Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 11 October 2016 and 04 November 2016
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
Identification: Diammonium phthalate
Batch: 7172002
Purity: 99.3%
Appearance: White crystalline powder
Expiry Date: August 2017
Storage Conditions: At room temperature, protected from moisture
Stability in Solvent: Not indicated by the Sponsor

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Details on animal used as source of test system:
The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
Justification for test system used:
In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
unchanged (no vehicle)
Details on test system:
Epi-200 SIT kit and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm Ø).
EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on November 01, 2016. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C, 5 ± 0.5 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: tissues were gently rinsed with DPBS at least 15 times

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Filter: 570 nm filter


NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
- The test substance is considered to be a non-irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL DPBS (MatTek)
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): as supplied

POSITIVE CONTROL SLS solution in deionised water (MatTek)
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hrs
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
77.7
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:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 77.7% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Any other information on results incl. tables

Results after treatment with Diammonium phthalate and the controls

Dose Group

Ex-po-sure Interval

Tissue No.

Absor-bance 570 nm
Well 1

Absor-bance 570 nm
Well 2

Absor-bance 570 nm
Well 3

Mean Absor-bance of 3 Wells

Mean-Absorbance

of three wells blank

corrected

Mean

Absor-bance

of 3 tissues

after blank correction *

Rel. Absor-bance [%] Tissue 1, 2 + 3**

Relative Standard Deviation

[%]

Mean Rel. Absorbance

[% of Negative Control]***

Blank

 

 

0.037

0.038

0.038

0.038

0.000

 

 

 

 

Negative Control

60 min

1

2.018

2.011

2.020

2.016

1.978

1.998

99.0

5.4

100.0

2

2.227

2.132

2.098

2.153

2.115

105.8

3

1.995

1.910

1.913

1.939

1.902

95.2

Test Item

60 min

1

1.526

1.471

1.490

1.496

1.458

1.553

73.0

5.4

77.7

2

1.655

1.598

1.603

1.619

1.581

79.1

3

1.679

1.649

1.641

1.656

1.619

81.0

Positive Control

60 min

1

0.098

0.110

0.097

0.102

0.064

0.061

3.2

5.8

3.1

2

0.099

0.099

0.100

0.099

0.062

3.1

3

0.096

0.094

0.095

0.095

0.057

2.9

 

* Mean of three replicate wells after blank correction

** relative absorbance per tissue

*** relative absorbance per treatment group

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, Diammonium phthalate is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of Diammonium phthalate by means of the Human Skin Model Test.

The white test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Each three tissues of the human skin model EpiDermwere treated with the test item, the negative control (DPBS) or the positive control (5% SLS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD

0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item Diammonium phthalate the mean relative absorbance value decreased to 77.7% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Diammonium phthalate is not irritant to skin.