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

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 march 2012 to 6 september 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed in accordance to OECD guideline 429

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximatively 12 weeks (preliminary test) and 8 weeks (main test)
- Weight at study initiation: 18.1 to 21.6 g
- Housing: group of 2 (preliminary test) or 4 (main test) in polycarbonate cages (techniplast 1145T, 435 cm2) containing autoclaved sawdust (SICSA, Alfortville, France). Each cage contained 2 enrichments (mouse hut and cocoon). In the main test, on day 6, animals were individually housed in disposable crystal polystyrene cages.
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiaten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum, filtered (0.22 µm) tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 50+/-20%
- Air changes (per hr): 12 cycles/hour of filtered , non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 25 april 2012 To: 07 may 2012

Vehicle:

propylene glycol

Remarks:

sigma batch S39776-337

Concentration:

50%

No. of animals per dose:

2 females per dose for the preliminary test
4 females per dose in the main test

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: homogeneous suspension to the naked eye at the concentration of 50% in propylene glycol

- Irritation potential: ear thickness measurement in a preliminary test in order to define the test item concentrations to be used in the main test. From day 1 to day 3 then on day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. On completion of the observation period, the animals were sacrificed then discarded without macroscopic post mortem examination.

- Lymph node proliferation response: five groups of mice received the test item at concentrations of 50, 25, 10, 5 or 2.5% . One negative control group received the vehicle (Propylene Glycol) under the same experimental conditions. Additionally, one positive control group received the positive control, α hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions.
From day 1 to day 3 then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6.
After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-TdR.
The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI). Stimulation indices (SI) = dpm per node of the treated group/dpm per node of the vehicle control group

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: auricular lymph node cell (ALNC) proliferation assay
- Criteria used to consider a positive response: SI> or = to 3 for a dose group with consideration of a dose-response relationship

TREATMENT PREPARATION AND ADMINISTRATION:
test item was administered as a homogeneous suspension in the vehicle. The test item was mixed under magnetic agitation with the required quantity of vehicle.
Mice received the test item, by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 50, 25, 10, 5 or 2.5% under a dose-volume of 25 µL.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

EC3 value (theorical concentration resulting in a SI value of 3) was determined by linear interpolation of points on the dose-response relation ship curve, immediately above and below the 3-fold threshold.

Positive control results:

the threshold positive value of 3 for the SI was reached in the positive control group (SI=13.00). the experiment was considered valid

Parameter:

SI

Remarks on result:

other: a dose-related increase in the SI was recorded and the threshold positive value of 3 was exceeded at concentrations >5% test item: 2.5% ->SI=1.30; 5% ->SI=2.78; 10%->SI=6.53; 25%->SI=10.40; 50%-> SI=8.12 EC3 value is equal to 5.3%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: negative control: 116.50 dpm per node test item: 2.5% ->151.75 dpm/node; 5% ->324.38 dpm/node; 10%->760.63 dpm/node; 25%->1212.00 dpm/node; 50%-> 946.13 dpm/node positive control HCA 25%: 1514.75 dpm/node

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

Concentration 2.5 %

Key result

Parameter:

SI

Value:

2.78

Test group / Remarks:

Concentration 5 %

Key result

Parameter:

SI

Value:

6.53

Test group / Remarks:

Concentration 10 %

Key result

Parameter:

SI

Value:

10.4

Test group / Remarks:

Concentration 25 %

Key result

Parameter:

SI

Value:

8.12

Test group / Remarks:

Concentration 50 %

Clinical history:

All animals of the preliminary test: no clinical signs and final sacrifice at day 6.

All animals of the main test: no clinical signs and final sacrifice at day 6.

Cutaneous reactions:

all animals of the main test treated by test item at 2.5%, 5%, 10% or 25%: no cutaneous reaction

animals treated by test item at 50%: blue discoloration of one ear (3 mice) at day 2 and dryness of the skin (2 mice) at day 6.

Ear thickness measurement (%):

groups	3	4	5	6	7	8
percentage of ear thickness increase at day 2 compared to day 1	6.19	2.00	3.06	2.06	3.06	1.98
percentage of ear thickness increase at day 2 compared to day 1	3.09	4.00	6.12	4.12	8.16	5.94
percentage of ear thickness increase at day 2 compared to day 1	3.09	3.00	4.08	6.19	5.10	8.91

Body weight and body weight change (g) Mean+/-SD:

groups	1	2	3	4	5	6	7	8	9
day 1	22.2 +/-1.1	21.2 +/-1.3	19.8 +/-0.7	20.4 +/-0.6	20.4 +/-0.7	19.2 +/-0.6	20.4 +/-1.0	19.4 +/-0.6	19.2 +/-1.1
day 6	22.1 +/-0.5	23.1 +/-1.1	19.4 +/-0.6	20.5 +/-0.2	20.0 +/-1.0	19.2 +/-0.7	20.0 +/-1.1	19.6 +/-1.1	19.8 +/-0.9
day6 -day1	-0.1 +/-0.6	1.9 +/-0.1	-0.4 +/-0.3	0.1 +/-0.5	-0.4 +/-0.4	-0.1 +/-0.4	-0.4 +/-0.4	0.1 +/-0.6	0.6 +/-0.5

dpm, SI and irritation level results summary

groups	treatment and concentrations	number of nodes par group	dpm per group	dpm per node	stimulation index (SI)	irritation level
3	negative control vehicle	8	932	116.5
4	test item 2.5%	8	1214	151.75	1.30	I
5	test item 5%	8	2595	324.38	2.78	I
6	test item 10%	8	6085	760.63	6.53	I
7	test item 25%	8	9696	1212.00	10.40	I
8	test item 50%	8	7569	946.13	8.12	I
9	positive control HCA 25%	8	12118	1514.75	13.00

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Migrated information

Conclusions:

According to EC3 value obtained, the test item should be considered as a moderate sensitizer.
The classification of the test item should be R43 (directive 67/548/EEC) and H317 (regulation 1272/2008/EEC)

Executive summary:

The objective of this study was to evaluate the potential of the test item, Copper Guanylurea Nitrate, to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA).

The study was based on theOECD Guideline No. 429 "Skin Sensitisation: Local Lymph Node Assay", 22nd July 2010 and on the Commission Regulation (EC) No. 440/2008, B.42, 30 May 2008.

 

Methods

To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item, by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 50, 25, 10 or 5% under a dose-volume of 25 µL.From day 1 to day 3 then on day 6, the thicknessof both ears of each animal was measured and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and thenon days 1 and 6.On completion of the observation period, the animals were sacrificedthen discarded without macroscopicpost‑mortemexamination. In the main test, five groups of four female mice received the test item by topical routeto the dorsal surface of both earson days 1, 2 and 3at concentrations of50, 25, 10, 5 or 2.5% under a dose-volume of 25 µL. One negative control group of four females received the vehicle (Propylene Glycol) under the same experimental conditions. Additionally, one positive control group of four females received the positive control, α‑hexylcinnamaldehyde (HCA), at 25%in a mixture acetone/olive oil (4/1; v/v)under the same experimental conditions. From day 1 to day 3 then on day 6, the thicknessof the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and thenon days 1 and 6. After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (³H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of³H-TdR.

The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

 

Results

 Mortality and clinical signs 

No unscheduled deaths and no clinical signs were observed during the observation period.

 Local irritation

A blue discoloration of the ears was observed on day 3 only in 3/4 females treated at the concentration of 50%. Dryness of ear skin and scabs were noted in 2/4 animals of the same group on day 6 only.

No notable increase in ear thickness was observed at any tested concentrations.

 Proliferation assay

The SI of the positive control was >3; this experiment was therefore considered valid.

A dose-related increase in the SI was recorded and the threshold positive value of 3 was exceeded at concentrations > 5%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

 The EC₃value is equal to 5.3%.

Conclusion

Under the experimental conditions of this study, the test item, Copper Guanylurea Nitrate, induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Therefore, the classification of the test item should be the following: R43 (Directive 67/548/EEC) and H317 (Regulation 1272/2008/EEC). According to the EC₃value obtained, the test item should be considered as a moderate sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

CuGUN show sensitising properties in mice. There is no reason to believe that results obtained in mice would not be applicable to humans.

Migrated from Short description of key information:
Murine Local Lymph Node Assay (LLNA): sensitising (OECD guideline 429 and EU method B.42), GLP.
No unscheduled deaths and no clinical signs were observed during the observation period.
- Local irritation: A blue discoloration of the ears was observed on day 3 only in 3/4 females treated at the concentration of 50%. Dryness of ear skin and scabs were noted in 2/4 animals of the same group on day 6 only. No notable increase in ear thickness was observed at any tested concentrations.
- Proliferation assay: The SI of the positive control was >3; this experiment was therefore considered valid. A dose-related increase in the SI was recorded and the threshold positive value of 3 was exceeded at concentrations > 5%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.
The EC3 value is equal to 5.3%.

Justification for selection of skin sensitisation endpoint:
The study was performed in accordance to OECD 429 and EC B.42 guideline, and to GLP. No deviation was observed.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No data are available for respiratory sensitisation.

As it was discussed for acute toxicity inhalation endpoint, CuGUN have a very low vapour pressure (0.0000009 Pa ) and only 10 % of particles are below 13.5 µM (more than 90 % of particles are non inhalable). However, this potential hazard is taken into account for risk assessment even if CuGUN is not classified as respiratory sensitizer.

Justification for classification or non-classification

According to 1272/2008 (CLP) and 286/2011 (2nd ATP) regulation, CuGUN is classified as Skin sensitizer, category 1B, based on the EC3 value which is higher than 2%.