Reaction mass of disodium [2,2'-(ethane-1,2-diylbis{[2-(hydroxy-kO)benzyl]imino-kN})diacetato-kO(4-)]zinc(2-) and sodium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 of August till 6 of December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well condcted and well documented study under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted in 2013 before the LLNA method has become prefered.

Test material

Test material information

Constituent 1

Reference substance name:

ZnHBED

IUPAC Name:

ZnHBED

Constituent 2

Reference substance name:

disodium [2,2'-(ethane-1,2-diylbis{[2-(hydroxy-kO)benzyl]imino-kN})diacetato-kO(4-)]zinc(2-)

Cas Number:

1263416-97-2

Molecular formula:

C20 H20 N2 Na2 O6 Zn

IUPAC Name:

disodium [2,2'-(ethane-1,2-diylbis{[2-(hydroxy-kO)benzyl]imino-kN})diacetato-kO(4-)]zinc(2-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

The experiment was conducted on the Dunkin-Hartley guinea pigs (outbred) coming from the husbandry of laboratory animals of the Charles River Laboratories in L’Arbresle cedex, France. Prior to the experiment, the animals were quarantined and observed daily for 5 days [SOP/T/6]. A general
medical-veterinary examination was performed on the day of the introduction of the animals to the quarantine, whereas a detailed medical-veterinary examination was performed prior to the beginning of the experiment [SOP/T/46]. Animals without any clinical signs were introduced to the experiment.
Two males whose average body weight was 489.5 g and two females whose average body weight was 476.0 g were used in the pilot study. They were 8-9 weeks old.
Fifteen males whose average body weight was 549.1 g and fifteen females whose average body weight was 466.6 g were used in the main study. They were 10 weeks old.

During the quarantine and the experiment, the animals were kept in ai r-conditioned rooms under the
following conditions:
- air temperature: 20 – 23 ºC;
- relative ai r humidity: 50 – 68%
- artificial fluorescent lighting; lighting cycle: 12 hours light/12 hours dark
- facility air exchange: about 16 times/hour [SOP/T/9].
The animals were individually housed 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.
The animals were given ad libitum access to the LSK standard granulated fodder and drinking tap water with ascorbic acid (a 0.6% solution). Water withascorbic acid was given to the animals during the quarantine period and the experiment. Ascorbic acid was not provided during skin observations taking place after stage III of the main study (challenge – topical application).

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Induction 2

Concentration / amount:

Pilot study:
I stage - 0,5%, 15, 2%
2%, 4%, 6%
II stage - 30% and 50%
405 and 50%

Main test: 50%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

water

Challenge 2

Concentration / amount:

Pilot study:
I stage - 0,5%, 15, 2%
2%, 4%, 6%
II stage - 30% and 50%
405 and 50%

Main test: 50%

No. of animals per dose:

Pilot study:
I stage - 0,5%, 15, 2% - 1 animal
2%, 4%, 6% - 1 animal
II stage - 30% and 50% - 1 animal
405 and 50% - 1 animal

Main test: 50% - 20 animals

Details on study design:

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: 4% 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 4% 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 int radermal injections. Since the 50% aqueous solution didnot cause skin irritation, on the day before stage II of the main study 10% sodium lauryl sulfate 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; it means that they were given a medium (aqua pro injectione) instead of the test item. In order to challenge sensitization, a 50% aqueous solution of the test item was applied to the right flanks of the treated andthe 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.

Positive control substance(s):

yes

Remarks:

mercaptobenzothiazole

Results and discussion

Positive control results:

Refrence test:
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 skinchanges 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)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no skin reaction

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no skin reaction.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no skin reaction

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no skin reaction.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

7

Total no. in group:

20

Clinical observations:

discrete or patchy erythema

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 7.0. Total no. in groups: 20.0. Clinical observations: discrete or patchy erythema.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

7

Total no. in group:

20

Clinical observations:

discrete or patchy erythema, skin dryness

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 7.0. Total no. in groups: 20.0. Clinical observations: discrete or patchy erythema, skin dryness.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

During the readings, allergic skin reactions were stated in seven treated animals, i.e. 35% of all treated animals. In the control group animals, no pathological skin changes were stated.
Taking the study results into consideration, the test item, i.e. Zn (II) HBED can be classified into the following categories:
- agents causing moderate sensitization – according to the Magnusson and Kligman classification,
- category 1, sub-category 1B – 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.