Nuclease, deoxyribo-

Endpoint summary

Description of key information

Regarding skin irritation:

Two batches (PPW42035 and U7F9Q) of deoxyribonuclease were tested.

- The mean viability of the EpiDerm™ reconstructed skin membranes was 88 ± 1 % compared to the negative control group. Based on the results obtained in the present study, deoxyribonuclease, batch PPW42035 was considered as non-irritant (UN GHS No Category).

- The mean viability of the SkinEthic™ human reconstructed epidermis was 96.2%. Based on the results obtained in the present study, the test substance deoxyribonuclease (PDE), batch U7F9Q was classified as non-irritant.

Regarding eye irritation:

- On the basis of the results obtained by slit-lamp examination and applying the classification criteria of the ICE, it was concluded that the tested deoxyribonuclease, batch PPW42035 was not irritating to eyes.

- Liquid deoxyribonuclease (batch U7F9Q - 20.2 mg AP/mL in PBS buffer) tested in human corneal epithelial cells revealed a viability of 98.2%. Therefore, it was concluded that it was not irritating to the eye (No Category), when tested in the present model under the test conditions.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-11-2016 to 01-12-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Adopted 28 July 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate issued by Health Care Inspectorate of the Ministry of Health, The Netherlands.

Test system:

human skin model

Remarks:

EpiDerm™ reconstructed skin membranes

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN PREPARATION
- Procedure used: EpiDerm™ reconstructed skin membranes. The keratinocytes were plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2).
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37ºC and 5% CO2
- Temperature of post-treatment incubation (if applicable): Same
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: each skin model was removed from the well, rinsed with PBS to remove the study substance (and mesh), blotted dry and transferred to a 6-well plate containing medium.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Yes, but not specified.
- Wavelength: 570 nm
- Filter: Not specified
- Filter bandwidth: Not specified
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
The in vitro skin irritation potential of the test substance was determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model:
Mean tissue viability (% of negative control): ≤ 50 %, classification and labelling required, irritant or corrosive.
Mean tissue viability > 50 %, Non-irritant (No Category).

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- Concentration (if solution): used as is (12.0% enzyme concentrate dry matter)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (12.0% enzyme concentrate dry matter)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (12.0% enzyme concentrate dry matter)

Duration of treatment / exposure:

60 min.

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

Triplicate tissues each for test substance, negative control Phosphate Buffered Saline and positive control 5% Sodium Dodecyl Sulphate.

Type of coverage:

open

Preparation of test site:

other: Not necessary since they are reconstructed skin membranes

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- Concentration (if solution): used as is (12.0% enzyme concentrate dry matter)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (12.0% enzyme concentrate dry matter)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): used as is (12.0% enzyme concentrate dry matter)

Duration of treatment / exposure:

60 minutes

Details on study design:

TEST SITE
- Area of exposure: 0.64 cm2
- % coverage: 100%
- Type of wrap if used: Nylon mesh

REMOVAL OF TEST SUBSTANCE
- Washing (if done): With PBS
- Time after start of exposure: Exposure time 60 min, post-exposure time about 42 h.

SCORING SYSTEM:
- Method of calculation: OD measurement with a spectrophotometer

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

0

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:

- OTHER EFFECTS:
- Direct-MTT reduction: No reduction was observed.
- Colour interference with MTT: None observed.

The experiment was considered valid if:
- the OD of the viable negative control was ≥ 0.8 and ≤ 2.8.
- skin models treated with the positive control showed mean tissue viability ≤ 20% compared to the negative control.
The test group was considered valid if the standard deviation (SD) calculated from individual tissue viability percentages of the three replicates was ≤ 18%.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean viability of the EpiDerm™ reconstructed skin membranes was 88 ± 1 % compared to the negative control group.
Based on the results obtained in the present study, deoxyribonuclease, batch PPW42035 was considered as non-irritant (UN GHS No Category).

Executive summary:

This study was performed to determine the in vitro skin irritation potential of deoxyribonuclease, batch PPW42035 using EpiDerm™ reconstructed skin membranes. The skin membranes were topically exposed to the test substances for 60 min. Viability of the epidermal cells was assessed using the MTT test at ca. 42 h post-exposure. Negative and positive controls were run in parallel. The general principle for the detection of viability via the MTT test is the conversion of the yellow tetrazolium salt (MTT) to the blue/purple coloured product formazan by mitochondrial enzymes. The formation of formazan was measured using a spectrophotometer. The acceptance criteria of the negative and positive control were met and therefore, the study was considered valid.

The mean viability of the skin membranes was 88 ± 1 % compared to the negative control group.

Based on the results obtained in the present study, deoxyribonuclease, batch PPW42035 was considered as non-irritant (UN GHS No Category).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 January 2017 – 27 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

GLP compliance:

no

Test system:

human skin model

Source species:

human

Cell type:

other: Reconstructed Human Epidermis

Cell source:

foreskin from a single donor

Vehicle:

other: PBS

Details on test system:

SKIN DISC PREPARATION
- Procedure used: SkinEthic™ Reconstituted Human Epidermis (RHE). The tissues were transferred on 6-well pates containing 1 mL medium and incubated overnight.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37ºC and 5% CO2
- Temperature of post-treatment incubation (if applicable): Same
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Place a funnel in a large beaker. The wells were rinsed 25mL x 1mL PBS at a 5-8 cm distance from the insert to remove all residual test substance from the epidermal surface.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Yes (Enspire)
- Wavelength: 570 nm
- Filter: Not specified
- Filter bandwidth: Not specified
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- The mean Optical Density (ODNC) at 570 nm of the tree replicates tissues treated with the NC is ≥ 1.4
- The mean Viability of the tree replicates tissues treated with the PC, expressed as % of the negative control is ≤30%
- The difference of viability between the tree replicates tissues of a single test chemical is ≤ 20 in the same run

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL
- Concentration (if solution): used as is

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): used as is
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): used as is

Duration of treatment / exposure:

42 min.

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

Triplicate tissues each for test substance, negative control Phosphate Buffered Saline and positive control 5% Sodium Dodecyl Sulphate.

Type of coverage:

open

Preparation of test site:

other: Not necessary since it is a human skin model.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL
- Concentration (if solution): used as is

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): used as is
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): used as is

Duration of treatment / exposure:

42 minutes

Details on study design:

TEST SITE
- Area of exposure: Reconstructed Human Epidermis Tissue (0.5 cm2)
- % coverage: 100%
- Type of wrap if used: Nylon mesh

REMOVAL OF TEST SUBSTANCE
- Washing (if done): With PBS
- Time after start of exposure: Exposure time 42 min, post-exposure time about 42 min.

SCORING SYSTEM:
- Method of calculation: OD measurement with a spectrophotometer

Irritation / corrosion parameter:

% tissue viability

Value:

96.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No reduction was observed.

The experiment was considered valid if:
- The mean Optical Density (ODNC) at 570 nm of the tree replicates tissues treated with the NC is ≥ 1.4
- The mean Viability of the tree replicates tissues treated with the PC, expressed as % of the negative control is ≤30%
- The difference of viability between the tree replicates tissues of a single test chemical is ≤ 20 in the same run

Interpretation of results:

GHS criteria not met

Conclusions:

The mean viability of the SkinEthic™ human reconstructed epidermis was 96.2%. Based on the results obtained in the present study, the test substance deoxyribonuclease, batch U7F9Q was classified as non-irritant.

Executive summary:

This study was performed to determine the in vitro skin irritation potential of deoxyribonuclease, batch U7F9Q, using SkinEthic™ human reconstructed epidermis model.

The study was conducted according to OECD Test Guideline No. 439 (2015), and Invittox protocol: SkinEthictm Skin Irritation Test -42bis. SkinEthic kit RHE, immortalized human epidermis cells was used containing 0.5 cm2 tissue constructs and maintenance medium for the tissues constructs/inserts, total of 9 inserts. Deoxyribonuclease batch U7F9Q was applied directly as is on the cells.16 μL was applied to the inserts in triplicate for 42 minutes.

PBS was used as a negative control and 5% SDS as positive control. The test substance is considered to be irritant to skin (Cat. 2), if the mean relative viability after 42 min exposure and 42 hours post incubations is less or equal to 50% of the negative control. The included positive and negative control performed as expected and the study is hence considered valid.

The mean viability of the SkinEthic™ human reconstructed epidermis was 96.2%. Based on the results obtained in the present study, the test substance deoxyribonuclease, batch U7F9Q was classified as non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-11-2016 to 10-01-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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)

Version / remarks:

adopted on 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: ROSS, spring chickens

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands
- Age at study initiation: 7 weeks old
- Weight at study initiation: 1.5-2.5 kg
- Housing: The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The chicken eye cornea was treated with 30 µL.
- Concentration (if solution): undiluted test sample, 12.0% enzyme concentrate dry matter.

Duration of treatment / exposure:

The exposure period was 10 seconds

Observation period (in vivo):

The eyes were examined at ca 0, 30, 75, 120, 180 and 240 minutes after treatment.

Number of animals or in vitro replicates:

3 eyes for the positive control and the test enzyme, and one eye for the negative control.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES:
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus.

EQUILIBRATION AND BASELINE RECORDINGS: Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

NUMBER OF REPLICATES: 1 eye for the negative control and 3 eyes for the negative and positive control, respectively.

NEGATIVE CONTROL USED: Isotonic saline.

POSITIVE CONTROL USED: Benzalkonium Chloride (BAC) 5%

APPLICATION DOSE AND EXPOSURE TIME: undiluted test sample, 12.0% enzyme concentrate dry matter for a treatment time of 30, 75, 120, 180 and 240 minutes.

OBSERVATION PERIOD: The eyes were examined at ca 0, 30, 75, 120, 180 and 240 minutes after treatment.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline.
- Indicate any deviation from test procedure in the Guideline: No deviation

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland
- Damage to epithelium based on fluorescein retention: Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland
- Swelling: Corneal thickness was measured using the Haag-Streit slit-lamp microscope, set at 0.095 mm.
- Macroscopic morphological damage to the surface: Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland
- Others (e.g, histopathology): The microscopic slides were not subjected to histopathological examination.

SCORING SYSTEM:
- Mean corneal swelling (%): 2%
- Mean maximum opacity score: 0.0
- Mean fluorescein retention score at 30 minutes post-treatment: 0.0

DECISION CRITERIA: decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No opacity or fluorescein retention were observed. Microscopic examination of the corneas did not reveal any abnormalities.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test has already been established in labs starting the ICE test, and does not need to be demonstrated again as mentioned in OECD guidelines.

ACCEPTANCE OF RESULTS:
The test was considered acceptable if the concurrent negative or vehicle/solvent and the concurrent positive controls are identified as UN-GHS Non-Classified and UN-GHS Category 1, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

On the basis of the results obtained by slit-lamp examination and applying the classification criteria of the ICE, it was concluded that the tested deoxyribonuclease batch was not irritating to eyes.

Executive summary:

Deoxyribonuclease, batch PPW42035 was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (BAC 5%). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 μL for 10 seconds followed by a 20 mL saline rinse. The following three main parameters were measured to disclose possible adverse eye effects: Corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Deoxyribonuclease, batch PPW42035 only caused very slight swelling of the cornea (mean of 2%). No opacity or fluorescein retention were observed. Microscopic examination of the corneas did not reveal any abnormalities.

Applying the classification criteria of the ICE, the following irritation classifications can be assigned:

Deoxyribonuclease, batch PPW42035:

- NC:“Not Classified” (UN-GHS and EU-CLP classifications).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

In vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 January 2017 to 16 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

Species:

human

Strain:

other: SkinEthic Human Corneal Epithelium (HCE)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 µL of undiluted test substance.

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

30 minutes ± 2 minutes at 37°C, 5% CO2, ≥ 95% humidity.

Number of animals or in vitro replicates:

2

Details on study design:

Performed according to In vitro Prediction Assay for Acure Ocular Irritation of Liquid Chemicals SkinEthic Human Corneal Epithelial Model (SkinEthicTM HCE).

Irritation parameter:

in vitro irritation score

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Liquid deoxyribonuclease (batch U7F9Q - 20.2 mg AP/mL in PBS buffer) tested in human corneal epithelial cells revealed a viability of 98.2%. Therefore, it was not irritating to the eye (No Category), when tested in the present model under the test conditions.

Executive summary:

The Human Corneal Epithelium Assay (HCE) from SkinEthic (France) was used to assess the eye irritation potential of the test substance. The MTT conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT to a blue formazan precipitate, was used to assess cellular metabolism after exposure to a test substance for the defined exposure time.

The test system and procedure described in In vitro Prediction Assay for Acute Ocular Irritation of Liquid Chemicals SkinEthic Human Corneal Epithelial Model (SkinEthicTM HCE) March 6, 2016 was used.

Liquid deoxyribonuclease (batch U7F9Q - 20.2 mg AP/mL in PBS buffer) tested in human corneal epithelial cells revealed a viability of 98.2%.

Therefore, it was not irritating to the eye (No Category), when tested in the present model under the test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Not classified.