Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
not specified
Principles of method if other than guideline:
- Principle of test: Skin permeability of DMF (100 %) and DMF/water mixtures was determined by an in vitro skin absorption study.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dimethylformamide
EC Number:
200-679-5
EC Name:
N,N-dimethylformamide
Cas Number:
68-12-2
Molecular formula:
C3H7NO
IUPAC Name:
N,N-dimethylformamide
Details on test material:
- Name of test material (as cited in study report): DMF obtained from Tedia Company Inc. (Fairfield, OH, USA)
- Substance type: organic
- Physical state: liquid
- Analytical purity: 99.5 % (HPLC grade)
Radiolabelling:
no

Test animals

Species:
other: lateral-abdomen porcine skin (2-4 months old)

Administration / exposure

Type of coverage:
other: in vitro study
Vehicle:
water
Duration of exposure:
24 h
Doses:
0.2 mL of DMF (undiluted), 50 % DMF/50 % water and 10 % DMF/90 % water
No. of animals per group:
not applicable
Details on study design:
DOSE PREPARATION: before application

APPLICATION OF DOSE: For each experiment, 0.2 ml of DMF pure (or DMF/water mixture) was applied to the epidermal side of the prepared skin
The receptor fluid was collected per 0.5,1.0, and 2.0 h during the periods of 0-4, 5-12. and 13-24 h to examine its penetration rates during the 24-h exposure period.

VEHICLE: water

REMOVAL OF TEST SUBSTANCE
Immediately after the above 24-h exposure, the skin sample was rinsed by 10 ml de-ion water for ten times to remove the DMF residual remaining on the surface of the skin sample. After the above rinsing procedure. 100 µL of de-ion water was applied on the epidermal side of the skin sample to maintain its moisture content Then a 48-h extending skin penetration experiment was conducted on each skin sample to examine its post penetration rate after the above 24-h exposure. Here, the receptor fluid was collected per 0.5, 1.0, 6.0, 8.0 and 12.0 h respectively during the post exposure periods of 0-1,1-2, 2-8, 8-24 and 24-48 h.

CALCULATIONS:
The amount of DMF containing in the rinsed de-ion water was regarded as the DMF remaining on the skin surface after the 24-exposure (denoted as the "unabsorbed"). The total amount of DMF containing in both the receptor fluid collected during the 48-h extending skin penetration experiment and the test skin after the 48-h extending experiment was regarded as the total amount of DMF retaining in the skin layer after the 24-h exposure (denoted as the "skin retention"). Finally, the amount of DMF containing in the receptor fluid collected during the 24-h exposure period was regarded as the amount of DMF penetrated through the skin layer during the 24-h exposure period (denoted as the "skin penetration"). The above three amounts were calculated and were used to check the recovery of DMF from the mass balance aspect.

ANALYSIS
- Method type(s) for identification: gas chromatography equipped with thermionic sensitive detector (Varian 3600 CX CC/TSD; GenTech Scientific, Inc Arcade, NY, USA) coupled to an auto-sampler (Varian 8200 CX; GenTech).

- Validation of analytical procedure: not reported
- Limits of detection and quantification: The limit of detection was 0.53 mg/mL

Results and discussion

Signs and symptoms of toxicity:
not examined
Remarks:
determination of skin permeabilities in vitro is the aim of the study
Dermal irritation:
not examined
Remarks:
determination of skin permeabilities in vitro is the aim of the study
Total recovery:
100 %
Recovery of applied dose acceptable: yes
Limit of detection (LOD): 0.53 mg/mL
Percutaneous absorptionopen allclose all
Dose:
0.2 mL
Parameter:
percentage
Absorption:
9.1 %
Remarks on result:
other: 24 h
Remarks:
Skin penetration (DMF pure)
Dose:
0.2 mL
Parameter:
percentage
Absorption:
2.6 %
Remarks on result:
other: 24 h
Remarks:
Skin penetration (DMF 50 %)
Dose:
0.2 mL
Parameter:
percentage
Absorption:
1.2 %
Remarks on result:
other: 24 h
Remarks:
Skin penetration (DMF 10 %)
Dose:
0.2 mL
Parameter:
percentage
Absorption:
5 %
Remarks on result:
other: 24 h
Remarks:
Skin retention (DMF pure)
Dose:
0.2 mL
Parameter:
percentage
Absorption:
0.8 %
Remarks on result:
other: 24 h
Remarks:
Skin retention (DMF 50 %)
Dose:
0.2 mL
Parameter:
percentage
Absorption:
0.2 %
Remarks on result:
other: 24 h
Remarks:
Skin retention (DMF 10 %)
Dose:
0.2 mL
Parameter:
percentage
Absorption:
86 %
Remarks on result:
other: 24 h
Remarks:
Unabsorbed (DMF pure)
Dose:
0.2 mL
Parameter:
percentage
Absorption:
97 %
Remarks on result:
other: 24 h
Remarks:
Unabsorbed (DMF 50 %)
Dose:
0.2 mL
Parameter:
percentage
Absorption:
99 %
Remarks on result:
other: 24 h
Remarks:
Unabsorbed
Conversion factor human vs. animal skin:
not applicable

Any other information on results incl. tables

Three skin penetration parameters, including flux. Kp and Tlag, were determined and shown in Table 1. For both DMF10 % and DMF50 % their penetration fluxes ( = 0.015 ± 0.007 and 0.126 ± 0.017 mg/cm2/h, respectively) were only -1.1 % and 15 % in magnitude as that of DMF100 % ( = 0.872 ± 0.231 mg/cm2/h). respectively. The same trend could also be found in Kp. The values of Kp for both DMF10 % and DMF50 % (= 1.57 ± 0.708 and 2.67 ± 0355 10-4cm/h, respectively) were only -17 % and 29 % in magnitude as that of DMF100 % ( = 924 ± 2.44 10-4cm/h). respectively. These results suggest that the higher water content would lead to the lower penetration rate of DMF. Thus, skin penetration rates are proportional to the DMF content in the DMF/water mixtures.

Table 1: Summary of skin penetration parameters for DMF 100 %, DMF 50 % and DMF 10 % (n=5).

DMF forms

Flux (mg/cm2/h)

Kp (0.0001 cm/h)

Tlag(h)

DMF 100 % Mean ± SD

0.872 ± 0.231

9.24 ± 2.44

5.54 ± 0.827

DMF 50 %  Mean ± SD

0.126 ± 0.017

2.67 ± 0.355

5.34 ± 0.367

DMF 10 %  Mean ± SD

0.015 ± 0.007

1.57 ± 0.708

7.82 ± 1.06

Kp- permeability coefficient;

Tlag - lag time

Table 1 also shows the estimated Tlag, for the three selected DMF concentrations during the 24-h exposure period. The longest Tlag, was found in DMF)(K (7.82 h), but quite similar Tlag were found in both DMF50 %, and DMF100 % ( = 5.34 and 5.54 h, respectively).

Percutaneous absorption rates (see above) show the distribution of DMF after the 24-h exposure period for the three selected DMF concentrations. They found that 85.9 %, 96.6 % and 98.7 % of applied doses were still remaining on the skin surface after the 24-h exposure (i.e, the "unabsorbed") for DMF100 %, DMF50 % and DMF10 %, respectively. They also found that 1.17 %, 2.61 % and 9.09 % of applied doses had penetrated through the skin layer (i.e, "skin penetration"), and only 0.181 %, 0.838 % and 4.98 % of applied doses were still remaining in the skin layer (i.e., "skin retention") for DMF10 %, DMF50 % and DMF 100 % respectively. As a result, the total penetration amounts of DMF during the 24-h exposure (Le, "skin penetration" + "skin retention") accounted for 1.35 %, 3.45 % and 14.1 % of applied doses for DMF10 %, DMF50 % and DMF100 %, respectively. The above results were not so surprising since the penetration rates of DMF were found to be proportional to its concentrations (Table 1). Here, it should be noted that almost most applied doses used in the present study were still remaining on the skin surface (i.e., the "unabsorbed" fraction are much greater than that of "skin penetration" and "skin retention").

The results of 48-h extending skin penetration experiment conducted after the 24-h exposure period show that the elimination rate of DMF100 % was significantly higher than that of DMF50 % and DMF10 %. The result inferred that the DMF might cause the perturbation effect on the skin layer and result in the increased elimination rate. Particularly, it should be noted that the perturbation effect of DMF was concentration dependent (i.e., the higher DMF concentration, the greater perturbation effect). After the 24-h exposure the amount of DMF retained in the skin layer for DMF100% was significantly higher than that of DMF50 % and DMF10 % (percutaneous absorption rates, see above). Therefore, we found that elimination rate of DMF100 % was significantly higher than that of DMF 50 % and DMF 10 % could be theoretically plausible.

All DMF originally remaining in the skin layer penetrated through the test skin into the receptor fluid in 8 h and 16 h for DMF10 % and DMF50 %, respectively. But residual DMF could still be detected in the skin layer after the 48-h extending period for DMF100 %. These results suggested that the higher DMF content in the skin layer would result in not only a higher elimination rate of DMF, but also a prolonger releasing of DMF from skin layer to the human body.

The "reservoir effects" of DMF were defined. This is the fraction of DMF amount originally remaining in skin layer after the 24-h exposure period (="skin retention" shown in "percutaneous absorption rates" (see above), denoted here as the "reservoir fraction") to those amount which penetrated through the test skin throughout the entire experiment period, including both periods of the 24-h exposure and 48-h extending penetration (= "skin retention" + "skin penetration", as shown in "percutaneous absorption rates" (see above) denoted here as the "total penetration"). Table 2 shows the resultant "reservoir fractions", "total penetrations" and the estimated reservoir effects for the three selected DMF concentrations. It can be seen that the estimated "reservoir effect" for DMF100 % (= 34.1 %) was higher than that of DMF 50 %; and DMF 10 % ( = 27.1 % and 14.1 %, respectively).

Table 2: The estimated skin reservoir effects for DMF 100 %, DMF 50 % and DMF 10 % (n = 5).

Parameters

DMF 100 %

DMF 50 %

DMF 10 %

Reservoir fraction, mg

8.81 ± 4.64

0.928 ± 0.361

0.339 ± 0.010

Total penetration, mg

24.8 ± 8.11

3.35 ± 0.53

0.288 ± 0.107

Estimated reservoir effect, %

34.1 ± 7.43

27.1 ± 5.84

14.1 ± 2.91

Applicant's summary and conclusion

Conclusions:
The results of the present study suggest that the impact associated with the internal burden of DMF could be prolonged even the external exposure of DMF is terminated, particularly for those dermal contact with DMF/water mixtures with a high DMF content.
Executive summary:

This study was set out to determine the skin permeabilities of neat N, N-dimethylformamide (DMF. denoted as DMF100 %) and DMF/water mixtures (including 50 % DMF/50 % water and 10 % DMF/90 % water mixtures (v/v), denoted as DMF50 % and DMF10 % respectively) and to assess their skin reservoir effects on the systemic absorption. The penetration fluxes for DMF10 % and DMF50 % ( = 0.015 and 0.126 mg/cm²/h. respectively) were only -1.1 % and 15 % in magnitude as that of DMF100 % ( = 0.872 ± 0.231 mg/cm²/h). respectively. The above results could be because the perturbation effect of the DMF content was much more significant than the rehydration effect of the water content on skin permeability. It was found that 85.9 %, 96.6 % and 98.7 % of applied doses were still remaining on the skin surface. 4.98 %, 0.838 % and 0.181 % were still remaining in the skin layer, and 9.09 %, 2.61 % and 1.17 % penetrated through the skin layer after the 24-h exposure for DMF100 %, DMF 50 % and DMF10 %, respectively. They found that the half-life (T/2) of DMF retaining in the skin layer were 12 .3, 4.07 and 1.24 h for DMF100 %, DMF50 % and DMF10 %, respectively. The estimated reservoir effect for DMF100 % (=34.1 %) was higher than that of DMF50 % and DMF10 % ( = 27.1 % and 14.1 %. respectively). The above results suggest that the impact associated with the internal burden of DMF could be prolonged even the external exposure of DMF is terminated, particularly for those dermal contact with DMF/water mixtures with high DMF contents.