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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
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:
Plase see the attachment.
Cross-reference
Reason / purpose for cross-reference:
read-across source

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
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 the Bureau for Chemical Substances
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of disodium [2,2'-(imino-kappaN)dibutanedioato-kappa2O1,O4(4-)]manganese(2-) and sodium sulphate
EC Number:
939-867-3
Cas Number:
666748-58-9
Molecular formula:
MnC8H7NO8Na2
IUPAC Name:
Reaction mass of disodium [2,2'-(imino-kappaN)dibutanedioato-kappa2O1,O4(4-)]manganese(2-) and sodium sulphate
Constituent 2
Reference substance name:
manganese chelate of sodium salt N-[1,2 dicarboxyethyl] D,L aspartic acid
IUPAC Name:
manganese chelate of sodium salt N-[1,2 dicarboxyethyl] D,L aspartic acid
Constituent 3
Reference substance name:
Mn (II) IDHA
IUPAC Name:
Mn (II) IDHA
Test material form:
other: granules solved in aqua pro injectione
Details on test material:
- Name of test material (as cited in study report): Mn (II) IDHA
- Substance type: chelate
- Physical state: solid (odourless, white microgranules)
- Composition of test material, percentage of components: 9.07 % of Cu2+, 56.7% of MnIDHA, 40% of Na2SO4, and 3% of H2O (humidity)
- Lot/batch No.: 7/13, produced in June, 2013
- Expiration date of the lot/batch: June, 2016
- Storage condition of test material: at temperature between 0 – 30°C

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: 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

Study design: in vivo (non-LLNA)

Inductionopen allclose all
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)
Challengeopen allclose all
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

Results and discussion

Positive control results:
The sensitivity and reliability of the procedure used to assess skin sensitization is monitored every six
months using a reference substance, i.e. mercaptobenzothiazole. The study was conducted between
13.03.2013 – 28.04.2013. The reagent’s purity was 97% (batch number: MKBH9652V; stability: until it is
used). It was produced by SIGMA-ALDRICH (Appendix 4). The following concentrations of the test item
were used: 1% causing mild skin changes at stage I (induction - intradermal injections) of the main study,
30% causing mild skin changes at stage II (induction – topical application), and 10% causing no skin
changes at stage III (challenge – topical application). During the readings, allergic skin reactions were
stated in nineteen treated animals, i.e. 95% of all treated animals. In the control group animals, no
pathological skin changes were stated.

In vivo (non-LLNA)

Resultsopen allclose all
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.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
No. with + reactions:
19
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
No. with + reactions:
19
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
positive control
No. with + reactions:
19
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.