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Description of key information

In vitro skin irritation (OECD 439), human skin model test (EpiSkin): not irritating

In vitro eye irritation (OECD 437), bovine corneal opacity test: not corrosive

In vitro eye irritation (OECD 439), EpiOcular eye irritation test: not irritating

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:
2017-04-05 to 2017-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human donors
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EpiDerm (MatTek)
- Tissue batch number(s): 25829

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: yes
- Wavelength: 540 nm
- Filter: no

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.857 ± 0.107
- Barrier function: 5.4 h
- Contamination: Screened for HIV-1, hepatits B and C, bacteria, yeast, and other fungi. No contaminates were detected.

NUMBER OF REPLICATE TISSUES: triplicate

PREDICTION MODEL / DECISION CRITERIA
The irritant potential was predicted from the relative mean tissue viabilities compared to the negative control. If the mean relative tissue viability is > 50% the test material is not considered an irritant. If the mean relative tissue viability is ≤ 50%, the test material is considered an irritant.

The test meets acceptance criteria if:
- mean absolute OD570 of the three negative control tissues is ≥ 1.0 and ≤ 2.5
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 mg
- Concentration: 25 mg test item were mixed with 300 μL sterile deionised water

NEGATIVE CONTROL
- Amount(s) applied: 30 μL

POSITIVE CONTROL
- Amount(s) applied: 30 μL
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 111
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: The standard deviation determined for all triplicates was below the limit of acceptance of 18 %. Hence, all acceptance criteria required were fulfilled.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1. Summary of Results

 

Optical density [OD540]

mean tissue (n = 3)

Mean Viability

(n = 3) [%]

CV

[%]

Dimelamine pyrophosphate

1.550

111.0

1.2

D-PBS (negative control)

1.396

100.0

7.6

5% SDS (positive control)

0.092

6.5

10.9

Interpretation of results:
other: CLP/EU GHS criteria are not met, no classification is required according to Regulations (EC) No 1272/2008.
Conclusions:
In an in vitro skin irritation human skin model test (EpiSkin) according to OECD guideline 439 and in compliance with GLP, a cell viability of 111% was measured when compared to the untreated control. Therefore, the test item should not be classified.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-17 to 2017-03-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
July 26, 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
December 09, 2010
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES

- Source: obtained from a slaughterhouse
- Storage, temperature and transport conditions of ocular tissue : To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution2 (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL
- Time interval prior to initiating testing: not reported
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: yes
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 μL
- Concentration: 20% suspension in 0.9% sodium chloride solution (w/v)
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle were completely submerged in Hanks’ Balanced Salt Solution containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL. The eyes were examined for defects (e.g., increased opacity, scratches, and neovascularisation), and only corneas from eyes free of defects were used. The closed-chamber method was then used where the dissected corneas (2 to 3 mm rim of sclera) were mounted to anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32 ± 1°C for at least one hour. After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% sodium chloride solution

POSITIVE CONTROL USED
20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
750 μL for 240 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes; 90 ± 5 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After exposure, the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: 1 mL sodium fluorescein9 solution (5 mg/mL in 0.9 % sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32 ± 1 °C for 90 ± 5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader. Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) ; IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: A test item that induces an IVIS ≤ 3 is defined as no category; > 3 and ≤ 55; no prediction can be made; and > 55 as a corrosive or severe irritant.

A test was considered acceptable if the positive control gave an IVIS that fell within two standard deviations of the current historical mean. The negative or solvent/vehicle control responses were less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. In cases of borderline results in the first testing run, a second testing run should be considered. A borderline result is defined as:

• 2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
• 1 of the 3 corneas gave discordant prediction from the mean of all 3 corneas AND the discordant result was > 10 IVIS units from the cut-off threshold of 55.
• If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing. If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a UN GHS Category 1 prediction.
Irritation parameter:
other: mean opacity score
Value:
ca. 2.536
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
other: mean permeability value
Value:
ca. 0.18
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
ca. 5.241
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
Interpretation of results:
other: non corrosive according to Regulations (EC) No 1272/2008.
Conclusions:
Under the present test conditions Dimelamine pyrophosphate tested in the in vitro BCOP test method, had an IVIS value of 5.241, which is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently no prediction concerning irritant or severely irritant potential of the test item can be made.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-12 to 2018-01-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
Version / remarks:
July 28, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz
Species:
human
Strain:
not specified
Details on test animals or tissues and environmental conditions:
- The protocol used was based on the protocol and usage of the EpiOcularTM Eye Irritation Test (OCL-200-EIT, Mattek) and was concordant with the OECD Guideline 492.
- EpiOcular™ RhCE tissue construct (Mattek) with certification analysis certificate.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 mg
Duration of treatment / exposure:
6 hours, followed by a 25-minute post-treatment immersion, and an 18-hours post-treatment incubation, prior to the MTT endpoint
Duration of post- treatment incubation (in vitro):
25-minute post-treatment immersion and an 18-hours post-treatment incubation
Number of animals or in vitro replicates:
3
Details on study design:
- Details of the test procedure used : The protocol used was based on the protocol and usage of the EpiOcularTM Eye Irritation Test (OCL-200-EIT, Mattek) and was concordant with the OECD Guideline 492.
- RhCE tissue construct used, including batch number : EpiOcular™ RhCE tissue construct (Mattek), batch number 23793
- Doses of test chemical and control substances used : 50 mg of the test item, 50 μL of the concurrent negative (sterile deionised water) control or 50 μL of the positive control (methyl acetate)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods : Treatment: incubated at 37°C, 5% CO2 and 95% relative humidity for 6 ± 0.25 hours; Post-exposure immersion: immediately transferred in 5 mL pre-warmed Assay Medium (room temperature) for 25 ± 2 minutes; Post-exposure incubation: incubated at 37 °C, 5% CO2 and 95% relative humidity for 18 ± 0.25 hours
- Number of tissue replicates used per test chemical and controls: 3
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : The extracted MTT formazan was quantified using a standard absorbance (Optical Density) measurement procedure in the range between 540 and 590 nm.
- Each run using EpiOcular™ tissue batches was accepted if:

The negative control OD was >0.8 and <2.5.
The mean relative viability of the positive control was below 50% of control viability.
The difference of viability between two replicate tissues was <20 %

If the test item-treated tissue viability is > 60 % relative to negative control-treated tissue viability, the test item is predicted to be non-irritant.
If the test item-treated tissue viability is ≤ 60 % relative to negative control-treated tissue viability, the test article is predicted to be irritant.
Irritation parameter:
other: corrected mean viability (%)
Value:
ca. 83.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: OD540 values (%)
Value:
> 60
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: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: The percent difference of all replicates determined was below the limit of acceptance of 20%. Hence, all acceptance criteria required were fulfilled.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
Interpretation of results:
other: CLP/EU GHS criteria are not met, no classification is required according to Regulations (EC) No 1272/2008.
Conclusions:
Under the present test conditions dimelamine pyrophosphate administered by topical application onto the EpiOcular RhCE tissue constructs as cultures for 6 hours, followed by a 25-minute post-treatment immersion, and a 18-hour post-treatment incubation to the MTT endpoint was non-cytotoxic and predicted to the non-irritant according to the UN GHS/CLP.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

To determine the test material’s irritating potential to skin, dimelamine pyrophosphate was evaluated in an OECD 439 study conducted in accordance with GLP (LPT, 2017). Dimelamine pyrophosphate was applied as solid test item to the model skin surface (EpiDerm(TM) Model), which was moistened with Dulbecco’s phosphate buffered saline (D-PBS). D-PBS was used as the negative control and 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive control. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

 

The mean viability of cells exposed to dimelamine pyrophosphate was 111.0 % of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50 %. Dimelamine pyrophosphate was considered to be non-cytotoxic and predicted to be non-irritant to skin.

Eye Irritation

In a study conducted in accordance to OECD 437 and GLP compliant, the ocular corrosivity was evaluated in a Bovine Corneal Opacity and Permeability Assay (BCOP) (LPT, 2017) for dimelamine pyrophosphate. For the assay, the solid test item was dissolved in a 0.9% sodium chloride solution with a final concentration of 20% dimelamine pyrophosphate. A solvent control (0.9 % NaCl solution) and positive control (20% Imidazole) also were evaluated. The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 240 minutes. The optical density (OD) was measured at a wavelength of 490 nm.

 

Following treatment with dimelamine pyrophosphate a mean opacity of 2.536 ± 0.449 and a mean permeability value of 0.180 ± 0.033 compared to the negative control were determined. The calculated IVIS of 5.241 ± 0.783 is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently no prediction concerning irritant or severely irritant potential of the test item could be made. Because no overall conclusions could be made from this assay, the test material was evaluated again in an OECD 492 assay.

To determine the test material’s irritating potential to eyes, dimelamine pyrophosphate was evaluated in an OECD 492 study conducted in accordance with GLP (LPT, 2018). For this study, dimelamine pyrophosphate (50 mg) was applied topically to three-dimensional Reconstructed human Cornea-like Epithelium (RhCE) tissue constructs and tissue viability was measured following exposure and a post-treatment incubation period. Exposures consisted of a 6 hour treatment period, followed by a 25-minute post-treatment immersion, and an 18-hours post-treatment incubation, prior to the MTT endpoint. The corrected mean viability of the cells exposed to dimelamine pyrophosphate was 83.1% of the mean negative control value. Hence, the OD540 values were above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 60%. Based on these results, the test item was considered to be non-cytotoxic and predicted to be non-irritant.

Justification for classification or non-classification

The available data on skin and eye irritation with dimelamine pyrophosphate do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.