Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation in vivo test:

- The mean erythema score over 3 animals in the time span 24-72h is 2.23.

- The mean oedema score over 3 animals in the time span 24-72h is 3.10

Eye irritation in vitro BCOP test:

- IVIS score: 1.26

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Type of coverage:
semiocclusive
Vehicle:
other: The test substance was applied as supplied
Amount / concentration applied:
537 mg (administered as 0.5 mL of test material)
Duration of treatment / exposure:
4 h
Number of animals:
3
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
1.7
Reversibility:
fully reversible within: 16 days
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
2
Reversibility:
fully reversible within: 16 days
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
3
Reversibility:
fully reversible within: 16 days
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
2.7
Reversibility:
fully reversible within: 13 dyas
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
3.3
Reversibility:
fully reversible within: 13 days
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
3.3
Reversibility:
fully reversible within: 13 days
Irritant / corrosive response data:
Reversibility of any observed effect: Changes fully reversible within 16 days.
Other effects:
Very slight or slight erythema (score 1-2) and severe oedema (score 4) were observed in all animals one hour after bandage removal. At the 24 hour examination, slight or moderate erythema (score 2-3) and severe oedema (score 4) were evident. There was a gradual resolution of the oedema and erythema from 48 or 72 hour after bandage removal up to Day 13. The test sites of all animals were overtly normal on Day 16.

Maximum duration of erythema was 16 days and of oedema 13 days.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The mean erythema score over 3 animals in the time span 24-72h is 2.23.
The mean oedema score over 3 animals in the time span 24-72h is 3.10
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:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:
09 October 2017
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
purity: 98.4%
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test system:
Freshly isolated bovine cornea obtained as a by-product from animals freshly slaughtered
- Source: A. Moksel AG, Buchloe, Germany
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item, the negative or positive control
Duration of treatment / exposure:
- 10 minutes incubation at 32 ± 1 °C, washed at least three times with MEM (containing phenol red)
Duration of post- treatment incubation (in vitro):
- illuminance measurement was performed after 2 hours incubation with RPMI at 32 ± 1 °C
- After the illuminance measurement the corneas were incubated for 90 minutes with RPMI and sodium fluorescein solution at 32 ± 1 °C for the optical density determination
Number of animals or in vitro replicates:
3 corneas/group
Details on study design:
Preparation of the Corneas
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS
containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2
to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the
corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring
of the posterior chamber, they had been visually examined for defects and any defective cornea had
been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws.
The chambers of the corneal holder were then filled with RPMI (without phenol red) containing
1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The
corneas were incubated for one hour at 32 ± 1 °C.

Treatment of the Corneas
After the equilibration period, the medium was removed from both chambers and replaced with fresh
complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer.
Three corneas with illuminance readings approximately equivalent to the median illuminance of all
corneas were selected as negative-control corneas. The illuminance of each cornea was read and re
corded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.
The medium was removed from the anterior chamber and replaced with the test item or control.
0.750 mL of the test substance or the control substance was introduced into the anterior chamber. Af
ter 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed
and the epithelium washed at least three times with MEM (containing phenol red). Once the medium
was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).
The anterior chamber was refilled with complete RPMI and an illuminance measurement was p
erformed after 2 hours incubation at 32 ± 1 °C. Also, each cornea was observed visually and pertinent
observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers
of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL
sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for
90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical
density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Test Groups
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with ethanol 100%

Validity of the assay:
The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations
of the current historical mean.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background
bovine corneas treated with the respective negative control.

Evaluation of Results:
The following formula was used to calculate the opacity, whereas the values a and b are equipmentspecific
variables empirically determined by the manufacturer: Opacity= ( I0/I-b)/a; with a = 0.025 and b = 0.9894.

The change in opacity for each cornea was calculated by subtracting the initial opacity
reading from the final opacity reading. These values were corrected by subtracting from
each the average change in opacity observed for the negative-control corneas. The
mean opacity value for each treatment was calculated by averaging the corrected
opacity values of each cornea for a given treatment.
The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the
negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490

The mean OD490 value of each treatment group was calculated by averaging the final
corrected OD490 values of the treated corneas for that treatment condition.
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3 corneas / 10 min incubation time
Value:
1.26
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The in vitro irritation score obtained with the positive control fell within the two standard
deviations of the current historical mean and therefore this assay is considered to be
valid.
The negative control responses resulted in opacity and permeability values that are less
than the established upper limits for background bovine corneas treated with the
respective negative control.

In Vitro Irritation Score

Cornea no. Test item Corrected opacity Corrected OD490 value IVIS
1 Negative control 0.11 0.004 0.48
2 0.68 0.005
3 0.47 0.003
MV 0.42 0.004
4 Positive control 22.29 1.178 52.16
5 35.57 1.776
6 23.62 2.046
MV 27.16 1.667
7 Test item 1.28 0.002 1.26
8 0.55 0.004
9 1.83 0.002
MV 1.22 0.003
Interpretation of results:
GHS criteria not met
Conclusions:
According to the evaluation criteria the test item 4-acetoxystyrene is classified into UN
GHS No Category.
Executive summary:

The eye irritancy potential of 4-acetoxystyrene was investigated in the bovine corneal opacity and permeability assay according to OECD 437 and in compliance to GLP.

All 3 corneas treated with 4-acetoxystyrene showed a very slight opacity of the tissue. The following mean in vitro irritation score (IVIS) was calculated: 1.26

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Therefore the test item was classified into UN GHS No Category.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Justification for classification or non-classification

Based on the mean oedema score (3.10 in 3 animals, 24 -72h time span) obtained in the available in vivo skin irritation test, classifciation of 4-acetoxystyrene as Skin Irritant Cat. 2 is warranted.

Based on the IVIS score (1.26) obtained in the available in vitro BCOP eye irritation study, classification of 4-acetoxystyrene as eye irritant is not required.