Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
guinea pig maximisation test
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
4/03/1997 - 28/03/1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The underlying hypothesis for the read-across is that chelates have the same mode of action based on their ability to chelate, remove or add metal cations to body causing perturbation of body’s micronutrients balance.
The source substance is a chelating agent in a target substance. The only difference between the target and the main source substance is presence of magnessium (Mg) cation instead two Na+ cations. As magnessium is an essential micro element required by all forms of life, is considered not to influence the toxicological activity.
Detailed information about the substances purity and data matric are available in RA Statement (IUCLID 13.2)
Cross-referenceopen allclose all
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
from 2010-12-14 to 2011-01-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented GLP-Guideline study
Justification for type of information:
A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment. The source substance has the same chelating agent as in a target substance (MgNa2IDHA). The only difference between the target and the source substance is presence of magnessium (Mg) cation instead Cu2+ cations. As magnessium is an essential micro element required by all forms of life, is considered not to influence the toxicological activity.
Detailed information about the substances purity and data matric are available in RA Statement (IUCLID 13.2)
Reason / purpose:
read-across source
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
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 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.

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.

Reason / purpose:
read-across: supporting information
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
April - June 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well-documented GLP-Guideline Study
Justification for type of information:
A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment. The source substance has the same chelating agent as in a target substance (MgNa2IDHA). The only difference between the target and the source substance is presence of magnessium (Mg) cation instead Fe3+ cations. As magnessium is an essential micro element required by all forms of life, is considered not to influence the toxicological activity.
Detailed information about the substances purity and data matric are available in RA Statement (IUCLID 13.2)
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: OECD no406
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was conducted in 2007 before the LLNA-method has become prefered method.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hodowla Zwierząt Laboratoryjnych Ilkowice 41, 32-218 Slaboszew, Poland
- Weight at study initiation: averge 376 g
- Housing:
- Diet (e.g. ad libitum): conventional laboratory diet (TOP DOVO Dobra Voda, supplier)
- Water (e.g. ad libitum): water with addition of ascorbic acid in concentration 0.05%
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): artificial, 12h light/ 12h dark

Cages
Housed in groups of 5 in cages in conformity with laboratory welfare legislation.

IN-LIFE DATES:
Deliver: 27.04.2007
Aclimatisation: 4.05.2007
Pilot study: 9-14.05.2007
Division of animals into groups and weighting: 14.05.2007
Induction phase: 1. intradermal injection: 15.05.2007
2. topical appliation: 22.05.2007
Rest period: 23.05.2007 - 4.06.2007
Challenge: 5.06.2007
Observations: 7-8.06.2007
End study: 8.06.2007
Route:
intradermal
Vehicle:
other: Freund's complete adjuvant (FCA), saline, aqua pro injectione
Concentration / amount:
Induction:
- intradermal injections - 5% Fe(III)IDHA in aqua pro injectione
- topical application - 75% Fe(III)IDHA in aqua pro injectione

Chellenge:
- topical application - 10% Fe(III)IDHA in in aqua pro injectione
Route:
epicutaneous, occlusive
Vehicle:
other: Freund's complete adjuvant (FCA), saline, aqua pro injectione
Concentration / amount:
Induction:
- intradermal injections - 5% Fe(III)IDHA in aqua pro injectione
- topical application - 75% Fe(III)IDHA in aqua pro injectione

Chellenge:
- topical application - 10% Fe(III)IDHA in in aqua pro injectione
No. of animals per dose:
treated group:20
control group: 10
Details on study design:
Pilot study
The ocncentration of test substance used for each induction exposure was selected well-tolerated systemically and the highest to cause mild - to - moderate skin irritation. The concentration for the challenge exposure was selected the highest non-irritant dose. The appropriate concentrations was determined using two animals for intradermal injections and three animals for topical application (occlusive dressing for 24 h).

Induction phase
1. Intradermal injections: three pairs of intradermal injection of 0.1 ml volume were givenin the shoulder region
Day 1 - treated group
injection 1: mixture FCA/saline 1:1
injection 2: the test substance in water at the selected concentration in pilot study
injection 3: mixture injection 2 and injection 1
Day 1 control group
injection 1: mixture FCA/saline 1:1
injection 2: the undiluted vehicle
injection 3: mixture injection 2 and injection 1

Topical application
Day 7 - treated and control groups
Because the substance is a skin irritant, the tet area, is not painted with 0.5 ml of 10% natrii lauryl sulfas in vaseline, in order to cerate a local irritation.
Day 8 - treated group
The test area was cleared of hair. A gauze 2x4 cm was fully loaded with vehicle and was applied to the test area and held in contact by an occlusive dressing for 48 h. During at least 14 days, no treatment was applied.

Challenge
Day 22 - treated and control groups
The flank of treated and control animals was cleared of hair. A patch loaded with the substance was applied to one flank of the animals and patch loaded with the vehicle was applied ti the other flank. The patch was held in contact by an occlusivve dressing for 24 h.

Obsrvations performed
Approximately 21 hours after removing the patch, the challenge area was cleaned and shaved. Approximalety 3 hours lated (48 h from the start of the challenge application) the skin reaction was observed and recorded according to the grades. Approximately 48 h after removing of the patch (72 h from the start of the challenge), the second observation was made and recorded.
Individual starting body weight and ending body weight were statistically evaluated by Statgraphics statistical program. To identify homogenity groups the Bartell test was used. In the case of homogenity One-Way Analysis of Variance was applied.
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 group
Dose level:
75%
No. with + reactions:
13
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75%. No with. + reactions: 13.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75%
No. with + reactions:
10
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75%. No with. + reactions: 10.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 4.0. 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.

Please see attachment.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Topical application of Fe(III)IDHA incuced the sensitisation rate 50%. According to this value the tet article Fe(III)IDHA belongs to "moderate sensitizer" (III).
Executive summary:

The skin sensitisation potential of Fe(III)IDHA was tested in the Guinea pig maximisation test (Magnusson and Kligman). 20 animals were used in the treated group and 10 animals in the control group. For induction treatment of animals treated group were intradermally injected 5% Fe(III)IDHA in Aqua pro injectione with complete FCA followed one week lated by epidermal application of 75% Fe(III)IDHA in Aqua pro injectione. For induction treatment of animals control group were intradermally injected with vehivle (Aqua pro injectione) with complete FCA followed one week lated by epidermal application of vehicle. Two weeks after epidermal application all animals were challenged with 10% Fe(III)IDHAa in Aqua pro injectione. Fe(III)IDHA induced the sensitisation rate 50%. According to this value the substnce Fe(III)IDHA belongs to "moderate sensitizer" (III) ( Skin Sens 1B, H3317 acc. to Regulation 1272/2008).

Reason / purpose:
read-across: supporting information
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
from 2013-08-06 to 2013-10-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented GLP-Guideline study
Justification for type of information:
A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment. The source substance has the same chelating agent as in a target substance (MgNa2IDHA). The only difference between the target and the source substance is presence of magnessium (Mg) cation instead Mn2+ cations. As magnessium is an essential micro element required by all forms of life, is considered not to influence the toxicological activity.
Detailed information about the substances purity and data matric are available in RA Statement (IUCLID 13.2)
Reason / purpose:
read-across source
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 the Bureau for Chemical Substances
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: Charles River Laboratories in Germany and France
- Age at study initiation: from 8 to 14 weeks old - Pilot study and 9 weeks - Main study
- Weight at study initiation: 590.3 g (males) and 524.7 g (females) - Pilot study
and 510.7 g (males) and 446.0 g (females) - Main study
- Housing: individually in plastic cages. The dimensions of the cages were 58 x 37 x 21 cm (length x width x height ). The cages were covered with wire bar lids. UV-sterilized wood shavings were used as bedding [SOP/T/16, SOP/T/48].
- Diet (e.g. ad libitum): ad libitum standard granulated LSK standard granulated 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: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24 ºC
- Humidity (%): 35 – 89 %
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Start of the study: 02.08.2013
Start of the experimental phase: 06.08.2013
End of the experimental phase: 19.10.2013
Route:
intradermal and epicutaneous
Vehicle:
other: aqua pro injectione
Concentration / amount:
induction - intradermal injections: 15 % (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
Concentration / amount:
induction - intradermal injections: 15 % (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 the day before the test, an electric razor was used to shave the dorsal and flank skin of the animals covering the area of about 4.5 x 6 cm. Only animals without any visible skin i rritation or abrading were used in the experiment. In order to prepare appropriate concent rations of the test item, aqua pro injectione was used as a medium.

Concentration determination for stage I of the main study:
In order to determine a proper concent ration for stage I, i.e. induction – intradermal injections, six intradermal injections in a volume of 0.1 mL each were given in the shoulder region of four guinea pigs. The injections were as follows: 2 with Freund’s Complete Adjuvant (FCA), 1 with the medium, and 3 with appropriate concentrations of the test item. Guinea pig no. 1 received the test item at the concentrations of 0.5%, 1%, and 2%, guinea pig no. 2 received the test item at the concentrations of 4%, 6%, and 8%, guinea pig no. 3 received the test item at the concentrations of 10%, 15%, and 20%, and guinea pig no. 4 received the test item at the concentrations of 15%, 17%, and 20%.

Concentration determination for stages II and III of the main study:
In order to determine proper concentrations to be used at stage II of the main study (induction – topical application) and stage III of the main study (challenge – topical application), the test item at the concentrations of 30% and 50% (guinea pig no. 1) and 40% and 50% (guinea pigs no. 2 and 3) was
applied to the shaved flanks of three guinea pigs. Two concentrations were tested on each animal. Aqueous solutions of the test item at the appropriate concentrations in a volume of 0.5 mL each were applied to gauze patches (2 x 2 cm). Then, the patches were laid on the flanks of the animals and covered with PVC foil and an elastic bandage. The exposure time was 24 hours. Following this period of time, the bandage and the gauze patches were removed from the skin

Clinical observations
These observations comprised the evaluation of general condition of the animals and detailed clinical observations of their skin. The general observations of all animals for mortality and morbidity were conducted twice a day or once a day in case of days off. In the pilot study, the detailed observations of the animals’ skin were performed 24, 48, 72, 96, and 120 hours after the intradermal injections and 24, 48, 72, 96, and 120 hours after the end of the 24-hour exposure following the test item application. Skin reactions to intradermal injections (the test item and the medium) were evaluated by measuring the diameter of erythema. All other skin changes were also recorded. Skin reactions to the test item were evaluated according to the grading scale based on the OECD Guideline No. 404, the EU Method B.4., and the Standard Operating Procedure SOP/T/24. After the observation period, the animals were euthanized by intraperitoneal administration of morbital at a dose of 200 mg/kg b.w. [SOP/T/39] and trans ferred to utilization.

Body weights
Body weights of the animals were determined on day 0 and on the day of the experiment termination (before euthanasia)

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 and control groups: In the I step of main study the animals of treated group were given intradermally 15% aqueous solution of test item with Freund’s Complete Adjuvant (FCA) and 15 % aqueous solution of test item without FCA.
In the II step of main study, 50% aqueous solution of test item was applied to skin in the site of intradermal injections. 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. During period of induction a group of control animals was subjected to sham treatment – they were given medium instead of test item.
- Site: dorsal skin of animals’ trunk and flanks
- Frequency of applications: one intradermal injection and one dermal application
- Duration: dermal application: 48 hours
- Concentrations: 15 % 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: In order to challenge sensitization the 50% aqueous solution of test item was applied to right flank of treated and control animals. Medium was applied to left flank. During period of induction a group of control animals was subjected to sham treatment – they were given medium instead of test item.
- Site: right flank
- Concentrations: 50 % aqueous solution
- Evaluation (hr after challenge): 24, 48 and 72 hours since the end of exposure
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 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.

Results of the pilot study

Clinical signs

The test item at the concentration of 15% causing mild skin changes was selected for stage I of the main study.

The results of the pilot study allowed for the determination of the test item concentration of 50% causing no skin changes to be used at stage II of the main study (induction – topical application). Because the test item was a solid, the maximum concentration of the test item applied to the skin could be 50%.

The concentration of 50% causing no skin changes was selected for stage III of the main study (challenge – topical application).

Body weights of the animals

After the termination of the pilot study, insignificant body weight loss was stated in three animals (no. 2/241, 3/134, and 2/228)

Results of the main study

Clinical observations

During the readings which took place 24, 48, and 72 hours after the end of the exposure, the control group animals did not exhibit any pathological changes in the sites of the test item and the medium application. During the readings which took place 24, 48, and 72 hours after the end of the exposure, the treated group animals did not exhibit any pathological changes in the sites of the test item and the medium application. On the grounds of the obtained results, it may be stated that no allergic skin reactions occurred in the treated animals. During the experiment, the control and the treated animals did not reveal any changes in behaviour or general clinical changes.

Table 3: Summary of results

Mn (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 accidently dead animals 0/10 0/20
Skin changes in the medium application site following challenge no changes no changes
Skin changes in the test item application site following challenge

no changes

no changes

No. of animals exhibiting allergic reactions 0/10 0/20
% of sensitized animals - 0 %

During the readings, the treated animals did not exhibit any allergic skin reactions.

Taking the study results into consideration, the test item, i.e. Mn (II) IDHA can be classified into the following categories:

- agents causing no sensitization – according to the Magnusson and Kligman classification,

- agents which are beyond categorization – according to the Commission Regulation (EU) No. 286/2011

of March 10, 2011 amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No. 1272/2008 of the European Parliament and of the Council on classification, labelling, and packaging of substances and mixtures.

Body weight loss stated in three animals was probably caused by stress resulting from the preparation of the animals for the experiment, the way of protecting the test item and/or the medium, and observations.

Table 4. Main study -challenge-topical application (patch test)-control group.
Test item: 50% flank: right
Medium: aqua pro injectione flank: left
Date of application: 15.10. 2013.
Date of removal: 16.10 .2013.
Evaluation of skin reactions according to the grading scale of Magnusson and Kligman
(based on theOECDGuideline No.406 / EUMethod B.6.)
Animal No. Evaluation after [h]
Sex comp. reg. 24 4S 72
left flank right flank left flank right flank left fank right flank
males 1 46 0 0 0 0 0 0
2 54 0 0 0 0 0 0
3 5S 0 0 0 0 0 0
4 65 0 0 0 0 0 0
5 66 0 0 0 0 0 0
females 6 106 0 0 0 0 0 0
7 10S 0 0 0 0 0 0
8 124 0 0 0 0 0 0
9 126 0 0 0 0 0 0
10 135 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

Table 5. Main study - challenge - topical application (patch test) - treated group.
Test item concentration: 50% flank: right
Medium: aqua pro injectione flank: left
Date of application: 15.10. 2013
Date of removal: 16.10. 2013
Evaluation of skin reactions according to the grading scale of Magnusson and Kligman
(based on the OECD Guideline No. 406 /EU Method B.6.)
Sex Animal No. Evaluation after [h] Animals with/without allergic reactions (+/-) %of
sensi tized animals
comp. reg. 24 4S 72
left flank right flank left flank right flank left flank right flank
males 1 36 0 0 0 0 0 0 - 0%
2 39 0 0 0 0 0 0 -
3 40 0 0 0 0 0 0 -
4 42 0 0 0 0 0 0 -
5 43 0 0 0 0 0 0 -
6 44 0 0 0 0 0 0 -
7 47 0 0 0 0 0 0 -
8 51 0 0 0 0 0 0 -
g 52 0 0 0 0 0 0 -
10 67 0 0 0 0 0 0 -
females 11 10g 0 0 0 0 0 0 -
12 114 0 0 0 0 0 0 -
13 118 0 0 0 0 0 0 -
14 120 0 0 0 0 0 0 -
15 125 0 0 0 0 0 0 -
16 128 0 0 0 0 0 0 -
17 129 0 0 0 0 0 0  
18 130 0 0 0 0 0 0 -
19  131 0 0 0 0 0 0 -
20 133 0 0 0 0 0 0 -
0 - no vsible changes, 1 - discrete or patchy erythema, 2 - moderate or confluent erythema, 3 - intense erythema and swelling

Body weights of the animals

During the main study, body weight gain was stated in all animals. The average body weight gain was 127.2 g in the control males, 115.4 g in the treated males, 73.0 g in the control females, and 77.3 g in the treated females .

Table 6. Main study - body weights [g] - control group.
Sex Animal No. Day of experiment Body weight gain [g]
comp. reg. 0 final
males 1 46 530 657 127
2 54 493 604 111
3 58 478 613 135
4 65 535 668 133
5 66 518 648 130
females 6 106 473 560 87
7 108 437 500 63
8 124 447 534 87
9 126 452 522 70
10 1135 425 483 58
 
Average body weight [g] Average body
  initial final weight gain [g]
males 510.8 638.0 127.2
femals 446.8 519.8 73.0

Table 7. Main study - body weights [g] - treated group.
Animal No. Day of experiment Body weight gain [g]
Sex comp. reg. 0 final
males 1 36 491 612 121
2 39 493 640 147
3 40 476 552 76
4 42 548 708 160
5 43 517 625 108
6 44 486 591 105
7 47 467 567 100
8 51 550 732 182
9 52 532 628 96
10 67 546 605 59
females 11 109 469 538 69
12 114 428 497 69
13 118 418 513 95
14 120 388 459 71
15 125 444 486 42
16 128 487 593 106
17 129 447 534 87
18 130 481 550 69
19 131 398 493 95
20 133 496 566 70
 
Average body weight [g] Average body
  initial final weight gain [g]
males 510.6 626.0 115.4
femals 445.6 522.9 77.3
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item Mn (II) IDHA caused no sensitization in animals. On the ground of the study Mn (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 Mn (II) IDHA in Dunkin Hartley guinea pigs (Kropidło, 2013, Study Code Al 9/13, according to OECD 406). The experiment commenced with a pilot study which allowed for the determination of the test item concentrations to be used in the main study. These were as follows: 15% causing mild skin changes at stage I (induction - intradermal injections), 50% causing no skin changes at stage II (induction – topical application), and 50% causing no skin changes at stage III (challenge – topical application). Because the test item was a solid, the maximum concentration of the test item applied to the skin could be 50%. There were 20 animals in the treated group and 10 animals in the control group used in the main study. The main study was comprised of three parts: a two-stage induction phase and a challenge phase. At stage I of the main study, the treated animals were given intradermal injections containing a 15% aqueous solution of the test item with Freund’s Complete Adjuvant (FCA). At stage II of the main study, a 50% aqueous solution of the test item was applied to the skin in the sites of the intradermal injections. Since the 50% aqueous solution did not cause skin irritation, on the day before stage II of the main study 10% sodium lauryl sul fate in vaseline was applied in the sites of the intradermal injections in order to create a local skin irritation.

During the induction period, the control group animals were subjected to sham treatment. In order to challenge sensitization, a 50% aqueous solution of the test item was applied to the right flanks of the treated and the control animals. The medium was applied to the left flanks. Following the challenge, i.e. 24, 48, and 72 hours after the end of the exposure, the treated and the control animals were observed for skin changes. General clinical observations were performed during the main study. Detailed skin observations were conducted 24, 48, and 72 hours after the end of the exposure. Body weights of the animals were determined on day 0 and on the day of the experiment termination (before euthanasia). All animals survived the experiment. After the observation period, the animals were euthanized.

During all readings, the treated animals did not exhibit any allergic skin reactions. In the cont rol group animals, no pathological skin changes were stated. During the experiment, the observed animals did not exhibit any general clinical signs. After the end of the experiment, body weight gain was stated in all animals. Based on the study results, the test item, i.e. Mn (II) IDHA can be classified as an agent causing no sensitization – according to the Magnusson and Kligman classification.

Reason / purpose:
read-across: supporting information
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A substantial body of evidence exists that the toxicity profiles of chelates depends mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body/environment. The source substance has the same chelating agent as in a target substance (MgNa2IDHA). The only difference between the target and the source substance is presence of magnessium (Mg) cation instead Zn2+ cations. As magnessium is an essential micro element required by all forms of life, is considered not to influence the toxicological activity.
Detailed information about the substances purity and data matric are available in RA Statement (IUCLID 13.2)
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
according to
Guideline:
other: SPR/T/24
GLP compliance:
yes (incl. certificate)
Remarks:
G-024
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
W doswiadczeniu wstepnym i głównym uŻyto samców i samic swinek morskich Dankin
Hartley, pochodzacych z Hodowli Zwierzat Laboratoryjnych w Ilkowicach, utrzymanej w typie
konwencjonalnym.

Zwierzeta przeszły 5-dniowa kwarantanne, w czasie której były codziennie obserwowane
[SPR/T/6]. W dniu przyjecia zwierzat do kwarantanny wykonano ogólne badanie lekarsko -
weterynaryjne, a przed wprowadzeniem do doswiadczenia u zwierzat wykonano szczegółowe
badanie lekarsko-weterynaryjne [SPR/T/46]. Do doswiadczenia wprowadzono zwierzeta nie
wykazujace żadnych objawów klinicznych.
Wszystkie zwierzeta były indywidualnie oznaczone [SPR/T/7].
W doswiadczeniu wstepnym użyto dwóch samców o sredniej masie ciała 596,5 g oraz
dwie samice o sredniej masie ciała 465 g. Zwierzeta w badaniu wstepnym były w wieku ok. 3
miesiecy. W doswiadczeniu własciwym użyto 3 samców w wieku ok. 3,5 miesiaca i sredniej
masie ciała 573 g oraz 19 samic w wieku ok. 3,5 miesiaca i sredniej masie ciała 489,5 g.
Route:
intradermal
Vehicle:
water
Concentration / amount:
Dla ustalenia steżenia do I etapu indukowania uczulenia - wstrzykniecia sródskórne, u
dwóch swinek morskich wygolono siersc w okolicy przedniej czesci grzbietu na powierzchni ok.
4,5 x 6 cm i wykonano wstrzykniecia sródskórne po 0,1 ml roztworu wodnego badanego
materiału. Testowano nastepujace ste>enia: 0,5%; 1%; 2%; 4% oraz 8%. Obserwacji odczynów
skórnych dokonano po 24, 48, 72, 96 i 120 godzinach od wstrzykniec. Wyniki obserwacji
zamieszczono w tabelach 1 - 2.
Do doswiadczenia głównego wybrano ste>enie 4%, dajace łagodne do umiarkowanego
działanie na skóre.
Route:
intradermal
Vehicle:
water
Concentration / amount:
Dla ustalenia steżenia do I etapu indukowania uczulenia - wstrzykniecia sródskórne, u
dwóch swinek morskich wygolono siersc w okolicy przedniej czesci grzbietu na powierzchni ok.
4,5 x 6 cm i wykonano wstrzykniecia sródskórne po 0,1 ml roztworu wodnego badanego
materiału. Testowano nastepujace ste>enia: 0,5%; 1%; 2%; 4% oraz 8%. Obserwacji odczynów
skórnych dokonano po 24, 48, 72, 96 i 120 godzinach od wstrzykniec. Wyniki obserwacji
zamieszczono w tabelach 1 - 2.
Do doswiadczenia głównego wybrano ste>enie 4%, dajace łagodne do umiarkowanego
działanie na skóre.
Positive control substance(s):
yes
Reading:
other: nn
Hours after challenge:
0
Group:
other: nn
Dose level:
0
No. with + reactions:
0
Total no. in group:
0
Clinical observations:
0
Remarks on result:
other: Reading: other: nn. . Hours after challenge: 0.0. Group: other: nn. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 0.0. Clinical observations: 0.
Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
PL:Na podstawie przeprowadzonego badania, biodegradowalny nawóz Zn (II) IDHA można
zaliczyc do substancji nie wykazujacych działania uczulajacego na skóre swinek morskich.
Executive summary:

PL:W trakcie odczytów po 24, 48 i 72 godzinach od zakonczenia narażania, na skórze zwierzat z grupy kontrolnej, w miejscu nałożenia badanego materiału, nie stwierdzono zmian patologicznych. W miejscu nałożenia medium również nie stwierdzono zmian patologicznych na skórze (tabela 5). W trakcie odczytu po 24, 48 i 72 godzinach od zakonczenia narażania, na skórze zwierzat grupy narażanej, w miejscu nałożenia badanego materiału nie stwierdzono zmian patologicznych. W miejscu nałożenia medium również nie stwierdzono zmian patologicznych (tabela 6). W oparciu o uzyskane wyniki można stwierdzic, że u zwierzat grupy narażanej nie wystapiły uczuleniowe odczyny skórne (tabela 6). Indywidualne masy ciała zwierzat narażanych i kontrolnych zestawiono w tabelach 7 i 8. W trakcie doswiadczenia stwierdzono niewielki spadek masy ciała u 1 samca z grupy kontrolnej oraz u 2 samic z grupy narażanej. U pozostałych zwierzat stwierdzono przyrost masy ciała. Sredni przyrost masy ciała samców grupy kontrolnej i grupy narażanej wyraznie sie różnił, ale jest to zwiazane z mała liczby samców w poszczególnych grupach oraz niewielkim spadkiem masy ciała samca grupy kontrolnej. Sredni przyrost masy ciała samic w grupie kontrolnej i grupie narażanej był na podobnym poziomie. W trakcie doswiadczenia u zwierzat grupy kontrolnej i narażanej nie obserwowano zmian w zachowaniu sie oraz zmian klinicznych.

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was conducted in 1997 before LLNA method become obligatory.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Hsd Poc:DH
Sex:
male
Details on test animals and environmental conditions:
Species and Strain

The animals used were SPF-bred guinea pigs of the strain Hsd Poc:DH supplied by the Harlan Winkelmann GmbH Laboratory Animal Breeders in 33176 Borchen. A total of 32 male animals were used in the study, including those employed in the range-finding test to determine the challenge concentration. In addition, 5 małe animals were used to determine the induction concentrations.
Appropriate sensitivity of this strain of guinea pig for sensitization tests and appropriate reliability of the experimental technique have been verified [3a, 3b] and are checked at regular intervals. The pertinent records are filed in the Bayer AG Fachbereich Toxikologie archive.

Acclimatization
Following receipt, the animals scheduled for this study were adapted to the maintenance conditions for a period of at least seven days before treatment, and their state of health was monitored.

State of Health
Only healthy animals exhibiting no clinical signs were used for the study. The animals were not vaccinated or treated against infections either before receipt, or during the adaptation or study periods.

Age and Body Weight
The guinea pigs exhibited a mean weight of 321 - 361 grams at the beginning of the study (see page 25). This weight corresponds to an age of 4 -6 weeks.

Animal Accommodation
During the adaptation and study period the animals were conventionally kept in type IV Makrolon® cages [4], in groups of five during the adaptation period and in groups of two or three per cage throughout the study period. The cages were exchanged for ones with clean bedding two times per week.
Low-dust wood shavings supplied by Ssniff Spezialdiaten GmbH, Soest, were used as bedding. The wood shavings were spotchecked for contaminant levels. Records of these test results are filed at BAYER AG. The analytical results provided no evidence for an effect on the study objective.

Animal Rooms
All animals used in this study were kept in the same animal room, one in which guinea pigs from other sensitization studies were also housed. Adequate separation, unambiguous cage markings and suitable organization of the week ensured that animals were not mixed up.

Cleaning. Disinfection
The animal room was swept out with a broom each day, and thoroughly cleaned with water once weekly. The room was disinfected at least once each month with Killavon® (1O grams benzalkonium chloride per 100 grams disinfectant; working dilution 1%). Contamination of the diet and contact with the experimental animals were avoided during this werk. No pest control measures were carried out in the animal rooms. KILLIGERM Roach Traps ®, a cockroach trap which uses no pesticides, was placed in the animal room.

Climatic Conditions

The room climate in the animal room was set as fellows:
Room temperatura:

Relative humidity: lighUdark cycle: Air exchange rate:

22 +/- 3°C (possibly drifting higher at outdoor temperatures above 24°C)
40-60%
Twelve hours; artificial lighting from 6 AM to 6 PM
>= 1O times per hour

Occasional deviations from these standards occurred at times including the period following the cleaning of the animal room. They had no apparent effect in the course of the study.

Diet
The diet consisted of "Altromin®3020 - Maintenance Diet for Guinea Pigs" supplied by Altromin GmbH in Lage, and of tap water. Food and water were provided ad libitum. The nutritive composition of Altromin®3020 is listed in the Annex on page
38. Pellets with a diameter of 3 mm (Altromin® 3022) were fed.
Two 750-ml polycarbonate bottles per cage were used for water during the test period [4, 5]. The tap water met drinking water standards [6]. Records of the analyses to monitor compliance with the drinking water specification are filed at Bayer AG. The available data provided no evidence for an effect on the study objective.

Results and discussion

Positive control results:
Using 2-mercaptobenzothiazole formulated with physiological NaCI, containing 2 % v/v Cremophor EL® the methodological reliability of the maximization test and the sensitivity of the strain was checked on female guinea pigs using concentrations as follows:

For the intradermal induction a 2.5% test substance formulation was used, and for the topical induction a 40% formulation.

After the challenge with a 40% test substance formulation 60% of the test animals exhibited dermal reactions. There was no reddening of the skin to be observed on control group animals.

The sensitivity as well as the reliability of the experimental technique is thus confirmed by this study.

The sensitivity of the used animal strain was also examined by a Buehler patch test performed in male guinea pigs using alpha-Hexylzimtaldehyd formulated in propylene glycol 400 with an epicutaneous induction concentration of 40% and a challenge concentration of 20%. Of the substance-group animals 65% exhibited dermal reactions following challenge with 20% test substance formulation. In the control group none of the animals reacted with skin effects with the 20% test substance formulations.

The sensitivity as well as the reliability of the experimental technique is thus confirmed by this study.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
0.5 ml 25% lminodisuccinat, Na-Salz formulated in demineralized water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Appearance and behaviour of the test substance group were not different from the control groups. At the end of the study, the body weight development of the treatment group animals corresponded to that of the control group animaIs
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
0.5 ml 25% lminodisuccinat, Na-Salz formulated in demineralized water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Appearance and behaviour of the test substance group were not different from the control groups. At the end of the study, the body weight development of the treatment group animals corresponded to that of the control group animaIs
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.5 ml demineralized water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Appearance and behaviour of the test substance group were not different from the control groups. At the end of the study, the body weight development of the treatment group animals corresponded to that of the control group animaIs
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0.5 ml demineralized water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Appearance and behaviour of the test substance group were not different from the control groups. At the end of the study, the body weight development of the treatment group animals corresponded to that of the control group animaIs

Any other information on results incl. tables

 General Examinations

 Appearance and behaviour of the test substance group were not different from the control groups.

 At the end of the study, the body weight development of the treatment group animals corresponded to that of the control group animaIs.

 

  Test for Skin-Sensitizing Effect

The incidence of skin reactions following the challenge is summarized in the following table:

 

Table: Number of animals exhibiting skin effects

    Test substance group (20 animals)              Control group (10 animals)           
      Test substance patch     Control patch    Test substance patch     Control patch
Hours  48 72  total   48 72   48 72  total   48 72 
Chalange 20%  0

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the maximization test and with respect to the evaluation criteria the test substance therefore thus exhibits no skin-sensitization potential.

Executive summary:

The Guinea Pig Maximization Test (GPMT) was performed on male guinea pigs to determine whether the test substance lminodisuccinat, Na-Salz exhibits skin­ sensitizing properties.

 

The study was conducted with the following test substance concentrations:

 

 

lntradermal induction:

1%

Topical induction:

25%

Challenge:

20%

 

 

 

For the induction and the challenge treatment the test substance was formulated in demineralized water solution to yield a solution.

 

The challenge using a 20% test substance formulation led to no skin effects in the animals in the treatment group and in the animals in the control group.

 

In summary, under the conditions of the maximization test and with respect to the evaluation criteria the test substance therefore thus exhibits no skin-sensitization potentiaI.