Trisodium nitrilotriacetate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October - November 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guidance 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 428 (Skin Absorption: In Vitro Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 28 for the conduct of skin absorption studies, March 2004

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium nitrilotriacetate

EC Number:

225-768-6

EC Name:

Trisodium nitrilotriacetate

Cas Number:

5064-31-3

Molecular formula:

C6H9NO6.3Na

IUPAC Name:

trisodium 2-[bis(carboxylatomethyl)amino]acetate

Details on test material:

- Name of test material (as cited in study report): 14C-Trisodium Nitrilotriacetate (aqueous Trilon A liquid)
- Physical state: aqueous liquid
- Purity test date: Oct. 2, 2007
- Lot/batch No.: 932-1003
- Expiration date of the lot/batch:
- Radiochemical purity (if radiolabelling): 99.4%
- Specific activity (if radiolabelling): 7.63 MBq/mg active ingredient
- Locations of the label (if radiolabelling): 2-C14
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:The radiochemical purity of the radiolabeled material was determined before the start of the study (for details see Certificate of Analysis). The test substance was regarded to be stable over the short test period under the storage conditions chosen.
- Storage condition of test material: At low temparatures and in the dark
- Other: application as 40 % and 1 % aqueous solutions

Radiolabelling:

yes

Test animals

Species:

other: human skin preparations (surgically removed skin from the abdomen)

Strain:

not specified

Sex:

not specified

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

water

Duration of exposure:

After application the openings of the donor compartments were covered with stretch adhesive fleece (semi-occlusive conditions).
The skin treated with the high dose was washed about 5 minutes after the application,
The skin treated with the low dose was washed after 6 hours after application.

Doses:

- Nominal doses: high (40% NTA) and low (1% NTA) aqueous solutions
- Actual doses: 381.02 mg/g and 10.2 mg/g
- Actual doses calculated as follows:
- Dose volume: 10 µg on a 1 cm skin surface area
- Rationale for dose selection: technical concentrate (concentration of active ingredient 40 weight-%, corresponding to the concentrated sales product) and in use dilution (concentration of active ingredient 1 weight-%, corresponding to a representative in use dilution)

Target concentration (active ingredient) of test-substance preparation Target amount of preparation applied Target amount of test substance (active ingredient) applied Exposure time
mg/g mg/cm² µg/cm²
High dose (concentrate) 400 10 4000 5 min
Low dose (use dilution) 10 10 100 6 h

No. of animals per group:

5 skin preparations per dose

Control animals:

no

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: The respective aliquot of the radiolabeled test substance solution was mixed with an adequate amount of the non-labeled test substances (concentrate and dilution).
- Method of storage: the preparations were producted the day before, stored over night and homogenized (stired and sonicated) just before application.

APPLICATION OF DOSE: applied in a modified Franz cell consisting of an upper donor compartment with a wide opening at the top and a lower reception compartment.

VEHICLE
- Justification for use and choice of vehicle (if other than water): aqueous solution
- Concentration (if solution): 40 weight-% and 1.03 weight-% Trisodium nitrilotriacetate
- Lot/batch no. (if required): 932-1003
- Purity: 99.4%

TEST SITE
- Preparation of test site: mounted in modified Franz cell.
- Area of exposure: The exposed skin area is about 1 cm²
- % coverage: no data

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: no (not applicable - in vitro test)

REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: diffusion cells were operated in the static mode with tap water as the receptor fluid.
- Washing procedures and type of cleansing agent: the skin was washed with water and washing fluid to mimic real world. Thoroughly rinsed by repeatedly pipetting about 250 ml washing fluid (Texapon N70, 1:140 w/w in bidistilled water) for two washes and a third using tap water.
- Time after start of exposure: 5 min for the high dose and 6 h for the low dose

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting (Wallac type 1409)

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Biopredic International
- Ethical approval if human skin: obtained from 4 donors
- Type of skin: human
- Preparative technique: supplied frozen; on receipt they were vacuum wrapped, labelled and frozen at -20°C until use. Each skin preparation was hydrated in physiological saline for about 10 min before mounding to the diffusion cell. Skin stretched between the donor and receptor compartments, which was stabilized and sealed using a metal clamp.
- Thickness of skin (in mm): 385-570
- Membrane integrity check: integrity of the skin preparation was determined by measuring its electrical resistance with a LCR bridge and in addition visual check for damage.
- Storage conditions: frozen at -20°C for maximum of 12 months; multiple freezing prohibited
- Justification of species, anatomical site and preparative technique: surgically removed skin from abdomen

PRINCIPLES OF ASSAY
- Diffusion cell: modified Franz Cell (Laboratory Glass Apparatus Inc., USA or BASF)
- Receptor fluid: tap water
- Solubility of test substance in receptor fluid: a purely aqueous receptor medium was appropriate as it guarantees sufficient solubility of the test substance.
- Static system: diffusion cells were operated in static mode
- Test temperature: receptor compartment was maintained at 32±1 °C by pumping temperature controlled water through a cell surrounding the receptor fluid container using a thermostat.
- Occlusion: after application the openings of the donor compartments were covered with stretch adhesive fleece (semi-occlusive)
- Reference substance(s):
- Other:

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

yes

Remarks:

high dose: due to the low pH of the substance severe skin damage is taken for granted

Absorption in different matrices:

- Skin wash: at low dose after 6 h and 24 h exposure; at high dose after 5 min, as it was taken granted that the low pH would result in severe skin damage which would greatly enhance penetration.
- Skin test site: approximately 1 cm skin from the abdomen
- Receptor fluid, receptor chamber, donor chamber (in vitro test system): tap water, receptor volume about 4 ml, sampling and replacement of the removed receptor fluid via an outlet (bottom) and inlet (top) pipe connection.
- Skin preparation (in vitro test system): Surgical removed skin from the abdomen were received frozen. These were hydrated in physiological saline for about 10 min before stretched between donor and receptor chambers.
- Stratum corneum (in vitro test system): The donor compartment was covered with sheet of Fixomul stretch (semi-occlusive adhesive fleece)

Total recovery:

The mean total recovery measured in diffusion cells at both high and low dose fulfilled the OECD quality criteria. The individual values ranged between 88 and 104 % and respectively 97 and 105 %.
At the high dose the amount of test substance was recovered from the skin membrane washings and tape strips. No test substance was found in the skin membrane.
Similar, in the low dose, nearly the total amount of test substance was recovered from the skin membrane washings and tape strips. Only 0,09 % of the test substance was found in the skin membrane and 0,11 % was recovered from the receptor fluid, receptor chamber washing and from the receptor samples including wash out.

Percutaneous absorption

Percutaneous absorption 1

Dose:

40 weight-%

Parameter:

percentage

Absorption:

ca. 0.001 %

Remarks on result:

other: 5 min

Remarks:

The applied dose was washed off after 5 min, so this experimental setting does not allow to draw conclusions on the skin-penetrating properties of the high dose (40% NTA) dilution.

Percutaneous absorption 2

Dose:

1 weigh-%

Parameter:

percentage

Absorption:

>= 0.042 - <= 0.472 %

Remarks on result:

other: 24 h

Any other information on results incl. tables

The mean total recoveries of radioactivity were 98.75% (high dose) and 101.8 % (low dose) of the applied radioactivity. The sum of radioactivity present in the skin plus radioactivity determined in the receptor fluid and receptor chamber washing was taken as the absorbed dose.

	High Dose		Low Dose
Target Applied Dose of Test substance [µg/cm²]	4000		100
Mean Nominal Applied Dose of Test substance [µg/cm²]	3932		100
	Mean Dose of Test substance found [µ]	% of Applied Dose	Mean Dose of Test substance found [µ]	% of Applied Dose
Non-absorbed Dose
Tape strips	0,282	0,007	0,104	0,105
Initial Membrane Washing*	3884,04	98,74	101,22	101,26
Membrane washing after 24 hours	0,085	0,002	0,252	0,253
Sum	3884,40	98,75	101,58	101,62
Skin Peparation	0,017	0,000	0,088	0,089
Absorbed Dose
Sum Receptor Samples 0-24 h including wash out	0,033	0,001	0,053	0,053
Receptor Fluid	0,000	0,000	0,051	0,051
Receptor chamber washing	0,025	0,001	0,006	0,006
Sum	0,058	0,001	0,110	0,110
Total	3884,48		101,78
Total Recovery		98,75		101,82
Note: *after 5 min.(high dose) and 6 hours (low dose)

Applicant's summary and conclusion

Conclusions:

Taking into account the sensitivity of the method and the variability of results with the low dose, no appreciable diffusion of the test substance (<= 0.1%) into or through skin was found under the conditions of this in vitro skin penetration study.

Executive summary:

Skin penetration of NTA has been investigated in vitro. Radiolabeled14C-Na3NTA was applied to human skin preparations in a Franz-type diffusion cells as 40 % and 1% aqueous dilution. At high concentration exposure was stopped after 5 min to avoid severe skin damage that would greatly enhance penetration. 0.001 % (n=5) of the substance was absorbed. However, the applied dose was washed off after 5 min in this setting, so this experimental setting does not allow to draw conclusions on elongated exposure to 40% Na3NTA. At low concentration exposure was stopped after 6 h. Absorption ranged between 0.042 and 0.472 % (n=5) during a sampling period of 24 hr. The presence of a lag time of absorption (about 1.6 h) underlines the functional diffusion barrier of human skin against the tested substance preparation.