs 2 minutes and of 2 eggs 5 minutes post application. No other effect was detected after treatment (8.8 scores of total 21

possible scores). No other effect was detected after treatment.

Thus, the test substance was graded as moderately irritating according to the evaluation scheme.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

For the assessment of the skin irritation potential of MDIPA Esterquat C18 unsatd. an in vitro skin irritation test as well as an in vivo test are available.

 

In a dermal irritation study according to OECD Guideline 439 (In Vitro Skin Irritation) (22 July 2010) and EU method B.46 (In vitro skin irritation: reconstructed human epidermis model test) (23 July 2009), 30 µL MDIPA Esterquat C18 unsatd. (100% a.i.) was applied in triplicates for 60 min to a three-dimensional human epidermis model (EpiDerm EPI-200-SIT, MatTek Corporation).

After 60 minutes exposure at 37°C, the tissues were rinsed 15 times with phosphate buffered saline to remove residual test substance. Subsequently the skin tissues were incubated for 42 ± 2 h at 37°C.

Cytotoxic (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Positive (5% SDS), negative (ultrapure water) and quality (1% Triton X-100) control gave the appropriate response.

The relative mean tissue viability obtained after 60 minutes treatment with MDIPA Esterquat C18 unsatd. compared to the negative control tissues was 82.7 ± 1.5%. Since the mean relative tissue viability for the test substance was above 50%, MDIPA Esterquat C18 unsatd. is considered to be not irritating.

 

Although the in vitro test was negative for skin irritation, an in vivo test was performed since for similar substances (fatty acid derivatives) it was shown that despite negative results in vitro the in vivo outcome may be positive (irritating).

In a primary dermal irritation study according to OECD guideline 404 (24 April 2002) and EU method B.4 (30 May 2008) young adult New Zealand White, CRL:KBL (NZW) rabbits (2 female, 1 male) were dermally exposed to 0.5 mL of undiluted MDIPA Esterquat C18 unsatd. (100% a.i.) for 4 hours to 6 cm² body surface area. Animals then were observed for 21 days.  Irritation was scored by the method of Draize.

Well-defined to moderate erythema and slight to moderate edema (grade 2 or 3) was observed within 72 hours after patch removal in all three animals. 7 days after patch removal very slight to slight erythema and edema (grading 1 or 2) was observed and 14 days after patch removal very slight erythema (grade 1) was still existent in all animals. 21 days after application all three animals were without any sign of skin irritation. Additionally, strong desquamation and atrophy of the skin was observed in all three animals beginning on day 7. These skin reactions declined with time as well. Grade 1 atrophy was still present in one animal on day 21. Mean erythema scores from observations at 24, 48 and 72 h after patch removal were >/=2.3 for 2/3 animals; mean edema scores were >/=2.3 for all three animals. In this study MDIPA Esterquat C18 unsatd. was irritating to the skin.

 

Furthermore an in vivo test with a test substance concentration of 6% is available:

In a primary dermal irritation study similar to US FHSA Federal regulation: 16 CFR 1500.41, 4 New Zealand White rabbits were dermally exposed to 0.5 mL of a 6% aqueous dispersion of MDIPA Esterquat C18 unsatd. Two application sites per animal were treated, one site was left intact, the other was abraded. Test sites were covered with an occlusive dressing for 24 hours. Animals were observed for 72 hours. Irritation was scored similar to FHSA Federal guideline 16 CFR 1500.41. This scoring system is similar to OECD guideline 404.  

24 h after application of the test item in 1/4 animals a very slight erythema (grade 1) was present at treated intact and abraded skin sites. This effect was reversible within 72 h. No other irritating effects were observed.

In this test a 6% aqueous dispersion of MDIPA Esterquat C18 unsatd. was not irritating

Study performance did not comply with requirements of the relevant recent EU and OECD guidelines, where a less vigorous application regime with semi-occlusive dressing, an exposure period of 4 hours, treatment of only intact skin is stipulated. Thus, it can be assumed, that a substance which shows only very slight irritating activity under the application regime of guideline US FHSA Federal regulation: 16 CFR 1500.41 will also not show a more than very slight irritating activity under the application regimes of the relevant recent EU and OECD guidelines.

 

In conclusion, MDIPA Esterquat C18 unsatd. is irritating to the skin (Category 2).

 

Supporting data on the source substances MDEA-Esterquat C16-18 and C18 unsatd. and MDIPA-Esterquat C16-18 and C18 unsatd. are included into the dossier to complete the toxicological profiles of the substances.

MDEA-Esterquat C16-18 and C18 unsatd. was not irritating to skin in a primary dermal irritation study comparable to OECD guideline 404 in rabbit as well as in an in vitro skin irritation study according to OECD Guideline 439.

The source substance MDIPA-Esterquat C16-18 and C18 unsatd. was not irritating in a study according to OECD Guideline 439, but showed signs of skin irritation in a dermal irritation study according to OECD guideline 404 in rabbit and is therefore classified as Skin Irrit. 2.

 

 

Eye irritation

For the assessment of the eye irritation potential of MDIPA Esterquat C18 unsatd. a HET-CAM and a BCOP assay with a concentration of 100% MDIPA Esterquat C18 unsatd. are available, as well as a BCOP assay and an in vivo test with 20% MDIPA Esterquat C18 unsatd. All tests are considered for hazard and risk assessment in a weight of evidence approach.

MDIPA Esterquat C18 unsatd. (100% a.i.) was tested in the hen's egg chorioallantoic membrane test (HET-CAM), an accepted alternative method to the Draize test, to evaluate the acute irritation potential. 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) of 6 fertilised and incubated eggs (strain SPAFAS).

200 µL of the test substance, negative (tap water) and positive (5% Texapon ASV 70 Special) control were applied onto the membrane. Observations for vascular injection, haemorrhage and coagulation were made after 30 seconds, 2 and 5 minutes.

Application of the negative control onto the CAM showed no effects; the irritation index was 0.0 of 21 total possible scores. Treatment with the positive control resulted in an irritation index of 10.3 of 21 total possible scores.

Treatment with the test substance resulted in the appearance of haemorrhage on the CAM of 5 eggs 5 minutes post application and in the appearance of coagulation on the CAM of 4 eggs 2 minutes and of 2 eggs 5 minutes post application. No other effect was detected after treatment (8.8 scores of total 21 possible scores). No other effect was detected after treatment.

Thus, the test substance was graded as moderately irritating according to the evaluation scheme.

A second in vitro study was performed to assess the corneal irritation and damage potential of MDIPA Esterquat C18 unsatd. (100% a.i.) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 7 September 2009 and EU method B.47, December 2010.

The corneae were incubated with the test substance and controls for 10 min. After rinsing with saline, the corneae 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 ≥ 55.1 is defined as a corrosive or severe irritant.

The test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro score was 99.30.

The positive control (2-Ethoxyethanol) increased the opacity and permeability of the corneae (mean in vitro score 56.02) corresponding to a classification as corrosive to the eye. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score 0.00).Since the mean in vitro irritancy score of the test substance was >55.1, MDIPA Esterquat C18 unsatd. is considered to be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

A further in vitro study was performed to assess the corneal irritation and damage potential of MDIPA Esterquat C18 unsatd. (20% in olive oil) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 7 September 2009 and EU method B.47, December 2010. The corneae were incubated with the test substance and controls for 10 min. After rinsing with saline, the corneae 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 ≥ 55.1 is defined as a corrosive or severe irritant. A 20% dilution of the test substance in olive oil caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 52.59. 

The positive control (2-Ethoxyethanol) increased the opacity and permeability of the corneae in both experiments (meanin vitroirritation score 65.46 (1st experiment) and 76.72 (2nd experiment)). 

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score 0.65 (1st experiment) and 0.21 (2nd experiment)). The solvent control olive control did not show relevant effects. The irritation score was 0.65. 

Since the mean in vitro irritancy score of the test substance was <55.1, a 20% dilution of MDIPA Esterquat C18 unsatd. in olive oil is considered to not be severely irritating/corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

As a 20% solution of the test substance was not corrosive on the BCOP, an in vivo test was conducted with 20% MDIPA Esterquat C18 unsatd. in olive oil:

In a primary eye irritation study according to OECD guideline 405 (24 April 2002) and EU method B.5 (31 May 2008) 0.1 mL of MDIPA Esterquat C18 unsatd. (20% in olive oil) was instilled into the conjunctival sac of the right eye of 2 male New Zealand White rabbits. The contralateral eye was treated with 0.1 mL olive oil as vehicle control. The eyes were not rinsed after application. Animals were then observed for 21 days.  Irritation was scored by the method of Draize. 

No corneal or iridial effects were noted in the vehicle control, only minimal conjunctival irritation was noted in both treated eyes one hour after treatment. Both eyes appeared normal at the 24 h observation. 

The test substance produced scattered or diffuse corneal opacity (grade 1) in one eye one hour after treatment and in both treated eyes for up to 7 days. Vascularisation with a localised ingrowth of vessels at the lower edge of the cornea, was noted in one treated eye at the 14 and 21 d observations. Iridial inflammation (grade 1) was observed, which was reversible within 7 or 14 days. Moderate conjunctival irritation (grade 2 redness and grade 2 chemosis) was noted in both treated eyes. This was reversible in one animal within 14 d. But minimal conjunctival irritation (grade 1 redness) was still present in one treated eye at the 14 d observation. One treated eye appeared normal at the 14 d observation. The corneal vascularisation observed in one treated eye was considered to be irreversible. 

Thus, a 20% dilution of MDIPA Esterquat C18 unsatd. in olive oil produced irreversible effects to the eye and thus has to be classified as Category 1 (irreversible effects on the eye).

In conclusion, MDIPA Esterquat C18 unsatd. causes irreversible effects on the eye (Category 1) at a concentration of 20%.

 

Supporting data on the source substances MDEA-Esterquat C16-18 and C18 unsatd. and MDIPA-Esterquat C16-18 and C18 unsatd. are included into the dossier to complete the toxicological profiles of the substances.

MDEA-Esterquat C16-18 and C18 unsatd.  was not irritatign to the eyes in a primary eye irritation study according to OECD guideline 405. 

In contrast, the source substance MDIPA-Esterquat C16-18 and C18 unsatd. is classified as Eye Irrit. 2 based on the results of an Isolated Chicken Eye (ICE) test. 

 

Respiratory irritation

No data on the respiratory irritation of MDIPA Esterquat C18 unsatd. 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.

Justification for classification or non-classification

Based on the available data MDIPA Esterquat C18 unsatd. has to be classified as irritating to the skin (Category 2) and causing irreversible effects on the eye (Category 1) and labelled with H315 (Causes skin irritation) and H318 (Causes serious eye damage) according to regulation (EC) 1272/2008.