Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th April 2017 - Experimental Termination 17th April 2017 Report Issued 5th June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

no

Remarks:

This study was not conducted in compliance with Good Laboratory Practice but was performed according to the approved protocol and any amendments, and followed applicable internal Cyprotex SOPs.

Test material

Test material information

Test material form:

liquid

Remarks:

Pale to yellow

Details on test material:

Chemical name: Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and Glycerol
Lot#: 1436088
Expiration Date: 10 August 2019
Molecular Weight: 147.17
Stability: 24 Months
Storage: RT in dark

Specific details on test material used for the study:

12.4.1. N-[2-(2-hydroxyethoxy)ethyl]acetamide
• CAS#: 118974-46-2
• Molecular Weight: 147.17
• Log Pow: The average measured log Pow value for the test substance, as determined from the test, was <0. As an estimation, the log Pow value was calculated to be 2.7 by extrapolation.
• Stability: 24 Months
• Solubility: At least 369.5 mg/mL in water as per the Sponsor.
12.4.2. Formulation
• 3H-N-[2-(2-hydroxyethoxy)ethyl]acetamide 1 mCi/mL in ethanol (see Appendix E)
o Radiochemical Purity: >99% by HPLC
o Chemical Purity: Product identity confirmed by HPLC co-elution with Authentic Standard and Mass Spectroscopy
o Specific Activity: 9.6 Ci/mMole by Mass Spec.
o Lot#: 173-048-000
o Source: ViTrax Co. (Placentia, CA 92870)
o Cat#: VC 100
• Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol
o Lot#: 1436088
o Expiration Date: 10 August 2019
12.4.4. Preparation: The radiolabeled test material was produced by and purchased from ViTrax Co. (Placentia, CA 92870) as requested by the Sponsor. A 10 mL aliquot of radio inert ElfaMoist AC was placed into a 50 mL conical tube. Enough radiolabeled test material was spiked into the aliquot to produce a stock that delivered approximately 243,763DPM per 6.4 µL dose (an addition of approximately 10-3 of original amount of test material). The spiked aliquot was vortexed for approximately 2 minutes and then tested for homogeneity of radiolabel by measuring the radioactivity, in DPM, in 10 µL aliquots of the mixture obtained from 3 different places in the mixture. The samples collected from the preparation were within ±5% of the mean; therefore, the preparation was considered homogeneous. The results are presented in the table below.
12.4.5. Justification of Use: The formulation that was used is the marketed formulation.

Radiolabelling:

yes

Administration / exposure

Details on in vitro test system (if applicable):

12.5. Other Materials
12.5.1. Human Cadaver Thigh Skin: Dermatomed (approximately 0.75 mm thickness) human cadaver thigh tissue from a single donor were received flash frozen from SciKon Innovation, Inc. (Durham, NC 27709) and stored at -80⁰C, as directed, until use. The barrier quality of the skin sections were assessed with transepithelial water loss (TEWL) measurements using a Vapometer SWL5 (Delfin Technologies) after placement onto the Franz Cells. All skin sections had an acceptable barrier function as indicated by TEWL readings of ≤ 11 g/m2/hour with values that ranged from 6.9 to 10.9 g/m2/hour.
• Skin Lot 151004: Scikon Innovation, Inc. Causcasian Female Aged 64, Exp: 09Mar2018
12.5.2. Miscellaneous Reagents:
• Phosphate Buffered Saline: Gibco, Cat # 10010-023, Lot# 1754017, Exp: October 2017; and Mattek, Cat # Dulbecco’s Phosphate Buffer, Lot# 041916PLA, Exp: 19April2017
• Sodium Azide: Sigma, Cat #S2002, Lot# MKBR3666V, Exp: Feb 2019
• Brij 98®: ACROS, Cat # 347181000, Lot # A0373841, Exp: Jun 2021
• Scintillation Fluid: Perkin-Elmer, Cat #: 6013329, Lot# 77-16191, Exp: 01Dec2017
• Ethanol: Spectrum, Cat #: E1424, Lot# 1FF0319, Exp: 22 Jan 2019
• Acetone: Sigma-Aldrich, Cat# 270725, Lot# SHBB8538V, Exp: July 2017
• Soluene-350: Perkin-Elmer, Cat#: 6003038, Lot#: 24-16131, Exp: 01Oct2017
12.6. Receiver Fluid
12.6.1. Test Material Receiver Fluid: PBS pH 7.4 with 0.1% Brij 98® and 1mM NaN3
12.6.2. Reference Control Receiver Fluid: PBS pH 7.4 with 0.1% Brij 98® and 1mM NaN3

Results and discussion

Percutaneous absorption

Percutaneous absorption 1

Time point:

12 h

Dose:

Absorbed dose

Parameter:

percentage

Absorption:

0.057

Percutaneous absorption 2

Key result

Time point:

24 h

Dose:

Absorbed dose

Parameter:

percentage

Absorption:

0.382

Percutaneous absorption 3

Key result

Time point:

24 h

Dose:

Absorbable dose

Parameter:

percentage

Absorption:

0.562

Any other information on results incl. tables

Mass Balance (%recovery)

For each Franz Cell, amounts of each test material(DPM)in the following compartments: initial donor (M_Donor(0)), tissue washes (M_Washes), tape strip extracts (M_{Tape strips}), skin extracts (M_skin), receiver fluid (M_Receiver) and Franz Cell rinse and Donor Swab (M_Rinse) at the end of the experimental period were measured for all test material and control chemicals. Test material mass balances in the system were calculated with Equation 1. 

Mass Balance = (M_Receiver) + (M_Washes) +(M_{Tape strips}) + (M_skin) + (M_Rinse) X 100 _______________________________________________

(M_Donor(0))

For each Franz Cell testing reference controls, amounts (DPM) of each reference control in the following compartments were measured: initial donor (M_Donor(0)), final dose (M_Donor(F)), tissue washes (M_Washes), skin extracts (M_skin), and receiver fluid (M_Receiver) at the end of the experimental period. Reference control mass balances in the system were calculated with Equation 2. 

Mass Balance = (M_Receivers) +(M_Washes) +(M_{Final Dose}) +(M_skin) X 100

_____________________________________

(M_Donor(0))

Table 1a and Appendices B and C present the mean and individual mass balance (percent recovery) values for the reference controls. Table 1b and Appendix D present the mean and individual mass balance values for N-[2-(2-hydroxyethoxy)ethyl]acetamide when formulated as ElfaMoist AC.  

Table 1a.  Mass Balance (% Recovery) for^14C-Benzoic Acid and^14C-Mannitol Treated-Franz Cells
Reference Controls Mass Balance (% Recovery) per Franz Cell
Reference	Replicate 2	Replicate 2	Replicate 3	Replicate 4	Mean (Std)
Benzoic acid	101.8%	101.9%	102.5%	99.7%	101.5% (1.2%)
Mannitol	98.0%	99.2%	98.1%	98.2%	98.4% (0.6%)
^§Arithmetic Mean, N=4

All four replicates treated with each of¹⁴C-Benzoic Acid or¹⁴C-Mannitol showed acceptable mass balance values of 100% ± 10%. When the mass balance falls into the acceptable range, the permeability data generated is regarded as being representative of the test material’s actual behavior in the test system.

Table 1b.  Mass Balance (% Recovery) for Franz Cells Treated with ^3H-N-[2-(2-hydroxyethoxy)ethyl]acetamide Formulated as ElfaMoist AC
Mass Balance (% Recovery) per Franz Cell				Mean^§(Std)
Replicate 1	Replicate 2	Replicate 3	Replicate 4
99.7%	103.5%	85.6%	97.6%	96.6% (7.8%)
^§Arithmetic Mean, N=4

Three of four Franz Cells treated with ElfaMoist AC had acceptable mass balances of 100% ± 10% for materials that were radiolabeled. The low mass balance for Replicate 3 was most likely due to variability in the dose volume applied since the greatest difference in material distribution between the Franz Cells was in the sum of the washes; replicate 3’s washes produced 81.9% of the initial dose while the other 3 replicates’ totals were all above 93%. 

Table 1c.  Mean§ Percent Initial Dose of All Franz Cell Compartments and Total Mass Balance* of All Franz Cells Treated with  3H-N-[2-(2-hydroxyethoxy)ethyl]acetamide Formulated as ElfaMoist AC
Mean % Initial Dose per Franz Cell Compartment					Total Mass Balance
Reciever	Washes	Tape Strips	Skin	Franz Cell Rinses
0.382%	92.853%	3.276%	0.094%	0.0040%
§Arithmetic Mean, N=4 *The sum of all mean percent intitial dose values of each Franz Cell compartment

Cumulative Transfer into the Receiver and Material Distribution

Table 2 presents the mean and individual mass of test material transferred into the receiver at each specified time point as a percent of initial dose. Table 3 presents the mean and individual test material distribution in each of the tape-strip groups and sum of all tape-strips as a percent of initial dose. Table 4 presents the mean and individual distribution in the skin extracts, the total absorbable dose (total test material present on or in the skin following washings), and total absorbed dose(amount of test material reaching the receiver buffer)as a percent of initial dose. 

Table 2. Mean^§and Individual Cumulative Percent of Initial Dose of ^3H-N-[2-(2-hydroxyethoxy)ethyl]acetamide Transferred Into Franz Cell Receivers When Formulated as Elfamoist AC over a 24-Hour Exposure Period on Human Cadaver Thigh Skin   (Mean^§± Standard Deviation)
Replicate	Cumulative % of Initial Dose in the Receiver Chamber at Each Time Point
	1 Hr	2 Hr	4 Hr	6 Hr	12 Hr	24 Hr
Replicate 1	0%	0%	0%	0%	0.051%	0.392%
Replicate 2	0%	0%	0%	0%	0.103%	0.384%
Replicate 3	0%	0%	0%	0%	0.000%	0.267%
Replicate 4	0%	0%	0.041%	0.002%	0.073%	0.487%
Mean^§	0%	0%	0.010%	0%	0.057%	0.382%
St Dev	0%	0%	0.021%	0.001%	0.043%	0.090%
§: Arithmetic Mean, N=4     Grayed replicate had a mass balance of 100% ± 10%

Table 3. Mean^§and Individual Mass as a Percent of Initial Dose of ^3H-N-[2-(2-hydroxyethoxy)ethyl]acetamide in each Tape-Strip Group and as the Sum of All Tape-Strip Groups over a 24-Hour Exposure Period on Human Cadaver Thigh Skin When Formulated as ElfaMoist AC (Mean^§± Standard Deviation)
Replicate	Tape-Strip Group 1	Tape-Strip Group 2	Tape-Strip Group 3	Tape-Strip Group 4	Sum of All Tape Strips
Replicate 1	1.401%	1.639%	0.030%	NS	3.069%
Replicate 2	1.344%	1.905%	0.042%	0.000%	3.291%
Replicate 3	2.668%	0.733%	0%	NS	3.402%
Replicate 4	2.560%	0.780%	0%	NS	3.340%
Mean^§	1.993%	1.265%	0.018%	0%	3.276%
St Dev	0.719%	0.596%	0.021%	0%	0.145%
§: Arithmetic Mean, N=4 Grayed replicate had a mass balance of 100% ± 10% NS: No Sample

Table 4. Mean^§and Individual Mass as a Percent of Initial Dose ^3H-N-[2-(2-hydroxyethoxy)ethyl]acetamide in Tape Stripped-Skin, the Total Absorbable Doseǂ, and the Absorbed Dose§ after a 24‑Hour Exposure Period on Human Cadaver Thigh Skin When Formulated as ElfaMoist AC (Mean^§± Standard Deviation)
Replicate	Skin	AbsorbableǂDose	Absorbed* Dose
Replicate 1	0.086%	3.156%	0.392%
Replicate 2	0.763%	3.367%	0.384%
Replicate 3	0.047%	3.448%	0.267%
Replicate 4	0.165%	3.505%	0.487%
Mean^§	0.094%	3.369%	0.382%
St Dev	0.050%	0.153%	0.090%
§: Arithmetic Mean, N=4
Grayed replicate had a mass balance of 100% ± 10%
ǂAbsorbable Dose: Total test material present on or in the skin following washings *Absorbed Dose: Amount of test material reaching the receiver buffer at 24-hours                           (See Table 2)

1.1.Permeability Coefficient (Papp, cm/h, reference controls only)

For each Franz Cell treated with reference controls, initial donor mass (M_Donor(0)) and final donor mass, cumulative mass in the receiver compartment (M_Receiver) over the 24-hour incubation period were determined.

Permeability coefficients (P, cm/hr) for the reference controls are presented in Table 5 and Appendices B and C and were calculated with Equation 3.

Equation 3:

P= M_R  [V_D]

    __ ___

M_D(0) [A^.Δt]

where V_Dis the volume of the donor compartment and A is the area of exposed tissue. To calculate the Papp values, the cumulative transfer of the test material and each reference control into the receiver (M_Receiver/M_Donor(0)) over time (Δt) was graphed and the slopes of the linear portions of the graphs (R²≥ 0.99) were multiplied by V_Dand divided by the area of exposed tissue.

Table 5. Permeability Coefficient (Papp, cm/h) for^14C-Benzoic Acid and^14C-Mannitol Treated Franz Cells
Reference Controls	Replicate 1	Replicate 2	Replicate 3	Replicate 4	Mean^§ (± Std)
Benzoic Acid	2.34x10^-4	5.47x10^-4	1.80x10^-3	4.69x10^-4	7.62x10^-4 (7.03x10^-4)
Mannitol	3.13x10^-5	3.91x10^-5	4.69x10^-5	2.34x10^-5	3.52x10^-5 (1.01x10^-5)
§Arithmetic Mean, N=4

 

The skin sections acted as expected with respect to the permeability characteristics of the reference controls. As expected,¹⁴C-Benzoic Acid, the high permeability reference, penetrated the tissue cultures at a faster rate than¹⁴C-Mannitol, the low permeability reference.

1.1.Flux Values (µg transferred/cm²of skin/hour)

The flux (µg transferred/cm²/h) for³H-N-[2-(2-hydroxyethoxy)ethyl]acetamide through the skin was calculated. To derive these values, the total amount of test material in the receiver based on percent of initial dose divided by the area of exposed skin over time was graphed. The flux values are the resulting slopes from the linear portions of these graphs (R² ≥ 0.99) and were calculated from data at time points starting at 6 hours. The flux values for the test material are presented in Table 6.

Table 6. Flux (µg transferred/cm^2/h) for Treated-Franz Cells over the 24-Hour Exposure Period
Replicate 1	Replicate 2	Replicate 3	Replicate 4	Mean^§(Std)
327.64	249.53	256.58	397.51	307.82 (69.43)
§Arithmetic Mean Grayed replicate had a mass balance of 100% ± 10%

Applicant's summary and conclusion

Conclusions:

In general, less than 0.5% of the initial dose was absorbed (mass of test substance reaching the receiver fluid) over the 24-hour exposure. There was at least a 4-hour lag phase where no material was found in the receiver chamber after which only small amounts of test material appeared. The overall mean flux rate, calculated from values after 6 hours of exposure, was 307.82 ± 69.43 µg transferred/cm2 skin/hour.

Executive summary:

The test substance “Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol” has been tested for skin absorption potential in an in vitro test performed according to OECD428, and using radiolabelled material.

The mass balance values for 8 of 8 Franz Cells treated with the two reference control materials were considered acceptable since they were within the OECD acceptable range for radiolabelled materials (100 ± 10%; OECD 2004).  Individual recoveries (Table 1a and Appendices B and C) for Franz Cells with acceptable mass balances ranged from 99.7% to 102.5% for 14C-Benzoic Acid and 98.0% to 99.2% for 14C-Mannitol.  Permeability coefficients generated by the high permeability reference control, 14C-Benzoic Acid (mean of 7.62x10-4 ± 7.03x10-4 cm/hour), and the low permeability reference control, 14C-Mannitol references (mean of 3.52x10-5 ± 1.01x10-5 cm/hour) indicated the skin samples behaved as expected when exposed to the reference controls (Table 5 and Appendices B and C). These Permeability Coefficient (Papp) values also fell within the historical range of Papp for this assay for Benzoic Acid (range of 1.56x10-5 to 1.75x10-2 cm/h, mean and standard deviation of 5.42x10-3 ± 3.99x10-3 cm/h; N=45) and Mannitol (range of 0.0 to 1.95x10-3 cm/h, mean and standard deviation of 2.73x10-4 ± 4.88x10-4 cm/h; N=40) Mass balance values that were within the OECD acceptable range indicated that the distribution of the test material in these Franz Cells are representative of the actual permeability behaviour of the material. Data should be interpreted with this in mind.

The mean mass balance for all Franz Cells was 96.6% (Table 1b); this was also the total mass balance value when the mean percents of initial dose of each of the Franz Cell compartments were added together (Table 1c).  Three of four Franz Cells treated with test substance had acceptable mass balances of 100% ± 10% for materials that were radiolabelled. The low mass balance for one replicate (replicate 3; 85.6%) was most likely due to variability in the dose volume applied since the greatest difference in material distribution between the Franz Cells was in the sum of the washes; replicate 3’s washes produced 81.9% of the initial dose while the other 3 replicates’ totals were all above 93%.  Since the material distribution of all the Franz Cells treated with test material was very similar except for the washes, the permeability behaviour of the test material in all four Franz Cells can be considered as the same.

The majority of the 3H N-[2-(2-hydroxyethoxy)ethyl]acetamide in the substance applied dose remained on the surface of the skin mounted on all four Franz Cells and was removed by the three washes at the end of the 24-hour exposure (Appendix D). Little to no test material penetrated into the receiver chamber.  For three of four Franz Cells, the test material was below detectable limits for the first 6 hours of the exposure; the remaining Franz Cell showed only 0.041% of the initial dose was absorbed into the receiver by 4 hours into the exposure.  At 12 hours, 3 of 4 Franz Cells showed 0.103% or less of the initial dose was absorbed into the receiver.  After 24-hours, the mean cumulative amount present in the receiver, also considered the absorbed dose, had increased to 0.382%±0.090% for all four Franz Cells.The flux was calculated from the amounts of test material appearing from 6 to 12 hours into the exposure and were similar for all Franz Cells ranging from 249.53 to 397.51 µg transferred/cm2/hour with a mean of 307.82±63.43 µg transferred/cm2/hour (Tables 2 and 6, Appendix D).The total amounts of the test material extracted from the tape strips as a percent of initial dose from each Franz Cell ranged from 3.069% to 3.402% (mean of 3.276%±0.145%) indicating little test material was available in the stratum corneum/epidermis for possible further absorption.

The amounts of test material in the tape-stripped skin was also small with individual values that ranged from 0.047% to 0.165% of initial dose (mean of 0.094%±0.050%, Table 4 and Appendix D). The total amounts present in the skin washes (Appendix D) also reflected these data in that washes from skin contained the majority of the dose with percents of initial dose ranging from 81.9% to 99.8%.

Conclusions:

Three of four Franz Cells treated with 3H-N-[2-(2-hydroxyethoxy)ethyl]acetamide when formulated as the product ‘Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol’ had acceptable mass balances of 100% ± 10% for test materials that were radiolabelled. The low mass balance for one replicate (replicate 3; 85.6%) was most likely due to variability in the dose volume applied since the greatest difference in material distribution between the Franz Cells was in the sum of the washes.  Since the material distribution of all the Franz Cells treated with the test substance was very similar except for the washes, the permeability behaviour of the test material in all four Franz Cells can be considered as the same.

In general, less than 0.5% of the initial dose was absorbed (mass of test substance reaching the receiver fluid) over the 24-hour exposure.  There was at least a 4-hour lag phase where no material was found in the receiver chamber after which only small amounts of test material appeared.  The overall mean flux rate, calculated from values after 6 hours of exposure, was 307.82 ± 69.43 µg transferred/cm2 skin/hour.  The amount of test material extracted from the tape strips representing material in the stratum corneum/epidermis ranged from 3.069% to 3.402% of the initial dose.  The mass of test material found in the tape-stripped skin ranged from 0.094%±0.050% of initial dose.  Overall, these data indicated little test material was absorbed into the receiver over the 24-hour exposure period and little test material was considered as absorbable (mass of material present in or on the washed skin); therefore, the majority of the test material in the initial dose remains on the surface of the skin over the 24-hour exposure period.