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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2010 - October 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully Guideline- and GLP-compliant
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD-Guideline 431, "In Vitro Skin Corrosion: Human Skin Model Test ", 13 April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: DRAFT: OECD Guideline In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, Paris 11 December 2009 (Version 4)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ESAC statement, 5 November 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Regulation (EC) 761/2009: B.46 "In vitro skin irritation: Reconstructed Human Epidermis Model Test". 23 July 2009
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
4-amino-2-methylpyrimidine-5-methylamine
EC Number:
202-384-7
EC Name:
4-amino-2-methylpyrimidine-5-methylamine
Cas Number:
95-02-3
Molecular formula:
C6H10N4
IUPAC Name:
5-(aminomethyl)-2-methylpyrimidin-4-amine
Details on test material:
Name of test material (as cited in study report): GREWE-DIAMIN
Chemical name: 4-amino-2-methyl-5-Pyrimidinemethanamine.
Molecular formula: C6H10N4.
Batch No.: M-622-035-200.
CAS No.: 95-02-3.
Appearance: White to yellowish crystals.
Solubility: Soluble.
pH: 10.3.
Conditions of storage: In the dark.
Stability at conditions of storage: Stable.
Date of expiry: February 2011.

Test animals

Species:
other: in vitro system
Strain:
other: MatTek´s EpiDerm System
Details on test animals or test system and environmental conditions:
MatTek´s EpiDerm System consists of normal, human-derived epidermal keratinocytes which
have been cultured form a multilayered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum
containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
The EpiDerm tissues (surface 0.6 cm²) are cultured on specially prepared cell culture
inserts (MILLICELLs, 10 mm ) and shipped as kits, containing 24 tissues on shipping agarose.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: n.a.
Amount / concentration applied:
The application spoon was filled with 25 mg finely grounded test substance.
The "spoonful" was levelled by gently scratching the excess material away, avoiding compression.
25 µL H2O were added for wetting of the test substance.
Duration of treatment / exposure:
Epiderm Skin Corrosivity Test:
3 minutes
1 hour
Epiderm Skin Irritation Test:
60 minutes, post-incubation: 42 hours
Observation period:
n.a.
Number of animals:
Epiderm Skin Corrosivity Test
Two tissue replicates were used for each treatment (exposure time), including distilled water
as negative and 8N KOH as positive control.
Epiderm Skin Irritation Test
Three tissue replicates were used, including distilled water as negative and 5 % SDS as
positive control.
Details on study design:
Epiderm Skin Corrosion Test
Two tissue replicates were used for each treatment (exposure time), including deionised water as negative and 8N KOH as positive control.
Exposure times:
3 minutes
1 hour
50 µL of each reference substance were dispensed directly atop the EpiDerm tissue.
The application spoon was filled with 25 mg finely grounded test substance.
The "spoonful" was levelled by gently scratching the excess material away, avoiding compression.
25 µL H2O were added for wetting of the test substance.

Epiderm Skin Irritation Test
Three tissue replicates were used, including distilled water as negative and 5 % SDS as
positive control.
Exposure time:
60 minutes, post-incubation: 42 hours
30 µL of each reference substance were dispensed directly atop the EpiDerm tissue.
The application spoon was filled with 25 mg finely grounded test substance.
The "spoonful" was levelled by gently scratching the excess material away, avoiding compression.
25 µL H2O were added for wetting of the test substance.


MTT-test
After incubation with the test substance and washing with PBS, the tissues were incubated
with MTT medium at 37°C and 5 % CO2. After 3 hours, the MTT medium was aspirated from
all wells and the tissues were gently rinsed with PBS (2 times). For extraction, the tissues
were incubated with extractant solution (isopropanol) for 2 hours with shaking.
After the extraction period, the tissues were pierced with an injection needle and the extract
(now a blue formazan solution) was allowed to run into the well from which the tissue was
taken. The 24-well plates were placed on a shaker for 15 minutes until the solutions were
homogeneous in colour.
For the Epiderm Skin Corrosivity Test per each tissue 3 × 200 µL aliquots, for the Epiderm Skin Irritation Test 2 × 200 µL aliquots
of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate and the OD was measured using
the extractant solution as blank in a plate spectrophotometer at 570 nm, without reference filter.

Calculations

Cell viability
Cell viability was calculated for each tissue as percent of the mean of the negative control
tissues. The skin corrosivity/irritation potential of the test substance was classified according
to remaining cell viability obtained after test substance treatment with either of the two
exposure times.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Mean tissue viability (%)
Basis:
mean
Time point:
other: 3 minutes
Score:
91.6
Reversibility:
other: n.a.
Remarks on result:
other: Non-Corrosive
Irritation parameter:
other: Mean tissue viability (%)
Basis:
mean
Time point:
other: 1 hour
Score:
90.7
Reversibility:
other: n.a.
Remarks on result:
other: Non-Corrosive
Irritation parameter:
other: Mean tissue viability (%)
Basis:
mean
Time point:
other: 60 minutes, 42 hours post-incubation
Score:
101.6
Reversibility:
other: n.a.
Remarks on result:
other: Non-Irritant
Irritant / corrosive response data:
Epiderm Skin Corrosivity Test:
The mean percentage viability of the treated skin discs after 3 minutes of exposure was
91.6 % which is above the threshold of 50 % for classification.
The mean percentage viability of the treated skin discs after 1 hour of exposure was
90.7 % which is above the threshold of 15 % for classification.

Epiderm Skin Irritation Test:
The mean percentage viability of the treated skin discs was 101.6 % which is above the
threshold of 50 % for classification.
Other effects:
Epiderm Skin Corrosivity Test:
Assay acceptance criteria according to the protocol INVITTOX n°119 by ECVAM:
The mean optical density (OD) of the tissues, treated with deionised water (negative
control) was 1.844 after 3 minutes, and 2.035 after 1 hour of exposure, that is higher than
0.8, as required by the assay acceptance criteria.
The mean tissue viability of the 3 minutes positive control was 15.3 %, that is lower than
30 %, as required by the assay acceptance criteria.
The maximum inter tissue viability differences of the "GREWE-DIAMIN" treated skin discs
were 15.0 % for 3 minutes and 3.4 % for 1 hour exposure, that is below 30 % as required
by the assay acceptance criteria.

Epiderm Skin Irritation Test:
Assay acceptance criteria according to the protocol used during the ECVAM validation study:
The mean OD of the tissues, treated with deionised water (negative control) was 2.061,
that is higher than 1.0 and lower than 2.5, as required by the assay acceptance criteria.
The mean tissue viability of the positive control was 7.2 %, that is lower than 20 %, as
required by the assay acceptance criteria.
The standard deviation calculated from individual percentual tissue viabilities of the
"GREWE-DIAMIN" treated skin discs was 3.7, that is below 18 as required by the assay
acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the results of this study and the Directive 2001/59/EC, the test substance
"GREWE-DIAMIN" is considered to be non-corrosive and non-irritant to skin.
Executive summary:

Aim and Method

The EpiDerm Skin Corrosivity/Irritation Test (Model EPI-200) was performed to reveal

possible irreversible tissue damages of the skin following the application of

"GREWE-DIAMIN".

Due to technical reasons the EpiDerm Skin Irritation Test was started previous to the

EpiDerm Skin Corrosivity Test.

EpiDerm Skin Irritation Test: The test substance was topically applied for 60 minutes to the

epidermal surfaces of three-dimensional human epidermis models. After a post-incubation of

42 hours, a cell viability test was performed.

EpiDerm Skin Corrosivity Test: During the post-incubation of 42 hours the test substance

was topically applied for 3 minutes and 1 hour to the epidermal surfaces of threedimensional

human epidermis models, followed by immediate determination of the cytotoxic

effect.

Investigations performed were in conformance with the Regulation (EC) 440/2008:

B.40.BIS. "In vitro skin corrosion: human skin model", the OECD-Guideline 431, "In Vitro

Skin Corrosion: Human Skin Model Test ", 13 April 2004, the DRAFT: OECD Guideline

In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, Paris 11

December 2009 (Version 4), the ESAC statement, 5 November 2008 and the Regulation

(EC) 761/2009: B.46 "In vitro skin irritation: Reconstructed Human Epidermis Model Test".

23 July 2009.

Results

EpiDerm Skin Corrosivity Test

Assay acceptance criteria according to the protocol INVITTOX n°119 by ECVAM:

The mean optical density (OD) of the tissues, treated with deionised water (negative

control) was 1.844 after 3 minutes, and 2.035 after 1 hour of exposure, that is higher than

0.8, as required by the assay acceptance criteria.

The mean tissue viability of the 3 minutes positive control was 15.3 %, that is lower than

30 %, as required by the assay acceptance criteria.

The maximum inter tissue viability differences of the "GREWE-DIAMIN" treated skin discs

were 15.0 % for 3 minutes and 3.4 % for 1 hour exposure, that is below 30 % as required

by the assay acceptance criteria.

"GREWE-DIAMIN":

The mean percentage viability of the treated skin discs after 3 minutes of exposure was

91.6 % which is above the threshold of 50 % for classification.

The mean percentage viability of the treated skin discs after 1 hour of exposure was

90.7 % which is above the threshold of 15 % for classification.

EpiDerm Skin Irritation Test

Assay acceptance criteria:

The mean OD of the tissues, treated with deionised water (negative control) was 2.061,

that is higher than 1.0 and lower than 2.5, as required by the assay acceptance criteria.

The mean tissue viability of the positive control was 7.2 %, that is lower than 20 %, as

required by the assay acceptance criteria.

The standard deviation calculated from individual percentual tissue viabilities of the

"GREWE-DIAMIN" treated skin discs was 3.7, that is below 18 as required by the assay

acceptance criteria.

"GREWE-DIAMIN":

The mean percentage viability of the treated skin discs was 101.6 % which is above the

threshold of 50 % for classification.

CONCLUSION

According to the results of this study and the Directive 2001/59/EC, the test substance

"GREWE-DIAMIN" is considered to be non-corrosive and non-irritant to skin.