Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-19-03 to 2015-08-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD 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)

Version / remarks:

adopted 26 July 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria

EC Number:

943-175-7

IUPAC Name:

Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria

Specific details on test material used for the study:

Substance was tested at different pH values and various concentrations:
pH 5.8; 100%, 50% and 10%
pH 7.0; 100%, 50% and 10%

Test animals / tissue source

Species:

chicken

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 at study initiation: Approximately 7 weeks old
- Weight at study initiation: body weight range 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.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

liquids used as supplied by the sponsor

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg for solid test substance; 30 µL for liquid preparations

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, 30 µL): 2
positive control (NaOH, 30 mg): 3
test group: 3 per concentration / pH

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 mg test item. 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 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.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

100% pH 5.8

Value:

33

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

100% pH 5.8

Value:

3.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

100% pH 5.8

Value:

2.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

percent corneal swelling

Run / experiment:

100 % pH 7

Value:

16

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

100 % pH 7

Value:

2.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 6

Irritation parameter:

fluorescein retention score

Run / experiment:

100 % pH 7

Value:

2.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 7

Irritation parameter:

percent corneal swelling

Run / experiment:

50% pH 5.8

Value:

28

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 8

Irritation parameter:

cornea opacity score

Run / experiment:

50% pH 5.8

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 9

Irritation parameter:

fluorescein retention score

Run / experiment:

50% pH 5.8

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
The test item at 50% / pH 5.8 and 100% / pH 5.8 caused corneal effects leading to Category 1 classification, consisting of moderate or severe corneal swelling (28 and 33%), severe or severe to very severe opacity (mean score of 3.0 or 3.5) and severe or moderate to severe fluorescein retention (mean score of 2.8 or 3.0). In addition, loosening (veil-like) of epithelium was observed.

Microscopic examination of the corneas treated with teh test item at 100% / pH 5.8 revealed very slight, slight or severe erosion and very slight vacuolation (one cornea top region; one cornea low region) of the epithelium. In addition, the epithelium top ceillayer was hypertrophic in one cornea.
Microscopic examination of the corneas treated with the test item at 50% / pH 5.8 revealed slight or moderate erosion and slight vacuolation (one cornea mid region) of the epithelium. In addition, the epithelium top cell layer was hypertrophic all corneas.
The AlSE histopathology criteria for upgrading to category 1 were met, but did not need to be applied, because it was already classified as category 1 on the basis of the slit-Iamp observations.

The test item at 100% / pH 7.0 caused corneal effects leading to Category 2/2A classification, consisting of slight corneal swelling (16%), moderate or moderate to severe opacity (mean score of 2.3) and moderate or moderate to severe fluorescein retention (mean score of 2.2). In addition, loosening (veil-like) of epithelium was observed.
Microscopic examination ofthe corneas treated with the test item at 100% / pH 7.0 revealed moderate or severe erosion, slight necrosis (one cornea) and very slight (one cornea low region) or slight (one cornea mid region) vacuolation of the epithelium.
According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is required.


DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

The negative control eyes did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control NaOH caused (very) severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused very slight, moderate or severe erosion and slight necrosis of the epithelium, and endothelial necrosis (one cornea).

Any other information on results incl. tables

Summary results of the slit-Iamp examination

Test material	Maximum mean score for:			Irritation categories^1	Irritation Index^2	Classifications (EU-CLP^3/UN-GHS^4)
	Swelling	Opacity	Fluorescin retention
test item(pH 5.8)_100%	33	3.5^5	2.8	IV;IV;IV	159	1/1
test item (pH 5.8)_50%	28	3.0^5	3.0	III;IV;IV	148	1/1
test item (pH 5.8)_10%	6	1.3^5	2.0	II;II;III	72	2/2B
test item (pH 7.0)_100%	16	2.3^5	2.2	II;III;III	106	1^7/1^7
test item (pH 7.0)_50%	8	1.3^5	2.0	II;II;III	74	2/2B
test item (pH 7.0)_10%	8	1.5^5	2.0	II;II;III	78	2/2B
NaOH (positive control)	46	4.0^6	3.0	IV;IV;IV	186	1/1

 

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 1272/2808 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Oirectives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2806.

4 UN-GHS: NC = not classified; Category 2B = mild irritant, causes eye irritation; Category 2A = 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 Loosening (veil-like) of epithelium

6 Severe loosening of epithelium

7 According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is required.

 

 

Individual histopathological findings

Test material	Eye no	Epithelium						Stroma			Endothelium
		Erosion	Necrosis	Vacuolation			Notes	Disorder of fibers	Pyknotic nuclei
				top	mid	low			outer region (adjacent to epithelium)	inner region (adjacent to endothelium)
test item 100% pH 5.8	4	3	-	-	-	-	-	-	-	-	-
	6	1	-	-	-	½	-	-	-	-	-
	8	½	-	½	-	-	‡	-	-	-	-
test item 100% pH 7.0	5	3	-	-	-	-	-	-	-	-	-
	7	2	1	-	1	-	-	-	-	-	-
	9	2	-	-	-	½	-	-	-	-	-
NaOH (positive control)	1	3	1	-	-	-	-	-	-	-	P
	2	2	1	-	-	-	-	-	-	-	-
	3	½	1	-	-	-	-	-	-	-	-
Saline (negative control)	10	-	-	-	-	-	-	-	-	-	-

- = not observed; P = present; 1/2 = very slight; 1 = slight; 2 = moderate; 3 = severe;

† = scored in the top/mid/low section of the epithelium; ‡ = epithelium top cell layer hypertrophic

 

Test material	Eye no	Epithelium						Stroma			Endothelium
		Erosion	Necrosis	Vacuolation			Notes	Disorder of fibers	Pyknotic nuclei
				Top	mid	low			outer region (adjacent to epithelium)	inner region (adjacent to endothelium)
test item 50% pH 5.8	21	2	-	-	-	-	‡	-	-	-	-
	25	1	-	-	-	-	‡	-	-	-	-
	29	2	-	-	1	-	‡	-	-	-	-
test item 50% pH 7.0	22	½	-	1	-	-	‡	-	-	-	-
	26	½	-	1	-	-	‡	-	-	-	-
	30	½	-	-	-	-	-	-	-	-	-
test item 10% pH 5.8	23	½	-	-	-	-	‡	-	-	-	-
	27	½	1	-	-	-	‡	-	-	-	-
	31	½	1	½	-	-	‡	-	-	-	-
test item 10% pH 7.0	24	½	½	½	-	-	‡	-	-	-	-
	28	½	1	-	-	-		-	-	-	-
	32	½	½	-	-	-	‡	-	-	-	-
Saline (negative control)	33	-	-	-	-	-	-	-	-	-	-

- = not observed; P = present; 1/2 = very slight; 1 = slight; 2 = moderate; 3 = severe;

† = scored in the top/mid/low section of the epithelium; ‡ = epithelium top cell layer hypertrophic

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the results from this Isolated Chicken Eye test, the testsubstance Rhamnolipids applied neat (100%) is considered "Irreversible effects on the eye/serious damage to the eye" (Category 1) under the experimental conditions described in this report (pH 7 and pH 5.8).

Based on the results from this Isolated Chicken Eye test, the testsubstance Rhamnolipids applied as a 50% dilution at pH 5.8 is considered "Irreversible effects on the eye/serious damage to the eye" (Category 1) under the experimental conditions described in this report.

Executive summary:

In an Isolated Chicken Eye (ICE) test according to OECD Guideline 438 (adopted 26 July 2013), the eye irritation potential of the testsubstance Rhamnolipids was assessed at different concentrations and pH vaues. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption.

The isolated chicken eyes were exposed to a single application of 30 mg 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. In addition, histopathology of the corneas was performed.

 

The test item at 50% / pH 5.8 and 100% / pH 5.8 caused corneal effects leading to Category 1 classification, consisting of moderate or severe corneal swelling (28 and 33%), severe or severe to very severe opacity (mean score of 3.0 or 3.5) and severe or moderate to severe fluorescein retention (mean score of 2.8 or 3.0). In addition, loosening (veil-like) of epithelium was observed.

Microscopic examination of the corneas treated with the test item at 100% / pH 5.8 revealed very slight, slight or severe erosion and very slight vacuolation (one cornea top region; one cornea low region) of the epithelium. In addition, the epithelium top ceillayer was hypertrophic in one cornea.

Microscopic examination of the corneas treated with the test item at 50% / pH 5.8 revealed slight or moderate erosion and slight vacuolation (one cornea mid region) of the epithelium. In addition, the epithelium top cell layer was hypertrophic all corneas.

The AlSE histopathology criteria for upgrading to category 1 were met, but did not need to be applied, because it was already classified as category 1 on the basis of the slit-Iamp observations.

The test item at 100% / pH 7.0 caused corneal effects leading to Category 2/2A classification, consisting of slight corneal swelling (16%), moderate or moderate to severe opacity (mean score of 2.3) and moderate or moderate to severe fluorescein retention (mean score of 2.2). In addition, loosening (veil-like) of epithelium was observed.

Microscopic examination of the corneas treated with the test item at 100% / pH 7.0 revealed moderate or severe erosion, slight necrosis (one cornea) and very slight (one cornea low region) or slight (one cornea mid region) vacuolation of the epithelium.

According to the AlSE histopathology criteria, upgrading to category 1 on basis of the histopathology of the corneas is required.

 

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.

The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.