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Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study with well characterized test material

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Radiolabelled substance; 14C in the cyanuric acid part.
Specific details on test material used for the study:
- Name of test material (as cited in study report): Melamin Cyanurat
- Substance type: white powder
- Physical state: solid
- Analytical purity: pure
- Impurities (identity and concentrations): free melamine < 0.01%, free cyanuric acid 0.02%, water 0.06%
- Purity test date: 17. Sept 2009
- Lot/batch No.: 03955XY9 (non-radioactive), 1015-1003 (radioactive)
- Expiration date of the lot/batch: 17. Sep 2014
- Radiochemical purity (if radiolabelling): 98.9%
- Specific activity (if radiolabelling): 6.8 MBq/mg
- Locations of the label (if radiolabelling): Cyanuric acid [2,4,6-14C]
- Expiration date of radiochemical substance (if radiolabelling): 13. Dec 2011
- Stability under test conditions: stable
- Storage condition of test material: < -10°C, in the dark
- Other: UV-area percentage of radiolabelled sample: 96.4%. Just prior to performing the study, the radiochemical purity was again determined (n =2) and found to be 99.4% (pre-dose) and 99.3% (post-dose).
Radiolabelling:
yes
Remarks:
14C at Cyanurate

Test animals

Species:
human
Strain:
other: dermatomed human skin from caucasian donors
Sex:
female
Details on test animals and environmental conditions:
not relevant

Administration / exposure

Type of coverage:
open
Vehicle:
water
Duration of exposure:
24h
Doses:
5.461 +/- 0.713 mg/cm2
No. of animals per group:
10 skin samples
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: [14C]-Melamin Cyanurat (0.5709 mg) and non-labelled melamine cyanurate
(499.4 mg) were weighed into a vessel, and dissolved with stirring by addition of deionised water, to give a final formulation weight of 0.97 g. The concentration of test substance in the dosing formulation, based on weighed amounts, was 517 mg/mL (observed as a white paste).
- Method of storage: not applicable

APPLICATION OF DOSE: The test substance was administered as a single dermal application (ca 6.4 mg of formulation) using a positive displacement pipette.

VEHICLE

- Amount(s) applied (volume or weight with unit): 0.007 mL (Formulation)
- Concentration (if solution): 517 mg/mL


TEST SITE
- Preparation of test site: none
- Area of exposure: 0.64 cm2, (0.9 cm diameter)
- % coverage: 100%


REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: not applicable
- Washing procedures and type of cleansing agent: detergent (Liquid Ivory Soap, ca 10% w/v, no organic solvent contained), then rinsing with deionised water
- Time after start of exposure:24h



SAMPLE PREPARATION
- Storage procedure: -20°C
- Preparation details:Skin and tape samples were solubilized. Portions of receptor-fluid, applicaiton site washings, solubilized skin, solubilized tape strips and diffusion chamber washings were added directly to liquid scintillant.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting
- Liquid scintillation counting results (cpm) converted to dpm as follows: no data
- Validation of analytical procedure:
- Limits of detection and quantification:

OTHER:
Solubilizing agent: Soluene-350 (Perkin Elmer LAS)
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Biopredic International via TCS Cellworks Ltd., Buckinghamshire, UK
- Ethical approval if human skin: yes
- Type of skin: abdominal
- Preparative technique: dermatomed
- Thickness of skin (in mm): 0.24 - 0.4
- Membrane integrity check: yes (triated water, cut off Kp of 15x10exp-4 cm/h, determined for 2h exposure)
- Storage conditions: -20°C
- Justification of species, anatomical site and preparative technique: no data

PRINCIPLES OF ASSAY
- Diffusion cell: See figure, stainless steel flow-through chamber
- Receptor fluid: Ethanol/Water (1:1)
- Solubility of test substance in receptor fluid: reported as acceptable as determined in preliminary experiment. No detailed information available.
- Static system: no
- Flow-through system: yes
- Test temperature: ca 35°C (receptor fluid)
- Humidity: no data
- Occlusion: open
- Reference substance(s): Triated Water
- Other: The flow-through cells were set to collect fractions for one hour pre-dose and at 0-15, 15-30, 30-45, 45-60, 75-90, 90-105 and 105-120 minutes plus hour long samples from 2 hours to 24 hours post dose.

Results and discussion

Signs and symptoms of toxicity:
not examined
Remarks:
not applicable
Dermal irritation:
not examined
Absorption in different matrices:
- Skin wash: Residual formulation was washed from the surface of the skin membrane with a solution of Liquid Ivory soap (ca 10% w/v; containing no organic solvent) and rinsed with deionised water. The washings were retained for analysis.
- Skin test site: abdominal

- Receptor fluid, receptor chamber, donor chamber (in vitro test system): ethanol:water, 1:1 v/v, Franz-type chamber
- Skin preparation (in vitro test system): Twelve 20 mm diameter discs were supplied as dermatomed sections of ca 320 μm thickness (range: 240 to 400 μm), and ten discs were used for this study.
- Stratum corneum (in vitro test system): tape strips
Total recovery:
- Total recovery: 94.94% (+/- 14.3%)
- Recovery of applied dose acceptable: yes
- Results adjusted for incomplete recovery of the applied dose:
- Limit of detection (LOD): The limit of quantification for each batch of samples analysed by direct counting was taken as twice the mean background disintegration rate obtained from vials containing an equivalent volume of an appropriate solvent in liquid scintillant. However, due to
the very low levels of radioactivity recovered in the receptor fluid samples, many of which were below twice background counts, net dpm counts for all receptor fluid samples were used to calculate concentrations.
- Quantification of values below LOD or LOQ: no applicable
Percutaneous absorption
Dose:
5 mg/cm2
Parameter:
percentage
Absorption:
ca. 0.04 %
Remarks on result:
other: 24h
Remarks:
human skin

Any other information on results incl. tables

Table 1: Overview

 

Administered dose: 5 mg/cm2(n=10)

 

Mean

SD

CV%

Max. rate of penetration over 0-24 h (µg/cm2/hour)

0.182

0.106

58.2

Permeability coefficient
(cm/hour)

1.987 x 10-7

0.223 x 10-7

11.2

Lag Time
(hours)

0.021

0.032

152

Table 2: Recovery of radioactivity in different samples

 

Percent of administered dose (± SD)

Sample

Mean

SD

CV%

Receptor fluid

0.040

0.009

22.5

Skin wash

93.44

14.30

15.3

Cell wash

0.103

0.025

24.3

Skin

0.307

0.194

63.2

Tape Strip 1

0.530

0.178

33.6

Tape Strip 2

0.223

0.133

59.6

Tape Strip 3-5

0.299

0.306

102

Total

94.94

14.38

15.1

Table 3: Mean absorption of radioactivity following a single application of [14C]‑Melamin Cyanurat to human dermatomed membranes

Time (h)

Cumulative absorption of [14C]-Melamin Cyanurat
(µg equivalents/cm2skin) (± SD) (n=10)

0.25

0.050 ± 0.014

0.5

0.103 ± 0.041

0.75

0.149 ± 0.060

1

0.189 ± 0.065

1.25

0.227 ± 0.064

1.5

0.265 ± 0.065

1.75

0.306 ± 0.070

2

0.341 ± 0.074

3

0.488 ± 0.092

4

0.634 ± 0.093

5

0.779 ± 0.100

6

0.896 ± 0.114

7

0.979 ± 0.114

8

1.069 ± 0.126

9

1.163 ± 0.131

10

1.252 ± 0.135

11

1.338 ± 0.139

12

1.431 ± 0.149

13

1.519 ± 0.152

14

1.602 ± 0.165

15

1.683 ± 0.173

16

1.766 ± 0.185

17

1.854 ± 0.193

18

1.950 ± 0.219

19

2.040 ± 0.228

20

2.115 ± 0.231

21

2.195 ± 0.231

22

2.275 ± 0.237

23

2.352 ± 0.245

24

2.431 ± 0.244

Max rate µg/cm2/hour

0.182 ± 0.106

Mean rate µg/cm2/hour

0.099 ± 0.011

Kp cm/hour (x10-7)

1.987 ± 0.223

Lag time h

0.021 ± 0.032

Table 4: Details of the rates of penetration of tritiated water through dermatomed membranes prepared from human skin

Cell
identification

Membrane
identification

Age/sex

Membrane
thickness (µm)

Permeability coefficient
Kp (cm/hour)

101

TRA002717-OC01

Female, 52 yr, Caucasian

340

5.003E-04

102

TRA002717-OC02

Female, 52 yr, Caucasian

310

2.744E-04

103

TRA002717-OC03

Female, 52 yr, Caucasian

320

3.161E-04

104

TRA002718-OC01

Female, 35 yr, Caucasian

270

1.320E-03

105

TRA002718-OC02

Female, 35 yr, Caucasian

320

6.069E-04

106

TRA002718-OC03

Female, 35 yr, Caucasian

260

5.265E-04

107

TRA002718-OC04

Female, 35 yr, Caucasian

240

6.241E-04

108

TRA002719-OC01

Female, 43 yr, Caucasian

360

2.837E-04

109

TRA002719-OC02

Female, 43 yr, Caucasian

400

3.653E-04

110

TRA002719-OC03

Female, 43 yr, Caucasian

360

5.257E-04

Table 5: Absorption of radioactivity following a single application of [14C]-Melamin Cyanurat to intact human dermatomed membranes at a nominal dose level of 5 mg/cm2

 

Cumulative absorption of [14C]-Melamin Cyanurat (µg equivalents/cm2skin)

Time (h)

101

102

103

104

105

106

107

108

109

110

Mean

SD

0.25

0.046

0.070

0.067

0.039

0.046

0.046

0.046

0.032

0.067

0.039

0.050

0.014

0.5

0.123

0.208

0.109

0.077

0.077

0.095

0.081

0.070

0.113

0.081

0.103

0.041

0.75

0.162

0.310

0.151

0.127

0.102

0.123

0.116

0.113

0.155

0.127

0.149

0.060

1

0.208

0.362

0.187

0.158

0.144

0.162

0.158

0.137

0.190

0.187

0.189

0.065

1.25

0.239

0.398

0.225

0.187

0.169

0.204

0.215

0.183

0.229

0.218

0.227

0.064

1.5

0.285

0.436

0.260

0.211

0.222

0.236

0.253

0.218

0.271

0.257

0.265

0.065

1.75

0.334

0.486

0.289

0.260

0.264

0.267

0.274

0.243

0.331

0.313

0.306

0.070

2

0.373

0.528

0.310

0.282

0.299

0.285

0.331

0.285

0.370

0.348

0.341

0.074

3

0.531

0.672

0.412

0.394

0.433

0.412

0.595

0.419

0.517

0.493

0.488

0.092

4

0.704

0.806

0.560

0.510

0.588

0.535

0.721

0.595

0.669

0.651

0.634

0.093

5

0.841

0.926

0.683

0.619

0.746

0.683

0.880

0.743

0.866

0.806

0.779

0.100

6

0.933

1.080

0.785

0.728

0.862

0.785

1.021

0.852

0.989

0.922

0.896

0.114

7

1.006

1.172

0.869

0.820

0.936

0.869

1.094

0.922

1.077

1.024

0.979

0.114

8

1.087

1.249

0.936

0.897

1.028

0.943

1.197

1.014

1.218

1.126

1.069

0.126

9

1.182

1.316

0.996

0.968

1.123

1.066

1.334

1.119

1.295

1.235

1.163

0.131

10

1.270

1.387

1.084

1.028

1.197

1.168

1.415

1.232

1.408

1.334

1.252

0.135

11

1.355

1.453

1.161

1.101

1.284

1.274

1.499

1.306

1.510

1.439

1.338

0.139

12

1.464

1.534

1.239

1.168

1.387

1.362

1.601

1.404

1.612

1.541

1.431

0.149

13

1.566

1.622

1.313

1.242

1.482

1.464

1.679

1.496

1.707

1.622

1.519

0.152

14

1.640

1.679

1.397

1.288

1.577

1.541

1.777

1.584

1.819

1.721

1.602

0.165

15

1.707

1.760

1.467

1.351

1.650

1.626

1.858

1.679

1.914

1.812

1.683

0.173

16

1.802

1.841

1.527

1.422

1.731

1.696

1.957

1.763

2.013

1.911

1.766

0.185

17

1.893

1.918

1.598

1.485

1.833

1.805

2.038

1.851

2.126

1.992

1.854

0.193

18

1.992

2.006

1.675

1.559

1.900

1.869

2.277

1.946

2.214

2.066

1.950

0.219

19

2.104

2.087

1.745

1.643

1.988

1.957

2.337

2.020

2.365

2.154

2.040

0.228

20

2.182

2.161

1.826

1.707

2.059

2.034

2.418

2.090

2.439

2.238

2.115

0.231

21

2.256

2.249

1.900

1.788

2.150

2.104

2.488

2.164

2.520

2.330

2.195

0.231

22

2.347

2.344

1.981

1.841

2.231

2.175

2.562

2.259

2.604

2.407

2.275

0.237

23

2.404

2.428

2.038

1.911

2.309

2.249

2.643

2.347

2.699

2.495

2.352

0.245

24

2.481

2.488

2.119

1.985

2.404

2.333

2.720

2.428

2.780

2.569

2.431

0.244

Max rate
µg/cm2/h

0.165

0.479

0.126

0.118

0.148

0.129

0.178

0.149

0.165

0.160

0.182

0.106

Mean rate
µg/cm2/h

0.100

0.094

0.084

0.080

0.099

0.097

0.113

0.100

0.114

0.106

0.099

0.011

Kp cm/h

(x10-7)

2.018

1.886

1.700

1.605

1.986

1.944

2.273

2.024

2.303

2.135

1.987

0.223

Lag time
(h)

0.000*

0.091

0.00*0

0.000*

0.012

0.000*

0.053

0.050

0.000*

0.000*

0.021

0.032

* - negative values taken as zero to calculate mean lag time

Applicant's summary and conclusion

Conclusions:
Absorption of melamine cyanurate as supplied in form of an aqueous paste is very poor (0.04%).