1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Version / remarks:

adopted in 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Environmental Health and Safety Publications Series on testing and Assessment N° 28. Guidance Document for the Conduct of Skin Absorption Studies

Version / remarks:

adopted in 2004

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: European Commission Guidance Document on Dermal Absorption- Sanco/222/2000 rev. 7

Version / remarks:

adopted in 2004

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

EC Number:

619-749-5

Cas Number:

730979-19-8

Molecular formula:

C16H20FN5

IUPAC Name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: Wistar Rj: WI (IOPS HAN)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 7 - 9 weeks
- Weight at study initiation: 259 - 391 g
- Housing: suspended, stainless steel and wire mesh cages
- Individual metabolism cages: yes (Jencon's metabowls Mk III or Radleys metabolism cages)
- Diet: Rodent diet A04C-10, pelleted and irradiated, (S.A.F.E. Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: filtered and softened tap water, ad libitum
- Acclimation period: at least 13 days in the room, 24 h in the metabolism cages

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Duration of exposure:

8 hours

Doses:

- Nominal doses: 0.05, 0.2 and 500 g/L (corresponding to 0.5, 2 and 5000 µg/cm²)
- Actual doses: 0.28-0.31 µg/cm², 1.5 - 1.8 µg/cm² and 5.67 - 6.14 mg/cm²
- Dose volume: approximately 10 µL/cm²
- Rationale for dose selection: neat product and 2 spray dilutions
Please refer to Table 1 under "Any other information on material and methods incl. tables").

No. of animals per group:

4 animals per dose and termination time point

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: Undiluted test substance or spray dilutions with water.
- Method of storage: at approximately -20°C in the dark

APPLICATION OF DOSE:

TEST SITE
- Preparation of test site: The test site was shaved approximately 24 hours prior to dosing. Just prior to dosing the animals were lightly anaesthetized and two plastic protective saddles were secured in place using Cyanoacrylate adhesive to define the site for application of the test substance.
- Area of exposure: dorsal skin, approximately 2 x 6 cm²
- Type of cover / wrap: a perforated plastic cover (to allow ventilation) held in place over the plastic saddle with surgical tape

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: (cover was held in place over the plastic saddle with surgical tape

REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: cover was removed
- Washing procedures and type of cleansing agent: Cover and application site were swabbed with freshly prepared 1% v/v Tween 80 in PBS using natural sponge swabs until no more radioactivity was detected on the swabs with a Geiger monitor. Animals that were required to provide samples beyond 8 hours were then fitted with a clean cover to capture any 14C lost by desquamation and replaced in the metabolism cage
- Time after start of exposure: 8 hours

SAMPLE COLLECTION
- Collection of blood: cardiac puncture at different termination time points under anaesthasia (Isofluran)
- Collection of urine and faeces: Urine and faeces were collected separately into receivers at 0 to 8, 8 to 24 and at 24-hour intervals up to sacrifice. At the end of each collection period all debris was removed from the metabolism cage and retained. At each sampling, the cage was carefully washed with distilled water. At study termination the cage was washed with an appropriate organic solvent in addition to water.
- Terminal procedure: The rats were exsanguinated whilst under anaesthesia (Isofluran). The treated skin was swabbed following sacrifice prior to removal. The skin was then shaved (shavings retained), if necessary, prior to tape-stripping to remove the stratum corneum. This procedure involved the application of an adhesive tape (CILS, France) for 5 seconds before the tape was carefully removed against the direction of hair growth. This process was continued until a 'shiny' appearance of the epidermis was evident, indicating that the stratum corneum had been removed.
- Analysis of organs: The treated area of skin was removed and taken for analysis. Further skin samples were also taken for analysis, including the area immediately surrounding the treated skin (approx. 1 cm wide) and a control sample (untreated skin: clearly separated from the application site). The residual carcass was also retained for analysis.

SAMPLE PREPARATION
- Storage procedure: Whenever possible samples were processed as they were collected. Remaining samples were stored at approximately -20°C in the dark until required.
- Preparation details:
Urine: The urine samples were thawed to about room temperature. The weights were recorded and duplicate weighed aliquots added directly to scintillation fluid and analysed.
Cage wash and debris: The cage washings were thawed to about room temperature, and weighed. Duplicate weighed aliquots were taken for analysis. The cage debris (if appropriate) was analysed by a suitable method depending on the nature of the debris.
Faeces: The faeces samples were thawed, then weighed and homogenised in a measured weight of distilled water using a top drive homogeniser (Ultra-Turrax). Triplicate aliquots of the homogenate were combusted directly after drying on Combusto-pads.
Whole blood: The samples of blood were thawed to about room temperature. Triplicate weighed aliquots of cardiac blood were combusted directly after drying of weighed aliquots on Combusto-pads contained in Combusto-cones.
Skin swabs: according to the type of samples the following procedures were used: Swabs 8h and at termination time and surrounding swabs: The swabs of the application site were weighed and were solubilised using Soluene®. Triplicate weighed aliquots were taken for LSC analysis. Swabs X, Y and Z (last three swabs): 2 mL of Soluene and scintillation fluid were directly added to each swab for LSC analysis.
Tape strips: All tape strips were solubilised using tetrahydrofuran. Scintillation fluid was directly added to each tape strip sample for LSC analysis
Skin samples: Skin samples were solubilised separately using alcoholic potassium hydroxide, the total weight determined and duplicate weighed aliquots taken for LSC analysis.
Saddle, gauze, tapes and cover (dressing): The saddle, surgical tape and cover were soaked in an appropriate solvent (acetonitrile). The washings were weighed and duplicate weighed aliquots taken for LSC analysis.
Fur samples: The fur samples removed from the treated skin prior to tape-stripping were soaked in an appropriate solvent (acetonitrile, 2 mL). Scintillation fluid was directly added to each fur sample for LSC analysis
Carcass: The residual carcass was thawed, then weighed and solubilised in alcoholic potassium hydroxide; the total weight determined and duplicated weighed aliquots taken for LSC analysis.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting
- Limits of detection: twice the background values for blank samples in appropriate scintillation cocktails

Results and discussion

Signs and symptoms of toxicity:

no effects

Dermal irritation:

not specified

Absorption in different matrices:

For detailed absorption in different matrices please refer to the result tables included under "Any other information on results incl. tables".

Total recovery:

- Total recovery of groups 1 to 4: 90.6 - 92.7% (500 g/L), 92.7 - 101.4% (0.2 g/L), 99.7 - 107.7% (0.05 g/L)
- Recovery of applied dose acceptable: no (groups 1 - 4, 500 g/L); yes (groups 5 and 6, 0.2 g/L); no (groups 7 and 8, 0.2 g/L); yes (groups 9 -12, 0.05 g/L)
- Results adjusted for incomplete recovery of the applied dose: yes (according to EFSA Guidance on dermal absorption, 2017)
- Limit of detection (LOD): The limit of detection was taken to be twice the background values for blank samples in appropriate scintillation cocktails.
- Quantification of values below LOD or LOQ: no

Percutaneous absorption

Percutaneous absorption 1

Time point:

8 h

Dose:

500 g/L

Parameter:

percentage

Absorption:

5.268 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 2

Time point:

8 h

Dose:

500 g/L

Parameter:

percentage

Absorption:

20 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 3

Time point:

24 h

Dose:

500 g/L

Parameter:

percentage

Absorption:

3.025 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 4

Time point:

24 h

Dose:

500 g/L

Parameter:

percentage

Absorption:

12 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 5

Time point:

72 h

Dose:

500 g/L

Parameter:

percentage

Absorption:

2.403 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 6

Time point:

72 h

Dose:

500 g/L

Parameter:

percentage

Absorption:

17 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 7

Time point:

168 h

Dose:

500 g/L

Parameter:

percentage

Absorption:

2.764 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 8

Time point:

168 h

Dose:

500 g/L

Parameter:

percentage

Absorption:

15 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 9

Time point:

8 h

Dose:

0.2 g/L

Parameter:

percentage

Absorption:

21.254 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 10

Time point:

8 h

Dose:

0.2 g/L

Parameter:

percentage

Absorption:

27 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 11

Time point:

24 h

Dose:

0.2 g/L

Parameter:

percentage

Absorption:

18.98 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 12

Time point:

24 h

Dose:

0.2 g/L

Parameter:

percentage

Absorption:

28 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 13

Time point:

72 h

Dose:

0.2 g/L

Parameter:

percentage

Absorption:

11.451 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 14

Time point:

72 h

Dose:

0.2 g/L

Parameter:

percentage

Absorption:

22 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 15

Time point:

168 h

Dose:

0.2 g/L

Parameter:

percentage

Absorption:

14.866 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 16

Time point:

168 h

Dose:

0.2 g/L

Parameter:

percentage

Absorption:

32 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 17

Time point:

8 h

Dose:

0.05 g/L

Parameter:

percentage

Absorption:

42.659 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 18

Time point:

8 h

Dose:

0.05 g/L

Parameter:

percentage

Absorption:

48 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 19

Time point:

24 h

Dose:

0.05 g/L

Parameter:

percentage

Absorption:

28.687 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 20

Time point:

24 h

Dose:

0.05 g/L

Parameter:

percentage

Absorption:

41 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 21

Time point:

72 h

Dose:

0.05 g/L

Parameter:

percentage

Absorption:

30.208 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 22

Time point:

72 h

Dose:

0.05 g/L

Parameter:

percentage

Absorption:

38 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Percutaneous absorption 23

Time point:

168 h

Dose:

0.05 g/L

Parameter:

percentage

Absorption:

23.467 %

Remarks on result:

other: mean potentially absorbable percentage

Percutaneous absorption 24

Time point:

168 h

Dose:

0.05 g/L

Parameter:

percentage

Absorption:

29 %

Remarks on result:

other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Any other information on results incl. tables

Table 2: Achieved doses

	High dose (500 g/L nominal)	Intermediate dose (0.2 g/L nominal)	Low dose (0.05 g/L nominal)
Radiochemical dose applied per animal	537.1 to 582.1 kBq	69.3 to 85.6 kBq	13.2 to 15.0 kBq
Compound dose applied per animal	67.98 to 73.68 mg	17.9 to 21.6 µg	3.3 to 3.8 µg
Compound dose applied per cm² *	5.67 to 6.14 mg/cm²	1.5 to 1.8 µg/cm²	0.28 to 0.31 µg/cm²

*The application rate of the three formulations was 10 µL/cm² and the area of application 12 cm².

Table 3: Mean distribution of radioactivity 8, 24, 72 and 168 h after a single topical application of the test material at the highest dose level (500 g/L; results expressed as % of applied dose; n=4 rats/group)

Group	1		2		3		4
Termination time	8 h		24 h		72 h		168 h
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
SURFACE COMPARTMENT
Skin swabs at 8 h	84.516	3.336	86.305	1.073	86.440	2.637	85.807	1.324
Skin swabs at termination time	-	-	0.925	1.081	0.571	0.519	0.734	0.280
Surrounding swabs	0.169	0.101	0.006	0.006	0.009	0.011	0.000	0.000
Total % swabs	84.685	3.266	87.236	1.349	87.019	2.886	86.541	1.130
Surface dose (Tape-strips 1 & 2)	0.283	0.140	0.952	0.418	0.652	0.357	0.508	0.136
Fur	-	-	-	-	-	-	1.421	0.335
Dressing	0.320	0.059	0.646	0.589	0.997	1.365	1.491	0.570
Total % non absorbed	85.287	3.160	88.834	0.530	88.669	3.323	89.961	1.817
WASHED SKIN COMPARTMENT
Stratum corneum^a	1.842	0.855	2.155	0.987	1.894	0.783	1.243	0.582
Treated skin^b	1.210	0.942	0.252	0.209	0.062	0.050	0.317	0.238
Surrounding skin	0.695	0.352	0.155	0.090	0.095	0.082	0.440	0.235
Total % at dose site	3.747	1.898	2562	1.208	2.051	0.878	2.001	0.428
SYSTEMIC COMPARTMENT
Urines	0.001	0.002	0.013	0.003	0.010	0.003	0.038	0.012
Faeces	0.019	0.002	0.070	0.019	0.101	0.010	0.202	0.041
Cage wash	0.012	0.015	0.018	0.015	0.000	0.001	0.093	0.067
Total % excreted	0.032	0.014	0.101	0.032	0.112	0.011	0.333	0.071
Cardiac blood	0.000	0.000	0.003	0.001	0.002	0.001	0.002	0.001
Non treated skin	1.020	0.788	0.111	0.060	0.076	0.009	0.246	0.122
Carcass	0.469	0.233	0.249	0.032	0.162	0.009	0.183	0.029
Total % directly absorbed	1.521	0.969	0.463	0.109	0.352	0.013	0. 763	0.218
Total % potentially absorbable^c	5.268	2.001	3.025	1.224	2.403	0.880	2.764	0.438
Overall Total % Recovery	90.555	2.075	91.859	1.362	91.072	2.553	92.724	1.888

^a: tape-strips excluding 1 & 2 which are considered to be non-absorbed dose

^b: skin after tape stripping procedure

^c: total % directly absorbed + total at dose site

SD: standard deviation

- = no sample

 

Table 4: Mean distribution of radioactivity 8, 24, 72 and 168 h after a single topical application of the test material at the intermediate dose level (0.2 g/L; results expressed as % of applied dose; n=4 rats/group)

Group	1		2		3		4
Termination time	8 h		24 h		72 h		168 h
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
SURFACE COMPARTMENT
Skin swabs at 8 h	72.008	4.972	56.794	6.427	75.583	4.192	71.019	9.816
Skin swabs at termination time	-	-	17.819	8.593	2.259	1.030	0.765	0.458
Surrounding swabs	0.124	0.158	0.011	0.009	0.016	0.017	0.008	0.001
Total % swabs	72.133	4.849	74.624	3.989	77.858	4.301	71.792	9.384
Surface dose (Tape-strips 1 & 2)	5.420	2.948	3.949	1.820	1.248	0.743	1.357	1.187
Fur	-	-	-	-	0.223	0.447	0.712	0.545
Dressing	2.573	2.041	0.715	0.271	1.951	3.193	5.368	2.268
Total % non absorbed	80.126	5.577	79.288	3.916	81.280	2.024	79.229	6.971
WASHED SKIN COMPARTMENT
Stratum corneum^a	13.877	3.301	10.255	4.339	2.570	1.093	2.466	2.389
Treated skin^b	3.702	2.688	0.763	0.439	0.291	0.100	0.611	0.583
Surrounding skin	0.443	0.256	0.174	0.033	0.190	0.054	0.563	0.381
Total % at dose site	18.023	3.521	11.192	4.062	3.052	1.166	3.640	3.204
SYSTEMIC COMPARTMENT
Urines	0.074	0.030	0.664	0.100	1.319	0.148	2.457	1.282
Faeces	0.014	0.029	1.565	0.383	3.354	0.892	5.887	2.511
Cage wash	0.000	0.000	0.122	0.200	0.372	0.247	0.520	0.388
Total % excreted	0.089	0.044	2.352	0.643	5.045	1.188	8.864	4.023
Cardiac blood	0.008	0.016	0.000	0.000	0.000	0.000	0.000	0.000
Non treated skin	0.644	0.108	0.379	0.044	0.460	0.083	0.564	0.109
Carcass	2.491	0.080	5.058	2.749	2.894	0.264	1.797	0.312
Total % directly absorbed	3.232	0.222	7.788	2.617	8.399	1.254	11.226	4.442
Total % potentially absorbable^c	21.254	3.573	18.980	5.654	11.451	2.068	14.866	7.517
Overall Total % Recovery	101.380	3.716	98.267	2.092	92.701	1.307	94.094	0.654

^a: tape-strips excluding 1 & 2 which are considered to be non-absorbed dose

^b: skin after tape stripping procedure

^c: total % directly absorbed + total at dose site

SD: standard deviation

- = no sample

 

Table 5: Mean distribution of radioactivity 8, 24, 72 and 168 h after a single topical application of the test material at the low dose level (0.05 g/L; results expressed as % of applied dose; n=4 rats/group)

Group	1		2		3		4
Termination time	8 h		24 h		72 h		168 h
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
SURFACE COMPARTMENT
Skin swabs at 8 h	56.565	7.240	60.826	13.318	62.481	11.227	64.537	6.532
Skin swabs at termination time	-	-	6.730	4.433	4.244	2.780	3.911	1.515
Surrounding swabs	0.237	0.215	0.028	0.019	0.009	0.008	0.035	0.035
Total % swabs	56.802	7.225	67.585	10.411	66.734	12.042	68.483	5.426
Surface dose (Tape-strips 1 & 2)	3.713	1.617	7.749	7.860	3.012	1.016	1.149	0.280
Fur	-	-	-	-	2.869	2.970	2.137	0.852
Dressing	1.780	2.084	0.746	1.492	4.896	3.981	4.500	1.967
Total % non absorbed	62.295	6.264	76.080	8.188	77.511	6.647	76.269	4.541
WASHED SKIN COMPARTMENT
Stratum corneum^a	21.447	3.650	10.978	6.837	7.253	1.700	3.285	0.328
Treated skin^b	7.063	6.602	2.402	2.553	0.834	0.218	0.933	0.491
Surrounding skin	1.214	0.702	0.421	0.122	1.675	1.072	0.687	0.060
Total % at dose site	29.725	5.557	13.801	9.319	9.761	2.649	4.904	0.810
SYSTEMIC COMPARTMENT
Urines	0.214	0.070	1.497	0.474	2.646	0.661	2.589	0.727
Faeces	0.000	0.000	2.879	1.147	5.844	1.681	6.430	0.882
Cage wash	0.000	0.000	0.658	0.660	0.115	0.230	0.721	1.191
Total % excreted	0.214	0.070	5.034	2.094	8.605	2.073	9.740	2.041
Cardiac blood	0.000	0.000	0.118	0.125	0.241	0.062	0.000	0.000
Non treated skin	2.575	0.283	1.820	0.177	3.018	0.620	2.814	0.276
Carcass	10.146	2.635	7.913	0.478	8.583	1.251	6.010	1.238
Total % directly absorbed	12.935	2.767	14.886	2.526	20.446	3.178	18.563	3.403
Total % potentially absorbable^c	42.659	3.584	28.687	7.656	30.208	4.586	23.467	3.593
Overall Total % Recovery	104.955	4.568	104.767	2.822	107.718	2.691	99.737	4.728

^a: tape-strips excluding 1 & 2 which are considered to be non-absorbed dose

^b: skin after tape stripping procedure

^c: total % directly absorbed + total at dose site

SD: standard deviation

- = no sample

Results (total potentially absorbed) were corrected for the missing material when recovery was below the set limit (mean over all animals < 95%) as well as for variability between animals according to the EFSA Guidance on dermal absorption (2017). Results are shown in the results table "Percutaneous absorption".