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Materials and methods

Principles of method if other than guideline:
Test procedure:
ne female Wistar strain rat (BK:Wistar) was twenty-eight to

thirty days old when used for skin disc preparation.

Skin Disc Preparation

Following an acclimatisation period of two days the animal
was shaved to remove hair from the dorsal surface. The
shaved area was washed using an antibiotic wash. After three

days a second antibiotic wash was performed. Four days
later the animal was humanely killed by inhalation of a
rising concentration of CO2 followed by cervical
dislocation. The animal was in the telogen phase of hair
growth and little or no hair growth was visible.

When the animal had been humanely killed the dorsal skin was

removed from the rat as a single pelt. Care was taken
during the procedure to avoid unnecessary damage to the
pelt. Excess fat was removed and the pelt mounted, epidermal
side uppermost, onto a polytetrafluoroethylene (PTFE) tube.

The tissue was secured in place using a rubber "0" ring.
Excess tissue was trimmed away and the "0" ring/PTFE
interface sealed with soft paraffin wax. The tube was
supported by a clamp inside a labelled 30 ml glass
receptacle containing 10 ml electrolyte solution (154 mM

Skin Disc Quality Control

Two skin discs of approximately 0.79 cm2 were taken from the

pelt and the TER measured as a quality control procedure.
Each disc had to give a resistance value of greater than l0
kohms) in order for the remainder of the pelt to be used in
the assay. If either disc fell below the lO kohms threshold,

the pelt was discarded. The quality control discs were then

discarded and new discs from the acceptable pelt were
mounted on the PTFE tubes.

Test Material Administration

The test material was applied to the epidermal surface of
three skin discs for a contact period of 24 hours.

The test material was used as supplied. A volume of 150 µl
was applied to the inner epidermal surface using an
automatic pipettor.

At the end of the exposure period, the test material was
removed by washing the skin disc with a jet of warm tap
water until no further test material could be removed.

Three positive and negative control discs were assayed. The
positive control was hydrochloric acid (approximately 36%)
and the negative control was sterile distilled water. The
contact period for the positive and negative controls was 24


Determination of Transcutaneous Electrical Resistance (TER)

The TER was measured using a Wheatstone Bridge with a low
voltage alternating current. Prior to measurement of the
resistance, the surface tension of the skin disc was reduced

by adding a sufficient volume of 70% ethanol to cover the
epidermis. The ethanol was removed by inverting the tube
after approximately 3 seconds. The PTFE tube was then placed

in the labelled receptor chamber and the tissue was
hydrated by the addition of 3 ml MgSO4 solution (154 mM) to
the inside of the PTFE tube. Any air bubbles present were
dislodged by tapping the tube.

The stainless steel electrodes of the databridge were placed

on either side of the skin disc. The measurement was taken
and a value in ohm/kohm per skin disc was displayed on the
databridge display. The mean TER for the skin discs was

Results and discussion

Details on results:
The mean TER recorded for the 24 hour test material contact
period was greater than 5 kohms.

The TER recorded for the positive and negative control discs
were as follows:

Positive control disc, Hydrochloric acid (approximately
36%): 638 ohms

Negative control disc, Sterile distilled water: 26.0 ohms

The negative control was slightly out of the range stated in
the Standard Test Method (10 - 25 kohms). This was
considered not to affect the purpose or integrity of the

Applicant's summary and conclusion