Isobutyl 4-chloro-3,5-diaminobenzoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-27 - 2017-05-05 (experimental phase)

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the testing of Chemicals 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original Guideline adopted July 28, 2015), and as described in detail in the Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200), Rev. 07/11/2014.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D65189 Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl 4-chloro-3,5-diaminobenzoate

EC Number:

251-311-5

EC Name:

Isobutyl 4-chloro-3,5-diaminobenzoate

Cas Number:

32961-44-7

Molecular formula:

C11H15ClN2O2

IUPAC Name:

2-methylpropyl 3,5-diamino-4-chlorobenzoate

Test material form:

solid: particulate/powder

Details on test material:

Storage Conditions: room temperature in the dark
Stability in Solvent: stable in water, not quantified

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human epidermis

Source strain:

other: not applicable

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. 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.

Justification for test system used:

specified in the guideline

Vehicle:

unchanged (no vehicle)

Remarks:

tissues wetted with 25 µL DPBS prior to application

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT kits and MTT-100 assay 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(R), 10 mm diameter).
- Tissue batch number(s): Lot 25810, Keratinocyte strain 00267
- Production date: 03 May 2017
- Shipping date: 03 May 2017
- Delivery date: 03 May 2017 (Wednesday)
- Date of initiation of testing: On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material.
- Observable damage in the tissue due to washing: None stated.
- Modifications to validated SOP: none stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Wavelength: 570 nm
- Filter: 570 nm filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA

Historical Data
Positive Control Negative Control [OD570]
Mean Viability 4.37% Mean Absorption 1.74
Rel. Standard Deviation 21.60% Rel. Standard Deviation 9.40%
Range of Viabilities 2.20% - 6.78% Range of Absorbance 1.34 – 2.00
Mean Absorption 0.08
Rel. Standard Deviation 20.12%
Range of Absorbance 0.03 - 0.11
Data of 103 studies performed from July 2015 until March 2017

NUMBER OF REPLICATE TISSUES: triplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
For the current test (1h treatment), an irritation potential of a test item leading to H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (6th) revision 2015) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Each 25 mg ± 2 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to triplicate tissues, which were wetted with 25 µL DPBS prior to application.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS (MatTek) to triplicate tissues

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% SLS (MatTek) solution in deionised water to triplicate tissues

Duration of treatment / exposure:

1h

Duration of post-treatment incubation (if applicable):

Tissues were incubated for 24.6 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation medium was changed (0.9 mL of pre-warmed fresh medium). Thereafter tissues were incubated for another 17.5 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was 42.1 hours.

Number of replicates:

3 replicates per item

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

expressed as Mean Rel. Absorbance [% of Negative Control ]

Run / experiment:

Mean

Value:

104.1

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:
- Visible damage on test system: none stated
- Direct-MTT reduction: no
- Colour interference with MTT: no

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Testing was performed via a GLP OECD 439 guideline study on the registered substance itself. The present in vitro method is recommended in a bottom up testing approach. The validity criteria are met, making the results sufficiently reliable to assess the irritating potential of the test item to the skin. The present in vitro method allows the identification of irritating chemical substances and mixtures. Compared to the relative absorbance value of the negative control the mean relative absorbance value was not reduced (104.1%; threshold for irritancy: ≤ 50%) after exposure of the skin tissues to the test item. Therefore, the test item is not considered to possess an irritant potential. Hence, the present result is sufficient to conclude that Isobutyl 4-chloro-3,5-diaminobenzoate does not need to be classified as skin irritant, and no further testing is required.

Executive summary:

This in vitro GLP OECD 439 study was performed to assess the irritation potential of Isobutyl 4-chloro-3,5-diaminobenzoate by means of the Human Skin Model Test.

The test item was not soluble in water and did not dye water when mixed with it (pre-test for colour interference), and it did not reduce MTT (pre-test for direct MTT reduction). Therefore, additional tests with viable (without MTT addition) or freeze-killed tissues were not necessary for determination of correction factors for calculating the true viability in the main experiment.

Each three tissues of the human skin model EpiDerm™ were 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 OD³0.8 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 Isobutyl 4-chloro-3,5-diaminobenzoate the mean relative absorbance value did not decrease (104.1%) compared to the relative absorbance value of the negative control. The threshold for irritancy of ≤ 50% was not affected. 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, Isobutyl 4-chloro-3,5-diaminobenzoate is not irritant to skin.