Registration Dossier

Administrative data

Description of key information

Skin irritation:    

Not irritating (OECD TG 439/EU method B.46; GLP), neat test substance,

relative mean tissue viability after 15 minutes was 114% compared to the negative control      

Irritating (OECD TG 404/EU method B.4; GLP), 30% (w/v) concentration in water applied to the intact skin of rabbits for 4 hours,  

mean erythema scores at 24, 48 and 72 h after patch removal were 3.0/2.7/3.0;  mean edema scores were 4.0/2.3/3.7      

histopathological examination revealed no evidence of full thickness destruction of the skin or scar tissue.

Eye irritation:    

Irritating (OECD 438; GLP), neat test substance,  

mean corneal swelling 9%, mean corneal opacity score 2.0,

mean fluorescein retention score 1.5;  vesicular appearance of the top-layer of the epithelium, irritation index 79.    

Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium only.  

No abnormalities of the stroma and endothelium were observed.

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted on 7 September 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: isolated chicken eye
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: ROSS, spring chickens from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands
- Age: Approximately 7 weeks old - Weight: approximately 1.5 - 2.5 kg

Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.

Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that showed opacity (score higher than 0.5), were unacceptably stained with fluorescein (score higher than 0.5), or showed any other signs of damage were rejected and were replaced.

After an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured once more to determine the zero reference value for corneal swelling calculations.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
Duration of treatment / exposure:
10 s
Observation period (in vivo):
eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment
Number of animals or in vitro replicates:
negative control (physiological saline): 1
positive control (BAC 5% (w/v)): 3
test group: 3
Details on study design:
REMOVAL OF TEST SUBSTANCE - Washing (if done): 20 mL saline
- Time after start of exposure: 10 s
SCORING SYSTEM: according to ICE classification criteria OECD TG 438
TOOL USED TO ASSESS SCORE: slit lamp microscope / fluorescein

At time t=0, i.e. immediately after the zero reference measurement, the following procedure was applied for each test eye: The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards. Next, three corneas were treated with 30 µL of neat MDIPA-Esterquat C16-18 and C18 unsatd. After an exposure period of 10 seconds, the corneal surface was rinsed thoroughly with 20 mL of isotonic saline at ambient temperature. After rinsing, each eye in the holder was returned to its chamber. The negative control eyes were treated with physiological saline only and the 3 positive control eyes with 5 % (w/v) BAC. The eyes were examined at 0, 30, 75, 120, 180 and 240 minutes after treatment. All examinations were performed with a slit-lamp microscope. Fluorescein retention was scored only at 30 minutes after treatment. After the final examination, the test and control eyes were preserved in a neutral aqueous phosphate-buffered solution of 4% formaldehyde. The tissues selected were embedded in paraffin wax, sectioned at 5 μm and stained with Periodic Acid-Schiff for histopathology examination. Ocular effects were evaluated using the endpoints of corneal thickness (swelling), corneal opacity and fluorescein retention.
Irritation parameter:
percent corneal swelling
Value:
9
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
2
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
1.5
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
79
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritant / corrosive response data:
The test substance caused slight corneal swelling (mean swelling of 9%), moderate corneal opacity (mean score of 2) and slight to moderate fluorescein retention (mean score of 1.5). In addition, the top-layer of epithelium had a vesicular appearance. The calculated Irritation Index was 79.
Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium only. No abnormalities of the stroma and endothelium were observed.
The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control eyes showed severe corneal effects and demonstrated the ICE test as meeting the acceptance criteria to be considered a valid study.
Microscopic examination of the one cornea treated with the negative control (saline) did not reveal any abnormalities. The positive control BAC 5% (w/v) caused moderate or severe erosion of the epithelium in the three corneas and vacuolation in the epithelium of two corneas.

Summary of results

Test material

 

Maximum mean score for:

Irritation

categories1

Irritation

Index2

Classifications

(EU-CLP3/UN-GHS4)

 

Swelling %

Opacity

Fluorescein

retention

test item

9

2.05

1.5

II;III;II

79

2/2B

Saline

(negative control)

0

0.0

0.0

Not applicable; one eye tested

BAC 5% (w/v) (positive control)

24

3.06

3.0

III;IV;IV

144

Category 1

1 I = no effect; II = slight effect; III = moderate effect; IV = severe effect.

2   Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)

3 EU-CLP: NC = not classified; Category 2 = Irritating to eyes; Category 1 = irreversible effects on the eye/serious damage to the eye. Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006.

4 UN-GHS: NC = not classified; Category2B = mild irritant, causes eye irritation;Category2A = irritant, causes eye irritation;Category 1 = irreversible effects on the eye/serious damage to the eye.United Nations-Economic Commission for Europe (UN/ECE) (2003). Globally Harmonised System of Classification and Labelling of Chemicals (GHS). UN, New York and Geneva, 2007.

5   Vesicular appearance of epithelium

6   Severe loosening of epithelium

Individual values test substance

Eye no. 1

Corneal thickness (instrument units)

 

 

Corneal opacity

 

Fluorescein Retention

T (min)= ->

-45

0

30

75

120

180

240

 

 

0

30

75

120

180

240

 

0

30

 

60

58

62

63

63

64

64

 

 

0

1.5

1.5

2

2

2

 

0

1.5

swelling %

6.9

8.6

8.6

10.3

10.3

 

 

 

 

 

 

 

 

 

 

 

Eye no. 3

Corneal thickness (instrument units)

 

 

Corneal opacity

 

Fluorescein Retention

T (min)= ->

-45

0

30

75

120

180

240

 

 

0

30

75

120

180

240

 

0

30

 

59

57

60

61

62

63

63

 

 

0

1.5

1.5

2

2

2

 

0

1.5

swelling %

5.3

7.0

8.8

10.5

10.5

 

 

 

 

 

Eye no. 5

Corneal thickness (instrument units)

 

 

Corneal opacity

 

Fluorescein Retention

T (min)= ->

-45

0

30

75

120

180

240

 

 

0

30

75

120

180

240

 

0

30

 

63

62

65

66

66

66

66

 

 

0

1.5

1.5

2

2

2

 

0

1.5

swelling %

4.8

6.5

6.5

6.5

6.5

 

 

 

 

mean

6

7

8

9

9

 

mean

1.5

1.5

2.0

2.0

2.0

 

mean

1.5

In all corneas the top-layer of epithelium had a vesicular appearance

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Based on the results from this Isolated Chicken Eye test, MDIPA-Esterquat C16-18 and C18 unsatd. is considered to be irritating to the eyes under the experimental conditions described in this report.
Executive summary:

For the assessment of the eye irritation potential of MDIPA-Esterquat C16-18 and C18 unsatd. Data from an Isolated Chicken Eye (ICE) test are available This in vitro study was performed in accordance with OECD Test Guideline 438 (Isolated Chicken Eye Test Method for Identifying

i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage) adopted on 7 September 2009.

In details the testing has already been performed according to the most recent version of the guideline adopted on July 26th2013.

 

Approximately 7 weeks old chickens (obtained from slaughter animals for human consumption) were used as eye-donors. A total of 7 eyes were selected for testing: 3 for the test substance, 3 for the positive control (5 % (w/v) Benzalkonium Chloride (BAC) and 1 for the negative control (physiological saline).

 

The isolated chicken eyes were exposed to a single application of 30 µL of the test sample for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells.

 

MDIPA-Esterquat C16-18 and C18 unsatd. tested neat causedcaused slight corneal swelling (mean swelling of 9%), moderate corneal opacity (mean score of 2) and slight to moderate fluorescein retention (mean score of 1.5). In addition, the top-layer of epithelium had a vesicular appearance. The calculated Irritation Index was 79.

Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium only. No abnormalities of the stroma and endothelium were observed

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control eyes showed moderate swelling (24%), severe corneal opacity (mean score of 3) and severe fluorescein retention (mean score of 3) and demonstrated the ICE test as meeting the acceptance criteria to be considered a valid study. The calculated Irritation Index of the positive control (5% (w/v) BAC) was 144. Microscopic examination of the one cornea treated with the negative control (saline) did not reveal abnormalities. The positive control 5% (w/v) BAC caused moderate or severe erosion of the epithelium in the three corneas and vacuolation in the epithelium of two corneas. 

According to the classification schemes identified within the ICE test protocol, MDIPA-Esterquat C16-18 and C18 unsatd. is classified as Category 2, Irritating to eyes. It is recognized that the prediction models adopted in the OECD Test Guideline 438 are foridentifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. However, further support for identification of MDIPA-Esterquat C16-18 and C18 unsatd. tested neat in this ICE study as a Category 2 irritant is derived from histopathology examination within the ICE test.

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 C16-18 and C18 unsatd. both an in vitro skin irritation test and an in vivo test are available.

 

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (22 July 2010) and EU method B.46 (In vitro skin irritation: reconstructed human epidermis model test) (20 July 2012), MDIPA-Esterquat C16-18 and C18 unsatd. (100% a. i.) was applied to the three-dimensional human epidermis model tissue (EPISKIN Small Model (EPISKIN-SM, 0.38 cm², Batch no.: 12-EKIN-039) for an exposure period of 15 minutes. The number of replicate tissues was three). The test substance was heated in advance to approximately 50°C to soften the waxy substance. To achieve optimal contact with the tissue without causing mechanical damage, the test substance was applied using a cotton swab. The exact amount of test substance applied to cover the entire skin surface could not be determined using this method. Since optimal contact was obtained using this method this deviation has no impact on the study integrity. After 15 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline pre-warmed to approximately 50°C, and residual test substance was carefully removed using a spatula. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (PBS) control gave responses that were within the acceptance criteria (please refer to the robust study summary for details) and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with MDIPA-Esterquat C16-18 and C18 unsatd. compared to the negative control tissues was 114%. Since the mean relative tissue viability for the test substance was above 50%, MDIPA-Esterquat C16-18 and C18 unsatd. is identified to be not irritating.

 

Data generated by fully validated in vitro methods can be used for REACH purposes provided that the information for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. Based on the result of the in vitro skin irritation study, MDIPA-Esterquat C16-18 and C18 unsatd. as neat substance would be Not Classified (NC) for skin irritation.

Although the in vitro test with neat substance was negative for skin irritation, a subsequent in vivo test with dispersed substance was performed because during the preliminary irritation testing and epidermal induction in the Guinea Pig Maximization Test (GPMT), signs of skin irritation were observed when the substance was applied as a dispersion in water.

It was decided to test the substance in vivo in a dispersed form, which ensures the best possible skin contact. For this purpose, formulation pre-tests have been performed. Water was chosen as the most suitable solvent for the hazard and risk assessment because during formulation and use the substance will also be dispersed in water.

 

In a mortar an amount of 2 g of MDIPA-Esterquat C16-18 and C18 unsatd. were weighed to the nearest 0.1 g and deionised water was added to achieve the desired concentrations (50%, 25% and 10% (w/w)). Using the pestle, the test substance and water were mixed for approx. 1 minute and the consistency and homogenicity qualitatively assessed. All of these procedures were conducted at Room Temperature (RT) as were all substances and liquids.

 

Substance

 

Solvent

 

Content[%] (w/w)

 

Description

 

MDIPA-Esterquat C16-18 and C18 unsatd.

deionised water

 

50

 

Very pasty, difficult to process

MDIPA-Esterquat C16-18 and C18 unsatd.

 

deionised water

 

25

Cream-like, homogeneous, non-flowing, could be applied on the skin

 

A concentration of 30% (w/v) was identified to be the highest concentration, which could be evenly applied to the skin. Dispersions of higher concentration were very pasty and not homogenous in appearance with the result that an even exposure over the whole patch area could not be achieved.

 

In a primary dermal irritation study performed in accordance with OECD guideline 404 (adopted 24th April 2002) and EU method B.4 (May 2008), three young adult New Zealand White rabbits were dermally exposed to 0.5 g of MDIPA-Esterquat C16-18 and C18 unsatd. as a 30% (w/v) dispersion in water. The substance was applied to separate skin-sites on intact, clipped skin using a semi-occlusive dressing. for 3 min, 1 h and 4 h to an area of 2 cm x 3 cm. After each removal of a dressing, the treated skin was cleaned of residual test substance using water. Animals then were observed for 14 days. Irritation was scored by the method of Draize.

A 3-minute exposure resulted in very slight oedema and/or erythema immediately after exposure in the treated skin area of the three rabbits.

A 1-hour exposure resulted in well-defined to severe erythema and very slight to severe oedema in the treated skin area of the three rabbits. For two rabbits erythema was scattered in nature at 7 days or between 24 hours and 7 days after exposure, and for one of these animals erythema was noted at the edges of the application area at 7 days after exposure. Fissuring and reduced flexibility of the skin was observed for one animal at 7 days after exposure. Scaling was observed on the treated skin site for all animals at 7 and/or 14 days after exposure, being present on the edges of the application area for one of the animals at 14 days after exposure.

A 4-hour exposure resulted in severe erythema and moderate or severe oedema in the treated skin area of the three rabbits. The maximum grade for erythema was generally achieved at 72 hours and/or 7 days after exposure. At 14 days after exposure, oedema had resolved and erythema remained present for two animals at well-defined grade. For one rabbit erythema was scattered in nature and present at the edges of the application area at 7 days after exposure. All animals showed reduced flexibility of the skin at 72 hours after exposure, and bald skin with scaling at 14 days after exposure. For one of these animals, scaling and bald skin was primarily present on the edges of the application area. Fissuring of the skin and yellow-brown superficial scabs were noted for two animals at 7 days after exposure, due to which oedema could not be scored. The mean 24, 48 and 72 hours value irritation scores after 4 hours of exposure were 3.0/2.7/3.0 for erythema and 4.0/2.3/3.7 for edema.

Sticky remnants of the test substance were present on all treated skin sites in all animals on Day 1.

One rabbit showed a pronounced inflammatory cell infiltrate in the 4 hour treated skin area consisting of a moderate lymphocytic infiltrate, a slight macrophage infiltrate with the presence of multinucleated giant cells containing hair debris (likely related to the clipping) and slight epidermal thickening. The minimal heterophilic or lymphocytic infiltrate observed in the other treated skin sites remained within the background pathology range.

The other two rabbits showed minimal inflammatory lesions consisting of a minimal lymphocytic and/or heterophilic infiltrate of the dermis in the skin sites treated for 3 minutes, one hour and four hours. The nature and incidence of these findings showed no apparent relation to the duration of treatment and remained within the background pathology range.

No evidence of full thickness destruction of the skin or scar tissue was observed during the observation period and at histopathological examination of the treated skin sites, indicating that no corrosion of the skin had occurred by dermal application of the test item to the intact rabbit skin.

Skin reactions (well-defined erythema, bald skin and/or scaliness) remained present in all animals up to the end of the 14-day observation period following a 4-hour exposure duration.

Histopathology showed a pronounced inflammatory cell infiltrate in the 4-hour treated skin site of one of the rabbits.

There were no signs of corrosion in any of the skin samples examined.

In this study, based on mean value scores for erythema and edema and taking into account reversibility criteria, MDIPA-Esterquat C16-18 and C18 unsatd. (30% dispersion in water) is a dermal irritant.

 

Taking all of the skin irritation data into account, and using a precautionary approach, although the in-vitro skin irritation test with the neat test substance identified MDIPA-Esterquat C16-18 and C18 unsatd. as Not Classified for skin irritation, the classification of Category 2 ((irritating to skin) will be applied also to the neat substance.

 

Eye irritation

For the assessment of the eye irritation potential of MDIPA-Esterquat C16-18 and C18 unsatd. Data from an Isolated Chicken Eye (ICE) test are available This in vitro study was performed in accordance with OECD Test Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage) adopted on 7 September 2009 In details the testing has already been performed according to the most recent version of the guideline adopted on July 26th 2013.

Approximately 7 weeks old chickens (obtained from slaughter animals for human consumption) were used as eye-donors. A total of 7 eyes were selected for testing: 3 for the test substance, 3 for the positive control (5 % (w/v) Benzalkonium Chloride (BAC) and 1 for the negative control (physiological saline).

The isolated chicken eyes were exposed to a single application of 30 µL of the test sample for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells.

MDIPA-Esterquat C16-18 and C18 unsatd. tested neat caused caused slight corneal swelling (mean swelling of 9%), moderate corneal opacity (mean score of 2) and slight to moderate fluorescein retention (mean score of 1.5). In addition, the top-layer of epithelium had a vesicular appearance. The calculated Irritation Index was 79.

Microscopic examination of the corneas treated with the test substance revealed very slight erosion of the epithelium only. No abnormalities of the stroma and endothelium were observed

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control eyes showed moderate swelling (24%), severe corneal opacity (mean score of 3) and severe fluorescein retention (mean score of 3) and demonstrated the ICE test as meeting the acceptance criteria to be considered a valid study. The calculated Irritation Index of the positive control (5% (w/v) BAC) was 144. Microscopic examination of the one cornea treated with the negative control (saline) did not reveal abnormalities. The positive control 5% (w/v) BAC caused moderate or severe erosion of the epithelium in the three corneas and vacuolation in the epithelium of two corneas. 

 

A second ICE-test confirmed this result:

Approximately 7 weeks old chickens (obtained from slaughter animals for human consumption) were used as eye-donors. A total of 7 eyes were selected for testing: 3 for the test substance MDIPA-Esterquat C16-18 and C18 unsatd., 3 for the positive control (5 % (w/v) Benzalkonium Chloride (BAC) and 1 for the negative control (physiological saline).

The isolated chicken eyes were exposed to a single application of approx. 30 µL of the test sample for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells.

MDIPA-Esterquat C16-18 and C18 unsatd. tested neat caused caused slight corneal swelling (mean swelling of 7%), severe opacity (mean score of 3.0) and moderate or severe fluorescein retention (mean score of 2.3). The calculated Irritation Index was 113. Microscopic examination of the corneas treated with the test item revealed very slight erosion and very slight vacuolation (top region) of the epithelium.

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control eyes showed moderate swelling (29%), severe corneal opacity (mean score of 3, with severe loosening of the epithelium) and severe fluorescein retention (mean score of 3) and demonstrated the ICE test as meeting the acceptance criteria to be considered a valid study. The calculated Irritation Index of the positive control (5% (w/v) BAC) was 149. Microscopic examination of the one cornea treated with the negative control (saline) did not reveal abnormalities. The positive control BAC 5% caused slight, moderate or severe erosion, very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane (one cornea).

 

According to the classification schemes identified within the ICE test protocol, MDIPA-Esterquat C16-18 and C18 unsatd. is classified as Category 2, Irritating to eyes. It is recognized that the prediction models adopted in the OECD Test Guideline 438 are for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. However, further support for identification of MDIPA-Esterquat C16-18 and C18 unsatd. tested neat in this ICE study as a Category 2 irritant is derived from histopathology examination within the ICE test.

 

Although histopathology is not a mandatory endpoint for classification purposes, it has been recognised by OECD as potentially useful when a more complete characterization of corneal damage is needed (OECD 2011 and OECD, 2013b). In particular the OECD Guidance Document 160 (OECD, 2011) which is a supplement to the Test Guideline 438 was developed to promote the use of histopathology evaluation as an additional endpoint for ocular toxicity testing. This Guidance Document states that: “Histopathological evaluation may be useful for: (i) assessment of the histological damage of chemical classes or formulations that are not well characterized in these test methods; (ii) assisting with determination of a mode of action where it cannot be easily predicted; (iii) assisting with determination of the likelihood of delayed effects; (iv) evaluation of the depth of injury, which has been proposed as a measure of reversibility or irreversibility (Maurer et al. 2002); (v) further characterization of the severity or scope of the damage as needed (Harbell et al. 2006; ICCVAM 2010b; Maurer et al. 2002); (vi) assisting with discrimination of cases where the response falls along the borderline between two categories based on the test method decision criteria”.

 

A recent publication by the European Detergent Industry (Cazelleet al., 2014) established a set of defined histopathology criteria for use of histopathology as an additional endpoint to the standard prediction model in the ICE test method as encouraged by and for the reasons identified by OECD. Such histopathology criteria were found to be suitable to identify EU CLP / UN GHS Category 1 non-extreme pH detergent and cleaning products and to allow a better discrimination from Category 2 products. Since this recent publication focuses on non-extreme pH detergent and cleaning products the majority of which are surfactant-based, the histopathology criteria defined in this paper are considered to be relevant to interpretation of the histopathology evaluation in the ICE conducted on MDIPA-Esterquat C16-18 and C18 unsatd. On this basis, the histopathology results for MDIPA-Esterquat C16-18 and C18 unsatd. confirmed this test substance as a non-Category 1 irritant i.e. Category 2.

 

In accordance with REACH Annex XI, 1.4 confirmatory in vivo testing is scientifically not necessary when an adequate internationally validated and regulatory accepted in vitro study is provided and the results are adequate for the purpose of classification and labelling. Adequate and reliable documentation of the applied method is provided in the respective Robust Study Summary. To date, the ICE test is not accepted as a stand-alone test for evaluation of eye irritation but rather for identifying chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. However, in combination with the use of histopathology as an additional endpoint to increase the robustness of identifying Category 1 irritants, testing of MDIPA-Esterquat C16-18 and C18 unsatd. in animals is identified as not warranted. 

 

There are several reasons that support this position. The very high variability of the Draize test with this type of substance. especially with sticky pastes and solids in the moderate to severe range of irritancy and with hydrophobic solutions is a key consideration (Prinsen MK, 2006).

 

Also, due to the solid/waxy nature of MDIPA-Esterquat C16-18 and C18 unsatd. it is very difficult to grind the neat test substance to a uniformly fine dust as requested by the OECD Test Guideline 405 for in vivo eye irriation/corrosion testing. Furthermore, the test substance is very poorly soluble. By instillation of such a poorly water soluble solid/waxy test material into the conjunctival cul-de-sac of the rabbit eye as requested by the OECD Test Guideline, the entrapped solid can rapidly cause a considerable and increasing degree of swelling of the conjunctivae, making it very difficult or even impossible for the test substance to be removed from the eye. This is explained in the paper by Prinsen 2006: “The enclosure of solid materials up to 24 h in the conjunctival cul-de-sac, sometimes in combination with mechanical damage, can have a devastating effect on the eye. In the case of poorly water-soluble solids with distinct cytotoxic properties, the entrapped solid can rapidly cause a considerable and increasing degree of swelling of the conjunctivae, making it even more difficult for the animal to remove the material. If, at the 1-h observation, the lower eye-lid is not pulled away far enough by the observer, it can stay unnoticed that a bulk of test material lays deeply hidden in the conjunctival cul-de-sac. Often, this forced continuous exposure for the next 24 h results in a complete closure of the eye-lids by the abundant production of colloidal discharge which often forms a sealing crust. The degree of swelling of the conjunctivae can be sufficiently severe such that removal of any remains of the test substance is hardly possible anymore. In the majority of these cases, the eye is permanently damaged or can only be saved by applying special care, such as regular daily cleaning and rinsing of the eye and eye-lids, often including cutting off the eye-lashes to prevent further sealing. In general, keeping the eye-lids open is essential for the recovery process, otherwise the enclosed inflammatory exudate will further damage the cornea. If no further extensive remedial treatment is given to the animal, the described exposure conditions can easily cause an initial opacity score of 1 or 2 to develop into a score of 3 or 4. Also, the eye can become vulnerable to microbiological infection (the so-called secondary inflammatory process), causing initial mild to moderate effects during the first days after exposure developing into more severe and prolonged effects during the 21 day observation period“ (Prinsen MK, 2006).

Even with application of the 1-h rinsing for solids now included in the revised OECD Test Guideline 405, for a nearly insoluble, waxy and sticky solid it will hardly be possible to remove all traces of the test material. A major anatomical difference between the human and the rabbit is the presence of a nictitating membrane (third eyelid) in the rabbit. Extension of the nictitating membrane during irrigation can lead to less effective rinsing of the ocular surface. Solid materials that cannot be solubilized by lacrimal fluid can additionally be trapped underneath this “third eyelid“ effectively making an occlusive exposure that results in making ocular damage even worse.

This scenario will likely lead to unrealistically long and continuous exposure of the test substance leading to an exposure situation that has little relevance for hazard identification and risk assessment, because it is highly unlikely to occur in humans, accidentally or intentionally (Prinsen MK, 2006). Based on questions of relevance based on technical considerations and not least on animal welfare considerations, anin-vivotest is identified as not warranted for this test substance. Instead the result of the ICE test, taken together with the additionally performed histopathology evaluation identifying very slight erosion of the epithelium only and no abnormalities of the stroma and endothelium, is considered relevant and adequate for classification and labelling of MDIPA-Esterquat C16-18 and C18 unsatd.

 

Respiratory irritation

No data on the respiratory irritation of MDIPA-Esterquat C16-18 and C18 unsatd. is 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.

 

References:

Cazelle, E., Eskes, C., Hermann, M., Jones, P., McNamee, P, Prinsen, M., Taylor, H., Wijnands, M.V.W. (2014).Suitability of Histopathology as an Additional Endpoint to the Isolated Chicken Eye Test to for Classification  of Detergents and Cleaning Products. ToxicologyIn Vitro. In Press.

Available at: http://dx.doi.org/10.1016/j.tiv.2014.01.010

 

OECD (2002). Acute dermal irritation/corrosion.OECD Guideline for the Testing of Chemicals No. 404, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

 

OECD (2011). Series on Testing and Assessment No. 160. Guidance Document on: The Bovine Corneal Opacity and Permeability (BCOP) and Isolated Chicken Eye (ICE) Test Methods: Collection of tissues for histological evaluation and collection of data on non-severe irritants. Paris, France: Organisation for Economic Cooperation and Development, 58pp. Available at:http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=ENV/JM/ MONO(2011)45&doclanguage=en.

 

OECD (2013a).In vitroskin irritation: Reconstructed HumanEpidermisTest Method. OECD Guideline for the Testing of Chemicals No. 439, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

 

OECD (2013b). Guideline for the Testing of Chemicals 438: Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage. Organisation for Economic Cooperation and Development, Paris, France, Available at:[http://www.oecd.org/env/testguidelines]

 

Prinsen. M. K. The Draize Eye Test and in vitro alternatives; a left-handed marriage?Toxicology in Vitro 20 (2006) 78–81

Justification for selection of skin irritation / corrosion endpoint:

No single key study has been selected since all available data (OECD guideline studies, no deviations, RL1, GLP) are considered together in an weight of evidence approach.

Justification for selection of eye irritation endpoint:

OECD guideline study, no deviations, RL1, GLP

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Based on the available data MDIPA-Esterquat C16-18 and C18 unsatd. should be classified as irritating to the skin (Category 2) and irritating to the eye (Category 2) and labelled with H315 (Causes skin irritation) and H319 (Causes serious eye irritation) according to regulation (EC) 1272/2008.

According to the former European directive on classification and labelling 67/548/EEC MDIPA Esterquat C18 unsatd.is classified as R38 (Irritating to skin) and R36 (Irritating to eyes).