Reaction mass of disodium [2,2'-(imino-κN)dibutanedioato-κ2O1,O4(4-)]cuprum(2-) and sodium sulphate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2010-12-14 to 2011-01-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented GLP-Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

issued by Biuro Do Spraw Substancji i Preparatow Chemicznych

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: conventional husbandry of Institute of Occupational Medicine, Łódź
- Age at study initiation: from 8 to 11 weeks old
- Weight at study initiation: 558.3 g (males) and 530.3 g (females)
- Housing: individually in plastic cages with dimensions (length x width x height): 58 x 37 x 21 cm, with wired lid. UV-sterilized wooden shavings were used as a bedding. Each cage was equipped with label containing information on study code, group mark, dates of commencement and expected termination of experiment, sex and number of animal
- Diet (e.g. ad libitum): ad libitum standard granulated LSK fodder produced by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL, Motycz
- Water (e.g. ad libitum): ad libitum tap drinking water with supply of ascorbic acid (0.6% solution)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23 ºC
- Humidity (%): 40 – 80 %
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Start of the study: 09.11.2010
The pilot study: from 23.11.2010 to 06.12.2010
The main study: from 14.12.2010 to 08.01.2011

Route:

intradermal and epicutaneous

Vehicle:

other: aqua pro injectione (intradermal injection), distilled water (topical application)

Concentration / amount:

induction - intradermal injections: 0.2 % (causing mild changes)
induction - topical application: 50 % (not causing changes on skin)
challenge - topical application: 50 % (not causing changes on skin)

Route:

epicutaneous, semiocclusive

Vehicle:

other: aqua pro injectione (intradermal injection), distilled water (topical application)

Concentration / amount:

induction - intradermal injections: 0.2 % (causing mild changes)
induction - topical application: 50 % (not causing changes on skin)
challenge - topical application: 50 % (not causing changes on skin)

No. of animals per dose:

treated group: 20
control group: 10

Details on study design:

RANGE FINDING TESTS:
The experiment was commenced by the pilot study in which concentrations of test item for the main study were established.
On day before test dorsal skin of animals’ trunk and flanks were shaved with electric razor on area about 4.5 x 6 cm. Only animals without any visible skin irritation or abrading were used for the experiments.
In order to determine concentration for I step of induction – intradermal injections, six intradermal injections were given to frontal part of two guinea pigs in volume of 0.1 mL each: 2 with Freund’s Complete Adjuvant (FCA); 1 with medium and 3 with the appropriate concentrations of test item. The following concentrations of test item were given to guinea pig No 1 – 0.5%, 1%, 2% and to guinea pig No 2 – 0.2%, 0.5% and 1%.

In order to determine concentration for II step of main study (induction – topical application) and concentration for III step of main study (challenge – topical application), the test item in concentrations of 40% and 50% was applied to shaved flanks of two guinea pigs. Two concentrations were tested on each animal. The aqueous solutions of appropriate concentrations of test item were applied in volume 0.5 mL to gauze patches with dimensions 2 x
2 cm which were subsequently laid on skin of flanks. The gauze patches were covered with PCV foil and elastic bandage. The exposure time was 24 hours. After this time the band and gauze patches were removed.

Clinical observations
Clinical observations comprised evaluation of general condition of animals as well as detailed clinical observations. General observations of all animals for mortality and morbidity were conducted twice a day and once a day (on days off). Observations of skin of animals in the pilot study were performed after 24, 48, 72, 96 and 120 hours since intradermal injections as well as 24, 48, 72, 96 and 120 hours since the end of 24-hour period of exposure following application of test item to skin. Skin reaction for intradermal injections of test item and medium was evaluated by measurement of diameter of formed erythema and other changes on skin were also described. Results of skin reaction to administration of test item were evaluated according to grading scale, on the ground of OECD Guideline No 404, EU Method B.4. and SPR/T/24.
The animals were sacrificed after observation period by peritoneal administration of pentobarbital in dose 200 mg/kg b.w. [15] and transferred to utilization.
Body weight of animals was determined individually for each animal directly before administration of test item (day 0) and on day of termination of experiment – prior to sacrifice of animals.

MAIN STUDY
The main study comprised three parts: double induction and a challenge.

A. INDUCTION EXPOSURE
- No. of exposures: 2 (on day 0 an intradermal injection and on day 7 a dermal application)
- Exposure period: 48 hours
- Test groups: intradermally: 0.2% aqueous solution of test item with Freund’s Complete Adjuvant (FCA). Due to no skin irritation after aplication of 50% aqueous solution,10% sodium lauryl sulfate in vaseline was applied in the site of intradermal injections on day before II step of experiment in order to create local skin irritation. Epicutan: 50% aqueous solution of test item was applied to skin in the site of intradermal injections.
- Control group: During period of induction a group of control animals was subjected to sham treatment – they were given medium instead of test item. The animals were treated also with 10% sodium lauryl sulfate in vaseline in the site of intradermal injections on day before II step of experiment in order to create local skin irritation.
- Site: dorsal skin of animals’ trunk and flanks
- Frequency of applications: one intradermal injection and one dermal application
- Duration: dermal application: 48 hours
- Concentrations: 0.2 % and 50 % aqueous solution

B. CHALLENGE EXPOSURE
- No. of exposures: once
- Day(s) of challenge: day 21
- Exposure period: 24 hours
- Test and control groups: The 50% aqueous solution of test item was applied in volume of 0.5 mL to prepared skin of right flank of animals of treated and control group.
- Site: right flank
- Concentrations: 50 % aqueous solution
- Evaluation (hr after challenge): 24, 48 and 72 hours

Challenge controls:

During period of induction a group of control animals was subjected to sham treatment – they were given medium instead of test item. During challenge, 50 % aqueous solution of test item was applied to right flank of control animals. Medium was applied to left flank.

Positive control substance(s):

no

Positive control results:

not applicable as no positive controls were used

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0.

All animals survived period of experiment.

Table 3: Summary of results

Cu (II) IDHA.Skin sensitization study
	CONTROL GROUP	TREATED GROUP
Number of animals in group	10	20
Number of animals subjected to the final assessment	10	20
Number of dead animals	0/10	0/20
Skin changes following challenge in the site of medium application	no changes	no changes
Changes on skin following challenge after test item application	no changes	no changes
Animals with allergic reaction	0/10	0/20
% of allergic animals	-	0 %

During the experiment no general clinical signs were stated in observed animals. After termination of experiment body weight loss was stated in two animals of treated group.

Pilot study

Clinical signs

The test item concentration of 0.2% causing mild changes on skin was selected for I step of main study. On the ground of obtained results the test item concentration of 50% not causing changes on skin was selected for II step of main study (induction – topical application). Since the test item was a solid form is the maximal concentration which could be given was 50%. For III step (challenge – topical application) the test item concentration of 50% not causing changes on skin was selected.

After termination of pilot study, body weight loss was stated in one animal (2/483).

Table 4: Results of observation of skin reactions (pilot study - intradermal inejctions) - Animal 1

Evaluation after hours since injection	Concentration	Changes
		Erythema(diameter in mm)	Other
24	1	3	visible site of insertion
	2	7	visible site of insertion
	3	7	visible site of insertion
	4	9	visible site of insertion
48	1	2	visible site of insertion
	2	5	visible site of insertion
	3	4	visible site of insertion
	4	6	visible site of insertion, blue field in the middle
72	1	2	visible site of insertion
	2	5	visible site of insertion
	3	3	visible site of insertion
	4	5	visible site of insertion, blue field in the middle
96	1	3	visible site of insertion
	2	5	visible site of insertion
	3	5	visible site of insertion
	4	8	visible site of insertion, blue field in the middle, scab
120	1	2	dryness of epidermis
	2	4	dryness of epidermis
	3	5	visible site of insertion, scab
	4	7	visible site of insertion, scab

concentration 1: 0 %, concentration 2: 0.5 %, concentration 3: 1 %, concentration 4: 2 %

Table 5: Results of observation of skin reactions (pilot study - intradermal inejctions) - Animal 2

Evaluation after hours since injection	Concentration	Changes
		Erythema	Other
		(diameter in mm)
24	1	4	visible site of insertion
	2	11	visible site of insertion
	3	10	visible site of insertion, grey field
	4	20	visible site of insertion in white edging, grey field in the middle
48	1	3	visible site of insertion
	2	5	visible site of insertion
	3	10	necrosis
	4	16	necrosis
72	1	3	visible site of insertion
	2	5	visible site of insertion
	3	8	scab
	4	11	scab
96	1	3	visible site of insertion
	2	4	visible site of insertion
	3	8	scab
	4	10	scab
120	1	3	visible site of insertion
	2	4	visible site of insertion
	3	8	scab
	4	10	scab

No changes were found after topical application of a 40 or a 50 % solution of Cu (II)IDHA.

Main study

Clinical observations

During readings after 24, 48 and 72 hours since the end of exposure, no pathological changes were stated on skin of animals of control group and animals of treated group, in the site of test item application. In the site of medium application no pathological changes were stated in

both groups.

On the ground of obtained results, one may state, that no allergic skin reactions were stated in animals of treated group.

During experiment no changes in behavior and no general clinical signs were observed in the remaining animals of control and treated group.

Table 6: Results of observations of skin reactions at challenge (main study - treated group)

TREATED GROUP
Concentration of test item:					50%		flank: right
Medium:					distilled wat		flank: left
Date of administration:					04.01.2011
Date of removal:					05.01.2011
Sex	Animal No		Evaluation after hours						Animals with/withou allergic reaction (+/-)	%of allergic animals
	comp.	reg.	24		48		72
			left flank	right flank	left flank	right flank	left flank	right flank
males	1	48s	0	0	0	0	0	0	-	0 %
	2	4s0	0	0	0	0	0	0	-
	3	504	0	0	0	0	0	0	-
	4	514	0	0	0	0	0	0	-
	5	515	0	0	0	0	0	0	-
	e	521	0	0	0	0	0	0	-
	7	523	0	0	0	0	0	0	-
	8	525	0	0	0	0	0	0	-
	s	52e	0	0	0	0	0	0	-
	10	53e	0	0	0	0	0	0	-
females	11	484	0	0	0	0	0	0	-
	12	488	0	0	0	0	0	0	-
	13	4s2	0	0	0	0	0	0	-
	14	4s3	0	0	0	0	0	0	-
	15	50e	0	0	0	0	0	0	-
	1e	507	0	0	0	0	0	0	-
	17	510	0	0	0	0	0	0	-
	18	528	0	0	0	0	0	0	-
	1s	52s	0	0	0	0	0	0	-
	20	530	0	0	0	0	0	0	-
0 - no visible changes, 1 - discrete or patchy erythema, 2 - moderate or confluent erythema, 3 -intense erythema and swelling

Body weight of animals

Body weight loss was stated in two animals of treated group (male 2/490 and female 12/488). Body weight gains were stated in the remaining animals of control and treated group. The average body weight gain of males of control group amounted 71.6 g, whereas in males of treated group 82.1 g. The average body weight gain of females of control group amounted 51.8 g and in females of treated group 49.3 g.

Table 7: Body weights (main study, g)

TREATED GROUP
Sex		Animal No			Day of experiment				Body weight gain [g]
		comp.	reg.		0		final
males		1	489		589		621		32
		2	490		591		585		-6
		3	504		548		568		20
		4	514		585		722		137
		5	515		519		602		83
		6	521		451		491		40
		7	523		618		697		79
		8	525		599		745		146
		9	526		554		683		129
		10	536		534		695		161
females		11	484		619		649		30
		12	488		539		531		-8
		13	492		615		684		69
		14	493		600		655		55
		15	506		476		521		45
		16	507		488		524		36
		17	510		558		611		53
		18	528		489		573		84
		19	529		474		547		73
		20	530		440		496		56
				Average body weight [g]				Average body weight gain [g]
				initial		final
males				558.8		640.9		82.1
females				529.8		579.1		49.3

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item Cu (II) IDHA caused no sensitization in animals. On the ground of the study Cu (II) IDHA may be included to the agents not causing sensitization of skin of guinea pigs.

Executive summary:

A study was undertaken to investigate the skin sensitisation potential of Cu (II) IDHA in Dunkin Hartley guinea pigs (OECD 406; Kropidło, 2011, Study Code Al 3/11). A test group consisted of 20 animals and the control group of 10 animals which were sham-exposed in the following. The test item was applied first intradermally (0.2 %) to the animals, and 7 days later via topical application (as a 50 % solution). These two applications were for induction purposes. Then the animals were left for 2 weeks and then challenged with a 50 % solution of the test item. Thereafter the readings were made (after 24, 48, and 72 hours). The test item Cu (II) IDHA caused no sensitization in animals. On the ground of the study Cu (II) IDHA may be included to the agents not causing sensitization of skin of guinea pigs. The recorded body weight losses in animals of both pilot and main study were probably caused by stress connected with preparations of animals for experiment and also with the manner of protection of administered test item or medium as well as observations themselves.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A study was undertaken to investigate the skin sensitisation potential of Cu (II) IDHA in Dunkin Hartley guinea pigs (Kropidło, 2011, Study Code Al 3/11, according to OECD 406). A test group consisted of 20 animals and the control group of 10 animals which were sham-exposed in the following. The concentrations of test item were determined in a pilot study. In the main study, the test item was applied first intradermally (0.2 %) to the animals, and 7 days later via topical application (as a 50 % solution). These two applications were for induction purposes. The animals were left for 2 weeks and then challenged with a 50 % solution of the test item. Thereafter the readings were made after 24, 48, and 72 hours. Body weight of animals was determined individually for each animal directly before administration of test item (day 0) and on day of termination of experiment – prior to sacrifice of animals. General clinical observations and detailed skin observations were performed during the study.

All animals survived period of experiment. The test item Cu (II) IDHA caused no sensitization in treated animals. During readings no allergic skin reactions were stated in animals of treated group. No pathological changes were stated on skin of animals of control group. During the experiment no general clinical signs were stated in observed animals. After termination of experiment body weight loss was stated in two animals of treated group. The body weight loss was also observed in the pilot study in one animal. The body weight loses were probably caused by stress connected with preparations of animals for experiment and also with the manner of protection of administered test item or medium as well as observations themselves.

On the ground of the study Cu (II) IDHA may be included to the agents not causing sensitization of skin of guinea pigs.

Migrated from Short description of key information:
- Skin sensitisation, OECD 406, guinea pigs, non sensitising

Justification for selection of skin sensitisation endpoint:
Only one study available

Justification for classification or non-classification

There were no dermal reactions in any of the test or control animals following the challenge with Cu(2Na)IDHA. Therefore, the substance is considered to be a non-sensitizer and does not need to be classified and labelled according to European Regulation (EC) No. 1272/2008.