1,3,5-triazine-2,4,6(1H,3H,5H)-trione, compound with 1,3,5-triazine-2,4,6-triamine (1:1)

Dermal absorption

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

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,3,5-triazine-2,4,6(1H,3H,5H)-trione, compound with 1,3,5-triazine-2,4,6-triamine (1:1)

EC Number:

253-575-7

EC Name:

1,3,5-triazine-2,4,6(1H,3H,5H)-trione, compound with 1,3,5-triazine-2,4,6-triamine (1:1)

Cas Number:

37640-57-6

Molecular formula:

C3H6N6.C3H3N3O3

IUPAC Name:

1,3,5-triazinane-2,4,6-trione - 1,3,5-triazine-2,4,6-triamine (1:1)

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 or test system 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/cm^2(n=10)
	Mean	SD	CV%
Max. rate of penetration over 0-24 h (µg/cm^2/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/cm^2skin) (± 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/cm^2/hour	0.182 ± 0.106
Mean rate µg/cm^2/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/cm^2skin)
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/cm^2/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/cm^2/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%).