Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
issued by Biuro Do Spraw Substancji i Preparatow Chemicznych
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: aqueous solution
Details on test material:
- Name of test material (as cited in study report): Cu (II) IDHA
- Substance type: chelate
- Physical state: solid (odourless, blue microgranules)
- Analytical purity: contained 9.82% of copper
- Lot/batch No.: 1/10, produced in April, 2010
- Expiration date of the lot/batch: April, 2013
- Storage condition of test material: in tightly closed package at temperature from -10ºC to +30ºC

In vivo test system

Test animals

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

Study design: in vivo (non-LLNA)

Inductionopen allclose all
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)
Challengeopen allclose all
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

Results and discussion

Positive control results:
not applicable as no positive controls were used

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
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 group
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 group
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.