Reaction mass of bisisopropyl peroxydicarbonate and bis-sec-butyl peroxydicarbonate and isopropyl-secbutylperoxydicarbonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-26 to 2017-07-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, no. 437 (adopted: 26 July 2013)

Deviations:

yes

Remarks:

Concerning: Calibration of the Opacitometer (see Test System)

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Munich, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bisisopropyl peroxydicarbonate and bis-sec-butyl peroxydicarbonate and isopropyl-secbutylperoxydicarbonate

EC Number:

931-536-1

Molecular formula:

mixture of C8H14O6, C9H16O6, C10H18O6

IUPAC Name:

Reaction mass of bisisopropyl peroxydicarbonate and bis-sec-butyl peroxydicarbonate and isopropyl-secbutylperoxydicarbonate

additive 1

Reference substance name:

Diallyl 2,2'-oxydiethyl dicarbonate

EC Number:

205-528-7

EC Name:

Diallyl 2,2'-oxydiethyl dicarbonate

Cas Number:

142-22-3

Molecular formula:

C12H18O7

IUPAC Name:

diallyl oxydiethane-2,1-diyl biscarbonate

Test material form:

liquid

Specific details on test material used for the study:

Name: Isopropyl sec-butyl peroxy dicarbonate (multi constituent)
Product Name: Trigonox ADC-NS30
Chemical Name: Reaction mass of bisisopropyl peroxydicarbonate and bis-sec-butyl peroxydicarbonate and isopropylsecbutylperoxydicarbonate
Composition: 71.5% w/w Diethylene glycol bis(allyl carbonate) [142-22-3]
28.0% w/w Isopropyl sec-butyl peroxy dicarbonate [79350-78-4],
Di-sec-butyl peroxydicarbonate [19910-65-7], and
Diisopropyl peroxydicarbonate [105-64-6]
Batch No.: 16041B0206
Physical State: liquid
Colour: colorless
Density: 1.1 g/mL
Storage Conditions: ≤-20°C, protected from light
Expiry Date: 08 August 2017
Safety Precautions: The routine hygienic procedures will be sufficient to assure personnel health and safety.
Peroxide handling
on the lab: The peroxide - water mixture is a 2 liquids phase system. Intensive mixing is required to get the right organic – water ratio in the sample applied for tox testing:
• place the polymer support on the bottom of the other beaker (to fix the inner beaker)
• fill the other glass beaker for 1/3 with ice
• place an inner beaker, with solvent and a magnetic stirring rod, in the outer beaker
• add the peroxide during stirring, apply a stirring speed of
• 200 rpm for peroxide - IPA mixtures, or 500 rpm for peroxide – water (or salt water) mixtures
• maximum handling time: 60 minutes

Test system

Vehicle:

physiological saline

Amount / concentration applied:

The test item was used as provided by the sponsor. Handling of the peroxide – water mixture before administration is described above . 750 µL of the test substance or the control substance physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 17031412, expiry date: 12/2019) was introduced into the anterior chamber.

Duration of treatment / exposure:

After 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).

Observation period (in vivo):

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.

Details on study design:

Test System

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
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.


Calibration of the Opacitometer:
The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to
1000 lux ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 95-105 lux. The calibration procedure was performed before each test, once before the first illuminance measurement and once before the second illuminance measurement and is documented in the raw data.

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 recorded. 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.
750 ± L of the test substance or the control substance was introduced into the anterior chamber. After 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 performed 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%
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 equipment-specific variables empirically determined by the manufacturer:
Opacity = (I0/I - b)/a

with a = 0.025 and b = 0.9894

The value I0 = I(zero) is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically.
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 mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. 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)
The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in the table below.

An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.
For this purpose further testing with another suitable method is required

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

3.65

In vivo

Irritant / corrosive response data:

The eye irritancy potential of Isopropyl sec-butyl peroxy dicarbonate (multi constituent) was investigated in the bovine corneal opacity and permeability assay.
The test item was used as provided by the sponsor. Handling of the peroxide – water mixture before administration is described above.
All 3 corneas treated with Isopropyl sec-butyl peroxy dicarbonate (multi constituent) showed slight opacity of the tissue.
The following mean in vitro irritation score was calculated:
3.65

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made regarding the classification of the test substance Isopropyl sec-butyl peroxy dicarbonate (multi constituent) according to the evaluation criteria. Further testing in another suitable method is required.

Executive summary:

Summary Results

The eye irritancy potential of Isopropyl sec-butyl peroxy dicarbonate (multi constituent) was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item:                      tested as provided by the sponsor

Visual Observation after treatment:         All 3 corneas treated with Isopropyl sec-butyl peroxy dicarbonate (multi constituent) showed slight opacity of the tissue.

Meanin vitroirritation score:                     3.65

Classification:     

 

X	No prediction can be made

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.