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Administrative data

Description of key information

The substance gave negative results in an in vitro skin irritation assay. It also gave negative results both in an in vitro eye irritation assay and an in vivo eye irritation assay.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
other: In vitro skin irritation guideline study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-31 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 439:In vitro skin irritation: Reconstructed Human Epidermis Test Method (adopted 22 July 2010)
Deviations:
yes
Remarks:
Listed below.
Qualifier:
according to
Guideline:
other: Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test ".
Deviations:
yes
Remarks:
Listed below.
Principles of method if other than guideline:
In vitro skin irritation test with Substance 3, NLP 500-090-6 using a human skin model.
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Skin Ethic
Justification for test system used:
As per OECD guideline.
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Saturated swabs with undiluted substance were placed into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μl PBS (negative control) and 3 tissues with 10 μl 5% SDS (positive control) respectively.
Duration of treatment / exposure:
The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. The substance could not be removed completely from the skin tissues. One of the tissues was damaged during removal of the substance. After rinsing the cell culture inserts were each dried carefully. The skin tissues were kept in new 12-well plates on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed.
Duration of post-treatment incubation (if applicable):
Subsequently the skin tissues were incubated for 42 hours at 37 °C.
Number of replicates:
The test was performed on a total of 3 tissues per test substance together with negative and positive controls.
Details on study design:
Test systemEPISKIN Standard ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 11-EKIN-004. This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.RationaleIn the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).SourceSkinEthic Laboratories, Nice, France.
Irritation / corrosion parameter:
% tissue viability
Value:
> 50
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.
Irritant / corrosive response data:
The substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. The substance was stained yellow/brownish. The MTT solution was slightly discoloured, but did not turn blue / purple and no blue / purple precipitate was observed. Therefore it was concluded that the substance did not interact with MTT. The mean absorption at 570 nm measured after treatment with the substance and controls are presented in Table 1. The individual OD570 measurements are presented in Table 3. Table 2 shows the mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissues was 94%. Since the mean relative tissue viability for the substance was above 50%, the substance is considered to be non-irritant. The positive control had a mean cell viability after 15 minutes exposure of 5%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Table 1 Mean absorption in the in vitro skin irritation test with Modified Small Vinyl Ester

 

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean    

(OD570)

SD

Negative control

1.132

1.144

1.070

1.115                       ±   0.040

Test substance

1.111

*

0.980

1.046**                     ±   0.093

Positive control

0.036

0.052

0.091

0.060                       ±   0.028

 

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

 

* The tissue was damaged during removal of the test substance, and therefore the OD570 of this tissue is not reliable

** Based on two individually treated tissues

Table 2 Mean tissue viability in the in vitro skin irritation test with Modified Small Vinyl Ester

 

Mean tissue viability

(percentage of control)

 

Negative control

100

Modified Small Vinyl Ester

94**

Positive control

5

 

** Based on two individually treated tissues

Table 3 Individual OD measurements at 570 nm

 

 

A

(OD570)

B

(OD570)

C

(OD570)

Negative control

OD570 measurement 1

OD570 measurement 2

 

1.087

0.178

 

1.108

1.180

 

1.044

1.096

Modified Small Vinyl Ester

OD570 measurement 1

OD570 measurement 2

1.078

1.145

 

*

*

 

1.024

0.937

Positive control

OD570 measurement 1

OD570 measurement 2

 

0.035

0.037

 

0.042

0.063

 

0.093

0.089

 

OD = Optical density

Triplicate exposures are indicated by A, B and C.

* The tissue was damaged during removal of the test substance, and therefore the OD570of this tissue is not reliable

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
It is concluded that this test is valid and that the substance is non-irritant in the in vitro skin irritation test under the experimental conditions.
Executive summary:

The substance was a clear light yellow-greenish to light brown very high viscous liquid. The substance was applied on a saturated cotton swab, which was placed directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the substance. The relative mean tissue viability of two tissues obtained after 15 minutes treatment with the substance compared to the negative control tissues was 94%. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment the substance is considered to be non-irritant. The positive control had a mean cell viability of 5% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly. Finally, it is concluded that this test is valid and that the substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS- Source:Charles River France, L’Arbresle Cedex, France- Age at study initiation:at least 6 weeks old- Weight at study initiation:at least 1.0 kg- Housing: Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).- Diet (e.g. ad libitum): Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period- Water (e.g. ad libitum): ad libitum- Acclimation period: At least five days.ENVIRONMENTAL CONDITIONS- Temperature (°C): 21.0 ± 3.0 ºC (actual range: 18.8-19.4 ºC),- Humidity (%):40-70% (actual range: 47-71%)- Air changes (per hr): 15- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL - Amount(s) applied (volume or weight with unit): 0.08 mL
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
21 days
Number of animals or in vitro replicates:
3 animals of one sex
Details on study design:
REMOVAL OF TEST SUBSTANCE- Washing (if done):Rinsed with tap water- Time after start of exposure: VariesSCORING SYSTEM:CORNEAL IRRITATIONOpacity: degree of density (area most dense taken for reading)No ulceration or opacity (may include slight dulling of normal luster) ............................................ 0Scattered or diffuse areas of opacity, details of iris clearly visible ................................................. 1Easily discernible translucent area, details of iris slightly obscured ............................................... 2Nacreous area, no details of iris visible, size of pupil barely discernible ........................................ 3Opaque cornea, iris not discernible through the opacity .............................................................. 4Area of cornea involved:No ulceration or opacity ................................................................................................................ 0One quarter or less but not zero .................................................................................................. 1Greater than one quarter, but less than half ................................................................................. 2Greater than half, but less than three quarters ............................................................................. 3Greater than three quarters, up to whole area .............................................................................. 4IRISNormal ....................................................................................................................................... 0Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or combination thereof, iris still reacting to light (sluggish reaction is positive) ....................................................................................................... 1No reaction to light, hemorrhage, gross destruction (any or all of these) ....................................... 2CONJUNCTIVAL IRRITATIONRedness (refers to palpebrae and sclera, excluding cornea and iris):Blood vessels normal .................................................................................................................. 0Some blood vessels definitely hyperaemic (injected) .................................................................... 1Diffuse, crimson color, individual vessels not easily discernible .................................................... 2Diffuse beefy red .......................................................................................................................... 3Chemosis (refers to lids and/or nictitating membranes):No swelling ………………………………………… ........................................................................ 0Any swelling above normal (includes nictitating membranes) ...................................................... 1Obvious swelling with partial eversion of lids ................................................................................ 2Swelling with lids about half closed ............................................................................................. 3Swelling with lids more than half closed ....................................................................................... 4Discharge:No discharge (may include small amounts observed in inner canthus of normal animals) ........... 0Any amount different from normal and/or lacrimation ................................................................... 1Discharge with moistening of the lids and hairs just adjacent to lids ............................................. 2Discharge with moistening of the lids and hairs (considerable area around the eye) ................... 3TOOL USED TO ASSESS SCORE: Ophthalmic examination lamp
Irritation parameter:
cornea opacity score
Remarks:
Cornea opacity
Basis:
mean
Time point:
other: 1, 24, 48, 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 1, 24, 48, 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Time point:
other: 1, 24, 48, 72 hours
Score:
0.4
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: Reversible in 2 animals within 24 hours, and 1 animal within 48 hours
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: 1, 24, 48, 72 hours
Score:
0
Max. score:
4
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
mean
Time point:
other: 1, 24, 48, 72 hours
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 24 hours
Irritant / corrosive response data:
Instillation of approximately 100.4 mg of Substance 3, NLP 500-090-6 (a volume of approximately 0.08 mL) into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness and discharge. The irritation had completely resolved within 24 hours on two animals and within 48 hours in the other animal. No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. Results are presented in Table 1 below.

Table 1. Individual eye irritation scores

Animal

Time after dosing

Cornea

Iris

Conjunctivae

 

Comments

Opacity

(0-4)

Area

(0-4)

Fluor area (%)2

 

Redness

(0-3)

Chemosis

(0-4)

Discharge

(03)

161

1 hour

0

0

-

0

1

0

1

b

24 hours

0

0

0

0

0

0

0

-

48 hours

0

0

-

0

0

0

0

-

72 hours

0

0

-

0

0

0

0

-

18

1 hour

0

0

-

0

2

0

1

c

24 hours

0

0

0

0

1

0

0

-

48 hours

0

0

-

0

0

0

0

-

72 hours

0

0

-

0

0

0

0

c

23

1 hour

0

0

-

0

1

0

1

-

24 hours

0

0

0

0

0

0

0

-

48 hours

0

0

-

0

0

0

0

-

72 hours

0

0

-

0

0

0

0

-

1Sentinel,1Green staining after fluorescein treatment (percentage of total corneal area)

Comments:

b                       Remnants of the test substance in the eye.

c                       Remnants of the test substance on the outside of the eyelids.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
Eye irritation of Modified Small Vinyl Ester was tested in a guideline study (OECD 405) in vivo in rabbits. No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after instillation of Modified Small Vinyl Ester revealed no corneal epithelial damage. Redness of the conjunctivae and discharge was observed at one hour after instillation that reversed completely within 24 hours except redness in one aminal that reversed completely within 48 hours. Based on these data, Modified Small Vinyl Ester is not eye irritating.
Executive summary:

Acute eye irritation/corrosion of Modified Small Vinyl Ester was tested in vivo in the rabbit in a the guideline study (OECD 405).

Single samples of approximately 100.4 mg of Modified Small Vinyl Ester (a volume of approximately 0.08 mL) were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours after instillation. Remnants of Modified Small Vinyl Ester were present in the eye of one animal and on the outside of the eyelids in the two other animals on Day 1.

Instillation of Modified Small Vinyl Ester resulted in irritation of the conjunctivae, which consisted of redness and discharge. The irritation had completely resolved within 24 hours on two animals and within 48 hours in the third animal. No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. Based on these results Modified Small Vinyl Ester is not irritating to the eyes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Modified Small Vinyl Ester was tested for skin irritation potential in a Reconstructed Human Epidermis assay (OECD 439). The relative mean tissue viability of two tissues obtained after 15 minutes treatment with Modified Small Vinyl Ester compared to the negative control tissues was 94%. Since the mean relative tissue viability for Modified Small Vinyl Ester was above 50% after 15 minutes treatment Modified Small Vinyl Ester is considered to be non-irritant in this assay.

Modified Small Vinyl Ester was tested for eye irritation potential using the Bovine Corneal Opacity and Permeability test (BCOP test). It did not induce ocular irritation or opacity. The mean in vitro irritancy score of 1.1 after 10 minutes of treatment. The positive control showed a mean score of approximately 130. It was also tested in vivo in the rabbit in a guideline study (OECD 405). Instillation of the substance resulted in irritation of the conjunctivae, which consisted of redness and discharge. The irritation had completely resolved within 24 hours on two animals and within 48 hours in the third animal. No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. Based on these results Modified Small Vinyl Ester is not irritating to the eyes.


Justification for selection of skin irritation / corrosion endpoint:
Only one study available

Justification for selection of eye irritation endpoint:
Two studies are available. The in vivo study has been selected as the more definitive study.

Justification for classification or non-classification

Modified Small Vinyl Ester was tested in vitro in a guideline study (OECD 439) using reconstructed human epidermis. It was not irritating under the experimental conditions of this test. Modified Small Vinyl Ester was tested in the Bovine Corneal Opacity and Permeability test based on the most recent OECD guideline. Modified Small Vinyl Ester was not irritating under the experimental conditions of this test. The substance was also tested in vivo in a guideline study (OECD 405) in rabbits. No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after instillation of Modified Small Vinyl Ester revealed no corneal epithelial damage. Redness of the conjunctivae and discharge was observed at one hour after instillation. The effects reversed completely within 24 hours except redness in one animal that reversed completely within 48 hours. Based on these data, Modified Small Vinyl Ester is not irritating to the eye. On the basis of this data there is no justification for classification for either skin or eye effects.