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Description of key information

In vitro studies of skin irritation (EpiSkin) and eye damage (ICE Test) are available for the submission substance.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 September 2009 to 21 January 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Draft version 6 second circulation followed (20 March 2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
The human keratinocytes come from mammary/abdominal samples obtained from healthy consenting donors during plastic surgery. HIV 1 & 2, HEP B and HEP C tests are carried out on the donors as well as verification of the bacteriological and fungal sterility of the cells. After a stage of immersed culture of the keratinocytes on the collagen support (3 days), during emersion (10 days) the epidermis differentiates and a horny layer is formed. The production of EpiSkin® is in accordance with the quality reference norms ISO 9001, ensuring traceability and reproducibility of the epidermal tissue as well as that of the quality control tests carried out on each batch of epidermis. The reproducibility of each batch is checked by histological analysis taking into account the general organisation, the stratification of the epidermis, the nucleation of the basal layer and the size of the intercellular spaces, adhesion of the basal layer to the support and the quantity of granular cells and the thickness of the horny layer. Each criterion is scored out of 4. The maximum score is 28. The minimum score for the criterion of acceptability of the model is 19.5. The score for the batch used on this study (09-EKIN-033) is 22.3 mg/mL; standard deviation of 0.3 and CV of 1.2%. The reproducibility of the response of each EpiSkin® batch is tested against a reference irritant: SDS. The IC50 of the surfactant is measured by the MTT assay after 18 h of contact. The minimum score for acceptability of the model is 1.5 mg/mL. The IC50 value for the batch used on this study (09-EKIN-033) is 2.3 mg/mL.

Prior to conducting the irritation assay, it was first necessary to determine if the test item was itself capable of reducing methylthiazoldiphenyl-tetrazolium bromide (MTT) to its formazan metabolite. Since the assay depends on the ability of viable skin cells to reduce MTT to formazan, any reduction by the test item would interfere with the integrity of the results. The test item (10 μL) was added to MTT (2 mL, ca 0.3 mg/mL) in PBS in a glass universal vial (3 replicates) and incubated at ca 37°C in a humidified atmosphere with 5% CO2 for ca 3 h. Formazan production was assessed by visual inspection. Three replicates of the positive control (eugenol, 10 μL) and the negative control (sterile, ultra-pure water, 10 μL) were tested in parallel to demonstrate the efficacy of the MTT solution.

After arrival at Charles River, the EpiSkin® kits were inspected for quality. The pH and temperature indicators were both acceptable. Each individual unit was then transferred to a single well of a 12 well microtiter plate containing maintenance medium (2 mL). The maintenance medium had been warmed to ca 37°C in a waterbath. Prior to dosing, all units were then incubated for ca 24 h at ca 37°C in a humidified atmosphere with 5% CO2.

DMP Tech was applied onto the exposed surface of three viable EpiSkin® reconstructed human epidermis units using a Gilson positive displacement pipette set to deliver 10 μL. The test item was gently spread over the entire exposed skin surface using the tip of the pipette, taking care to avoid damaging the epidermis. The surface area of the EpiSkin® is ca 0.38 cm2 and therefore the application rate was ca 26.3 μL/cm2.

After exposure to the test item or positive (5% sodium dodecyl sulphate) and negative controls (PBS solution) for ca 15 min, the EpiSkin® units were washed by rinsing with Dulbecco’s phosphate buffered saline (25 mL). After washing, all skin units were blotted dry with tissue paper, transferred to fresh maintenance medium and incubated for ca 42 h at ca 37°C in a humidified atmosphere with 5% CO2.

After the recovery period, all EpiSkin® units were transferred to a new 12 well microtiter plate containing MTT solution in assay medium (ca 0.3 mg/mL, 2 mL per well). Each unit was tapped gently on tissue paper to remove residual moisture before transferring to the MTT solution. The skin units were then incubated for ca 3 h at ca 37°C in a humidified atmosphere with 5% CO2.

After incubation, each skin unit was removed from the MTT solution and gently tapped dry on tissue paper to remove any excess moisture. The exposed skin was then completely removed from the unit using a specially designed biopsy punch. The epidermis was separated from the collagen matrix and both layers added to an appropriately labelled microfuge tube containing acidic isopropanol (500 μL). Samples were then stored at ca 4°C protected from light.

After ca 72 h storage, the samples were removed from the refrigerator and mixed by vortexing to ensure a homogenous mixture. Two aliquots (200 μL) were then added to a 96-well flat bottom microtiter plate for each sample, ensuring that no solid material was removed from the sample tube. Plates were analysed using an MRX plate reader using wavelength measurement at 550 nm. Absorbance values were calculated against the background acidified isopropanol sample contained on the plate.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL of neat test material over a surface area of approximately 0.38 cm2, giving an application rate of 26.3 µL/cm2.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
Approximately 42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
87.91
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
In the MTT direct reduction test, the visual assessment of the formation of the purple-coloured formazan was assessed. The positive control (eugenol) reduced the MTT solution to formazan almost immediately, generating a dark purple colour before incubation. The negative control (sterile, ultra-pure water) and DMP Tech did not reduce MTT to formazan after 3 hours incubation.
Interpretation of results:
GHS criteria not met
Conclusions:
The results of this study do not indicate that Dimethylolpropane Tech (DMP Tech) is a skin irritant.
Executive summary:

An in vitro assessment of irritation potential of skin was conducted using the EpiSkin test system. The method followed was the draft version of OECD guideline 439. Dimethylolpropane Tech (DMP Tech) was exposed for 15 minutes at an application rate of 23.3 µL/cm2. DMP Tech showed a viability of 87.91% when compared to the concurrent negative control.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
03 February 2015 to 09 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
GLP compliance:
yes (incl. certificate)
Species:
chicken
Strain:
other: Ross 308
Details on test animals or tissues and environmental conditions:
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 µL/eye
Duration of treatment / exposure:
10 seconds and then rinsed with physiological saline
Duration of post- treatment incubation (in vitro):
30, 75, 120, 180 and 240 minutes after post-treatment rinse.
Number of animals or in vitro replicates:
3 eyes for test material and positive control
1 eye for negative control
Details on study design:
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within approximately 45 minutes before the start of application. Slight changes in thickness (-1.6% to 0.0%) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test item was applied onto the centre of the cornea. The test item was applied in a volume of 30 μL onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, taking care not to damage or touch the cornea.
The positive control eyes were treated in a similar way with 30 μL benzalkonium chloride solution 5 % (w/v). The negative control eye was treated with 30 μL of physiological saline (Salsol solution 0.9 %). One eye was treated with physiological saline, three eyes with the test item and another three with benzalkonium chloride solution 5 % (w/v).

The time of application was observed, then after an exposure period of 10 seconds from the end of the application, the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item. The eye was returned to the chamber after rinsing. The time while the eye was out of the chamber was limited to the minimum.

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable. Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
Irritation parameter:
percent corneal swelling
Remarks:
at up to 75 minutes
Value:
0.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Class I
Irritation parameter:
percent corneal swelling
Remarks:
at up to 240 minutes
Value:
0.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Class I
Irritation parameter:
cornea opacity score
Value:
0.67
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Class II
Irritation parameter:
fluorescein retention score
Value:
2.17
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Class III

Treatment

Replicate

Corneal thickness (instrumental units)/Corneal swelling (%)

Corneal opacity score

Fluorescein retention

Observation time (min)

Observation time (min)

Maximum change in opacity

Observation time (min)

Change in fluorescein retention

0

30

75

120

180

240

0

30

75

120

180

240

0

30

Physiological saline

A

60

60/0.0

60/0.0

60/0.0

60/0.0

60/0.0

0

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.0

DMP tech

A

64

64/0.0

64/0.0

64/0.0

64/0.0

63/‑1.6

0

0.5

0.5

1.0

1.0

1.0

1.0

0

2

2.0

B

61

62/1.6

62/1.6

62/1.6

62/1.6

62/1.6

0

0.5

0.5

0.5

0.5

0.5

0.5

0

2

2.0

C

61

61/0.0

61/0.0

60/‑1.6

60/1.6

60/‑1.6

0

0.5

0

0.5

0.5

0.5

0.5

0.5

3

2.5

Mean

 

-/0.5

-/0.5

-/0.0

-/0.0

-/0.5

 

0.67

 

2.17

Benzalkonium chloride

A

64

67/4.7

69/7.8

72/12.5

76/18.8

78/21.9

0

4

4

4

4

4

4.0

0

3

3.0

B

64

67/4.7

70/9.4

72/12.5

77/20.3

79/23.4

0

4

4

4

4

4

4.0

0.5

3

2.5

C

62

64/3.2

66/6.5

68/9.7

72/16.1

76/22.6

0

3

4

4

4

4

4.0

0

3

3.0

Mean

 

-/4.2

-7.9

-/11.6

-/18.4

-/22.6

 

4.0

 

2.83

Interpretation of results:
study cannot be used for classification
Conclusions:
The test material treated eyes showed no corneal swelling, a mean maximum change in opactiy score of 0.67 and a mean change in fluorescein of 2.17. The test item could not be classified as either a severe irritant or as a non-irritant on the basis of these data.
Executive summary:

An ex vivo eye irritation study has been conducted in isolated chicken's eyes with the submission substance in accordance with OCED guideline 438. Zero reference values were taken for corneal thickness, corneal opacity and fluorescein retention for each eye before 10 µL of test material, physiological saline (negative control) or benzalkonium chloride (positive control) was added. The test material and positive control were tested in triplicate. The negative control was tested in a single eye. After a 10 second exposure, the eyes were rinsed with saline. Corneal swelling, corneal opacity change and fluorescein retention change was noted for all eyes. The test material treated eyes showed no corneal swelling, a mean maximum change in opactiy score of 0.67 and a mean change in fluorescein of 2.17. The test item could not be classified as either a severe irritant or as a non-irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

An in vitro skin irritation study was conducted using the EpiSkin test system. The method followed was the draft version of OECD guideline 439. Dimethylolpropane Tech (DMP Tech) was exposed for 15 minutes at an application rate of 23.3 µL/cm2. DMP Tech showed a viability of 87.91% when compared to the concurrent negative control.

An ex vivo study for eye damage was conducted using isolated chicken eyes. Treated eyes showed no corneal swelling, a mean maximum change in opactiy score of 0.67 and a mean change in fluorescein of 2.17. The test item could not be classified as either a severe irritant or as a non-irritant on the basis of this study.

Justification for classification or non-classification

The substance is not classifed as a skin irritant based on the data from the in vitro skin irritation study in EpiSkin. The isolated chicken eye test indicates that the substance is not classified for serious eye damage; however no conclusion can be made for classification as an eye irritant on the basis of this study. The impurity NEO is classified for eye damage (Category 1), therefore as a precautionary approach the submission substance is also classified in Category 1 (serious eye damage).