Copper(2+), bis[N-[amino(imino-.kappa.N)methyl]urea-.kappa.O]-, nitrate (1:2)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 february 2012 to 26 june 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed in accordance to OECD guideline 439

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

during the preliminary test for the detection of colouring potential of the test item, an additional colour evaluation after 1 hour of incubation was performed. It was considered not to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

copper(2+) bis(carbamimidoylurea) dinitrate

EC Number:

800-038-5

Cas Number:

1071838-81-7

Molecular formula:

Cu(C2H6N4O)2 (NO3)2

IUPAC Name:

copper(2+) bis(carbamimidoylurea) dinitrate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

other: human skin model

Strain:

other: EpiskinTM reconstituted human epidermis

Details on test animals or test system and environmental conditions:

TEST CELLS
- Source: SkinEthic Laboratories, Nice, FRANCE
- batch No. 12-EKIN-014
- Passage: second
- origin: adult donor
- quality control: histology scoring 21.7+/-0.3 (>or=19.5) and IC50 determination (SDS concetration, MTT test) 2.1 mg/ml (> or equal 1.5 mg/ml)
- expiration date: april 9, 2012

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C
- Humidity (%): saturated
- CO2: 5%

EXPERIMENT DATES: From: march 27, 2012 To: april 6, 2012

Test system

Type of coverage:

not specified

Preparation of test site:

other: no preparation was needed because of in vitro test

Vehicle:

unchanged (no vehicle)

Remarks:

original form

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied to each tissue: 10 mg +/- 2 mg

Duration of treatment / exposure:

test item: 15 (+/-1) minutes
recovery time after treatment: 42 (+/- 1) hours

Observation period:

no observation period. MTT viability assay was assessed after the end of the recovery period

Number of animals:

each condition was assessed in triplicate tissue

Details on study design:

NEGATIVE CONTROL
- Phosphate-Buffered saline 1X
- Lot/batch no. (if required): prepared by CiToxLAB, 020412A
-dose: 10 µl applied to negative control tissue

POSITIVE CONTROL
- sodium dodecyl sulphate (SDS) as a 5% (w/v) aqueous solution
- Lot/batch no. (if required): Bio-Rad, 111011A
-dose: 10 µl applied to positive control tissue

REMOVAL OF TEST SUBSTANCE
- at the end of incubation period (15 minutes)
- Washing (if done): test item was removed from the well of treatment plate and rinced with D-PBS.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

16

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 min exposure + 42h recovery. Reversibility: other: not relevant: in vitro test. (migrated information)

Other effects / acceptance of results:

irritant
no other effect

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Migrated information

Conclusions:

CuGUN is considered to be irritant to skin as the mean relative viability was <50% relative to the negative control. According to these results, the classification is irritant (R38), category 2 (H315)

Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item,Copper Bis Guanylurea Nitrate (CuGUN),using the Episkin^TMreconstituted human epidermis model. The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 640/2012, B.46) and the study was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practice.

 

Methods

Preliminary tests were performed to detect the ability of the test item to directly reduce MMT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 14 to 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 41 hours and 55 minutes at, 5% CO₂in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

 

Results

Preliminary test

In the preliminary test, theMTT solution containing the test item was found blue when compared with the negative control. However according to the NSMTT% results of the CIT study No. 38756 TIC for the same test item, it was considered that the test item did not interact with MTT. The blue colouration of the MTT solution containing the test item was probably due to the intrinsic colour of the test item, consequently no additional controls were performed on water-killed tissues in parallel to the main test. The test item was found to have a colouring potential in the preliminary test as the water solution containing the test item was coloured. As a result, additional controls were used in parallel to the main test (two viable tissues were used: one was treated with the test item and the other with the negative control; these two tissues were not incubated with MTT)for the evaluation of the non specific OD.

Main test

The acceptance criteria for this test were:

.            the mean cOD of the three negative controls (PBS) was≥0.6 and the standard deviation value of the % viability was≤18%,

.            relative mean viability of the positive control (SDS) was ≤ 40% of the relative mean viability of the negative control and the standard deviation value of the % viability was ≤ 18%. All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid. Following a 15 minutes exposure and a 42-hour recovery period, the true relative mean viability of the tissues treated with the test item was 16%.

 

Conclusion

 Under the experimental conditions of this study, the test item,Copper Bis Guanylurea Nitrate (CuGUN), is considered to be irritant to skin as the mean relative viability was<50%relative to the negative control (see the classification table below). According to these results, the classification of the test item is the following:

.            irritant R38 (EU classification); Category 2 (H315) (Regulation EC No. 1272/2008 (CLP Regulation)).

 

Criteria forin vitroclassification	Interpretation	Directive 67/548/EEC	Regulation EC No. 1272/2008 (CLP Regulation)
Mean relative viability is ≤ 50%	Irritant	Irritant (R38)	Category 2 (H315)
Mean relative viability is > 50%	Non-irritant	Not classified	No category