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Endpoint summary

Description of key information

PLS Green 5 was negative for skin and eye irritation when tested in key in vivo studies in New Zealand rabbits according to standard OECD 404 and OECD 405 methods, respectively. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to valid methods and considered reliable, adequate and relevant, but it was non-GLP and only limited data were provided.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: male young healthy adults
- Acclimation period: at least 5 days

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: . The areas not tested on the animal itself, serve as a control

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

The readings of the skin reactions are carried out in periods of 1, 24, 48, 72 hours and 14 days, as applicable.

Number of animals:

preliminary test: 1
main test: 2

Details on study design:

TEST SITE
- Area of exposure: dorsal thoracic region in an area of approximately 6 cm2
- Type of wrap if used:This area is covered with sterile gauze and secured with the help of hypoallergenic adhesive tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period (4 hours), residual test substance is removed using water or appropriate solvent, as necessary.
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Evaluation of Edema formation:
Grade 0: No edema
Grade 1: Very light edema (little perceptive)
Grade 2: Mild edema (border area well-defined and more perceptive)
Grade 3: Moderate edema (increase of approximately 1 mm)
Grade 4: Severe edema (of more than 1 mm increase, including the area higher than the area in contact with the product)

Evaluation of Erythema formation:
Grade 0: Normal skin (usually white in color, can be pink)
Grade 1: Mild erythema (slightly reddened skin where realizes a tint unlike the control area)
Grade 2: Well-defined erythema (the skin becomes red, usually in every area)
Grade 3: Moderate to severe erythema (the skin presents with intense and diffuse redness)
Grade 4: Severe erythema of the skin (the skin presents mild eschar formation -injuries in depth)

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

"PLS Green 5" (pH = 5.0) was held in the shaved region on the thoracic back of test animals (pre-test). After exposure of the test substance to the skin of the animal for a period of four hours, slight erythema (grade 1) was observed. However, after 24 hours after the start of the test, the skin presents with normal appearance and coloration featuring the complete reversibility of the observed reactions (grade 0). We also point out that the animal not presented any systemic signs of toxicity during the days of observation. The same pattern of skin reaction was observed in both animals used for the main test and there is also full reversibility of initial erythema observed. Since there were not detected any skin reactions for a period of 72 hours after removal of the semi-occlusive bandages, the test was terminated and the test substance classified as non-irritating.

 Table 1- Values ​​attributed to skin reactions after exposure of the animal to "PLS Green 5" in the pretest.

Cage	Animal	Parameters	4h exposure	1h	24h	48h	72h	14d
1	1	Edema	0	0	0	0	0	-
		Erythema	1	0	0	0	0	-

 

 

Table 2- Values ​​attributed to skin reactions after exposure of the animal to "PLS Green 5" in the main test.

Cage	Animal	Parameters	1h	24h	48h	72h	14d
2	2	Edema	0	0	0	0	-
		Erythema	0	0	0	0	-

 

Table 3- Values ​​attributed to skin reactions after exposure of the animal to "PLS Green 5" in the main test.

Cage	Animal	Parameters	1h	24h	48h	72h	14d
3	3	Edema	0	0	0	0	-
		Erythema	0	0	0	0	-

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

"PLS Green 5" was not irritating to rabbit skin under the conditions of this test.

Executive summary:

PLS Green 5 (Ph = 5.0) was administered in three New Zealand rabbits for 4h on 6 cm2 skin surface by semi-occlusive exposure. Scoring was done after 1h, 24h, 48h, 72h and 14 days for edema and erythema. Values for edema and erythema were 0 in all three annimals up to 72h. In conclusion, PLS Green 5 was not irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to valid methods and considered reliable, adequate and relevant, but it was non-GLP and only limited data were provided.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: young healthy adults
- Acclimation period: at least 5 days

Vehicle:

unchanged (no vehicle)

Controls:

other: • The untreated eye of the animal serves as control.

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

The observation period should be sufficient to evaluate the reversibility of the effects observed.
To determine the reversibility of eye effects, the animals should be observed for 21 days after the administration of test substance. If it is possible to determine the reversibility of these effects before 21 days, the test can be completed at this time, this period being not less than three days. The test was completed after 72 h observation.

Number of animals or in vitro replicates:

preliminary test: 1
main test: 2

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): . The eyes of the animal should not be washed for the first 24 hours after application of the test substance, but the washing with saline solution 0.9% for better visualization of ocular reactions is allowed during readings. However, in the case of solid samples if the test substance has not been removed by physiological mechanisms one hour after administration, the eye of the animal may
be washed with 0.9% saline solution or distilled water
- Time after start of exposure: 24 hours

SCORING SYSTEM:
The readings of ocular reactions should be recorded in periods of 1, 24, 48, 72 hours and 7, 14 and 21 days (where applicable). A graduate of the intensity of ocular reactions will be recorded according to the following criteria:

Cornea opacity: intensity degree (readings should be made in the most dense areas)
Grade 0: Normal cornea: transparent with characteristic brilliance.
Grade 1: The loss of normal brightness is observed. The details of the iris are clearly visible, with diffuse opacity area.
Grade 2: Defined opacity areas are observed. The iris details are visible
Grade 3: Necrosis areas are observed. The iris details are not visible (the observer is still able to graduate iritis and pupillary response to light).
Grade 4: Corneal opacity is observed. The iris cannot be displayed (the observer is not able to graduate iritis or pupillary response to light).
* The corneal opacity area should be measured and recorded.

Iris
Grade 0: Normal iris
Grade 1: Sharply deep wrinkles, congestion, swelling, moderate hyperemia around the cornea. The animal exhibits reaction to light (pupillary reflex).
Grade 2: bleeding, complete destruction. The animal has no reaction to light (pupillary reflex).

Conjunctiva: hyperaemia
Grade 0: Normal. The conjunctiva is presented pink perilimbic without congestion. The vessels of the eyelid and bulbar conjunctiva are easily observable.
Grade 1: Hyperemic blood vessels of the conjunctiva of the eyelid. There is some congestion perilimbic.
Grade 2: Palpebral vessels of the conjunctiva are not easily discernible, with diffuse redness and at least 75% of the perilimbic region is congested.
Grade 3: Dark and intense redness across the conjunctiva. Presence of petechiae (points bleeding).

Chemosis: edema (refers to lids and / or nictitating membranes)
Grade 0: Absence of edema.
Grade 1: Mild edema without eversion of the eyelids (upper and lower eyelids are positioned as the normal eye).
Grade 2: Swelling with eversion of the eyelids, often the upper and lower eyelids are misaligned.
Grade 3: Swelling with eversion of the two eyelids (the borders of the eyelids don’t get together ).
Grade 4: Edema with upper eyelid eversion more pronounced than the lower eyelid (it is difficult to retract the eyelids and observe the perilimbic region).

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

2

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

3

Irritant / corrosive response data:

"PLS Green 5" (pH = 5.0)was applied in the left eye of experimental animals. One hour after ocular application of the test substance hyperemia of the vessels of the palpebral conjunctiva (grade 1) was observed. The complete reversal of that eye reaction occurred 24 hours after initiation of the test (preliminary test). Since any additional ocular reactions were not detected for a period of 72 hours after the start of the test, the preliminary test was terminated. It is noteworthy that during the experimental period there were not observed any systemic reactions indicative of toxicity. In order to confirm the results obtained in the preliminary test, two additional animals were used undergoing similar experimental procedure (main test). As in the preliminary test, one hour after ocular application of the test substance hyperemia of the vessels of the palpebral conjunctiva (grade 1) was observed.
A complete reversal of that eye reaction occurred 48 hours after initiation of the test and there were not any systemic signs of toxicity observed . The product was therefore classified as slightly irritating and the test terminated 72 hours after application of the test substance.

Table 1. Values attributed to ocular reactions after exposure of the animal to “PLS Green 5” in the preliminary eye irritation test

Cage	Animal	Parameters	1h	24h	48h	72h	14d	21d
4	4	Cornea	0	0	0	0	-	-
		Iris	0	0	0	0	-	-
		Conjunctiva redness	1	0	0	0	-	-
		Chemosis	0	0	0	0	-	-

 

Table 2. Values attributed to ocular reactions after exposure of the animal to “PLS Green 5” in the main eye irritation test

Cage	Animal	Parameters	1h	24h	48h	72h	14d	21d
5	5	Cornea	0	0	0	0	-	-
		Iris	0	0	0	0	-	-
		Conjunctiva redness	1	0	0	0	-	-
		Chemosis	0	0	0	0	-	-

 

Table 3. Values attributed to ocular reactions after exposure of the animal to “PLS Green 5” in the main eye irritation test

Cage	Animal	Parameters	1h	24h	48h	72h	14d	21d
6	6	Cornea	0	0	0	0	-	-
		Iris	0	0	0	0	-	-
		Conjunctiva redness	1	0	0	0	-	-
		Chemosis	0	0	0	0	-	-

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

"PLS Green 5" was slightly irritating to the rabbit eye under the conditions of this test.

Executive summary:

PLS Green 5 (pH = 5.0) was applied in the left eye of three New Zealand rabbits. One hour after ocular application of the test substance hyperemia of the vessels of the palpebral conjunctiva (grade 1) was observed. The complete reversal of that eye reaction occurred 24 hours after initiation of the test (preliminary test). Since any additional ocular reactions were not detected for a period of 72 hours after the start of the test, the preliminary test was terminated. It is noteworthy that during the experimental period there were not observed any systemic reactions indicative of toxicity. In order to confirm the results obtained in the preliminary test, two additional animals were used undergoing similar experimental procedure (main test). As in the preliminary test, one hour after ocular application of the test substance hyperemia of the vessels of the palpebral conjunctiva (grade 1) was observed. A complete reversal of that eye reaction occurred 48 hours after initiation of the test and there were not any systemic signs of toxicity observed. Although the product case concluded to be slightly irritating, the mean scores of 24 -72h were 0 for all observation criteria, therefore classification is not needed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a key in vivo study, PLS Green 5 (pH = 5.0) was administered in three New Zealand rabbits for 4h on 6 cm² skin surface by semi-occlusive exposure (de Carvalho, 2010). Scoring was done after 1h, 24h, 48h, 72h for edema and erythema. Values for edema and erythema were 0 in all three animals up to 72h. In conclusion, PLS Green 5 was not irritating.

 

In a key in vivo study, PLS Green 5 (pH = 5.0) was applied in the left eye of three New Zealand rabbits (de Carvalho, 2010). One hour after ocular application of the test substance hyperaemia of the vessels of the palpebral conjunctiva (grade 1) was observed. The complete reversal of that eye reaction occurred 24 hours after initiation of the test (preliminary test). Since any additional ocular reactions were not detected for a period of 72 hours after the start of the test, the preliminary test was terminated. Two additional animals were used undergoing similar experimental procedure (main test). As in the preliminary test, one hour after ocular application of the test substance hyperaemia of the vessels of the palpebral conjunctiva (grade 1) was observed. A complete reversal of that eye reaction occurred 48 hours after initiation of the test and there were not any systemic signs of toxicity observed. Although the product case concluded to be slightly irritating, the mean scores of 24 - 72h were 0 for all observation criteria, therefore classification is not needed.

Justification for classification or non-classification

Based on the results and according to the EC criteria for classification and labelling according to CLP regulation (EC No. 1272/2008 of 16 December 2008), 'fatty acids, C16 -18 and C18-unsaturated isopentyl esters, epoxidized' does not have to be classified and has no obligatory labelling requirement for skin and eye irritation.